Sample records for wild type group
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Zheng, H; Peret, T C; Randolph, V B; Crowley, J C; Anderson, L J
1996-01-01
Candidate live-virus vaccines for respiratory syncytial virus are being developed and are beginning to be evaluated in clinical trials. To distinguish candidate vaccine strains from wild-type strains isolated during these trials, we developed PCR assays specific to two sets of candidate vaccine strains. The two sets were a group A strain (3A), its three attenuated, temperature-sensitive variant strains, a group B strain (2B), and its four attenuated, temperature-sensitive variant strains. The PCR assays were evaluated by testing 18 group A wild-type strains, the 3A strains, 9 group B wild-type strains, and the 2B strains. PCR specific to group A wild-type strains amplified only group A wild-type strains, and 3A-specific PCR amplified only 3A strains. PCR specific to group B wild-type strains amplified all group A and group B strains but gave a 688-bp product for group B wild-type strains, a 279-bp product for 2B strains, a 547-bp product for all group A strains, and an additional 688-bp product for some group A strains, including 3A strains. These types of PCR assays can, in conjunction with other methods, be used to efficiently distinguish candidate vaccine strains from other respiratory syncytial virus strains. PMID:8789010
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fat-1 mice prevent high-fat plus high-sugar diet-induced non-alcoholic fatty liver disease.
Guo, Xiao-Fei; Gao, Jin-Long; Li, Jiao-Mei; Li, Duo
2017-11-15
High-fat and high-sugar (HFS) diets have been suggested to play a causal role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). This study aimed to investigate whether fat-1 transgenic mice with a higher tissue content of n-3 polyunsaturated fatty acids (PUFAs) could prevent HFS diet-induced NAFLD, compared with wild-type mice. The fat-1 and wild-type littermates had free access to a 15% fructose solution plus high-fat diet, a 15% glucose solution plus high-fat diet, or a 15% sucrose solution plus high-fat diet, respectively. Caloric intake, weight gain, biochemical parameters, histology, and gene and protein expression levels were measured after 8 weeks of intervention. Liquid intake in glucose- or sucrose-fed mice was about 2-fold compared with that in fructose-fed mice. The wild-type mice given glucose showed the highest total caloric intake and weight gain compared to the other groups. The serum concentrations of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and alanine transaminase (ALT) were significantly lowered in fat-1 groups compared with their paired wild-type groups. Histological analysis showed that the wild-type groups fed the HFS diets developed hepatic lipid accumulation and steatosis, compared with the fat-1 groups. The gene and protein expression levels involved in fatty acid synthesis and the toll-like receptor (TLR)-4 signaling pathway were significantly inhibited in the fat-1 groups compared with the wild-type groups. The endogenously synthesized n-3 PUFAs of the three fat-1 groups, which inhibit fatty acid synthesis and the TLR-4 signaling pathway, prevent HFS diet-induced NAFLD.
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[Upregulation of P2X3 receptors in dorsal root ganglion of TRPV1 knockout female mice].
Fang, Xiao; Shi, Xiao-Han; Huang, Li-Bin; Rong, Wei-Fang; Ma, Bei
2014-08-25
The study was aimed to investigate the changes in mechanical pain threshold in the condition of chronic inflammatory pain after transient receptor potential vanilloid 1 (TRPV1) gene was knockout. Hind-paw intraplantar injection of complete freund's adjuvant (CFA, 20 μL) produced peripheral inflammation in wild-type and TRPV1 knockout female mice. The mechanical pain thresholds were measured during the 8 days after injection and pre-injection by using Von-Frey hair. Nine days after injection, mice were killed and the differences of expression of c-Fos and P2X3 receptor in the dorsal root ganglia (DRG) and spinal cord dorsal horn were examined by Western blotting between the two groups. Compared with that in wild-type mice, the mechanical pain threshold was increased significantly in TRPV1 knockout mice (P < 0.05); 3 days after CFA injection, the baseline mechanical pain threshold in the TRPV1 knockout mice group was significantly higher than that in the wild-type mice group (P < 0.05); The result of Western blotting showed that the expression of c-Fos protein both in DRG and spinal cord dorsal horn of TRPV1 knockout mice group was decreased significantly compared with that in wild-type mice group (P < 0.01, P < 0.05), while the expression of P2X3 receptor in DRG of TRPV1 knockout mice group was increased significantly compared with that in wild-type mice group (P < 0.05). Our findings indicate that TRPV1 may influence the peripheral mechanical pain threshold by mediating the expression of c-Fos protein both in DRG and spinal cord dorsal horn and changing the expression of P2X3 receptor in DRG.
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Miyabara, Elen H; Nascimento, Tabata L; Rodrigues, Débora C; Moriscot, Anselmo S; Davila, Wilmer F; AitMou, Younss; deTombe, Pieter P; Mestril, Ruben
2012-04-01
Heat shock proteins play a key regulatory role in cellular defense. To investigate the role of the inducible 70-kDa heat shock protein (HSP70) in skeletal muscle atrophy and subsequent recovery, soleus (SOL) and extensor digitorum longus (EDL) muscles from overexpressing HSP70 transgenic mice were immobilized for 7 days and subsequently released from immobilization and evaluated after 7 days. Histological analysis showed that there was a decrease in cross-sectional area of type II myofiber from EDL and types I and II myofiber from SOL muscles at 7-day immobilization in both wild-type and HSP70 mice. At 7-day recovery, EDL and SOL myofibers from HSP70 mice, but not from wild-type mice, recovered their size. Muscle tetanic contraction decreased only in SOL muscles from wild-type mice at both 7-day immobilization and 7-day recovery; however, it was unaltered in the respective groups from HSP70 mice. Although no effect in a fatigue protocol was observed among groups, we noticed a better contractile performance of EDL muscles from overexpressing HSP70 groups as compared to their matched wild-type groups. The number of NCAM positive-satellite cells reduced after immobilization and recovery in both EDL and SOL muscles from wild-type mice, but it was unchanged in the muscles from HSP70 mice. These results suggest that HSP70 improves structural and functional recovery of skeletal muscle after disuse atrophy, and this effect might be associated with preservation of satellite cell amount.
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Rocha, Natália Galito; Neves, Fabricia Junqueira; Silva, Bruno Moreira; Sales, Allan Robson Kluser; Nóbrega, Antonio Claudio
2012-03-01
Nitric oxide is the primary mediator of vasodilation during mental stress. Since genetic polymorphisms in the nitric oxide synthase (eNOS) gene seem to impair the production of NO, this study aimed to evaluate the effect of an exercise bout on hemodynamic responses to mental stress in subjects with the 894G>T polymorphism of eNOS. Subjects without (wild-type group; n = 16) or with (polymorphic-type group; n = 19) the 894G>T polymorphism underwent a mental stress challenge before and after a maximal cardiopulmonary exercise test. Blood pressure was measured by auscultation and forearm blood flow by venous occlusion plethysmography. The groups were similar regarding anthropometric, metabolic, resting blood pressure and exercise variables. Before exercise, systolic blood pressure response during mental stress was higher in the polymorphic-type group (∆wild-type: 8.0 ± 2.0% vs. ∆polymorphic-type: 12.5 ± 1.8%, P = 0.01), while the increase in forearm vascular conductance was similar between the groups (∆wild-type 90.8 ± 26.4% vs. ∆polymorphic-type: 86.3 ± 24.1%, P = 0.44). After exercise, the systolic blood pressure at baseline and during mental stress was lower than before exercise in the whole group (P < 0.05), but the pressure response during mental stress was still higher in the polymorphic-type group (∆wild-type: 5.8 ± 1.5% vs. ∆polymorphic-type: 10.2 ± 1.4%, P = 0.01). The increase in forearm vascular conductance was inhibited only in the polymorphic-type group (∆before exercise 86.3 ± 24.1% vs. ∆after exercise: 41.5 ± 12.6%, P = 0.04). In conclusion, these results suggest the 894G>T eNOS polymorphism is associated with altered hemodynamic responses to mental stress both before and after a single bout of dynamic exercise with potential clinical implications.
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White, Samantha L; Sakhrani, Dionne; Danzmann, Roy G; Devlin, Robert H
2013-10-02
Release of domesticated strains of fish into nature may pose a threat to wild populations with respect to their evolved genetic structure and fitness. Understanding alterations that have occurred in both physiology and genetics as a consequence of domestication can assist in evaluating the risks posed by introgression of domesticated genomes into wild genetic backgrounds, however the molecular causes of these consequences are currently poorly defined. The present study has examined levels of mRNA in fast-growing pure domesticated (D), slow-growing age-matched pure wild (Wa), slow-growing size-matched pure wild (Ws), and first generation hybrid cross (W/D) rainbow trout (Oncorhynchus mykiss) to investigate the influence of genotype (domesticated vs. wild, and their interactions in hybrids) and developmental stage (age- or size-matched animals) on genetic responses (i.e. dominant vs. recessive) and specific physiological pathways. Highly significant differences in mRNA levels were found between domesticated and wild-type rainbow trout genotypes (321 mRNAs), with many mRNAs in the wild-domesticated hybrid progeny showing intermediate levels. Differences were also found between age-matched and size-matched wild-type trout groups (64 mRNAs), with unique mRNA differences for each of the wild-type groups when compared to domesticated trout (Wa: 114 mRNAs, Ws: 88 mRNAs), illustrating an influence of fish developmental stage affecting findings when used as comparator groups to other genotypes. Analysis of differentially expressed mRNAs (found for both wild-type trout to domesticated comparisons) among the genotypes indicates that 34.8% are regulated consistent with an additive genetic model, whereas 39.1% and 26.1% show a recessive or dominant mode of regulation, respectively. These molecular data are largely consistent with phenotypic data (growth and behavioural assessments) assessed in domesticated and wild trout strains. The present molecular data are concordant with domestication having clearly altered rainbow trout genomes and consequent phenotype from that of native wild populations. Although mainly additive responses were noted in hybrid progeny, the prevalence of dominant and non-additive responses reveals that introgression of domesticated and wild genotypes alters the type of genetic control of mRNA levels from that of wild-type, which may lead to disruption of gene regulation systems important for developing phenotypes for optimal fitness in nature. A clear influence of both fish age and size (developmental stage) on mRNA levels was also noted in this study, which highlights the importance of examining multiple control samples to provide a comprehensive understanding of changes observed between strains possessing differences in growth rate.
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Antonio de Luis, Daniel; Sagrado, Manuel Gonzalez; Aller, Rocio; Conde, Rosa; Izaola, Olatz; de la Fuente, Beatriz; Primo, David
2012-03-01
A silent intragenic polymorphism (1359 G/A) of the cannabinoid receptor 1 gene resulting in the substitution of the G to A at nucleotide position 1359 in codon 435 (Thr) was reported as a common polymorphism in Caucasian populations. Intervention studies with this polymorphism have not been realized. We decide to investigate the role of missense polymorphism (G1359A) of cannabinoid receptor 1 gene on adipocytokines response and weight loss secondary to a low-fat versus a low-carbohydrate diet in obese patients. A population of 249 patients was analyzed. A nutritional evaluation was performed at the beginning and at the end of a 3-month period in which subjects received one of two diets (diet I: low fat vs. diet II: low carbohydrate). One hundred forty three patients (57.4%) had the genotype G1359G (wild-type group), and 106 (42.6%) patients had G1359A (92 patients, or 36.9%) or A1359A (14 patients, or 5.6%; mutant-type group). With both diets in wild-type and mutant-type groups, body mass index (BMI), weight, fat mass, waist circumference and systolic blood pressure levels decreased. With both diets and in wild-type group, glucose, total cholesterol and insulin levels and homeostasis model assessment test score decreased. No metabolic effects were observed in mutant-type group. Leptin levels decreased significantly in the wild-type group with both diets (diet I: 10.8% vs. diet II: 28.9%; P<.05). The novel finding of this study is the lack of metabolic improvement of the mutant-type groups G1359A and A1359A after weight loss with both diets. Decrease in leptin level was higher with low-carbohydrate diet than low-fat diet. Copyright © 2012 Elsevier Inc. All rights reserved.
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Characterizing genomic differences of human cancer stratified by the TP53 mutation status.
Wang, Mengyao; Yang, Chao; Zhang, Xiuqing; Li, Xiangchun
2018-06-01
The key roles of the TP53 mutation in cancer have been well established. TP53 is the most frequently mutated gene, and its inactivation is widespread among human cancer types. However, the landscape of genomic alterations in human cancers stratified by the TP53 mutation has not yet been described. We obtained somatic mutation and copy number change data of 6551 regular-mutated samples from the Cancer Genome Atlas (TCGA) and compared significantly mutated genes (SMGs), copy number alterations, mutational signatures and mutational strand asymmetries between cancer samples with and without the TP53 mutation. We identified 126 SMGs, 30 of which were statistically significant in both the TP53 mutant and wild-type groups. Several SMGs, such as VHL, SMAD4 and PTEN, showed a mutation bias towards the TP53 wild-type group, whereas ATRX, IDH1 and RB1 were more prevalent in the TP53 mutant group. Five mutational signatures were extracted from the combined TCGA dataset on which mutational asymmetry analysis was performed, revealing that the TP53 mutant group exhibited substantially greater replication and transcription biases. Furthermore, we found that alterations of multiple genes in a merged mutually exclusive network composed of BRAF, EGFR, PAK1, PIK3CA, PTEN, APC and TERT were related to shortened survival in the TP53 wild-type group. In summary, we characterized the genomic differences and similarities underlying human cancers stratified by the TP53 mutation and identified multi-gene alterations of a merged mutually exclusive network to be a poor prognostic factor for the TP53 wild-type group.
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NADPH Oxidase Inhibition Improves Neurological Outcomes in Surgically-Induced Brain Injury
Lo, Wendy; Bravo, Thomas; Jadhav, Vikram; Zhang, John H.; Tang, Jiping
2007-01-01
Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91phox subunit of NADPH oxidase (gp91phox KO) and apocynin, a specific inhibitor of NADPH oxidase. Neurological function and brain edema were evaluated at 24 hours post-SBI in gp91phox KO and wild-type littermates grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into vehicle- and apocynin-treated (5mg/kg, i.p. 30 minutes before SBI) groups. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 hours post SBI. The gp91phox KO mice, but not the apocynin-treated mice showed significantly improved neurological scores. Brain edema was observed in both gp91phox KO and wild-type groups after SBI; however, there was no significant difference between these two groups. Brain edema was also not affected by apocynin-pretreatment. LPO levels were significantly higher in SBI group in both gp91phox KO and wild-type groups as compared to sham group. A trend, although without statistical significance, was noted towards attenuation of LPO in the gp91phox KO animals as compared to wild-type group. LPO levels were significantly attenuated at 3 hours post-SBI by apocynin pretreatment but not at 24 hours post-SBI. These results suggest that chronic and acute inhibition of NADPH oxidase activity does not reduce brain edema after SBI. Long-term inhibition of NADPH oxidase, however improves neurological functions after SBI. PMID:17317004
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Stimulus control by 5methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice
Winter, J. C.; Amorosi, D. J.; Rice, Kenner C.; Cheng, Kejun; Yu, Ai-Ming
2011-01-01
In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT. PMID:21624387
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Spontaneous Gac Mutants of Pseudomonas Biological Control Strains: Cheaters or Mutualists? ▿
Driscoll, William W.; Pepper, John W.; Pierson, Leland S.; Pierson, Elizabeth A.
2011-01-01
Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are “cheaters” that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions. PMID:21873476
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Atp1a3-deficient heterozygous mice show lower rank in the hierarchy and altered social behavior.
Sugimoto, H; Ikeda, K; Kawakami, K
2018-06-01
Atp1a3 is the Na-pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3-deficient heterozygous mice (Atp1a3 +/- ) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3 +/- and compared the rank and hierarchy structure between Atp1a3 +/- and wild-type mice within a housing cage using the round-robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3 +/- than wild-type mice, and Atp1a3 +/- approached Atp1a3 +/- mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3 +/- group than in the wild-type group, while no difference was observed in the mixed-genotype housing condition. (3) Hierarchy formation was not different between Atp1a3 +/- and wild type. (4) Atp1a3 +/- showed a lower rank in the mixed-genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3 +/- showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.
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Effects of Elevated Pax6 Expression and Genetic Background on Mouse Eye Development
Chanas, Simon A.; Collinson, J. Martin; Ramaesh, Thaya; Dorà, Natalie; Kleinjan, Dirk A.; Hill, Robert E.; West, John D.
2009-01-01
Purpose To analyze the effects of Pax6 overexpression and its interaction with genetic background on eye development. Methods Histologic features of eyes from hemizygous PAX77+/− transgenic (high Pax6 gene dose) and wild-type mice were compared on different genetic backgrounds. Experimental PAX77+/−↔wild-type and control wild-type↔wild-type chimeras were analyzed to investigate the causes of abnormal eye development in PAX77+/− mice. Results PAX77+/− mice showed an overlapping but distinct spectrum of eye abnormalities to Pax6+/− heterozygotes (low Pax6 dose). Some previously reported PAX77+/− eye abnormalities did not occur on all three genetic backgrounds examined. Several types of eye abnormalities occurred in the experimental PAX77+/−↔wild-type chimeras, and they occurred more frequently in chimeras with higher contributions of PAX77+/− cells. Groups of RPE cells intruded into the optic nerve sheath, indicating that the boundary between the retina and optic nerve may be displaced. Both PAX77+/− and wild-type cells were involved in this ingression and in retinal folds, suggesting that neither effect was cell-autonomous. Cell-autonomous effects included failure of PAX77+/− and wild-type cells to mix normally and overrepresentation of PAX77+/− in the lens epithelium and RPE. Conclusions The extent of PAX77+/− eye abnormalities depended on PAX77+/− genotype, genetic background, and stochastic variation. Chimera analysis identified two types of cell-autonomous effects of the PAX77+/− genotype. Abnormal cell mixing between PAX77+/− and wild-type cells suggests altered expression of cell surface adhesion molecules. Some phenotypic differences between PAX77+/−↔wild-type and Pax6+/−↔wild-type chimeras may reflect differences in the levels of PAX77+/− and Pax6+/− contributions to chimeric lenses. PMID:19387074
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Farchoukh, Lama; Kuan, Shih-Fan; Dudley, Beth; Brand, Randall; Nikiforova, Marina; Pai, Reetesh K
2016-10-01
Between 10% and 15% of colorectal carcinomas demonstrate sporadic DNA mismatch-repair protein deficiency as a result of MLH1 promoter methylation and are thought to arise from sessile serrated adenomas, termed the serrated neoplasia pathway. Although the presence of the BRAF V600E mutation is indicative of a sporadic cancer, up to 30% to 50% of colorectal carcinomas with MLH1 promoter hypermethylation will lack a BRAF mutation. We report the clinicopathologic and molecular features of MLH1-deficient colorectal carcinoma with wild-type BRAF and MLH1 promoter hypermethylation (referred to as MLH1-hypermethylated BRAF wild-type colorectal carcinoma, n=36) in comparison with MLH1-deficient BRAF-mutated colorectal carcinoma (n=113) and Lynch syndrome-associated colorectal carcinoma (n=36). KRAS mutations were identified in 31% of MLH1-hypermethylated BRAF wild-type colorectal carcinomas compared with 0% of MLH1-deficient BRAF-mutated colorectal carcinomas and 37% of Lynch syndrome-associated colorectal carcinomas. When a precursor polyp was identified, MLH1-hypermethylated BRAF wild-type colorectal carcinomas arose from precursor polyps resembling conventional tubular/tubulovillous adenomas in contrast to MLH1-deficient BRAF-mutated colorectal carcinomas, which arose from precursor sessile serrated adenomas (P<0.001). Both MLH1-hypermethylated BRAF wild-type colorectal carcinoma and MLH1-deficient BRAF-mutated colorectal carcinoma had a predilection for the right colon compared with Lynch syndrome-associated colorectal carcinoma (86% vs. 92% vs. 49%, P<0.001). There was no significant difference in mucinous differentiation, tumor-infiltrating lymphocytes, Crohn-like reaction, and medullary differentiation between the 3 tumor groups. Using Kaplan-Meier survival functions, there was no significant difference in disease-specific survival between the 3 patient groups (P>0.05). In conclusion, our results indicate that MLH1-hypermethylated BRAF wild-type colorectal carcinomas can harbor KRAS mutations and arise from precursor polyps resembling conventional tubular/tubulovillous adenomas.
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Kelly, Lorna; Bryan, Kenneth; Kim, Su Young; Janeway, Katherine A.; Killian, J. Keith; Schildhaus, Hans-Ulrich; Miettinen, Markku; Helman, Lee; Meltzer, Paul S.; van de Rijn, Matt; Debiec-Rychter, Maria; O’Sullivan, Maureen
2013-01-01
In contrast to adult mutant gastrointestinal stromal tumors [GISTs], pediatric/wild-type GISTs remain poorly understood overall, given their lack of oncogenic activating tyrosine kinase mutations. These GISTs, with a predilection for gastric origin in female patients, show limited response to therapy with tyrosine kinase inhibitors and generally pursue a more indolent course, but still may prove fatal. Defective cellular respiration appears to underpin tumor development in these wild-type cases, which as a group lack expression of succinate dehydrogenase [SDH] B, a surrogate marker for respiratory chain metabolism. Yet, only a small subset of the wild-type tumors show mutations in the genes coding for the SDH subunits [SDHx]. To explore additional pathogenetic mechanisms in these wild-type GISTs, we elected to investigate post-transcriptional regulation of these tumors by conducting microRNA (miRNA) profiling of a mixed cohort of 73 cases including 18 gastric pediatric wild-type, 25 (20 gastric, 4 small bowel and 1 retroperitoneal) adult wild-type GISTs and 30 gastric adult mutant GISTs. By this approach we have identified distinct signatures for GIST subtypes which correlate tightly with clinico-pathological parameters. A cluster of miRNAs on 14q32 show strikingly different expression patterns amongst GISTs, a finding which appears to be explained at least in part by differential allelic methylation of this imprinted region. Small bowel and retroperitoneal wild-type GISTs segregate with adult mutant GISTs and express SDHB, while adult wild-type gastric GISTs are dispersed amongst adult mutant and pediatric wild-type cases, clustering in this situation on the basis of SDHB expression. Interestingly, global methylation analysis has recently similarly demonstrated that these wild-type, SDHB-immunonegative tumors show a distinct pattern compared with KIT and PDGFRA mutant tumors, which as a rule do express SDHB. All cases with Carney triad within our cohort cluster together tightly. PMID:23717541
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Kelly, Lorna; Bryan, Kenneth; Kim, Su Young; Janeway, Katherine A; Killian, J Keith; Schildhaus, Hans-Ulrich; Miettinen, Markku; Helman, Lee; Meltzer, Paul S; van de Rijn, Matt; Debiec-Rychter, Maria; O'Sullivan, Maureen
2013-01-01
In contrast to adult mutant gastrointestinal stromal tumors [GISTs], pediatric/wild-type GISTs remain poorly understood overall, given their lack of oncogenic activating tyrosine kinase mutations. These GISTs, with a predilection for gastric origin in female patients, show limited response to therapy with tyrosine kinase inhibitors and generally pursue a more indolent course, but still may prove fatal. Defective cellular respiration appears to underpin tumor development in these wild-type cases, which as a group lack expression of succinate dehydrogenase [SDH] B, a surrogate marker for respiratory chain metabolism. Yet, only a small subset of the wild-type tumors show mutations in the genes coding for the SDH subunits [SDHx]. To explore additional pathogenetic mechanisms in these wild-type GISTs, we elected to investigate post-transcriptional regulation of these tumors by conducting microRNA (miRNA) profiling of a mixed cohort of 73 cases including 18 gastric pediatric wild-type, 25 (20 gastric, 4 small bowel and 1 retroperitoneal) adult wild-type GISTs and 30 gastric adult mutant GISTs. By this approach we have identified distinct signatures for GIST subtypes which correlate tightly with clinico-pathological parameters. A cluster of miRNAs on 14q32 show strikingly different expression patterns amongst GISTs, a finding which appears to be explained at least in part by differential allelic methylation of this imprinted region. Small bowel and retroperitoneal wild-type GISTs segregate with adult mutant GISTs and express SDHB, while adult wild-type gastric GISTs are dispersed amongst adult mutant and pediatric wild-type cases, clustering in this situation on the basis of SDHB expression. Interestingly, global methylation analysis has recently similarly demonstrated that these wild-type, SDHB-immunonegative tumors show a distinct pattern compared with KIT and PDGFRA mutant tumors, which as a rule do express SDHB. All cases with Carney triad within our cohort cluster together tightly.
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de Luis Roman, Daniel; de la Fuente, Rocio Aller; Sagrado, Manuel Gonzalez; Izaola, Olatz; Vicente, Rosa Conde
2006-10-01
Human obesity is characterized by high levels of leptin, and it has been suggested that obese patients may be leptin resistant. The aim of our study was to investigate the influence of Lys656Asn polymorphism in the leptin receptor gene on leptin response and weight loss secondary to a lifestyle modification (Mediterranean hypocaloric diet and exercise) in obese patients. A population of 67 obese (body mass index >30) nondiabetic outpatients was analyzed in a prospective way. Before and after 3 months of lifestyle modification program, bipolar electrical bioimpedance, blood pressure, and a serial assessment of nutritional intake with 3 days written food records and biochemical analysis were performed. The lifestyle modification program consisted of a hypocaloric diet (1520 kcal, 52% carbohydrates, 25% lipids and 23% proteins). The exercise program consisted of aerobic exercise for at least three times per week (60 min each). Statistical analysis was performed for the combined Lys656/Asn656 and Asn656/Asn656 as mutant group and type Lys656/Lys 656 as wild-type second group. Sixty seven patients gave informed consent and were enrolled in the study. The mean age was 45.7 +/- 16.6 years and the mean BMI 34.1 +/- 5.1, with 18 males (26.9%) and 49 females (73.1%). Thirty six patients (10 males/26 females) (46.8%) had the genotype Lys656/Lys 656 (wild-type group) and 31 patients (8 males/23 females) (46.3%) Lys656/Asn656 (n = 28, 41.8%) or Asn656/Asn656 (n = 3, 4.5%) (mutant group). The percentage of responders (weight loss) was similar in both groups (91.7 vs. 87.1%). In wild-type group (responders and nonresponders), BMI, weight, fat mass, systolic blood pressure and waist circumference decreased. In mutant group, BMI, weight and waist circumference decreased. No differences were detected between basal values in both groups. Only leptin levels decreased significantly in wild-type group (11.5%; p <0.05) (57.3 +/- 31.5 ng/mL vs. 45.8 +/- 29.3 ng/mL; p <0.05). In mutant group, leptin increased without statistical differences (0.44%; ns). Patients with Asn656 allele of LEPR gene have a different response than wild-type patients, and Lys656Lys patients have a significant decrease in weight, BMI, fat mass, waist circumference, systolic blood pressure and leptin levels.
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Stimulus control by 5-methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice.
Winter, J C; Amorosi, D J; Rice, Kenner C; Cheng, Kejun; Yu, Ai-Ming
2011-09-01
In previous studies we have observed that, in comparison with wild type mice, Tg-CYP2D6 mice have increased serum levels of bufotenine [5-hydroxy-N,N-dimethyltryptamine] following the administration of 5-MeO-DMT. Furthermore, following the injection of 5-MeO-DMT, harmaline was observed to increase serum levels of bufotenine and 5-MeO-DMT in both wild-type and Tg-CYP2D6 mice. In the present investigation, 5-MeO-DMT-induced stimulus control was established in wild-type and Tg-CYP2D6 mice. The two groups did not differ in their rate of acquisition of stimulus control. When tested with bufotenine, no 5-MeO-DMT-appropriate responding was observed. In contrast, the more lipid soluble analog of bufotenine, acetylbufotenine, was followed by an intermediate level of responding. The combination of harmaline with 5-MeO-DMT yielded a statistically significant increase in 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice; a comparable increase occurred in wild-type mice. In addition, it was noted that harmaline alone was followed by a significant degree of 5-MeO-DMT-appropriate responding in Tg-CYP2D6 mice. It is concluded that wild-type and Tg-CYPD2D6 mice do not differ in terms of acquisition of stimulus control by 5-MeO-DMT or in their response to bufotenine and acetylbufotenine. In both groups of mice, harmaline was found to enhance the stimulus effects of 5-MeO-DMT. Copyright © 2011 Elsevier Inc. All rights reserved.
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2012-01-01
Background/Aim Efforts have been made to eliminate wild poliovirus transmission since 1988 when the World Health Organization began its global eradication campaign. Since then, the incidence of polio has decreased significantly. However, serotype 1 and serotype 3 still circulate endemically in Pakistan and Afghanistan. Both countries constitute a single epidemiologic block representing one of the three remaining major global reservoirs of poliovirus transmission. In this study we used genetic sequence data to investigate transmission links among viruses from diverse locations during 2005-2007. Methods In order to find the origins and routes of wild type 1 poliovirus circulation, polioviruses were isolated from faecal samples of Acute Flaccid Paralysis (AFP) patients. We used viral cultures, two intratypic differentiation methods PCR, ELISA to characterize as vaccine or wild type 1 and nucleic acid sequencing of entire VP1 region of poliovirus genome to determine the genetic relatedness. Results One hundred eleven wild type 1 poliovirus isolates were subjected to nucleotide sequencing for genetic variation study. Considering the 15% divergence of the sequences from Sabin 1, Phylogenetic analysis by MEGA software revealed that active inter and intra country transmission of many genetically distinct strains of wild poliovirus type 1 belonged to genotype SOAS which is indigenous in this region. By grouping wild type 1 polioviruses according to nucleotide sequence homology, three distinct clusters A, B and C were obtained with multiple chains of transmission together with some silent circulations represented by orphan lineages. Conclusion Our results emphasize that there was a persistent transmission of wild type1 polioviruses in Pakistan and Afghanistan during 2005-2007. The epidemiologic information provided by the sequence data can contribute to the formulation of better strategies for poliomyelitis control to those critical areas, associated with high risk population groups which include migrants, internally displaced people, and refugees. The implication of this study is to maintain high quality mass immunization with oral polio vaccine (OPV) in order to interrupt chains of virus transmission in both countries to endorse substantial progress in Eastern-Mediterranean region. PMID:22353446
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Alternative Pathways for Production of Beta-Amyloid Peptides of Alzheimer’s Disease
Hook, Vivian; Schechter, Israel; Demuth, Hans-Ulrich; Hook, Gregory
2009-01-01
This highlight article describes three Alzheimer’s disease (AD) presentations made at the 5th General Meeting of the International Proteolysis Society that address enzymatic mechanisms that produce neurotoxic beta-amyloid (Aβ) peptides. One group described the poor kinetic properties of the BACE 1 β-secretase for cleaving the wild-type β-secretase site in the APP found in most AD patients. They demonstrated that cathepsin D displays BACE 1-like specificity, is 280-fold more abundant in human brain than BACE 1, and pepstatin A inhibits cleavage of β-secretase site peptides by brain extracts and cathepsin D, but not by BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the wild type β-secretase site, they suggested continuing the search for additional β-secretase candidate(s). The second group reported that cathepsin B is such an alternative β-secretase candidate possessing excellent kinetic efficiency and specificity for cleaving the wild-type β-secretase site. Significantly, they demonstrated that inhibitors of cathepsin B improved memory function with reduced amyloid plaque neuropathology and decreased brain Aβ(40/42) and β-secretase activity in AD animal models expressing APP containing the wild-type β-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Aβ that are present in AD brains, with evidence that cathepsin B, but not BACE 1, efficiently cleaves the wild-type β-secretase site containing isoaspartate. They also found that cyclization of N-terminal Glu by glutaminyl cyclase generates pGluAβ(3-40/42) peptides that are highly amyloidogenic. These presentations suggested that cathepsin B and glutaminyl cyclase are potential new AD therapeutic targets. PMID:18979625
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Effect of waxy (Low Amylose) on Fungal Infection of Sorghum Grain.
Funnell-Harris, Deanna L; Sattler, Scott E; O'Neill, Patrick M; Eskridge, Kent M; Pedersen, Jeffrey F
2015-06-01
Loss of function mutations in waxy, encoding granule bound starch synthase (GBSS) that synthesizes amylose, results in starch granules containing mostly amylopectin. Low amylose grain with altered starch properties has increased usability for feed, food, and grain-based ethanol. In sorghum, two classes of waxy (wx) alleles had been characterized for absence or presence of GBSS: wx(a) (GBSS(-)) and wx(b) (GBSS(+), with reduced activity). Field-grown grain of wild-type; waxy, GBSS(-); and waxy, GBSS(+) plant introduction accessions were screened for fungal infection. Overall, results showed that waxy grains were not more susceptible than wild-type. GBSS(-) and wild-type grain had similar infection levels. However, height was a factor with waxy, GBSS(+) lines: short accessions (wx(b) allele) were more susceptible than tall accessions (undescribed allele). In greenhouse experiments, grain from accessions and near-isogenic wx(a), wx(b), and wild-type lines were inoculated with Alternaria sp., Fusarium thapsinum, and Curvularia sorghina to analyze germination and seedling fitness. As a group, waxy lines were not more susceptible to these pathogens than wild-type, supporting field evaluations. After C. sorghina and F. thapsinum inoculations most waxy and wild-type lines had reduced emergence, survival, and seedling weights. These results are valuable for developing waxy hybrids with resistance to grain-infecting fungi.
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de Luis, D A; Aller, R; Izaola, O; Sagrado, M Gonzalez; Conde, R
2006-01-01
It has been found that the expression of fatty acid binding protein 2 (FABP2) mRNA is under dietary control. A G-to-A transition at codon 54 of FABP2 results in an amino acid substitution (from Ala 54 to Thr 54). This polymorphism was associated with high insulin resistance and high fasting insulin concentrations. The aim of our study was to investigate the influence of Thr54 polymorphism in the FABP2 protein on the response to a lifestyle modification (Mediterranean hypocaloric diet and exercise) in obese patients. A population of 69 obese (body mass index > 30) nondiabetic outpatients was analyzed in a prospective way. Before and after 3 months of the lifestyle modification program, indirect calorimetry, tetrapolar electrical bioimpedance measurement, blood pressure recording, serial assessment of the nutritional intake (3 days of written food records), and biochemical analysis were performed. The lifestyle modification program consisted of a hypocaloric diet (1,520 kcal; 52% carbohydrates, 25% lipids, and 23% proteins). The exercise program consisted of aerobic exercise for at least three times/week (60 min each). Statistical analysis was performed for combined Ala54/Thr54 and Thr54/Thr54 as a mutant group and wild-type Ala54/Ala54 as second group. The mean age was 45.5 +/- 16.7 years, the mean body mass index was 34.1 +/- 5.1, and there were 14 males (20.3%) and 55 females (79.7%) with a weight loss of 3.17 +/- 3.5 kg (3.5%). Thirty-seven patients (53.6%) had the genotype Ala54/Ala54 (wild-type group) and 32 (46.4%) patients either the genotype Ala54/Thr54 (26 patients, 30.2%) or the genotype Thr54/Thr54 (6 patients, 16.2%). The percentage of responders (weight loss) was similar in both groups (89.2 vs. 90.6%). In the wild-type group, body mass index, weight, fat mass, low-density lipoprotein cholesterol level, and waist circumference decreased, whereas the VO2 (oxygen consumption) increased. In the mutant group, glucose, body mass index, weight, waist circumference, and systolic blood pressure decreased, and VO2 increased. No differences were detected between basal values in both groups. Only the leptin levels showed a significant decrease in the wild-type group (23.85%; p < 0.05), with no statistically significant difference in the mutant group (2.59%; NS). Resistin, tumor necrosis factor alpha, interleukin 6, insulin, and C-reactive protein remained without changes in both groups. Weight loss is associated with different changes, depending on the FABP2 genotype. Carriers of the Thr54 allele have a different response than wild-type obese subjects, with a significant decrease of systolic blood pressure and glucose levels in Thr54 carriers and a significant decrease in fat mass, low-density lipoprotein cholesterol, and leptin in wild-type patients. Copyright 2006 S. Karger AG, Basel.
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Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis.
Nylund, A; Devold, M; Plarre, H; Isdal, E; Aarseth, M
2003-08-15
The present study describes the use of molecular methods in studying infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Canada, USA and Chile. The nucleotide sequences of the haemagglutinin gene (HA) from 70 ISAV isolates have been analysed for phylogenetic relationship and the average mutation rate of nucleotide substitutions calculated. The isolates constitute 2 major groups, 1 European and 1 North American group. The isolate from Chile is closely related to the North American isolates. The European isolates can be further divided into 3 separate groups reflecting geographical distribution, time of collection, and transmission connected with farming activity. Based on existing information about infectious salmon anaemia (ISA) and new information emerging from the present study, it is hypothesised that: (1) ISAV is maintained in wild populations of trout and salmon in Europe; (2) it is transmitted between wild hosts mainly during their freshwater spawning phase in rivers; (3) wild salmonids, mainly trout, possibly carry benign wild-type ISAV isolates; (4) a change (mutation) in virulence probably results from deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates; (5) ISA emerges in farmed Atlantic salmon when mutated isolates are transmitted from wild salmonids or, following mutation of benign isolates, in farmed salmon after transmission from wild salmonids; (6) farming activity is an important factor in transmission of ISAV between farming sites in addition to transmission of ISAV from wild salmonids to farmed salmon; (7) transmission of ISAV from farmed to wild salmonids probably occurs less frequently than transmission from wild to farmed fish due to lower frequency of susceptible wild individuals; (8) the frequency of new outbreaks of ISA in farmed salmon probably reflects natural variation in the prevalence of ISAV in wild populations of salmonids.
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Verdijk, Pauline; Rots, Nynke Y; van Oijen, Monique G C T; Weldon, William C; Oberste, M Steven; Okayasu, Hiromasa; Sutter, Roland W; Bakker, Wilfried A M
2014-09-03
An inactivated poliovirus vaccine (IPV) based on attenuated poliovirus strains (Sabin-1, -2 and -3) was developed for technology transfer to manufacturers in low- and middle-income countries in the context of the global polio eradication initiative. Safety and immunogenicity of Sabin-IPV (sIPV) was evaluated in a double-blind, randomized, controlled, dose-escalation trial in the target population. Healthy infants (n=20/group) aged 56-63 days, received a primary series of three intramuscular injections with low-, middle- or high-dose sIPV with or without aluminum hydroxide or with the conventional IPV based on wild poliovirus strains (wIPV). Virus-neutralizing titers against both Sabin and wild poliovirus strains were determined before and 28 days after three vaccinations. The incidence of local and systemic reactions was comparable with the wIPV. Seroconversion rates after three vaccinations were 100% for type 2 and type 3 polioviruses (both Sabin and wild strains) and 95-100% for type 1 polioviruses. Median titers were high in all groups. Titers were well above the log2(titer) correlated with protection (=3) for all groups. Median titers for Sabin-2 were 9.3 (range 6.8-11.5) in the low-dose sIPV group, 9.2 (range 6.8-10.2) in the low-dose adjuvanted sIPV group and 9.8 (range 5.5-15.0) in the wIPV group, Median titers against MEF-1 (wild poliovirus type 2) were 8.2 (range 4.8-10.8) in the low-dose sIPV group, 7.3 (range 4.5-10.2) in the low-dose adjuvanted Sabin-IPV group and 10.3 (range 8.5-17.0) in the wIPV group. For all poliovirus types the median titers increased with increasing dose levels. sIPV and sIPV adjuvanted with aluminum hydroxide were immunogenic and safe at all dose levels, and comparable with the wIPV. EudraCTnr: 2011-003792-11, NCT01709071. Copyright © 2014. Published by Elsevier Ltd.
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Xia, Jia-Qi; Song, Jie; Zhang, Yi; An, Ni-Na; Ding, Lei; Zhang, Zheng
2015-01-01
Background: Nitroglycerin (NTG) is one of the few immediate treatments for acute angina. Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme in the human body that facilitates the biological metabolism of NTG. The biological mechanism of NTG serves an important function in NTG efficacy. Some reports still contradict the results that the correlation between ALDH2 gene polymorphisms and NTG and its clinical efficacy is different. However, data on NTG measurement by pain relief are subjective. This study aimed to investigate the influence of ALDH2 gene polymorphism on intervention with sublingual NTG using noninvasive hemodynamic parameters of cardiac output (CO) and systemic vascular resistance (SVR) in Northern Chinese Han population. Methods: This study selected 559 patients from the Affiliated Hospital of Qingdao University. A total of 203 patients presented with coronary heart disease (CHD) and 356 had non-CHD (NCHD) cases. All patient ALDH2 genotypes (G504A) were detected and divided into two types: Wild (GG) and mutant (GA/AA). Among the CHD group, 103 were wild-type cases, and 100 were mutant-type cases. Moreover, 196 cases were wild-type, and 160 cases were mutant type among the NCHD volunteers. A noninvasive hemodynamic detector was used to monitor the CO and the SVR at the 0, 5, and 15 minute time points after medication with 0.5 mg sublingual NTG. Two CO and SVR indicators were used for a comparative analysis of all case genotypes. Results: Both CO and SVR indicators significantly differed between the wild and mutant genotypes at various time points after intervention with sublingual NTG at 5 and 15 minutes in the NCHD (F = 16.460, 15.003, P = 0.000, 0.000) and CHD groups (F = 194.482, 60.582, P = 0.000, 0.000). All CO values in the wild-type case of both NCHD and CHD groups increased, whereas those in the mutant type decreased. The CO and ΔCO differences were statistically significant (P < 0.05; P < 0.05). The SVR and ΔSVR changed between the wild- and mutant-type cases at all-time points in both NCHD and CHD groups had statistically significant differences (P < 0.05; P < 0.05). Conclusion: ALDH2 (G504A) gene polymorphism is associated with changes in noninvasive hemodynamic parameters (i.e. CO and SVR) after intervention with sublingual NTG. This gene polymorphism may influence the effect of NTG intervention on Northern Chinese Han population. PMID:25591559
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USDA-ARS?s Scientific Manuscript database
Acidovorax citrulli is a seed-borne pathogen that causes bacterial fruit blotch of cucurbits including melon and watermelon. We investigated the roles of quorum sensing in the wild-type group II strain Aac-5 of A. citrulli by generating aacR and aacI knockout mutants and their complementation strain...
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Franceschi, Sara; Lessi, Francesca; Aretini, Paolo; Ortenzi, Valerio; Scatena, Cristian; Menicagli, Michele; La Ferla, Marco; Civita, Prospero; Zavaglia, Katia; Scopelliti, Claudia; Apollo, Alessandro; Carbone, Francesco Giovanni; Vannozzi, Riccardo; Bevilacqua, Generoso; Pasqualetti, Francesco; Naccarato, Antonio Giuseppe; Mazzanti, Chiara Maria
2018-01-01
Glioblastoma is a devastating disease that despite all the information gathered so far, its optimal management remains elusive due to the absence of validated targets from clinical studies. A better clarification of the molecular mechanisms is needed. In this study, having access to IDH1 wild-type glioblastoma of patients with exceptionally long recurrence free survival (RFS), we decided to compare their mutational and gene expression profile to groups of IDH1 wild-type glioblastoma of patients with shorter RFS, by using NGS technology. The exome analysis revealed that Long-RFS tumors have a lower mutational rate compared to the other groups. A total of 158 genes were found differentially expressed among the groups, 112 of which distinguished the two RFS extreme groups. Overall, the exome data suggests that shorter RFS tumors could be, chronologically, in a more advanced state in the muli-step tumor process of sequential accumulation of mutations. New players in this kind of cancer emerge from the analysis, confirmed at the RNA/DNA level, identifying, therefore, possible oncodrivers or tumor suppressor genes. PMID:29844869
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Chandra, Saurav B; Mohan, Sumathy; Ford, Bridget M; Huang, Lei; Janardhanan, Preethi; Deo, Kaiwalya S; Cong, Linlin; Muir, Eric R; Duong, Timothy Q
2016-06-01
Reduced bioavailability of nitric oxide due to impaired endothelial nitric oxide synthase (eNOS) activity is a leading cause of endothelial dysfunction in diabetes. Enhancing eNOS activity in diabetes is a potential therapeutic target. This study investigated basal cerebral blood flow and cerebrovascular reactivity in wild-type mice, diabetic mice (Ins2(Akita+/-)), nondiabetic eNOS-overexpressing mice (TgeNOS), and the cross of two transgenic mice (TgeNOS-Ins2(Akita+/-)) at six months of age. The cross was aimed at improving eNOS expression in diabetic mice. The major findings were: (i) Body weights of Ins2(Akita+/-) and TgeNOS-Ins2(Akita+/-) were significantly different from wild-type and TgeNOS mice. Blood pressure of TgeNOS mice was lower than wild-type. (ii) Basal cerebral blood flow of the TgeNOS group was significantly higher than cerebral blood flow of the other three groups. (iii) The cerebrovascular reactivity in the Ins2(Akita+/-) mice was significantly lower compared with wild-type, whereas that in the TgeNOS-Ins2(Akita+/-) was significantly higher compared with the Ins2(Akita+/-) and TgeNOS groups. Overexpression of eNOS rescued cerebrovascular dysfunction in diabetic animals, resulting in improved cerebrovascular reactivity. These results underscore the possible role of eNOS in vascular dysfunction in the brain of diabetic mice and support the notion that enhancing eNOS activity in diabetes is a potential therapeutic target. © The Author(s) 2015.
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Male and Female Mice Lacking Neuroligin-3 Modify the Behavior of Their Wild-Type Littermates.
Kalbassi, Shireene; Bachmann, Sven O; Cross, Ellen; Roberton, Victoria H; Baudouin, Stéphane J
2017-01-01
In most mammals, including humans, the postnatal acquisition of normal social and nonsocial behavior critically depends on interactions with peers. Here we explore the possibility that mixed-group housing of mice carrying a deletion of Nlgn3 , a gene associated with autism spectrum disorders, and their wild-type littermates induces changes in each other's behavior. We have found that, when raised together, male Nlgn3 knockout mice and their wild-type littermates displayed deficits in sociability. Moreover, social submission in adult male Nlgn3 knockout mice correlated with an increase in their anxiety. Re-expression of Nlgn3 in parvalbumin-expressing cells in transgenic animals rescued their social behavior and alleviated the phenotype of their wild-type littermates, further indicating that the social behavior of Nlgn3 knockout mice has a direct and measurable impact on wild-type animals' behavior. Finally, we showed that, unlike male mice, female mice lacking Nlgn3 were insensitive to their peers' behavior but modified the social behavior of their littermates. Altogether, our findings show that the environment is a critical factor in the development of behavioral phenotypes in transgenic and wild-type mice. In addition, these results reveal that the social environment has a sexually dimorphic effect on the behavior of mice lacking Nlgn3 , being more influential in males than females.
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Male and Female Mice Lacking Neuroligin-3 Modify the Behavior of Their Wild-Type Littermates
Kalbassi, Shireene; Cross, Ellen
2017-01-01
Abstract In most mammals, including humans, the postnatal acquisition of normal social and nonsocial behavior critically depends on interactions with peers. Here we explore the possibility that mixed-group housing of mice carrying a deletion of Nlgn3, a gene associated with autism spectrum disorders, and their wild-type littermates induces changes in each other’s behavior. We have found that, when raised together, male Nlgn3 knockout mice and their wild-type littermates displayed deficits in sociability. Moreover, social submission in adult male Nlgn3 knockout mice correlated with an increase in their anxiety. Re-expression of Nlgn3 in parvalbumin-expressing cells in transgenic animals rescued their social behavior and alleviated the phenotype of their wild-type littermates, further indicating that the social behavior of Nlgn3 knockout mice has a direct and measurable impact on wild-type animals’ behavior. Finally, we showed that, unlike male mice, female mice lacking Nlgn3 were insensitive to their peers’ behavior but modified the social behavior of their littermates. Altogether, our findings show that the environment is a critical factor in the development of behavioral phenotypes in transgenic and wild-type mice. In addition, these results reveal that the social environment has a sexually dimorphic effect on the behavior of mice lacking Nlgn3, being more influential in males than females. PMID:28795135
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Rose, Tatiana L; Miagostovich, Marize P; Leite, José Paulo G
2010-12-01
Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix®. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix® vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.
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Siah, Ahmed; Morrison, Diane B.; Fringuelli, Elena; Savage, Paul S.; Richmond, Zina; Purcell, Maureen K.; Johns, Robert; Johnson, Stewart C.; Sakasida, Sonja M.
2015-01-01
Piscine reovirus (PRV) is a double stranded non-enveloped RNA virus detected in farmed and wild salmonids. This study examined the phylogenetic relationships among different PRV sequence types present in samples from salmonids in Western Canada and the US, including Alaska (US), British Columbia (Canada) and Washington State (US). Tissues testing positive for PRV were partially sequenced for segment S1, producing 71 sequences that grouped into 10 unique sequence types. Sequence analysis revealed no identifiable geographical or temporal variation among the sequence types. Identical sequence types were found in fish sampled in 2001, 2005 and 2014. In addition, PRV positive samples from fish derived from Alaska, British Columbia and Washington State share identical sequence types. Comparative analysis of the phylogenetic tree indicated that Canada/US Pacific Northwest sequences formed a subgroup with some Norwegian sequence types (group II), distinct from other Norwegian and Chilean sequences (groups I, III and IV). Representative PRV positive samples from farmed and wild fish in British Columbia and Washington State were subjected to genome sequencing using next generation sequencing methods. Individual analysis of each of the 10 partial segments indicated that the Canadian and US PRV sequence types clustered separately from available whole genome sequences of some Norwegian and Chilean sequences for all segments except the segment S4. In summary, PRV was genetically homogenous over a large geographic distance (Alaska to Washington State), and the sequence types were relatively stable over a 13 year period.
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Siah, Ahmed; Morrison, Diane B.; Fringuelli, Elena; Savage, Paul; Richmond, Zina; Johns, Robert; Purcell, Maureen K.; Johnson, Stewart C.; Saksida, Sonja M.
2015-01-01
Piscine reovirus (PRV) is a double stranded non-enveloped RNA virus detected in farmed and wild salmonids. This study examined the phylogenetic relationships among different PRV sequence types present in samples from salmonids in Western Canada and the US, including Alaska (US), British Columbia (Canada) and Washington State (US). Tissues testing positive for PRV were partially sequenced for segment S1, producing 71 sequences that grouped into 10 unique sequence types. Sequence analysis revealed no identifiable geographical or temporal variation among the sequence types. Identical sequence types were found in fish sampled in 2001, 2005 and 2014. In addition, PRV positive samples from fish derived from Alaska, British Columbia and Washington State share identical sequence types. Comparative analysis of the phylogenetic tree indicated that Canada/US Pacific Northwest sequences formed a subgroup with some Norwegian sequence types (group II), distinct from other Norwegian and Chilean sequences (groups I, III and IV). Representative PRV positive samples from farmed and wild fish in British Columbia and Washington State were subjected to genome sequencing using next generation sequencing methods. Individual analysis of each of the 10 partial segments indicated that the Canadian and US PRV sequence types clustered separately from available whole genome sequences of some Norwegian and Chilean sequences for all segments except the segment S4. In summary, PRV was genetically homogenous over a large geographic distance (Alaska to Washington State), and the sequence types were relatively stable over a 13 year period. PMID:26536673
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Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S; Damborsky, Jiri; Smatanova, Ivana Kuta
2011-02-01
Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.
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Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin
2014-05-16
Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin
2014-01-01
Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs. PMID:24695732
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Gupta, Sonal; Nawaz, Kashif; Parween, Sabiha; Roy, Riti; Sahu, Kamlesh; Kumar Pole, Anil; Khandal, Hitaishi; Srivastava, Rishi; Kumar Parida, Swarup; Chattopadhyay, Debasis
2017-02-01
Cicer reticulatum L. is the wild progenitor of the fourth most important legume crop chickpea (C. arietinum L.). We assembled short-read sequences into 416 Mb draft genome of C. reticulatum and anchored 78% (327 Mb) of this assembly to eight linkage groups. Genome annotation predicted 25,680 protein-coding genes covering more than 90% of predicted gene space. The genome assembly shared a substantial synteny and conservation of gene orders with the genome of the model legume Medicago truncatula. Resistance gene homologs of wild and domesticated chickpeas showed high sequence homology and conserved synteny. Comparison of gene sequences and nucleotide diversity using 66 wild and domesticated chickpea accessions suggested that the desi type chickpea was genetically closer to the wild species than the kabuli type. Comparative analyses predicted gene flow between the wild and the cultivated species during domestication. Molecular diversity and population genetic structure determination using 15,096 genome-wide single nucleotide polymorphisms revealed an admixed domestication pattern among cultivated (desi and kabuli) and wild chickpea accessions belonging to three population groups reflecting significant influence of parentage or geographical origin for their cultivar-specific population classification. The assembly and the polymorphic sequence resources presented here would facilitate the study of chickpea domestication and targeted use of wild Cicer germplasms for agronomic trait improvement in chickpea. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.
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HFE Gene Mutations and Iron Status in 100 Healthy Polish Children.
Kaczorowska-Hac, Barbara; Luszczyk, Marcin; Antosiewicz, Jedrzej; Ziolkowski, Wieslaw; Adamkiewicz-Drozynska, Elzbieta; Mysliwiec, Malgorzata; Milosz, Ewa; Kaczor, Jan J
2017-07-01
Iron participates in oxygen transport, energetic, metabolic, and immunologic processes. There are 2 main causes of iron overload: hereditary hemochromatosis which is a primary cause, is a metabolic disorder caused by mutations of genes that control iron metabolism and secondary hemochromatosis caused by multitransfusions, chronic hemolysis, and intake of iron rich food. The most common type of hereditary hemochromatosis is caused by HFE gene mutation. In this study, we analyzed iron metabolism in 100 healthy Polish children in relation to their HFE gene status. The wild-type HFE gene was predominant being observed in 60 children (60%). Twenty-five children (25%), presented with heterozygotic H63D mutation, and 15 children (15%), presented with other mutations (heterozygotic C282Y and S65C mutation, compound heterozygotes C282Y/S65C, C282Y/H63D, H63D homozygote). The mean concentration of iron, the level of ferritin, and transferrin saturation were statistically higher in the group of HFE variants compared with the wild-type group. H63D carriers presented with higher mean concentration of iron, ferritin levels, and transferrin saturation compared with the wild-type group. Male HFE carriers presented with higher iron concentration, transferrin saturation, and ferritin levels than females. This preliminary investigation demonstrates allelic impact on potential disease progression from childhood.
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Lin, Bowen; Hasegawa, Yu; Takane, Koki; Koibuchi, Nobutaka; Cao, Cheng; Kim-Mitsuyama, Shokei
2016-06-13
The high-fat Western diet is postulated to be associated with the onset and progression of Alzheimer's disease (AD). However, the role of high-fat-diet consumption in AD pathology is unknown. This study was undertaken to examine the role of high-fat-diet intake in AD. 5XFAD mice, a useful mouse model of AD, and control wild-type mice were fed (1) high-fat diet or (2) control diet for 10 weeks. The effects on cerebral AD pathology, cognitive function, and metabolic parameters were compared between each group of mice. High-fat diet significantly enhanced cerebrovascular β-amyloid (Aβ) deposition (P<0.05) and impaired cognitive function (P<0.05) in 5XFAD mice, but not in wild-type mice. High-fat diet enhanced hippocampal oxidative stress (P<0.05) and NADPH oxidase subunits, gp91(phox) (P<0.01) and p22(phox) (P<0.01) in 5XFAD mice, but not in wild-type mice. Furthermore, high-fat diet reduced cerebral occludin (P<0.05) in 5XFAD mice, but not in wild-type mice. Thus, 5XFAD mice exhibited greater susceptibility to high-fat diet than wild-type mice regarding cerebrovascular injury and cognitive impairment. On the other hand, 5XFAD mice fed high-fat diet exhibited much less increase in body weight, white adipose tissue weight, and adipocyte size than their wild-type counterparts. High-fat diet significantly impaired glucose tolerance in wild-type mice but not in 5XFAD mice. Thus, 5XFAD mice had much less susceptibility to high-fat-diet-induced metabolic disorders than wild-type mice. High-fat diet, independently of metabolic disorders, significantly promotes the progression of AD-like pathology through enhancement of cerebral amyloid angiopathy and oxidative stress. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.
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DNA barcoding of wild edible mushrooms consumed by the ethnic tribes of India.
Khaund, Polashree; Joshi, S R
2014-10-15
Wild edible mushrooms are consumed by the tribes of Meghalaya in the North-Eastern region of India, as part of their ethnic cuisine because of their favored organoleptic characteristics and traditionally known health benefits. Majority of these mushrooms have not yet been characterized in detail and are slowly shrinking in their natural habitats owing to anthropogenic factors and climate change. In the present study, representative specimens of ten morphologically distinct groups of wild edible mushrooms available in the traditional markets and their respective forest habitats, were subjected to multi-loci molecular characterization using SSU, ITS, RPB1 and RPB2 markers. The species identities inferred for the ten mushroom types using the SSU marker matched their morphological description in the case of four morphological groups only whereas the ITS marker successfully resolved the species identity for nine out of the ten mushroom groups under study. Both the protein coding gene markers RPB1 and RPB2 successfully resolved the species identity for three out of the ten morphologically distinct groups. Finally the most likely identity of the wild edible mushrooms under study has been suggested by matching their unique morphological characteristics with the generated DNA barcoding data. The present molecular characterization reveals the ten widely consumed wild mushroom types of Meghalaya, India to be Gomphus floccosus, Lactarius deliciosus, Lactarius volemus, Cantharellus cibarius, Tricholoma viridiolivaceum, Inocybe aff. sphaerospora, Laccaria vinaceoavellanea, Albatrellus ellisii, Ramaria maculatipes and Clavulina cristata. The final species identity generated by the ITS marker matched more accurately with the morphological characteristics/appearance of the specimens indicating the ITS region as a reliable barcode for identifying wild edible mushrooms. Copyright © 2014 Elsevier B.V. All rights reserved.
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Kulikova, Vitalia V; Zakomirdina, Ludmila N; Dementieva, Irene S; Phillips, Robert S; Gollnick, Paul D; Demidkina, Tatyana V; Faleev, Nicolai G
2006-04-01
Tyr72 is located at the active site of tryptophanase (Trpase) from Proteus vulgaris. For the wild-type Trpase Tyr72 might be considered as the general acid catalyst at the stage of elimination of the leaving groups. The replacement of Tyr72 by Phe leads to a decrease in activity for L-tryptophan by 50,000-fold and to a considerable rearrangement of the active site of Trpase. This rearrangement leads to an increase of room around the alpha-C atom of any bound amino acid, such that covalent binding of alpha-methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase). The changes in reactivities of S-alkyl-L-cysteines provide evidence for an increase of congestion in the proximity of their side groups in the mutant enzyme as compared to wild-type enzyme. The observed alteration of catalytic properties in a large degree originates from a conformational change in the active site. The Y72F Trpase retains significant activity for L-serine, which allowed us to conclude that in the mutant enzyme, some functional group is present which fulfills the role of the general acid catalyst in reactions associated with elimination of small leaving groups.
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Proteome profiling of virus-host interactions of wild type and attenuated measles virus strains.
Billing, Anja M; Kessler, Julia R; Revets, Dominique; Sausy, Aurélie; Schmitz, Stephanie; Barra, Claire; Muller, Claude P
2014-08-28
Quantitative gel-based proteomics (2D DIGE coupled to MALDI-TOF/TOF MS) has been used to investigate the effects of different measles virus (MV) strains on the host cell proteome. A549/hSLAM cells were infected either with wild type MV strains, an attenuated vaccine or a multiple passaged Vero cell adapted strain. By including interferon beta treatment as a control it was possible to distinguish between the classical antiviral response and changes induced specifically by the different strains. Of 38 differentially expressed proteins in total (p-value ≤0.05, fold change ≥2), 18 proteins were uniquely modulated following MV infection with up to 9 proteins specific per individual strain. Interestingly, wt strains displayed distinct protein patterns particularly during the late phase of infection. Proteins were grouped into cytoskeleton, metabolism, transcription/translation, immune response and mitochondrial proteins. Bioinformatics analysis revealed mostly changes in proteins regulating cell death and apoptosis. Surprisingly, wt strains affected the cytokeratin system much stronger than the vaccine strain. To our knowledge, this is the first study on the MV-host proteome addressing interstrain differences. In the present study we investigated the host cell proteome upon measles virus (MV) infection. The novelty about this study is the side-by side comparison of different strains from the same virus, which has not been done at the proteome level for any other virus including MV. We used different virus strains including a vaccine strain, wild type isolates derived from MV-infected patients as well as a Vero cell adapted strain, which serves as an intermediate between vaccine and wild type strain. We observed differences between vaccine and wild type strains as well as common features between different wild type strains. Perhaps one of the most surprising findings was that differences did not only occur between wild type and vaccine or Vero cell adapted strains but also between different wild type strains. In fact our study suggests that besides the cytokeratin and the IFN system wild type viruses seem to differ as much among each other than from vaccine strains. Thus our results are suggestive of complex and diverse virus-host interactions which differ considerably between different wild type strains. Our data indicate that interstrain differences are prominent and have so far been neglected by proteomics studies. Copyright © 2014 Elsevier B.V. All rights reserved.
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Laser Interferometer Measurements of the Viscoelastic Properties of Tectorial Membrane Mutants
NASA Astrophysics Data System (ADS)
Jones, Gareth; Russell, Ian; Lukashkin, Andrei
2011-11-01
The visco-elastic properties of the tectorial membrane (TM) can be determined by measuring the propagation velocity of travelling waves over a range of frequencies. This study presents a new method using laser interferometry and compares the TM's material properties (sheer storage modulus, G' and viscosity, η) at basal and apical locations in wild-type mice and basal locations of three mutant groups (TectaY1870C/+, Tectb-/- and Otoa-/-). The G' and η values calculated for the wild-type mice are similar to estimates derived using other methods whereas the mutant groups all exhibit slower wave propagation velocities and reduced longitudinal coupling.
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Patterns of Genetic Structure and Linkage Disequilibrium in a Large Collection of Pea Germplasm
Siol, Mathieu; Jacquin, Françoise; Chabert-Martinello, Marianne; Smýkal, Petr; Le Paslier, Marie-Christine; Aubert, Grégoire; Burstin, Judith
2017-01-01
Pea (Pisum sativum, L.) is a major pulse crop used both for animal and human alimentation. Owing to its association with nitrogen-fixing bacteria, it is also a valuable component for low-input cropping systems. To evaluate the genetic diversity and the scale of linkage disequilibrium (LD) decay in pea, we genotyped a collection of 917 accessions, gathering elite cultivars, landraces, and wild relatives using an array of ∼13,000 single nucleotide polymorphisms (SNP). Genetic diversity is broadly distributed across three groups corresponding to wild/landraces peas, winter types, and spring types. At a finer subdivision level, genetic groups relate to local breeding programs and type usage. LD decreases steeply as genetic distance increases. When considering subsets of the data, LD values can be higher, even if the steep decay remains. We looked for genomic regions exhibiting high level of differentiation between wild/landraces, winter, and spring pea, respectively. Two regions on linkage groups 5 and 6 containing 33 SNPs exhibit stronger differentiation between winter and spring peas than would be expected under neutrality. Interestingly, QTL for resistance to cold acclimation and frost resistance have been identified previously in the same regions. PMID:28611254
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Nisa-Martínez, Rafael; Jiménez-Zurdo, José I.; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás
2007-01-01
RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase–maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (ΔORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic ΔORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature. PMID:17158161
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Nisa-Martínez, Rafael; Jiménez-Zurdo, José I; Martínez-Abarca, Francisco; Muñoz-Adelantado, Estefanía; Toro, Nicolás
2007-01-01
RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase-maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (DeltaORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic DeltaORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.
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Tang, Ning; Zhang, Qianqian; Fang, Shu; Han, Xiao; Wang, Zhehai
2017-01-01
Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and recorded, and at the end of experiments, tumor xenografts were removed for Western blot and immunohistochemical analyses. Compared to control groups (negative control, regular-dose icotinib [IcoR], high-dose icotinib [IcoH], and docetaxel [DTX]) and regular icotinib dose (60 mg/kg) with docetaxel, treatment of mice with a high-dose (1200 mg/kg) of icotinib plus sequential docetaxel for 3 weeks (IcoH-DTX) had an additive effect on suppression of tumor xenograft size and volume (P < 0.05). Icotinib-containing treatments markedly reduced phosphorylation of EGFR, mitogen activated protein kinase (MAPK), and protein kinase B (Akt), but only the high-dose icotinib-containing treatments showed an additive effect on CD34 inhibition (P < 0.05), an indication of reduced microvessel density in tumor xenografts. Moreover, high-dose icotinib plus docetaxel had a similar effect on mouse weight loss (a common way to measure adverse reactions in mice), compared to the other treatment combinations. The study indicate that the high dose of icotinib plus sequential docetaxel (IcoH-DTX) have an additive effect on suppressing the growth of wild-type EGFR NSCLC cell nude mouse xenografts, possibly through microvessel density reduction. Future clinical trials are needed to confirm the findings of this study. PMID:27852073
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[Phenotype and mechanism of inducible ppp2r1a knockout mouse model].
Fan, J L; Wang, F P; Wang, S; Liu, X L; Wu, X N; Chen, W; Chen, L P; Li, W X
2018-05-06
Objective: Investigate the effects of inducible ppp2r1a knockout on main physiological function in adult mice and study the mechanism. Methods: Ppp2r1a(flox/flox) mice and CAGG-CreER mice were hybridized to obtain 20 CAGG-CreER ppp2r1a(flox/flox) and 20 mice in homozygous group. Two groups of mice were divided into 4 groups respectively, finally we got 8 groups with 5 mice in each group. Tamoxifen was injected intraperitoneally to acquire inducible ppp2r1a knockout mice. The knockout efficiency of PP2A Aα in vital organs was measured by Western blot. At 0, 2, 4 and 6 days after injection, we measured body weight, histopathological change, peripheral blood cell counts and blood biochemical. Real-time PCR was performed to measure expression of liver glucolipid metabolism genes. Results: After tamoxifen injection for 6 days, the knockout efficiency of PP2A Aα in vital organs was 35%, 12%, 15%, 60%, 69% and 72%, respectively in heart, liver, spleen, lung, kidney and brain. After tamoxifen injection for 6 days, the weight of homozygous mice was lower than that of wild type mice, with values of (17.42±1.76) g and (21.69±1.82) g, respectively ( P< 0.05). Moreover, the activity level, abdominal and renal fat were significantly decreased in homozygous mice. Homozygous mice survived no more than 7 days. Compared with wild type mice, the organ coefficient of spleen of homozygous mice was decreased at the 6th day, with values of (0.59±0.10)% and (0.36±0.05)% respectively ( P< 0.05). Obvious spleen atrophy and marked decrease of nucleated cells were showed by performing HE staining. Tunel staining revealed increased apoptosis ratio of splenic lymphocytes in homozygous mice. The levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) of homozygous mice were higher than wild type mice ( P< 0.05). The values of ALT and AST in homozygous mice were (153.68±62.80) U/L and (193.2±44.28) U/L. The corresponding values in wild type mice were (41.02±12.91) U/L and (69.40±9.55) U/L. The above results indicated that ppp2r1a knockout caused liver damage. Blood sugar level of homozygous mice was lower than in wild type mice ( P< 0.05), with values of (4.20±1.99) mmol/L and (8.88±0.65) mmol/L respectively. Plasma total cholesterol (TC), high density lipoprotein (HDL) and β-hydroxybutyric acid (β-HB) level of homozygous mice were higher than those of wild type mice ( P< 0.05). The values of TC, HDL and β-HB in homozygous mice were (3.12±0.39), (1.53±0.38) and (2.49±0.89) mmol/L. The corresponding values in wild type mice were (1.69±0.92), (0.78±0.50) and (0.45±0.30) mmol/L respectively. The above results indicated that ppp2r1a loss interfered glucose and cholesterol metabolism. In addition, we also found that the white blood cell count (WBC) and lymphocyte count (LYM) of homozygous mice were lower than in wild type mice ( P< 0.05). The values of WBC and LYM in homozygous mice were (1.88±0.89)×10(9)/L and (0.92±0.37)×10(9)/L respectively. The corresponding values in wild type mice were (3.91±0.80)×10(9)/L and (2.74±0.52)×10(9)/L respectively. The mRNA levels of glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) of homozygous were lower than wild type mice ( P< 0.05). The fold change of G6P and PEPCK in homozygous mice was 0.46±0.11 and 0.72±0.07 respectively. The corresponding fold change in wild type mice was 1.02±0.07 and 1.02±0.06 respectively. Conclusion: Whole body ppp2r1a is essential for the survival of adult mice, due to the important role in maintaining the metabolism of glucose and cholesterol of liver.
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African swine fever virus infection in Classical swine fever subclinically infected wild boars.
Cabezón, Oscar; Muñoz-González, Sara; Colom-Cadena, Andreu; Pérez-Simó, Marta; Rosell, Rosa; Lavín, Santiago; Marco, Ignasi; Fraile, Lorenzo; de la Riva, Paloma Martínez; Rodríguez, Fernando; Domínguez, Javier; Ganges, Llilianne
2017-08-01
Recently moderate-virulence classical swine fever virus (CSFV) strains have been proven capable of generating postnatal persistent infection (PI), defined by the maintenance of viremia and the inability to generate CSFV-specific immune responses in animals. These animals also showed a type I interferon blockade in the absence of clinical signs. In this study, we assessed the infection generated in 7-week-old CSFV PI wild boars after infection with the African swine fever virus (ASFV). The wild boars were divided in two groups and were infected with ASFV. Group A comprised boars who were CSFV PI in a subclinical form and Group B comprised pestivirus-free wild boars. Some relevant parameters related to CSFV replication and the immune response of CSFV PI animals were studied. Additionally, serum soluble factors such as IFN-α, TNF-α, IL-6, IL-10, IFN-γ and sCD163 were analysed before and after ASFV infection to assess their role in disease progression. After ASFV infection, only the CSFV PI wild boars showed progressive acute haemorrhagic disease; however, the survival rates following ASFV infection was similar in both experimental groups. Notwithstanding, the CSFV RNA load of CSFV PI animals remained unaltered over the study; likewise, the ASFV DNA load detected after infection was similar between groups. Interestingly, systemic type I FN-α and IL-10 levels in sera were almost undetectable in CSFV PI animals, yet detectable in Group B, while detectable levels of IFN-γ were found in both groups. Finally, the flow cytometry analysis showed an increase in myelomonocytic cells (CD172a + ) and a decrease in CD4 + T cells in the PBMCs from CSFV PI animals after ASFV infection. Our results showed that the immune response plays a role in the progression of disease in CSFV subclinically infected wild boars after ASFV infection, and the immune response comprised the systemic type I interferon blockade. ASFV does not produce any interference with CSFV replication, or vice versa. ASFV infection could be a trigger factor for the disease progression in CSFV PI animals, as their survival after ASFV was similar to that of the pestivirus-free ASFV-infected group. This fact suggests a high resistance in CSFV PI animals even against a virus like ASFV; this may mean that there are relevant implications for CSF control in endemic countries. The diagnosis of ASFV and CSFV co-infection in endemic countries cannot be ruled out and need to be studied in greater depth.
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Chemical and histological studies of wild and hatchery salmon in fresh water
Wood, E.M.; Yasutake, W.T.; Halver, J.E.; Woodall, A.N.
1960-01-01
In a study of coho salmon (Oncorhynchus kisutch), the gross chemical and histological changes occurring over a 14-month period spent in fresh water were determined. The determinations were made at 3-month intervals on: 1) hatchery-reared fish, 2) fish hatchery-reared for 3, 6, 9, and 12 months and then planted in a controlled stream for the remainder of the period; and 3) an indigenous group of wild fish in this stream. Wild fish showed high incidence of tissue damage from spinose hairs of the moth larva, Halisidota argentata. Hatchery fish were similarly affected with the severity and incidence of lesions varying directly with the time of exposure of the larvae in the wild environment. Both groups of fish were heavily parasitized by sporozoan organisms in the kidney and spinal cord. Kidney disease appeared in both wild and planted hatchery fish. The gross chemical composition of hatchery fish transformed rapidly after planting to that of the wild fish. Although the initial rate of fat loss is essentially constant for all hatchery groups after planting, fish that were hatchery reared for 9 to 12 months did not complete the transformation to the wild-type body composition by the time of downstream migration at 14 months.
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Han, Tae Kyung
2013-01-01
The purpose of the current study was to investigate whether or not the FABP2 gene polymorphism modulated obesity indices, hemodynamic factor, blood lipid factor, and insulin resistance markers through 12-week aerobic exercise training in abdominal obesity group of Korean mid-life women. A total of 243 abdominally obese subjects of Korean mid-life women voluntarily participated in aerobic exercise training program for 12 weeks. Polymerase Chain Reaction with Restriction Fragment Length Polymorphism (PCR-RFLP) assay was used to assess the FABP2 genotype of the participants (117 of AA homozygotes, 100 of AT heterozygotes, 26 of TT homozygotes). Prior to the participation of the exercise training program, baseline obesity indices, hemodynamic factor, blood lipid factor, and insulin resistance markers were measured. All the measurements were replicated following the 12-week aerobic exercise training program, and then the following results were found. After 12-week aerobic exercise training program, wild type (Ala54Ala) and mutant type (Ala54Thr+Thr54Thr) significantly decreased weight (P > .001), BMI (P > .001), %bf (P > .001), waist circumference (P > .001), WHR (P > .001), muscle mass (wild type p < .022; mutant type P > .001), RHR (P > .001), viseceral adipose area (wild type p < .005; mutant type P > .001), subcutaneous area (P > .001), insulin (wild type p < .005; mutant type P > .001) and significantly increased VO2max (P > .001). And wild type significantly decresed NEFA (P > .05), glucose (P > .05), OGTT 120min glucose (P > .05) and significantly increased HDLC (p > .005). Mutant type significantly decreased SBP (P > .001), DBP (P > .01), TC (P > .01), LPL (P > .05), LDL (P > .001), HOMA index (P > .01). The result of the present study represents that regular aerobic exercise training may beneficially prevent obesity index, blood pressure, blood lipids and insulin resistance markers independent of FABP Ala54Thr wild type and mutant type. PMID:25566432
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The effect of IDH1 mutation on the structural connectome in malignant astrocytoma.
Kesler, Shelli R; Noll, Kyle; Cahill, Daniel P; Rao, Ganesh; Wefel, Jeffrey S
2017-02-01
Mutation of the IDH1 gene is associated with differences in malignant astrocytoma growth characteristics that impact phenotypic severity, including cognitive impairment. We previously demonstrated greater cognitive impairment in patients with IDH1 wild type tumor compared to those with IDH1 mutant, and therefore we hypothesized that brain network organization would be lower in patients with wild type tumors. Volumetric, T1-weighted MRI scans were obtained retrospectively from 35 patients with IDH1 mutant and 32 patients with wild type malignant astrocytoma (mean age = 45 ± 14 years) and used to extract individual level, gray matter connectomes. Graph theoretical analysis was then applied to measure efficiency and other connectome properties for each patient. Cognitive performance was categorized as impaired or not and random forest classification was used to explore factors associated with cognitive impairment. Patients with wild type tumor demonstrated significantly lower network efficiency in several medial frontal, posterior parietal and subcortical regions (p < 0.05, corrected for multiple comparisons). Patients with wild type tumor also demonstrated significantly higher incidence of cognitive impairment (p = 0.03). Random forest analysis indicated that network efficiency was inversely, though nonlinearly associated with cognitive impairment in both groups (p < 0.0001). Cognitive reserve appeared to mediate this relationship in patients with mutant tumor suggesting greater neuroplasticity and/or benefit from neuroprotective factors. Tumor volume was the greatest contributor to cognitive impairment in patients with wild type tumor, supporting our hypothesis that greater lesion momentum between grades may cause more disconnection of core neurocircuitry and consequently lower efficiency of information processing.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yokohira, Masanao; Arnold, Lora L.; Pennington, Karen L.
Arsenic (+ 3 oxidation state) methyltransferase (As3mt) catalyzes reactions which convert inorganic arsenic to methylated metabolites. This study determined whether the As3mt null genotype in the mouse modifies cytotoxic and proliferative effects seen in urinary bladders of wild type mice after exposure to inorganic arsenic. Female wild type C57BL/6 mice and As3mt KO mice were divided into 3 groups each (n = 8) with free access to a diet containing 0, 100 or 150 ppm of arsenic as arsenite (As{sup III}). During the first week of As{sup III} exposure, As3mt KO mice exhibited severe and lethal systemic toxicity. At termination,more » urinary bladders of both As3mt KO and wild type mice showed hyperplasia by light microscopy. As expected, arsenic-containing granules were found in the superficial urothelial layer of wild type mice. In As3mt KO mice these granules were present in all layers of the bladder epithelium and were more abundant and larger than in wild type mice. Scanning electron microscopy of the bladder urothelium of As3mt KO mice treated with 100 ppm As{sup III} showed extensive superficial necrosis and hyperplastic changes. In As3mt KO mice, livers showed severe acute inflammatory changes and spleen size and lymphoid areas were decreased compared with wild type mice. Thus, diminished arsenic methylation in As3mt KO mice exacerbates systemic toxicity and the effects of As{sup III} on the bladder epithelium, showing that altered kinetic and dynamic behavior of arsenic can affect its toxicity.« less
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Analyzing Cold Tolerance Mechanism in Transgenic Zebrafish (Danio rerio)
Wang, Qian; Tan, Xungang; Jiao, Shuang; You, Feng; Zhang, Pei-Jun
2014-01-01
Low temperatures may cause severe growth inhibition and mortality in fish. In order to understand the mechanism of cold tolerance, a transgenic zebrafish Tg (smyd1:m3ck) model was established to study the effect of energy homeostasis during cold stress. The muscle-specific promoter Smyd1 was used to express the carp muscle form III of creatine kinase (M3-CK), which maintained enzymatic activity at a relatively low temperature, in zebrafish skeletal muscle. In situ hybridization showed that M3-CK was expressed strongly in the skeletal muscle. When exposed to 13°C, Tg (smyd1:m3ck) fish maintained their swimming behavior, while the wild-type could not. Energy measurements showed that the concentration of ATP increased in Tg (smyd1:m3ck) versus wild-type fish at 28°C. After 2 h at 13°C, ATP concentrations were 2.16-fold higher in Tg (smyd1:m3ck) than in wild-type (P<0.05). At 13°C, the ATP concentration in Tg (smyd1:m3ck) fish and wild-type fish was 63.3% and 20.0%, respectively, of that in wild-type fish at 28°C. Microarray analysis revealed differential expression of 1249 transcripts in Tg (smyd1:m3ck) versus wild-type fish under cold stress. Biological processes that were significantly overrepresented in this group included circadian rhythm, energy metabolism, lipid transport, and metabolism. These results are clues to understanding the mechanisms underlying temperature acclimation in fish. PMID:25058652
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Morodomi, Yosuke; Takenoyama, Mitsuhiro; Inamasu, Eiko; Toyozawa, Ryo; Kojo, Miyako; Toyokawa, Gouji; Shiraishi, Yoshimasa; Takenaka, Tomoyoshi; Hirai, Fumihiko; Yamaguchi, Masafumi; Taguchi, Kenichi; Seto, Takashi; Sugio, Kenji; Ichinose, Yukito
2014-07-01
Although patients with the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase gene (EML4-ALK) re-arrangement and epidermal growth factor gene EGFR mutations have proven sensitive to specific inhibitors, there is currently no consensus regarding the sensitivity of non-small cell lung cancer (NSCLC) patients with such mutations to cytotoxic chemotherapy. The responses to first-line cytotoxic chemotherapy were retrospectively compared between advanced or postoperative recurrent patients with non-squamous NSCLC who harbor the EML4-ALK fusion gene (ALK+), EGFR mutation (EGFR+), or neither abnormality (wild-type). Data for 22 ALK+, 30 EGFR+, and 60 wild-type patients were analyzed. The ALK+ group had a significantly lower response rate than the other two groups. Progression-free survival was significantly shorter in the ALK+ cohort compared to the EGFR+ (p<0.001) and wild-type cohorts (p=0.0121). NSCLC patients with the EML4-ALK fusion gene might be relatively insensitivite to cytotoxic chemotherapy. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation.
Dominici, P.; Moore, P. S.; Castellani, S.; Bertoldi, M.; Voltattorni, C. B.
1997-01-01
Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111S mutant enzymes exhibit Kcat values of about 50% and 15%, respectively, at pH 6.8, while the K(m) values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (L-5-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-111-->Ala mutant displays a reduced affinity for pyridoxal 5'-phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild-type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420-->390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Borri Voltattorni C. 1991. Eur J Biochem 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation. PMID:9300500
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Identification of Anion Channels Responsible for Fluoride Resistance in Oral Streptococci
Takeshita, Toru; Yamashita, Yoshihisa
2016-01-01
Recently, it has been reported that eriC and crcB are involved in bacterial fluoride resistance. However, the fluoride-resistance mechanism in oral streptococci remains unclear. BLAST studies showed that two types of eriCs (eriC1 and eriC2) and two types of crcBs (crcB1 and crcB2) are present across 18 oral streptococci, which were identified in ≥ 10% of 166 orally healthy subjects with ≥ 0.01% of the mean relative abundance. They were divided into three groups based on the distribution of these four genes: group I, only eriC1; group II, eriC1 and eriC2; and group III, eriC2, crcB1, and crcB2. Group I consisted of Streptococcus mutans, in which one of the two eriC1s predominantly affected fluoride resistance. Group II consisted of eight species, and eriC1 was responsible for fluoride resistance, but eriC2 was not, in Streptococcus anginosus as a representative species. Group III consisted of nine species, and both crcB1 and crcB2 were crucial for fluoride resistance, but eriC2 was not, in Streptococcus sanguinis as a representative species. Based on these results, either EriC1 or CrcBs play a role in fluoride resistance in oral streptococci. Complementation between S. mutans EriC1 and S. sanguinis CrcB1/CrcB2 was confirmed in both S. mutans and S. sanguinis. However, neither transfer of S. sanguinis CrcB1/CrcB2 into wild-type S. mutans nor S. mutans EriC1 into wild-type S. sanguinis increased the fluoride resistance of the wild-type strain. Co-existence of different F− channels (EriC and CrcB) did not cause the additive effect on fluoride resistance in oral Streptococcus species. PMID:27824896
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[Use of ITS and ISSR markers in the molecular characterisation of Pleurotus djamor hybrid strains].
Aguilar Doroteo, Leticia; Zárate Segura, Paola Berenice; Villanueva Arce, Ramón; Yáñez Fernández, Jorge; Garín Aguilar, María Eugenia; Guadarrama Mendoza, Paula Cecilia; Valencia Del Toro, Gustavo
Molecular characterisation of wild type Pleurotus species is important for germplasm conservation and its further use for genetic improvement. No molecular studies have been performed with monokaryons used for producing hybrid strains, either with the reconstituted strains obtained by pairing those monokaryons. The molecular characterisation of parental dikaryons, hybrid, and reconstituted strains as well as monokaryotic strains, is therefore of utmost importance. To carry out the molecular identification of Pleurotus djamor strains, i.e. dikaryotic wild type strains, hybrid strains, and the monokaryotic strains used for the hybrid formation. Five wild type strains of P. djamor from different states in Mexico were collected and molecularly identified by sequencing the ITS1-5.8-ITS2 region using ITS1 and ITS4 universal oligonucleotides. Four hybrid strains were obtained by pairing neohaplonts of two wild type strains selected. Six ISSR markers were used for the molecular characterisation of monokaryotic and dikaryotic strains. Using the ITS markers, an amplified product of 700bp was obtained in five wild type strains, with a 99-100% similarity with P. djamor. A total of 95 fragments were obtained using the ISSR markers, with 99% of polymorphism. Wild type strains were identified as P. djamor, and were clearly grouped with Mexican strains from other states of Mexico. ISSR markers allowed the generation of polymorphic bands in monokaryotic and dikaryotic strains, splitting both types of strains. The high degree of polymorphism indicates the genetic diversity of P. djamor, an advantage in mushroom production and in the improving of the species. Copyright © 2017 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Fang Cheng; Behr, Melissa; Xie Fang
2008-02-15
Chloroform causes hepatic and renal toxicity in a number of species. In vitro studies have indicated that chloroform can be metabolized by P450 enzymes in the kidney to nephrotoxic intermediate, although direct in vivo evidence for the role of renal P450 in the nephrotoxicity has not been reported. This study was to determine whether chloroform renal toxicity persists in a mouse model with a liver-specific deletion of the P450 reductase (Cpr) gene (liver-Cpr-null). Chloroform-induced renal toxicity and chloroform tissue levels were compared between the liver-Cpr-null and wild-type mice at 24 h following differing doses of chloroform. At a chloroform dosemore » of 150 mg/kg, the levels of blood urea nitrogen (BUN) were five times higher in the exposed group than in the vehicle-treated one for the liver-Cpr-null mice, but they were only slightly higher in the exposed group than in the vehicle-treated group for the wild-type mice. Severe lesions were found in the kidney of the liver-Cpr-null mice, while only mild lesions were found in the wild-type mice. At a chloroform dose of 300 mg/kg, severe kidney lesions were observed in both strains, yet the BUN levels were still higher in the liver-Cpr-null than in the wild-type mice. Higher chloroform levels were found in the tissues of the liver-Cpr-null mice. These findings indicated that loss of hepatic P450-dependent chloroform metabolism does not protect against chloroform-induced renal toxicity, suggesting that renal P450 enzymes play an essential role in chloroform renal toxicity.« less
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Rhodes, M E; Rubin, R T; McKlveen, J M; Karwoski, T E; Fulton, B A; Czambel, R K
2008-05-01
Both within the brain and in the periphery, M(1) muscarinic receptors function primarily as postsynaptic receptors and M(2) muscarinic receptors function primarily as presynaptic autoreceptors. In addition to classical parasympathetic effectors, cholinergic stimulation of central muscarinic receptors influences the release of adrenocorticotrophic hormone (ACTH) and corticosterone. We previously reported that oxotremorine administration to male and female M(2) receptor knockout and wild-type mice increased ACTH to a significantly greater degree in knockout males compared to all other groups, and that M(2) knockout mice of both sexes were significantly more responsive to the mild stress of saline injection than were wild-type mice. These results accord with the primary function of M(2) receptors as presynaptic autoreceptors. In the present study, we explored the role of the M(1) receptor in pituitary-adrenal responses to oxotremorine and saline in male and female M(1) knockout and wild-type mice. Because these mice responded differently to the mild stress of saline injection than did the M(2) knockout and wild-type mice, we also determined hormone responses to restraint stress in both M(1) and M(2) knockout and wild-type mice. Male and female M(1) knockout and wild-type mice were equally unresponsive to the stress of saline injection. Oxotremorine increased both ACTH and corticosterone in M(1) wild-type mice to a significantly greater degree than in knockout mice. In both M(1) knockout and wild-type animals, ACTH responses were greater in males compared to females, and corticosterone responses were greater in females compared to males. Hormone responses to restraint stress were increased in M(2) knockout mice and decreased in M(1) knockout mice compared to their wild-type counterparts. These findings suggest that M(1) and M(2) muscarinic receptor subtypes differentially influence male and female pituitary-adrenal responses to cholinergic stimulation and stress. The decreased pituitary-adrenal sensitivity to oxotremorine and restraint stress noted in M(1) knockout mice is consistent with M(1) being primarily a postsynaptic receptor. Conversely, the increased pituitary-adrenal sensitivity to these challenges noted in M(2) knockout mice is consistent with M(2) being primarily a presynaptic autoreceptor.
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Bearson, Bradley L.; Bearson, Shawn M. D.; Kich, Jalusa D.; Lee, In Soo
2014-01-01
Swine are often asymptomatic carriers of Salmonella spp., and interventions are needed to limit colonization of swine to enhance food safety and reduce environmental contamination. We evaluated the attenuation and potential vaccine use in pigs of a Salmonella enterica serovar Typhimurium mutant of rfaH, the gene encoding the RfaH antiterminator that prevents premature termination of long mRNA transcripts. Pigs inoculated with wild-type S. Typhimurium exhibited a significant elevation in average body temperature (fever) at 1 and 2 days post-inoculation; rfaH-inoculated pigs did not (n = 5/group). During the 7-day trial, a significant reduction of Salmonella in the feces, tonsils, and cecum were observed in the rfaH-inoculated pigs compared to wild-type inoculated pigs. To determine whether vaccination with the rfaH mutant could provide protection against wild-type S. Typhimurium challenge, two groups of pigs (n = 14/group) were intranasally inoculated with either the rfaH mutant or a PBS placebo at 6 and 8 weeks of age and challenged with the parental, wild-type S. Typhimurium at 11 weeks of age. The average body temperature was significantly elevated in the mock-vaccinated pigs at 1 and 2 days post-challenge, but not in the rfaH-vaccinated pigs. Fecal shedding at 2 and 3 days post-challenge and colonization of intestinal tract tissues at 7 days post-challenge by wild-type S. Typhimurium was significantly reduced in the rfaH-vaccinated pigs compared to mock-vaccinated pigs. Serological analysis using the IDEXX HerdChek Swine Salmonella Test Kit indicated that vaccination with the rfaH mutant did not stimulate an immune response against LPS. These results indicate that vaccination of swine with the attenuated rfaH mutant confers protection against challenge with virulent S. Typhimurium but does not interfere with herd level monitoring for Salmonella spp., thereby allowing for differentiation of infected from vaccinated animals (DIVA). PMID:26664915
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Kolettis, Theofilos M; Kontonika, Marianthi; La Rocca, Vassilios; Vlahos, Antonios P; Baltogiannis, Giannis G; Kyriakides, Zenon S
2017-04-01
We investigated the effects of autonomic dysfunction and endothelin on local conduction and arrhythmogenesis during myocardial infarction. We recorded ventricular tachyarrhythmias, monophasic action potentials, and activation sequences in wild-type and ET B -deficient rats displaying high endothelin levels. Central sympathetic inputs were examined after clonidine administration. Clonidine mitigated early and delayed arrhythmogenesis in ET B -deficient and wild-type rats, respectively. The right ventricular activation delay increased in clonidine-treated ET B -deficient rats and slightly decreased in wild-type rats. The left ventricular voltage rise decreased in all groups, whereas the activation delay increased mainly in clonidine-treated ET B -deficient rats. Central sympathetic activation and endothelin modulate ischemia-induced arrhythmogenesis. Ischemia alters excitability, whereas endothelin impairs local conduction, an action partly counterbalanced by central sympathetic activity.
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Genomic and environmental selection patterns in two distinct lettuce crop–wild hybrid crosses
Hartman, Yorike; Uwimana, Brigitte; Hooftman, Danny A P; Schranz, Michael E; van de Wiel, Clemens C M; Smulders, Marinus J M; Visser, Richard G F; van Tienderen, Peter H
2013-01-01
Genomic selection patterns and hybrid performance influence the chance that crop (trans)genes can spread to wild relatives. We measured fitness(-related) traits in two different field environments employing two different crop–wild crosses of lettuce. We performed quantitative trait loci (QTL) analyses and estimated the fitness distribution of early- and late-generation hybrids. We detected consistent results across field sites and crosses for a fitness QTL at linkage group 7, where a selective advantage was conferred by the wild allele. Two fitness QTL were detected on linkage group 5 and 6, which were unique to one of the crop–wild crosses. Average hybrid fitness was lower than the fitness of the wild parent, but several hybrid lineages outperformed the wild parent, especially in a novel habitat for the wild type. In early-generation hybrids, this may partly be due to heterosis effects, whereas in late-generation hybrids transgressive segregation played a major role. The study of genomic selection patterns can identify crop genomic regions under negative selection across multiple environments and cultivar–wild crosses that might be applicable in transgene mitigation strategies. At the same time, results were cultivar-specific, so that a case-by-case environmental risk assessment is still necessary, decreasing its general applicability. PMID:23789025
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Genomic and environmental selection patterns in two distinct lettuce crop-wild hybrid crosses.
Hartman, Yorike; Uwimana, Brigitte; Hooftman, Danny A P; Schranz, Michael E; van de Wiel, Clemens C M; Smulders, Marinus J M; Visser, Richard G F; van Tienderen, Peter H
2013-06-01
Genomic selection patterns and hybrid performance influence the chance that crop (trans)genes can spread to wild relatives. We measured fitness(-related) traits in two different field environments employing two different crop-wild crosses of lettuce. We performed quantitative trait loci (QTL) analyses and estimated the fitness distribution of early- and late-generation hybrids. We detected consistent results across field sites and crosses for a fitness QTL at linkage group 7, where a selective advantage was conferred by the wild allele. Two fitness QTL were detected on linkage group 5 and 6, which were unique to one of the crop-wild crosses. Average hybrid fitness was lower than the fitness of the wild parent, but several hybrid lineages outperformed the wild parent, especially in a novel habitat for the wild type. In early-generation hybrids, this may partly be due to heterosis effects, whereas in late-generation hybrids transgressive segregation played a major role. The study of genomic selection patterns can identify crop genomic regions under negative selection across multiple environments and cultivar-wild crosses that might be applicable in transgene mitigation strategies. At the same time, results were cultivar-specific, so that a case-by-case environmental risk assessment is still necessary, decreasing its general applicability.
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Bahl, Sunil; Gary, Howard E; Jafari, Hamid; Sarkar, Bidyut K; Pathyarch, Surendra K; Sethi, Raman; Deshpande, Jagadish
2014-11-01
Despite intensified use of monovalent oral poliovirus type 1 vaccine and improved coverage of immunization campaigns, wild poliovirus type 1 persisted in Indian states of Uttar Pradesh and Bihar during 2006 to 2009. A serosurvey was conducted among cases of acute flaccid paralysis in the 25 high-polio-incidence districts of western Uttar Pradesh. Children were recruited by age group (6-11 months, 12-24 months, and 25-69 months) from among cases reported through the acute flaccid paralysis surveillance system between November 2008 and August 2009. Seroprevalence for type 1 wild poliovirus was >96.4% for each age group. The seroprevalence of wild poliovirus types 2 and 3 increased with age, from 36.7% to 73.4% for type 2 and from 39.0% to 74.1% for type 3. In addition to the number of type-specific vaccine doses, father's level of education, being from a Muslim family, height for age, and female sex were the socioeconomic risk factors associated with seronegativity to poliovirus. The seroprevalence and risk factors identified in this study were consistent with the epidemiology of polio, and the findings were instrumental in optimizing vaccination strategy in western Uttar Pradesh with respect to the choice of OPV types, the frequency of supplementary immunization campaigns, and the urgency to improve routine immunization services. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Finiguerra, Michael; Avery, David E.; Dam, Hans G.
2015-01-01
The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST). Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI) or wild-type isoforms (PWI), while most individuals express relatively equal amounts of each (EI). There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR), ingestion rate (I), and gross growth efficiency (GGE) for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed. PMID:26075900
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Prince, Esther; Lazare, Farrah B.; Treem, William R.; Xu, Jiliu; Iqbal, Jahangir; Pan, Xiaoyue; Josekutty, Joby; Walsh, Meghan; Anderson, Virginia; Hussain, M. Mahmood; Schwarz, Steven M.
2015-01-01
Objectives ω-3 Fatty acids (FAs), natural ligands for the peroxisome proliferator-activated receptor–α (PPAR-α), attenuate parenteral nutrition–associated liver disease (PNALD). However, the mechanisms underlying the protective role of ω-3 FAs are still unknown. The aim of this study was to determine the effects of ω-3 FAs on hepatic triglyceride (TG) accumulation in a murine model of PNALD and to investigate the role of PPAR-α and microsomal triglyceride transfer protein (MTP) in this experimental setting. Methods 129S1/SvImJ wild-type or 129S4/SvJaePparatm/Gonz/J PPAR-α knockout mice were fed chow and water (controls); oral, fat-free PN solution only (PN-O); PN-O plus intraperitoneal (IP) ω-6 FA-predominant supplements (PN–ω-6); or PN-O plus IP ω-3 FA (PN–ω-3). Control and PN-O groups received sham IP injections of 0.9% NaCl. Hepatic histology, TG and cholesterol, MTP activity, and PPAR-α messenger RNA were assessed after 19 days. Results In all experimental groups, PN feeding increased hepatic TG and MTP activity compared with controls. Both PN-O and PN–ω-6 groups accumulated significantly greater amounts of TG when compared with PN–ω-3 mice. Studies in PPAR-α null animals showed that PN feeding increases hepatic TG as in wild-type mice. PPAR-α null mice in the PN-O and PN–ω-6 groups demonstrated variable degrees of hepatic steatosis, whereas no evidence of hepatic fat accumulation was found after 19 days of oral PN plus IP ω-3 FAs. Conclusions PN induces TG accumulation (steatosis) in wild-type and PPAR-α null mice. In PN-fed wild-type and PPAR-α null mice given IP ω-3 FAs, reduced hepatic TG accumulation and absent steatosis are found. Prevention of steatosis by ω-3 FAs results from PPAR-α–independent pathways. PMID:23757305
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Variation in biological properties of cauliflower mosaic virus clones.
al-Kaff, N; Covey, S N
1994-11-01
Infectious clones were prepared from virion DNA of three cauliflower mosaic virus (CaMV) isolates, 11/3, Xinjiang (XJ), and Aust, to investigate pathogenic variation in virus populations. Of 10 infectious clones obtained for isolate 11/3, four pathotypes were identified, each producing symptoms in turnip that differed from those of the 11/3 wild-type. Virus from two clonal groups of 11/3 was transmissible by aphids whereas that from two others was not. Of the five infectious clones obtained from isolate XJ, two groups were identified, one of which differed symptomatically from the wild-type. Only one infectious clone was obtained from isolate Aust and this had properties similar to the wild-type. Restriction enzyme polymorphisms were found in some clonal groups and these correlated with symptoms. Other groups with different pathogenic properties could not be distinguished apart by restriction site polymorphisms. Further variation was observed in the nucleotide sequences of gene II (coding for aphid transmission factor) from these viruses as compared with other CaMV isolates. In the aphid non-transmissible clones of isolate 11/3, one had a Gly to Arg mutation in gene II similar to that of other non-deleted non-transmissible CaMV isolates. The second had a 322 bp deletion at the site of a small direct repeat similar to that of isolate CM4-184 although occurring in a different position. The gene II deletion of isolate 11/3 produced a frame-shift that separated genes II and III by 60 bp. Most CaMV clones studied remained biologically stable producing similar symptoms during subsequent passages. However, one clone (11/3-7) produced two new biotypes during its first passage suggesting that it was relatively unstable. Our results show that wild-type populations of CaMV contain a range of infectious genome variants with contrasting biological properties and differing stability. We suggest that a variety of significant viral phenotypic changes can occur during each infection cycle resulting from relatively small genome changes.
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De Luis, D A; González Sagrado, M; Aller, R; Conde, R; Izaola, O; de la Fuente, B; Primo, D
2011-01-01
A intragenic biallelic polymorphism (1359 G/A) of the CB1 gene resulting in the substitution of the G to A at nucleotide position 1359 in codon 435 (Thr), was reported as a common polymorphism in Caucasian populations. Intervention studies with this polymorphism have not been realized. We decided to investigate the role of the polymorphism (G1359A) of CB1 receptor gene on adipocytokines response and weight loss secondary to a lifestyle modification (Mediterranean hypocaloric diet and exercise) in obese patients. A population of 94 patients with obesity was analyzed. Before and after 3 months on a hypocaloric diet, an anthropometric evaluation, an assessment of nutritional intake and a biochemical analysis were performed. The statistical analysis was performed for the combined G1359A and A1359A as a group and wild type G1359G as second group, with a dominant model. Forty seven patients (50%) had the genotype G1359G (wild type group) and 47 (50%) patients G1359A (41 patients, 43.6%) or A1359A (6 patients, 6.4%) (mutant type group) had the genotype. In wild and mutant type groups, weight, body mass index, fat mass, waist circumference and systolic blood pressure decreased. In mutant type group, resistin (4.15 ± 1.7 ng/ml vs. 3.90 ± 2.1 ng/ml: P < 0.05), leptin (78.4 ± 69 ng/ml vs 66.2 ± 32 ng/ml: P < 0.05) and IL-6 (1.40 ± 1.9 pg/ml vs 0.81 ± 1.5 pg/ml: P < 0.05) levels decreased after dietary treatment. The novel finding of this study is the association of the mutant allele (A1359) with a decrease of resistin, leptin and interleukin-6 secondary to weight loss.
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Microbiota-induced obesity requires farnesoid X receptor.
Parséus, Ava; Sommer, Nina; Sommer, Felix; Caesar, Robert; Molinaro, Antonio; Ståhlman, Marcus; Greiner, Thomas U; Perkins, Rosie; Bäckhed, Fredrik
2017-03-01
The gut microbiota has been implicated as an environmental factor that modulates obesity, and recent evidence suggests that microbiota-mediated changes in bile acid profiles and signalling through the bile acid nuclear receptor farnesoid X receptor (FXR) contribute to impaired host metabolism. Here we investigated if the gut microbiota modulates obesity and associated phenotypes through FXR. We fed germ-free (GF) and conventionally raised (CONV-R) wild-type and Fxr-/- mice a high-fat diet (HFD) for 10 weeks. We monitored weight gain and glucose metabolism and analysed the gut microbiota and bile acid composition, beta-cell mass, accumulation of macrophages in adipose tissue, liver steatosis, and expression of target genes in adipose tissue and liver. We also transferred the microbiota of wild-type and Fxr -deficient mice to GF wild-type mice. The gut microbiota promoted weight gain and hepatic steatosis in an FXR-dependent manner, and the bile acid profiles and composition of faecal microbiota differed between Fxr-/- and wild-type mice. The obese phenotype in colonised wild-type mice was associated with increased beta-cell mass, increased adipose inflammation, increased steatosis and expression of genes involved in lipid uptake. By transferring the caecal microbiota from HFD-fed Fxr-/- and wild-type mice into GF mice, we showed that the obesity phenotype was transferable. Our results indicate that the gut microbiota promotes diet-induced obesity and associated phenotypes through FXR, and that FXR may contribute to increased adiposity by altering the microbiota composition. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
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Schlegel, Victoria; Thieme, Markus; Holzmann, Carsten; Witt, Martin; Grittner, Ulrike; Rolfs, Arndt; Wree, Andreas
2016-11-09
Niemann-Pick Type C1 (NPC1) is an autosomal recessive inherited disorder characterized by accumulation of cholesterol and glycosphingolipids. Previously, we demonstrated that BALB/c-npc1 nih Npc1 -/- mice treated with miglustat, cyclodextrin and allopregnanolone generally performed better than untreated Npc1 -/- animals. Unexpectedly, they also seemed to accomplish motor tests better than their sham-treated wild-type littermates. However, combination-treated mutant mice displayed worse cognition performance compared to sham-treated ones. To evaluate effects of these drugs in healthy BALB/c mice, we here analyzed pharmacologic effects on motor and cognitive behavior of wild-type mice. For combination treatment mice were injected with allopregnanolone/cyclodextrin weekly, starting at P7. Miglustat injections were performed daily from P10 till P23. Starting at P23, miglustat was embedded in the chow. Other mice were treated with miglustat only, or sham-treated. The battery of behavioral tests consisted of accelerod, Morris water maze, elevated plus maze, open field and hot-plate tests. Motor capabilities and spontaneous motor behavior were unaltered in both drug-treated groups. Miglustat-treated wild-type mice displayed impaired spatial learning compared to sham- and combination-treated mice. Both combination- and miglustat-treated mice showed enhanced anxiety in the elevated plus maze compared to sham-treated mice. Additionally, combination treatment as well as miglustat alone significantly reduced brain weight, whereas only combination treatment reduced body weight significantly. Our results suggest that allopregnanolone/cyclodextrin ameliorate most side effects of miglustat in wild-type mice.
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Vaccination Against Porcine Circovirus-2 Reduces Severity of Tuberculosis in Wild Boar.
Risco, David; Bravo, María; Martínez, Remigio; Torres, Almudena; Gonçalves, Pilar; Cuesta, Jesús; García-Jiménez, Waldo; Cerrato, Rosario; Iglesias, Rocío; Galapero, Javier; Serrano, Emmanuel; Gómez, Luis; Fernández-Llario, Pedro; Hermoso de Mendoza, Javier
2018-03-09
Tuberculosis (TB) in wild boar (Sus scrofa) may be affected by coinfections with other pathogens, such as porcine circovirus type 2 (PCV2). Therefore, sanitary measures focused on controlling PCV2 could be useful in reducing the impact of TB in this wild suid. The aim of this study was to explore whether vaccination against PCV2 targeting young animals affects TB prevalence and TB severity in wild boar. The study was conducted on a game estate in mid-western Spain. Seventy animals of ages ranging from 4 to 8 months were captured, individually identified, vaccinated against PCV2 and released, forming a vaccinated group. Not-captured animals cohabiting with the vaccinated wild boar constituted the control group. Animals from both groups were hunted between 2013 and 2016 and a TB diagnosis based on pathological assessment and microbiological culture was made in all of them. The effect of PCV2 vaccination on TB prevalence and severity was explored using generalized lineal models. Whereas TB prevalence was similar in vaccinated and control groups (54.55 vs. 57.78%), vaccinated animals showed less probabilities to develop generalized TB lesions. Furthermore, mean TB severity score was significantly lower in vaccinated animals (1.55 vs. 2.42) suggesting a positive effect of PCV2 vaccination.
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den Hartog, Carolina R; Beckley, Jacob T; Smothers, Thetford C; Lench, Daniel H; Holseberg, Zack L; Fedarovich, Hleb; Gilstrap, Meghin J; Homanics, Gregg E; Woodward, John J
2013-01-01
Ethanol's action on the brain likely reflects altered function of key ion channels such as glutamatergic N-methyl-D-aspartate receptors (NMDARs). In this study, we determined how expression of a mutant GluN1 subunit (F639A) that reduces ethanol inhibition of NMDARs affects ethanol-induced behaviors in mice. Mice homozygous for the F639A allele died prematurely while heterozygous knock-in mice grew and bred normally. Ethanol (44 mM; ∼0.2 g/dl) significantly inhibited NMDA-mediated EPSCs in wild-type mice but had little effect on responses in knock-in mice. Knock-in mice had normal expression of GluN1 and GluN2B protein across different brain regions and a small reduction in levels of GluN2A in medial prefrontal cortex. Ethanol (0.75-2.0 g/kg; i.p.) increased locomotor activity in wild-type mice but had no effect on knock-in mice while MK-801 enhanced activity to the same extent in both groups. Ethanol (2.0 g/kg) reduced rotarod performance equally in both groups but knock-in mice recovered faster following a higher dose (2.5 g/kg). In the elevated zero maze, knock-in mice had a blunted anxiolytic response to ethanol (1.25 g/kg) as compared to wild-type animals. No differences were noted between wild-type and knock-in mice for ethanol-induced loss of righting reflex, sleep time, hypothermia or ethanol metabolism. Knock-in mice consumed less ethanol than wild-type mice during daily limited-access sessions but drank more in an intermittent 24 h access paradigm with no change in taste reactivity or conditioned taste aversion. Overall, these data support the hypothesis that NMDA receptors are important in regulating a specific constellation of effects following exposure to ethanol.
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den Hartog, Carolina R.; Beckley, Jacob T.; Smothers, Thetford C.; Lench, Daniel H.; Holseberg, Zack L.; Fedarovich, Hleb; Gilstrap, Meghin J.; Homanics, Gregg E.; Woodward, John J.
2013-01-01
Ethanol's action on the brain likely reflects altered function of key ion channels such as glutamatergic N-methyl-D-aspartate receptors (NMDARs). In this study, we determined how expression of a mutant GluN1 subunit (F639A) that reduces ethanol inhibition of NMDARs affects ethanol-induced behaviors in mice. Mice homozygous for the F639A allele died prematurely while heterozygous knock-in mice grew and bred normally. Ethanol (44 mM; ∼0.2 g/dl) significantly inhibited NMDA-mediated EPSCs in wild-type mice but had little effect on responses in knock-in mice. Knock-in mice had normal expression of GluN1 and GluN2B protein across different brain regions and a small reduction in levels of GluN2A in medial prefrontal cortex. Ethanol (0.75–2.0 g/kg; IP) increased locomotor activity in wild-type mice but had no effect on knock-in mice while MK-801 enhanced activity to the same extent in both groups. Ethanol (2.0 g/kg) reduced rotarod performance equally in both groups but knock-in mice recovered faster following a higher dose (2.5 g/kg). In the elevated zero maze, knock-in mice had a blunted anxiolytic response to ethanol (1.25 g/kg) as compared to wild-type animals. No differences were noted between wild-type and knock-in mice for ethanol-induced loss of righting reflex, sleep time, hypothermia or ethanol metabolism. Knock-in mice consumed less ethanol than wild-type mice during daily limited-access sessions but drank more in an intermittent 24 h access paradigm with no change in taste reactivity or conditioned taste aversion. Overall, these data support the hypothesis that NMDA receptors are important in regulating a specific constellation of effects following exposure to ethanol. PMID:24244696
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2012-01-01
Background Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial. Methods Eighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7) or asymptomatic (11), based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females. Results The study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers. Conclusions This is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was observed, suggesting that the two mechanisms are regulated independently. Moreover, neither the total DMD transcript level, nor the relative proportion of the wild-type transcript do correlate with the symptomatic phenotype. PMID:22894145
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Impaired ventilatory acclimatization to hypoxia in mice lacking the immediate early gene fos B.
Malik, Mohammad T; Peng, Ying-Jie; Kline, David D; Adhikary, Gautam; Prabhakar, Nanduri R
2005-01-15
Earlier studies on cell culture models suggested that immediate early genes (IEGs) play an important role in cellular adaptations to hypoxia. Whether IEGs are also necessary for hypoxic adaptations in intact animals is not known. In the present study we examined the potential importance of fos B, an IEG in ventilatory acclimatization to hypoxia. Experiments were performed on wild type and mutant mice lacking the fos B gene. Ventilation was monitored by whole body plethysmography in awake animals. Baseline ventilation under normoxia, and ventilatory response to acute hypoxia and hypercapnia were comparable between wild type and mutant mice. Hypobaric hypoxia (0.4 atm; 3 days) resulted in a significant elevation of baseline ventilation in wild type but not in mutant mice. Wild type mice exposed to hypobaric hypoxia manifested an enhanced hypoxic ventilatory response compared to pre-hypobaric hypoxia. In contrast, hypobaric hypoxia had no effect on the hypoxic ventilatory response in mutant mice. Hypercapnic ventilatory responses, however, were unaffected by hypobaric hypoxia in both groups of mice. These results suggest that the fos B, an immediate early gene, plays an important role in ventilatory acclimatization to hypoxia in mice.
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Overexpression of NGF ameliorates ethanol neurotoxicity in the developing cerebellum.
Heaton, M B; Mitchell, J J; Paiva, M
2000-11-05
Transgenic mice overexpressing NGF in the central nervous system under the control of the glial fibrillary acidic protein (GFAP) promoter were exposed to ethanol via vapor inhalation on postnatal days 4 and 5 (P4-5), the period of maximal cerebellar Purkinje cell sensitivity to ethanol. Wild-type controls were exposed in a similar manner. There were no differences in body weight or size following these procedures, but the transgenic brain weights at this age were significantly greater than wild-type controls. In the wild-type animals, a significant 33.3% ethanol-mediated loss of Purkinje cells in lobule I was detected via unbiased three-dimensional stereological counting on P5. In the GFAP-NGF transgenic animals, however, the 17.6% difference in Purkinje cell number in control and ethanol-exposed animals was not significant. There was a similar difference in Purkinje cell density in both groups, which did reach statistical significance (-32.7% in wild-type ethanol-treated animals, -17% in transgenic ethanol-exposed animals). These results suggest that endogenous overexpression of neurotrophic factors, which have previously been shown to protect against ethanol neurotoxicity in culture, can serve a similar protective function in the intact animal. Copyright 2000 John Wiley & Sons, Inc.
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Elwen, Simon Harvey; Nastasi, Aurora
2014-01-01
A signature whistle type is a learned, individually distinctive whistle type in a dolphin's acoustic repertoire that broadcasts the identity of the whistle owner. The acquisition and use of signature whistles indicates complex cognitive functioning that requires wider investigation in wild dolphin populations. Here we identify signature whistle types from a population of approximately 100 wild common bottlenose dolphins (Tursiops truncatus) inhabiting Walvis Bay, and describe signature whistle occurrence, acoustic parameters and temporal production. A catalogue of 43 repeatedly emitted whistle types (REWTs) was generated by analysing 79 hrs of acoustic recordings. From this, 28 signature whistle types were identified using a method based on the temporal patterns in whistle sequences. A visual classification task conducted by 5 naïve judges showed high levels of agreement in classification of whistles (Fleiss-Kappa statistic, κ = 0.848, Z = 55.3, P<0.001) and supported our categorisation. Signature whistle structure remained stable over time and location, with most types (82%) recorded in 2 or more years, and 4 identified at Walvis Bay and a second field site approximately 450 km away. Whistle acoustic parameters were consistent with those of signature whistles documented in Sarasota Bay (Florida, USA). We provide evidence of possible two-voice signature whistle production by a common bottlenose dolphin. Although signature whistle types have potential use as a marker for studying individual habitat use, we only identified approximately 28% of those from the Walvis Bay population, despite considerable recording effort. We found that signature whistle type diversity was higher in larger dolphin groups and groups with calves present. This is the first study describing signature whistles in a wild free-ranging T. truncatus population inhabiting African waters and it provides a baseline on which more in depth behavioural studies can be based. PMID:25203814
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Kristen, Arnt V; Ackermann, Katrin; Buss, Sebastian; Lehmann, Lorenz; Schnabel, Philipp A; Haunstetter, Armin; Katus, Hugo A; Hardt, Stefan E
2013-01-01
The detailed molecular mechanisms following activation of apoptosis in ischemia-reperfusion injury are unknown. This study using different transgenic mouse models provided first evidence that apoptosis in myocardial ischemia-reperfusion injury is rather linked to the mitochondrial pathway than to death receptor pathway. There is a wealth of evidence for activation of apoptosis in ischemia-reperfusion injury. However, the understanding of detailed molecular mechanism is lacking. The extent of myocardial infarction after ligation of the left anterior descending artery in mice carrying different transgenes for inhibition of either the intrinsic or the extrinsic or a combination of both apoptotic cascades was evaluated. The extent of myocardial damage was assessed by echocardiographic determination of left ventricular (LV) ejection fraction, LV hemodynamics, troponin T, and histology. The rate of apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3 staining. Highest perioperative rate of death was observed in the dominant-negative form of a truncated Fas-associated death domain (FADD-DN) group. Infarction size by 2,3,5-triphenyltetrazolium chloride (TTC) staining was smaller in the Bcl-2, but not in the other groups as compared to wild-type mice. This was accompanied by lower troponin T values in Bcl-2 transgenic mice as compared to the all other groups. Troponin T correlated well with macroscopic extent of myocardial infarction by TTC staining. A lower decline of LV ejection fraction was seen in the Bcl-2 as compared to wild-type or FADD-DN mice. A smaller number of TUNEL- and caspase-3-positive myocyte nuclei were observed in the Bcl-2 and FADD-DN group as compared to wild-type mice. We provide first evidence for protective effects on the myocardium in a transgenic mouse model of myocardial ischemia-reperfusion due to inhibition of the Bcl-2, but not the FADD pathway despite that reduced apoptotic cells were observed in both groups as compared to wild-type mice. Copyright © 2013 Elsevier Inc. All rights reserved.
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Hurko, Orest; Boudonck, Kurt; Gonzales, Cathleen; Hughes, Zoe A.; Jacobsen, J. Steve; Reinhart, Peter H.; Crowther, Daniel
2010-01-01
Mice transgenic for production of excessive or mutant forms of beta-amyloid differ from patients with Alzheimer's disease in the degree of inflammation, oxidative damage, and alteration of intermediary metabolism, as well as the paucity or absence of neuronal atrophy and cognitive impairment. Previous observers have suggested that differences in inflammatory response reflect a discrepancy in the state of the locus coeruleus (LC), loss of which is an early change in Alzheimer's disease but which is preserved in the transgenic mice. In this paper, we extend these observations by examining the effects of the LC on markers of oxidative stress and intermediary metabolism. We compare four groups: wild-type or Tg2576 Aβ transgenic mice injected with DSP4 or vehicle. Of greatest interest were metabolites different between ablated and intact transgenics, but not between ablated and intact wild-type animals. The Tg2576_DSP4 mice were distinguished from the other three groups by oxidative stress and altered energy metabolism. These observations provide further support for the hypothesis that Tg2576 Aβ transgenic mice with this ablation may be a more congruent model of Alzheimer's disease than are transgenics with an intact LC. PMID:20981353
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Ohno, Y; Egawa, T; Yokoyama, S; Nakai, A; Sugiura, T; Ohira, Y; Yoshioka, T; Goto, K
2015-12-01
Effects of heat shock transcription factor 1 (HSF1) deficiency on heat stress-associated increase in slow soleus muscle mass of mice were investigated. Both HSF1-null and wild-type mice were randomly assigned to control and heat-stressed groups. Mice in heat-stressed group were exposed to heat stress (41 °C for 60 min) in an incubator without anaesthesia. Significant increase in wet and dry weights, and protein content of soleus muscle in wild-type mice was observed seven days after the application of the heat stress. However, heat stress had no impact on soleus muscle mass in HSF1-null mice. Neither type of mice exhibited much effect of heat stress on HSF mRNA expression (HSF1, HSF2 and HSF4). On the other hand, heat stress upregulated heat shock proteins (HSPs) at the mRNA (HSP72) and protein (HSP72 and HSP110) levels in wild-type mice, but not in HSF1-null mice. The population of Pax7-positive nuclei relative to total myonuclei of soleus muscle in wild-type mice was significantly increased by heat stress, but not in HSF1-null mice. Furthermore, the absence of HSF1 gene suppressed heat stress-associated phosphorylation of Akt and p70 S6 kinase (p-p70S6K) in soleus muscle. Heat stress-associated increase in skeletal muscle mass may be induced by HSF1 and/or HSF1-mediated stress response that activates muscle satellite cells and Akt/p70S6K signalling pathway. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.
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Okadera, Kota; Abe, Masako; Ito, Naoto; Morikawa, Shigeki; Yamasaki, Ari; Masatani, Tatsunori; Nakagawa, Keisuke; Yamaoka, Satoko; Sugiyama, Makoto
2013-12-01
Group A rotaviruses (RVAs) are a major cause of acute dehydrating diarrhea in infants and young animals worldwide. RVAs have also been detected in several wild and zoo animals, indicating wide susceptibility of wild animals. However, the role of wild animals in the infection cycle of RVAs is unclear. Wild boars are indigenous in many countries in the world. Japanese wild boars (Sus scrofa leucomystax) have been migrating close to human habitats in Japan, indicating the possibility of natural transmission between domestic animals or humans and wild boars. We investigated infection of RVAs in wild boars in Japan to identify types of RVAs infecting wild animals. We obtained stool samples from 90 wild boars and detected a VP4 gene of RVAs by RT-semi-nested PCR. RVAs were detected in samples from four of the 90 wild boars. Nucleotide analyses of VP7 and VP4 genes revealed that the four strains belong to G9P[23], G4P[23], G9P[13] and G4P[6], suggesting a relation to porcine and human RVAs. We therefore characterized RVAs circulating among domestic pigs living in the same area as the wild boars. We collected stool samples from 82 domestic pigs. RVAs were detected in samples from 49 of the 82 domestic pigs. Phylogenetic and similarity analyses provided evidence for natural transmission between domestic pigs and wild boars. The results also suggested that natural reassortment events occurred before or after transmission between domestic pigs and wild boars. Our findings indicate the possibility that RVAs circulate among wild animals, humans and domestic animals in nature. Copyright © 2013 Elsevier B.V. All rights reserved.
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Abernathy, Jason; Panserat, Stéphane; Welker, Thomas; Plagne-Juan, Elisabeth; Sakhrani, Dionne; Higgs, David A; Audouin, Florence; Devlin, Robert H; Overturf, Ken
2015-12-01
Growth hormone (GH) transgenic salmon possesses markedly increased metabolic rate, appetite, and feed conversion efficiency, as well as an increased ability to compete for food resources. Thus, the ability of GH-transgenic fish to withstand periods of food deprivation as occurs in nature is potentially different than that of nontransgenic fish. However, the physiological and genetic effects of transgenic GH production over long periods of food deprivation remain largely unknown. Here, GH-transgenic coho salmon (Oncorhynchus kisutch) and nontransgenic, wild-type coho salmon were subjected to a 3-month food deprivation trial, during which time performance characteristics related to growth were measured along with proximate compositions. To examine potential genetic effects of GH-transgenesis on long-term food deprivation, a group of genes related to muscle development and liver metabolism was selected for quantitative PCR analysis. Results showed that GH-transgenic fish lose weight at an increased rate compared to wild-type even though proximate compositions remained relatively similar between the groups. A total of nine genes related to muscle physiology (cathepsin, cee, insulin-like growth factor, myostatin, murf-1, myosin, myogenin, proteasome delta, tumor necrosis factor) and five genes related to liver metabolism (carnitine palmitoyltransferase, fatty acid synthase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glucokinase) were shown to be differentially regulated between GH-transgenic and wild-type coho salmon over time. These genetic and physiological responses assist in identifying differences between GH-transgenic and wild-type salmon in relation to fitness effects arising from elevated growth hormone during periods of long-term food shortage.
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Characterisation of a type 1 Avian Paramyxovirus belonging to a divergent group.
Briand, François-Xavier; Massin, Pascale; Jestin, Véronique
2014-01-10
Newcastle disease, induced by a type 1 Avian Paramyxovirus (APMV-1), is one of the most serious poultry diseases. APMV-1 are divided into two classes based on genetic analysis: class II strains have been recovered from wild or domestic birds and include virulent and avirulent isolates whereas class I strains have been mainly isolated from wild birds and are avirulent. Within class I, a new proposed genotype has recently been reported. The only full genome strain of this group is presently characterised from the point of view of codon usage with reference to class I and class II specificities. Class-specific residues were identified on HN and F proteins that are the two major proteins involved in cell attachment and pathogenicity. Comparison of protein patterns and codon usage for this newly identified APMV-1 strain indicates it is similar to class I viruses but contains a few characteristics close to the viruses of class II. Transmission of viruses from this recently identified divergent group from wild birds to domestic birds could have a major impact on the domestic poultry industry. Copyright © 2013 Elsevier B.V. All rights reserved.
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Cristóvão, Filipe; Alonso, Carla Andrea; Igrejas, Gilberto; Sousa, Margarida; Silva, Vanessa; Pereira, José Eduardo; Lozano, Carmen; Cortés-Cortés, Gerardo; Torres, Carmen; Poeta, Patrícia
2017-03-01
The clonal diversity of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolates from nine different species of wild animals from distinct regions of Portugal and Spain and their content in replicon plasmids were analyzed. Among the initial 53 ESBL-producing E. coli isolates that were studied (from previous studies), 28 were selected, corresponding to different animal origins with distinct ESBL types and pulsed-field gel electrophoresis (PFGE) patterns. These 28 isolates produced different ESBLs ascribed to the following families: CTX-M, SHV and TEM. The isolates were classified into three phylogenetic groups: B1 (n = 11), A (n = 10) and D (n = 7). The seven E. coli of phylogroup D were then typed by multilocus sequence typing and ascribed to four distinct sequence types: ST117, ST115, ST2001 and ST69. The clonal diversity and relationship between isolates was studied by PFGE. Lastly, the plasmids were analyzed according to their incompatibility group using the PCR-based-replicon-typing scheme. A great diversity of replicon types was identified, with up to five per isolate. Most of the CTX-M-1 and SHV-12 producing E. coli isolates carried IncI1 or IncN replicons. The diversity of ESBL-producing E. coli isolates in wild animals, which can be disseminated in the environment, emphasizes the environmental and health problems that we face nowadays. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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de Luis, Daniel Antonio; Izaola, Olatz; Primo, David; de la Fuente, Beatriz; Mulero, Ines; Aller, Rocío
2016-01-01
Polymorphisms of a single nucleotide in RETN have been associated with indexes of insulin resistance. Our aim was to analyze the effects of the rs1862513 RETN gene polymorphism on insulin resistance, insulin levels, and resistin levels changes after 3 months of a low-fat hypocaloric diet. A Caucasian population of 133 obese patients was analyzed before and after 3 months on a low-fat hypocaloric diet. Fifty-six patients (42.1%) had the genotype GG (wild group) and 77 (57.9%) patients had the other genotypes; GC (59 patients, 44.4%) or CC (18 patients, 13.5%; mutant group). In wild and mutant genotype groups, weight, body mass index, fat mass, waist circumference, and systolic blood pressure decreased. In the wild genotype group, the decrease in total cholesterol was -13.1 ± 25.3 mg/dL (vs. -4.4 ± 13.7 mg/dL in mutant group: p = 0.004 for group deltas), low density lipoprotein (LDL)-cholesterol was -13.0 ± 21.5 mg/dL (-4.3 ± 10.5 mg/dL: p = 0.007), glucose -7.2 ± 3.5 mg/dL (-0.8 ± 0.2 mg/dL: p = 0.01), insulin -5.6 ± 2.5 mUI/L (-2.9 ± 1.2 mUI/L: p = 0.03) and homeostasis model assessment-insulin resistance (HOMA-IR) -2.5 ± 1.1 (-0.6 ± 1.4: p = 0.02). Leptin levels decreased in both genotypes (-10.1 ± 9.5 ng/dL in wild type group vs. -13.1 ± 0.2 ng/dL in mutant type group: p > 0.05). The present study suggests that the G/G genotype of RETN rs1862513 could be a predictor of the reduction of HOMA-IR, insulin, fasting glucose and LDL cholesterol secondary to a hypocaloric diet in obese subjects. © 2016 S. Karger AG, Basel.
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Toll-Like Receptor 3 Is Critical for Coxsackievirus B4-Induced Type 1 Diabetes in Female NOD Mice
Thuma, Jean R.; Courreges, Maria C.; Benencia, Fabian; James, Calvin B.L.; Malgor, Ramiro; Kantake, Noriko; Mudd, William; Denlinger, Nathan; Nolan, Bret; Wen, Li; Schwartz, Frank L.
2015-01-01
Group B coxsackieviruses (CVBs) are involved in triggering some cases of type 1 diabetes mellitus (T1DM). However, the molecular mechanism(s) responsible for this remain elusive. Toll-like receptor 3 (TLR3), a receptor that recognizes viral double-stranded RNA, is hypothesized to play a role in virus-induced T1DM, although this hypothesis is yet to be substantiated. The objective of this study was to directly investigate the role of TLR3 in CVB-triggered T1DM in nonobese diabetic (NOD) mice, a mouse model of human T1DM that is widely used to study both spontaneous autoimmune and viral-induced T1DM. As such, we infected female wild-type (TLR3+/+) and TLR3 knockout (TLR3−/−) NOD mice with CVB4 and compared the incidence of diabetes in CVB4-infected mice with that of uninfected counterparts. We also evaluated the islets of uninfected and CVB4-infected wild-type and TLR3 knockout NOD mice by immunohistochemistry and insulitis scoring. TLR3 knockout mice were markedly protected from CVB4-induced diabetes compared with CVB4-infected wild-type mice. CVB4-induced T-lymphocyte-mediated insulitis was also significantly less severe in TLR3 knockout mice compared with wild-type mice. No differences in insulitis were observed between uninfected animals, either wild-type or TLR3 knockout mice. These data demonstrate for the first time that TLR3 is 1) critical for CVB4-induced T1DM, and 2) modulates CVB4-induced insulitis in genetically prone NOD mice. PMID:25422874
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Gao, Xiao-Ming; Dilley, Rodney J; Samuel, Chrishan S; Percy, Elodie; Fullerton, Meryl J; Dart, Anthony M; Du, Xiao-Jun
2002-10-01
This paper addresses whether the enhanced left ventricular (LV) contractility and heart rate, seen in transgenic mice overexpressing beta -adrenergic receptor in the heart, might raise the incidence of LV rupture after myocardial infarct. Transgenic and wild-type mice underwent left coronary artery occlusion. Postinfarct deaths that occurred 1-7 days after surgery were analyzed. Hemodynamics, morphologic parameters, and collagen content in the LV were determined. A significantly lower incidence of LV rupture was observed in transgenic than in wild-type mice 3-5 days after myocardial infarct (2.5 versus 19.7%, p < 0.05), despite a similar infarct size between the two groups and better hemodynamic function in transgenic mouse hearts. Morphologic analysis showed a more severe infarct expansion in wild-type versus transgenic mice or in mice dying of rupture versus those that died of acute heart failure. Collagen content was higher in the LV of sham-operated transgenic than wild-type mice (p < 0.01) with both type I and type III collagen elevated. Such difference in collagen content between transgenic and wild-type mice was maintained in noninfarcted and infarcted LV. In conclusion, transgenic mice overexpressing beta -adrenergic receptor had a lower risk of cardiac rupture during the acute phase after infarction despite the markedly enhanced LV contractility and heart rate. As a hyperdynamic function due to beta-adrenergic activation would likely increase the risk of cardiac rupture and infarct expansion, the lack of rupture in this transgenic mouse model suggests that the interstitial collagen level is a more important factor than functional status in the pathogenesis of rupture and infarct expansion.
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Adedeji, A O; Okonko, I O; Adu, F D
2012-09-01
Sensitive poliovirus surveillance to detect vaccine-derived-polioviruses will continue to increase in importance. Isolating and identifying poliovirus strains from children of pediatrics age in Nigeria. A total of 120 fecal samples were randomly collected from children under the age of five who presented with acute flaccid paralysis. Samples were tested by tissue culture technique and further characterized by intratypic differentiation testing using ELISA and PCR methods. The study confirmed the presence of 22(18.3%) enteroviral isolates comprising 19(86.4%) polioviruses and 3(13.6%) non-polio enteroviruses. These 19 polioviruses include: Sabin-type poliovirus-1 (15.8%), poliovirus-2 (10.5%), poliovirus-3 (10.5%) and wild-type poliovirus-1 (63.2%) isolates. It showed that poliovirus infection was higher in children ages 6-11 months (18.9%), females (18.4%), northern states (91.0%) with no vaccination record (75.0%). Wild-type poliovirus-1 was isolated from the stool samples of 12(54.6%) children from northern states and in all age groups except 18-23 months. No significant differences (P >0.05) between poliovirus infection and age (18.9% vs. 17.7%; 81.9% vs. 18.2%) and sex (18.3% vs. 18.4%). There was significant differences (P<0.05) between poliovirus infection and location (91.0% vs. 9.0%) and history of polio vaccination (75.0% vs. 0.0%). No wild-type poliovirus was found in those with complete vaccination. This study further confirms the presence of Sabin and wild-type poliovirus among children in Nigeria. The isolation of Sabin strain of poliovirus is advantageous to the polio eradication program as it is capable of inducing natural immunity in susceptible hosts. Transmission of wild-type poliovirus among children with incomplete vaccination poses a serious threat to polio eradication program in Nigeria. Environmental and serological surveillance with larger sample size are important for monitoring poliovirus circulation in Nigeria.
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Pleasant, LaTawnya; Ma, Qing; Devarajan, Mahima; Parameswaran, Priyanka; Drake, Keri; Siroky, Brian; Shay-Winkler, Kritton; Robbins, Jeffrey; Devarajan, Prasad
2017-09-01
The early events that signal renal dysfunction in presymptomatic heart failure are unclear. We tested the hypothesis that functional and mechanistic changes occur in the kidney that precede the development of symptomatic heart failure. We employed a transgenic mouse model with cardiomyocyte-specific overexpression of mutant α-B-crystallin that develops slowly progressive cardiomyopathy. Presymptomatic transgenic mice displayed an increase in serum creatinine (1.17 ± 0.34 vs. wild type 0.65 ± 0.16 mg/dl, P < 0.05) and in urinary neutrophil gelatinase-associated lipocalin (NGAL; 278.92 ± 176.24 vs. wild type 49.11 ± 22.79 ng/ml, P < 0.05) but no renal fibrosis. Presymptomatic transgenic mouse kidneys exhibited a twofold upregulation of the Ren1 gene, marked overexpression of renin protein in the tubules, and a worsened response to ischemia-reperfusion injury based on serum creatinine (2.77 ± 0.66 in transgenic mice vs. 2.01 ± 0.58 mg/dl in wild type, P < 0.05), urine NGAL (9,198.79 ± 3,799.52 in transgenic mice vs. 3,252.94 ± 2,420.36 ng/ml in wild type, P < 0.05), tubule dilation score (3.4 ± 0.5 in transgenic mice vs. 2.6 ± 0.5 in wild type, P < 0.05), tubule cast score (3.2 ± 0.4 in transgenic mice vs. 2.5 ± 0.5 in wild type, P < 0.05), and TdT-mediated dUTP nick-end labeling (TUNEL)-positive nuclei (10.1 ± 2.1 in the transgenic group vs. 5.7 ± 1.6 per 100 cells counted in wild type, P < 0.01). Our findings indicate functional renal impairment, urinary biomarker elevations, and induction of renin gene and protein expression in the kidney that occur in early presymptomatic heart failure, which increase the susceptibility to subsequent acute kidney injury. Copyright © 2017 the American Physiological Society.
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Most, D; Efron, D T; Shi, H P; Tantry, U S; Barbul, A
2001-10-01
Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test. Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05). Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.
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Yoshihara, Ryouhei; Li, ZhengHao; Ishimori, Keisuke; Kuwabara, Kazuki; Hatakeyama, Shin; Tanaka, Shuuitsu
2017-08-01
To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2weeks of cultivation, despite that of the wild-type continuing for over 2years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhg1 KO strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhg1 KO strain than in the wild-type. mtDNA aberration was also observed in the mhg1 KO strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhg1 KO strain. Copyright © 2017 Elsevier Inc. All rights reserved.
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Fine-tuning the space, time, and host distribution of mycobacteria in wildlife
2011-01-01
Background We describe the diversity of two kinds of mycobacteria isolates, environmental mycobacteria and Mycobacterium bovis collected from wild boar, fallow deer, red deer and cattle in Doñana National Park (DNP, Spain), analyzing their association with temporal, spatial and environmental factors. Results High diversity of environmental mycobacteria species and M. bovis typing patterns (TPs) were found. When assessing the factors underlying the presence of the most common types of both environmental mycobacteria and M. bovis TPs in DNP, we evidenced (i) host species differences in the occurrence, (ii) spatial structuration and (iii) differences in the degree of spatial association of specific types between host species. Co-infection of a single host by two M. bovis TPs occurred in all three wild ungulate species. In wild boar and red deer, isolation of one group of mycobacteria occurred more frequently in individuals not infected by the other group. While only three TPs were detected in wildlife between 1998 and 2003, up to 8 different ones were found during 2006-2007. The opposite was observed in cattle. Belonging to an M. bovis-infected social group was a significant risk factor for mycobacterial infection in red deer and wild boar, but not for fallow deer. M. bovis TPs were usually found closer to water marshland than MOTT. Conclusions The diversity of mycobacteria described herein is indicative of multiple introduction events and a complex multi-host and multi-pathogen epidemiology in DNP. Significant changes in the mycobacterial isolate community may have taken place, even in a short time period (1998 to 2007). Aspects of host social organization should be taken into account in wildlife epidemiology. Wildlife in DNP is frequently exposed to different species of non-tuberculous, environmental mycobacteria, which could interact with the immune response to pathogenic mycobacteria, although the effects are unknown. This research highlights the suitability of molecular typing for surveys at small spatial and temporal scales. PMID:21288321
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Teruya, Tsuyoshi; Nakachi, Atsushi; Shimabukuro, Nobuhiro; Toritsuka, Daisuke; Azuma, Yasuharu; Hanashiro, Kiyotoshi; Nishiki, Takehiro; Ota, Morihito; Shimabuku, Masamori; Shiroma, Hiroshi
2015-05-01
Irinotecan is an effective drug in the treatment of colorectal cancer. However, there are reports of an association between certain UGT1A1 genetic polymorphisms and the development of adverse reactions(such as neutropenia)related to irinotecan metabolism. We retrospectively investigated UGT1A1 genetic polymorphisms and the occurrences of irinotecan-induced neutropenia in 25 patients of colorectal cancer at our hospital. Analysis of UGT1A1 genetic polymorphisms in these patients yielded the following classifications: a wild-type group( *1/*1)comprising 13 patients(52%), a heterozygous group(*1/ *28, *1/*6)of 10 patients(40%), and a homozygous group(*28/*28, *6/*6)of 2 patients(8%). The frequency of neutropenia was 15.4%(2/13)in the wild-type group, 30%(3/10)in the heterozygous group, and 100%(2/2)in the homozygous group. Grade 4 neutropenia only occurred in the homozygous group. These results suggest that a dose reduction of irinotecan should be considered for patients who fall into the homozygous group upon analysis of their UGT1A1 genetic polymorphisms, as such patients might be susceptible to grade 4 neutropenia.
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An agent-based model of dialect evolution in killer whales.
Filatova, Olga A; Miller, Patrick J O
2015-05-21
The killer whale is one of the few animal species with vocal dialects that arise from socially learned group-specific call repertoires. We describe a new agent-based model of killer whale populations and test a set of vocal-learning rules to assess which mechanisms may lead to the formation of dialect groupings observed in the wild. We tested a null model with genetic transmission and no learning, and ten models with learning rules that differ by template source (mother or matriline), variation type (random errors or innovations) and type of call change (no divergence from kin vs. divergence from kin). The null model without vocal learning did not produce the pattern of group-specific call repertoires we observe in nature. Learning from either mother alone or the entire matriline with calls changing by random errors produced a graded distribution of the call phenotype, without the discrete call types observed in nature. Introducing occasional innovation or random error proportional to matriline variance yielded more or less discrete and stable call types. A tendency to diverge from the calls of related matrilines provided fast divergence of loose call clusters. A pattern resembling the dialect diversity observed in the wild arose only when rules were applied in combinations and similar outputs could arise from different learning rules and their combinations. Our results emphasize the lack of information on quantitative features of wild killer whale dialects and reveal a set of testable questions that can draw insights into the cultural evolution of killer whale dialects. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Zhang, Xiaolong; Jiang, Quanlong; Xu, Xingli; Wang, Yongrong; Liu, Lei; Lian, Yaru; Li, Hao; Wang, Lichun; Zhang, Ying; Jiang, Guorun; Zeng, Jieyuan; Zhang, Han; Han, Jing-Dong Jackie; Li, Qihan
2018-04-25
Herpes simplex virus is a prevalent pathogen of humans of various age groups. The fact that no prophylactic or therapeutic vaccine is currently available suggests a significant need to further investigate the immune mechanisms induced by the virus and various vaccine candidates. We previously generated an HSV-1 mutant strain, M3, with partial deletions in ul7, ul41 and LAT that produced an attenuated phenotype in mice. In the present study, we performed a comparative analysis to characterize the immune responses induced by M3 versus wild-type HSV-1 in a mouse model. Infection with wild-type HSV-1 triggered an inflammatory-dominated response and adaptive immunity suppression and was accompanied by severe pathological damage. In contrast, infection with M3 induced a systematic immune response involving full activation of both innate and adaptive immunity and was accompanied by no obvious pathological changes. Furthermore, the immune response induced by M3 protected mice from lethal challenge with wild-type strains of HSV-1 and restrained virus proliferation and impaired latency. These data are useful for further HSV-1 vaccine development using a mutant strain construction strategy. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Prince, Esther; Lazare, Farrah B; Treem, William R; Xu, Jiliu; Iqbal, Jahangir; Pan, Xiaoyue; Josekutty, Joby; Walsh, Meghan; Anderson, Virginia; Hussain, M Mahmood; Schwarz, Steven M
2014-07-01
ω-3 Fatty acids (FAs), natural ligands for the peroxisome proliferator-activated receptor-α (PPAR-α), attenuate parenteral nutrition-associated liver disease (PNALD). However, the mechanisms underlying the protective role of ω-3 FAs are still unknown. The aim of this study was to determine the effects of ω-3 FAs on hepatic triglyceride (TG) accumulation in a murine model of PNALD and to investigate the role of PPAR-α and microsomal triglyceride transfer protein (MTP) in this experimental setting. 129S1/SvImJ wild-type or 129S4/SvJaePparatm/Gonz/J PPAR-α knockout mice were fed chow and water (controls); oral, fat-free PN solution only (PN-O); PN-O plus intraperitoneal (IP) ω-6 FA-predominant supplements (PN-ω-6); or PN-O plus IP ω-3 FA (PN-ω-3). Control and PN-O groups received sham IP injections of 0.9% NaCl. Hepatic histology, TG and cholesterol, MTP activity, and PPAR-α messenger RNA were assessed after 19 days. In all experimental groups, PN feeding increased hepatic TG and MTP activity compared with controls. Both PN-O and PN-ω-6 groups accumulated significantly greater amounts of TG when compared with PN-ω-3 mice. Studies in PPAR-α null animals showed that PN feeding increases hepatic TG as in wild-type mice. PPAR-α null mice in the PN-O and PN-ω-6 groups demonstrated variable degrees of hepatic steatosis, whereas no evidence of hepatic fat accumulation was found after 19 days of oral PN plus IP ω-3 FAs. PN induces TG accumulation (steatosis) in wild-type and PPAR-α null mice. In PN-fed wild-type and PPAR-α null mice given IP ω-3 FAs, reduced hepatic TG accumulation and absent steatosis are found. Prevention of steatosis by ω-3 FAs results from PPAR-α-independent pathways. © 2013 American Society for Parenteral and Enteral Nutrition.
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Ren, J; Youssoufian, H
2001-01-01
Fanconi anemia (FA) is an autosomal recessive disorder manifested by chromosomal breakage, birth defects, and susceptibility to bone marrow failure and cancer. At least seven complementation groups have been identified, and the genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene products defective in FA cells are not completely understood, a clue to the function of the FA group A gene product (FANCA) was provided by the detection of limited homology in the amino terminal region to a class of heme peroxidases. We evaluated this hypothesis by mutagenesis and functional complementation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on known biochemical and cellular functions of FANCA. While the substitution mutants were comparable to wild-type FANCA with regard to their stability, subcellular localization, and interaction with FANCG, only the Trp(183)-to-Ala substitution (W183A) abolished the ability of FANCA to complement the sensitivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apoptosis after exposure to H2O2 showed no differences between parental FA group A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis for the expression of the peroxide-sensitive heme oxygenase gene showed appropriate induction after H2O2 exposure. Thus, W183A appears to be essential for the in vivo activity of FANCA in a manner independent of its interaction with FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears to alter the peroxide-induced apoptosisor peroxide-sensing ability of FA group A cells. Copyright 2001 Academic Press.
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Nielsen, Line; Jensen, Trine Hammer; Kristensen, Birte; Jensen, Tove Dannemann; Karlskov-Mortensen, Peter; Lund, Morten; Aasted, Bent; Blixenkrone-Møller, Merete
2012-10-01
Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.
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[State of collective immunity against poliomyelitis in some regions of Russia].
Seĭbil', V B; Malyshkina, L P; Khishtova, S N; Lesnikova, M V; Baryshnikova, A S; Konopleva, T N; Mnozhina, E G; Agafonova, T V; Vladimirova, L A
2013-01-01
Study the state of collective immunity against poliomyelitis in 7 regions of Russia in the last 3 years. 2579 sera were studied for antibodies against poliomyelitis virus. Antibodies (AT) against 3 types of viruses were determined in neutralization reaction in RD cell culture, the state of collective immunity in the examined individuals was evaluated by the percent of individuals with AT against a type of poliovirus and geometric mean AT titer. The circulation of wild polioviruses was judged by the presence of strain specific AT against wild and vaccine viruses in the examined children (311 sera were studied). The indicators of collective immunity against poliomyelitis in both select examined regions and select age groups were generally high. The data obtained allow to make a conclusion that the quality of vaccine prophylaxis in the examined regions is good. Introduction of wild poliovirus type 1 from Tajikistan in 2010 caused disease in 7 residents of Russia whereas an epidemic that had affected more than 700 individuals emerged in Tajikistan. The studies carried out confirmed the necessity to continue qualitative poliomyelitis vaccine prophylaxis in the country despite the lack of circulation of wild polioviruses that can be introduced at any time.
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Li, Maoyin; Bahn, Sung Chul; Guo, Liang; Musgrave, William; Berg, Howard; Welti, Ruth; Wang, Xuemin
2011-01-01
The release of fatty acids from membrane lipids has been implicated in various plant processes, and the patatin-related phospholipases (pPLAs) constitute a major enzyme family that catalyzes fatty acid release. The Arabidopsis thaliana pPLA family has 10 members that are classified into three groups. Group 3 pPLAIII has four members but lacks the canonical lipase/esterase consensus catalytic sequences, and their enzymatic activity and cellular functions have not been delineated. Here, we show that pPLAIIIβ hydrolyzes phospholipids and galactolipids and additionally has acyl-CoA thioesterase activity. Alterations of pPLAIIIβ result in changes in lipid levels and composition. pPLAIIIβ-KO plants have longer leaves, petioles, hypocotyls, primary roots, and root hairs than wild-type plants, whereas pPLAIIIβ-OE plants exhibit the opposite phenotype. In addition, pPLAIIIβ-OE plants have significantly lower cellulose content and mechanical strength than wild-type plants. Root growth of pPLAIIIβ-KO plants is less sensitive to treatment with free fatty acids, the enzymatic products of pPLAIIIβ, than wild-type plants; root growth of pPLAIIIβ-OE plants is more sensitive. These data suggest that alteration of pPLAIIIβ expression and the resulting lipid changes alter cellulose content and cell elongation in Arabidopsis. PMID:21447788
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Narendran, Gopalan; Kavitha, Dhanasekaran; Karunaianantham, Ramesh; Gil-Santana, Leonardo; Almeida-Junior, Jilson L; Reddy, Sirasanambatti Devarajulu; Kumar, Marimuthu Makesh; Hemalatha, Haribabu; Jayanthi, Nagesh Nalini; Ravichandran, Narayanan; Krishnaraja, Raja; Prabhakar, Angamuthu; Manoharan, Tamizhselvan; Nithyananthan, Lokeswaran; Arjunan, Gunasundari; Natrajan, Mohan; Swaminathan, Soumya; Andrade, Bruno B
2016-01-01
Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory phenomenon complicating HIV management in coincidental tuberculosis (TB) infection, upon immune reconstitution driven by antiretroviral therapy (ART). Leukotriene A4 hydroxylase (LTA4H), an enzyme which converts LTA4 to LTB4, regulates the balance between the anti-inflammatory lipoxins and pro-inflammatory LTB4, with direct implications in TB-driven inflammation. In humans, a single nucleotide polymorphism (SNP) in the LTA4H promoter which regulates its transcriptional activity (rs17525495) has been identified and described to impact clinical severity of TB presentation and response to corticosteroid therapy. Notably, the role of LTA4H on TB-IRIS has not been previously evaluated. Here, we performed an exploratory investigation testing the association of LTA4H polymorphism with respect to frequency of TB-IRIS occurrence and severity of TB-IRIS presentation in HIV-TB co-infected individuals. Genotypic evaluation of the LTA4H enzyme from available samples was retrospectively correlated with clinical data captured in case sheets including IRIS details. The cohort included patients recruited from a prospective cohort study nested within a randomized clinical trial (NCT0933790) of ART-naïve HIV+ patients with newly diagnosed rifampicin sensitive pulmonary TB in South India. Frequency of the wild type genotype (CC), as well as of the mutant genotypes (CT or TT) in the IRIS and non-IRIS patients was estimated. Comparative analyses were performed between wild genotype (CC) and the mutant genotypes (CT or TT) and tested for association between the LTA4H polymorphisms and IRIS incidence and clinical severity. A total of 142 eligible ART-naïve patients were included in the analyses. Eighty-six individuals exhibited the wild genotype (CC) while 56 had mutant genotypes (43-CT and only 13-TT). Variant allele frequency was 0.23 and 0.26 in non-IRIS group and in IRIS group, respectively. Upon ART initiation, 51 patients developed IRIS while 91 did not. IRIS incidence was 34% and 37% in the wild (CC) and mutant type (CT/TT), respectively (p = 0.858) with a higher frequency of severe IRIS presentation in the mutant genotype group compared to the wild type genotype (p = 0.0006). A logistic regression model confirmed the association between the presence of CT/TT genotypes and occurrence of severe IRIS. Corticosteroid therapy successfully resolved IRIS in all cases irrespective of the LTA4H genotype. A higher incidence of severe IRIS among patients with mutant LTA4H genotypes (CT and TT) was observed compared to the wild type, despite similar IRIS incidence and immune restoration in both groups. Steroids were effective in alleviating IRIS in all the genotypes.
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Ullah, Anwar; de Giuseppe, Priscila Oliveira; Murakami, Mario Tyago; Trevisan-Silva, Dilza; Wille, Ana Carolina Martins; Chaves-Moreira, Daniele; Gremski, Luiza Helena; da Silveira, Rafael Bertoni; Sennf-Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Silvio Sanches; Arni, Raghuvir Krishnaswamy
2011-01-01
Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P1211, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5 Å, β = 105.9°. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60 Å, respectively. PMID:21301094
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Perrone, Danielle; Bender, Scott; Niewiesk, Stefan
2010-07-01
Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals.
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Perrone, Danielle; Bender, Scott; Niewiesk, Stefan
2010-01-01
Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals. PMID:20885846
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Mutations of the KISS1 Gene in Disorders of Puberty
Silveira, L. G.; Noel, S. D.; Silveira-Neto, A. P.; Abreu, A. P.; Brito, V. N.; Santos, M. G.; Bianco, S. D. C.; Kuohung, W.; Xu, S.; Gryngarten, M.; Escobar, M. E.; Arnhold, I. J. P.; Mendonca, B. B.; Kaiser, U. B.; Latronico, A. C.
2010-01-01
Context: Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP). Objective: Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH. Patients: Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development. Methods: The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells. Results: Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group. Conclusion: Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype. PMID:20237166
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Mutations of the KISS1 gene in disorders of puberty.
Silveira, L G; Noel, S D; Silveira-Neto, A P; Abreu, A P; Brito, V N; Santos, M G; Bianco, S D C; Kuohung, W; Xu, S; Gryngarten, M; Escobar, M E; Arnhold, I J P; Mendonca, B B; Kaiser, U B; Latronico, A C
2010-05-01
Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP). Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH. Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development. The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells. Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group. Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype.
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Use of multivariate analysis to suggest a new molecular classification of colorectal cancer
Domingo, Enric; Ramamoorthy, Rajarajan; Oukrif, Dahmane; Rosmarin, Daniel; Presz, Michal; Wang, Haitao; Pulker, Hannah; Lockstone, Helen; Hveem, Tarjei; Cranston, Treena; Danielsen, Havard; Novelli, Marco; Davidson, Brian; Xu, Zheng-Zhou; Molloy, Peter; Johnstone, Elaine; Holmes, Christopher; Midgley, Rachel; Kerr, David; Sieber, Oliver; Tomlinson, Ian
2013-01-01
Abstract Molecular classification of colorectal cancer (CRC) is currently based on microsatellite instability (MSI), KRAS or BRAF mutation and, occasionally, chromosomal instability (CIN). Whilst useful, these categories may not fully represent the underlying molecular subgroups. We screened 906 stage II/III CRCs from the VICTOR clinical trial for somatic mutations. Multivariate analyses (logistic regression, clustering, Bayesian networks) identified the primary molecular associations. Positive associations occurred between: CIN and TP53 mutation; MSI and BRAF mutation; and KRAS and PIK3CA mutations. Negative associations occurred between: MSI and CIN; MSI and NRAS mutation; and KRAS mutation, and each of NRAS, TP53 and BRAF mutations. Some complex relationships were elucidated: KRAS and TP53 mutations had both a direct negative association and a weaker, confounding, positive association via TP53–CIN–MSI–BRAF–KRAS. Our results suggested a new molecular classification of CRCs: (1) MSI+ and/or BRAF-mutant; (2) CIN+ and/or TP53– mutant, with wild-type KRAS and PIK3CA; (3) KRAS- and/or PIK3CA-mutant, CIN+, TP53-wild-type; (4) KRAS– and/or PIK3CA-mutant, CIN–, TP53-wild-type; (5) NRAS-mutant; (6) no mutations; (7) others. As expected, group 1 cancers were mostly proximal and poorly differentiated, usually occurring in women. Unexpectedly, two different types of CIN+ CRC were found: group 2 cancers were usually distal and occurred in men, whereas group 3 showed neither of these associations but were of higher stage. CIN+ cancers have conventionally been associated with all three of these variables, because they have been tested en masse. Our classification also showed potentially improved prognostic capabilities, with group 3, and possibly group 1, independently predicting disease-free survival. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:23165447
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Hong, Ji-Young; F. Sato, Eisuke; Nishikawa, Tomoko; Hiramoto, Keiichi; Inoue, Masayasu
2009-01-01
We previously reported that the plasma level of endotoxin and colonic expression of IgA in the mouse increased with obstructive jaundice induced by bile duct ligation (BDL). To elucidate the mechanism of the BDL-induced increase, we analyzed the effect of BDL on intestinal flora in wild type and inducible nitric oxide synthase (iNOS)-deficient mice (iNOS−/−) using the terminal restriction fragment length polymorphism analysis (T-RFLP) and 16S rDNA clone libraries. The amounts of bacterial DNA detected in fecal samples from both animal groups pretreated with antibiotics were extremely low as compared with untreated groups. We found that the profiles of enteric bacteria changed markedly after BDL. The bacterial composition is significantly different between not only wild type and iNOS−/− mice but also those before and after BDL, respectively. Among enteric bacteria examined, Lactobacillus murinus was found to increase markedly after BDL in rectum of both animal groups. However, Escherichia coli markedly increased after BDL in the iNOS−/− mice. These findings suggest that profiles of enteric flora change markedly in animals during obstructive jaundice by some mechanism that is affected by bile constituents and iNOS-derived NO. PMID:19308270
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Azadeh, Payam; Mortazavi, Nafiseh; Tahmasebi, Arezoo; Hosseini Kamal, Farnaz; Novin, Kambiz
2016-01-01
The aim of this study was to compare the efficacy and hematologic toxicity of cetuximab combined with various types of chemotherapy regimens in patients with KRAS wild-type metastatic colorectal cancer (mCRC). The response rate, progression-free survival (PFS) and overall survival of the patients were analyzed. In total, 45 patients were included in the study. The overall response rate for the combination of cetuximab and FOLFOX, FOLFIRI and CAPOX was 20, 46 and 30%, respectively, but the differences were not statistically significant. The median PFS for the three groups were 8, 6 and 3.5 months, respectively, but again these differences were not significant. All-grade leukopenia and anemia for the cetuximab plus FOLFOX group were significantly higher than for the other chemotherapy regimens. Our findings suggest that the combination of cetuximab and the three standard chemotherapy regimens resulted in the same outcomes in our patient population of mCRC, with higher hematologic toxicities among the FOLFOX subgroup. © 2015 S. Karger AG, Basel.
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Schunko, Christoph; Grasser, Susanne; Vogl, Christian R
2015-06-30
Wild plant gathering becomes again a popular and fashionable activity in Europe after gathering practices have been increasingly abandoned over the last decades. Recent ethnobotanical research documented a diversity of gathering practices from people of diverse socio-economic and cultural backgrounds who gather in urban and rural areas. Few efforts were though made to study the motivations for gathering wild plants and to understand the resurgent popularity of wild plant gathering. This paper addresses the following research questions: (1) which motivations activate wild plant gatherers? (2) which motivation-types of gatherers exist in the Grosses Walsertal? (3) how do the motivations for gathering relate to the socio-demographic background of gatherers? Field research was conducted in the Grosses Walsertal, Austria in the years 2008 and 2009 in two field research periods. Thirty-six local farmers were first interviewed with semi-structured interviews. The motivations identified in these interviews were then included in a structured questionnaire, which was used to interview 353 residents of the valley. Pupils of local schools participated in the data collection as interviewers. Principal Component Analysis was used to categorize the motivations and to identify motivation-types of wild plant gatherers. Generalized Linear Models were calculated to identify relations between motivations and the socio-demographic background of gatherers. The respondents listed 13 different motivations for gathering wild plants and four motivations for not gathering. These 17 motivations were grouped in five motivation-types of wild plant gatherers, which are in decreasing importance: product quality, fun, tradition, not-gathering, income. Women, older respondents and homegardeners gather wild plants more often for fun; older respondents gather more often for maintaining traditions; non-homegardeners more frequently mention motivations for not gathering. The resurgent popularity of wild plant gathering comes along with an internalization of motivations: the main motivations for wild plant gathering changed from the external extrinsic motivation of gathering because of necessity towards the internalized extrinsic motivation of gathering for the highly esteemed product quality and the intrinsic motivation of gathering for the pleasure of the activity itself. This internalization of motivations supports the persistence of wild plant gathering, a positive self-perception of gatherers and good quality of engagement with wild plant gathering.
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Pío-León, Juan Fernando; Delgado-Vargas, Francisco; Murillo-Amador, Bernardo; León-de-la-Luz, José Luís; Vega-Aviña, Rito; Nieto-Garibay, Alejandra; Córdoba-Matson, Miguel; Ortega-Rubio, Alfredo
2017-10-01
Ethnobotany is an adequate tool to identify the most import wild edible plants used by local people and to develop strategies for its sustainable use and policy making. This research records the wild edible plants used by the inhabitants of the Sierra la Laguna Biosphere Reserve, in Mexico, and identified priority species for sustainable development. The employed quantitative approach included the application of a food-oriented index named Food Significance Index (FSI), which integrated cultural, agricultural and food parameters; moreover, it used multivariate analysis to identify priority edible species for use and/or conservation. Fifty taxa were identified as the most important wild edible plants in the reserve, integrated into five priority groups. Foods in priority group 1 had the highest culinary diversity by grouping three fruits (Ficus petiolaris, Stenocereus thurberi, and Cyrtocarpa edulis), one almond-like seed (Cnidoscolus maculatus), one vegetable (Matelea cordifolia), and one condiment (Capsicum annuum). Priority groups 2-5 were selective for one or two types of food, such as fruits, teas, or seeds. Since group 1 was the most diverse, the FSI and the employed strategy permitted to identify the priority wild edible plant species with the highest potential for food security. The selected six species should be included in the future management program of the reserve as the priority wild edible plants to develop strategies for conservation, sustainable use, and improvement of the local population income. General policies to manage the selected six species are outlined. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Comparative Response of the Hepatic Transcriptomes of Domesticated and Wild Turkey to Aflatoxin B₁.
Reed, Kent M; Mendoza, Kristelle M; Abrahante, Juan E; Coulombe, Roger A
2018-01-13
The food-borne mycotoxin aflatoxin B₁ (AFB₁) poses a significant risk to poultry, which are highly susceptible to its hepatotoxic effects. Domesticated turkeys ( Meleagris gallopavo ) are especially sensitive, whereas wild turkeys ( M. g. silvestris ) are more resistant. AFB₁ toxicity entails bioactivation by hepatic cytochrome P450s to the electrophilic exo-AFB₁-8,9-epoxide (AFBO). Domesticated turkeys lack functional hepatic GST-mediated detoxification of AFBO, and this is largely responsible for the differences in resistance between turkey types. This study was designed to characterize transcriptional changes induced in turkey livers by AFB₁, and to contrast the response of domesticated (susceptible) and wild (more resistant) birds. Gene expression responses to AFB₁ were examined using RNA-sequencing. Statistically significant differences in gene expression were observed among treatment groups and between turkey types. Expression analysis identified 4621 genes with significant differential expression (DE) in AFB₁-treated birds compared to controls. Characterization of DE transcripts revealed genes dis-regulated in response to toxic insult with significant association of Phase I and Phase II genes and others important in cellular regulation, modulation of apoptosis, and inflammatory responses. Constitutive expression of GSTA3 was significantly higher in wild birds and was significantly higher in AFB₁-treated birds when compared to controls for both genetic groups. This pattern was also observed by qRT-PCR in other wild and domesticated turkey strains. Results of this study emphasize the differential response of these genetically distinct birds, and identify genes and pathways that are differentially altered in aflatoxicosis.
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Ramalingam, Suresh S; Jänne, Pasi A; Mok, Tony; O'Byrne, Kenneth; Boyer, Michael J; Von Pawel, Joachim; Pluzanski, Adam; Shtivelband, Mikhail; Docampo, Lara Iglesias; Bennouna, Jaafar; Zhang, Hui; Liang, Jane Q; Doherty, Jim P; Taylor, Ian; Mather, Cecile B; Goldberg, Zelanna; O'Connell, Joseph; Paz-Ares, Luis
2014-11-01
Dacomitinib is an irreversible pan-EGFR family tyrosine kinase inhibitor. Findings from a phase 2 study in non-small cell lung cancer showed favourable efficacy for dacomitinib compared with erlotinib. We aimed to compare dacomitinib with erlotinib in a phase 3 study. In a randomised, multicentre, double-blind phase 3 trial in 134 centres in 23 countries, we enrolled patients who had locally advanced or metastatic non-small-cell lung cancer, progression after one or two previous regimens of chemotherapy, Eastern Cooperative Oncology Group (ECOG) performance status of 0-2, and presence of measurable disease. We randomly assigned patients in a 1:1 ratio to dacomitinib (45 mg/day) or erlotinib (150 mg/day) with matching placebo. Treatment allocation was masked to the investigator, patient, and study funder. Randomisation was stratified by histology (adenocarcinoma vs non-adenocarcinoma), ethnic origin (Asian vs non-Asian and Indian sub-continent), performance status (0-1 vs 2), and smoking status (never-smoker vs ever-smoker). The coprimary endpoints were progression-free survival per independent review for all randomly assigned patients, and for all randomly assigned patients with KRAS wild-type tumours. The study has completed accrual and is registered with ClinicalTrials.gov, number NCT01360554. Between June 22, 2011, and March 12, 2013, we enrolled 878 patients and randomly assigned 439 to dacomitinib (256 KRAS wild type) and 439 (263 KRAS wild type) to erlotinib. Median progression-free survival was 2·6 months (95% CI 1·9-2·8) in both the dacomitinib group and the erlotinib group (stratified hazard ratio [HR] 0·941, 95% CI 0·802-1·104, one-sided log-rank p=0·229). For patients with wild-type KRAS, median progression-free survival was 2·6 months for dacomitinib (95% CI 1·9-2·9) and erlotinib (95% CI 1·9-3·0; stratified HR 1·022, 95% CI 0·834-1·253, one-sided p=0·587). In patients who received at least one dose of study drug, the most frequent grade 3-4 adverse events were diarrhoea (47 [11%] patients in the dacomitinib group vs ten [2%] patients in the erlotinib group), rash (29 [7%] vs 12 [3%]), and stomatitis (15 [3%] vs two [<1%]). Serious adverse events were reported in 52 (12%) patients receiving dacomitinib and 40 (9%) patients receiving erlotinib. Irreversible EGFR inhibition with dacomitinib was not superior to erlotinib in an unselected patient population with advanced non-small-cell lung cancer or in patients with KRAS wild-type tumours. Further study of irreversible EGFR inhibitors should be restricted to patients with activating EGFR mutations. Pfizer. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Smith, P; Endris, R; Kronvall, G; Thomas, V; Verner-Jeffreys, D; Wilhelm, C; Dalsgaard, I
2016-02-01
Epidemiological cut-off values were developed for application to antibiotic susceptibility data for Flavobacterium psychrophilum generated by standard CLSI test protocols. The MIC values for ten antibiotic agents against Flavobacterium psychrophilum were determined in two laboratories. For five antibiotics, the data sets were of sufficient quality and quantity to allow the setting of valid epidemiological cut-off values. For these agents, the cut-off values, calculated by the application of the statistically based normalized resistance interpretation method, were ≤16 mg L(-1) for erythromycin, ≤2 mg L(-1) for florfenicol, ≤0.025 mg L(-1) for oxolinic acid (OXO), ≤0.125 mg L(-1) for oxytetracycline and ≤20 (1/19) mg L(-1) for trimethoprim/sulphamethoxazole. For ampicillin and amoxicillin, the majority of putative wild-type observations were 'off scale', and therefore, statistically valid cut-off values could not be calculated. For ormetoprim/sulphadimethoxine, the data were excessively diverse and a valid cut-off could not be determined. For flumequine, the putative wild-type data were extremely skewed, and for enrofloxacin, there was inadequate separation in the MIC values for putative wild-type and non-wild-type strains. It is argued that the adoption of OXO as a class representative for the quinolone group would be a valid method of determining susceptibilities to these agents. © 2014 John Wiley & Sons Ltd.
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Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein.
Prajapati, R S; Lingaraju, G M; Bacchawat, Kiran; Surolia, Avadhesha; Varadarajan, Raghavan
2003-12-01
Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in T(m) values of P48A and P48S of 0.4 degrees C and 0.2 degrees C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein. Copyright 2003 Wiley-Liss, Inc.
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Casabuono, Adriana; Petrocelli, Silvana; Ottado, Jorgelina; Orellano, Elena G.; Couto, Alicia S.
2011-01-01
Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo2Hex6GalA3Fuc3NAcRha4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response. PMID:21596742
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Casabuono, Adriana; Petrocelli, Silvana; Ottado, Jorgelina; Orellano, Elena G; Couto, Alicia S
2011-07-22
Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo₂Hex₆GalA₃Fuc3NAcRha₄ and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.
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Maughan, Timothy S; Adams, Richard A; Smith, Christopher G; Meade, Angela M; Seymour, Matthew T; Wilson, Richard H; Idziaszczyk, Shelley; Harris, Rebecca; Fisher, David; Kenny, Sarah L; Kay, Edward; Mitchell, Jenna K; Madi, Ayman; Jasani, Bharat; James, Michelle D; Bridgewater, John; Kennedy, M John; Claes, Bart; Lambrechts, Diether; Kaplan, Richard; Cheadle, Jeremy P
2011-01-01
Summary Background In the Medical Research Council (MRC) COIN trial, the epidermal growth factor receptor (EGFR)-targeted antibody cetuximab was added to standard chemotherapy in first-line treatment of advanced colorectal cancer with the aim of assessing effect on overall survival. Methods In this randomised controlled trial, patients who were fit for but had not received previous chemotherapy for advanced colorectal cancer were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B), or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomisation. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and C is described in a companion paper. Here, we present the comparison of arm A and B, for which the primary outcome was overall survival in patients with KRAS wild-type tumours. Analysis was by intention to treat. Further analyses with respect to NRAS, BRAF, and EGFR status were done. The trial is registered, ISRCTN27286448. Findings 1630 patients were randomly assigned to treatment groups (815 to standard therapy and 815 to addition of cetuximab). Tumour samples from 1316 (81%) patients were used for somatic molecular analyses; 565 (43%) had KRAS mutations. In patients with KRAS wild-type tumours (arm A, n=367; arm B, n=362), overall survival did not differ between treatment groups (median survival 17·9 months [IQR 10·3–29·2] in the control group vs 17·0 months [9·4–30·1] in the cetuximab group; HR 1·04, 95% CI 0·87–1·23, p=0·67). Similarly, there was no effect on progression-free survival (8·6 months [IQR 5·0–12·5] in the control group vs 8·6 months [5·1–13·8] in the cetuximab group; HR 0·96, 0·82–1·12, p=0·60). Overall response rate increased from 57% (n=209) with chemotherapy alone to 64% (n=232) with addition of cetuximab (p=0·049). Grade 3 and higher skin and gastrointestinal toxic effects were increased with cetuximab (14 vs 114 and 67 vs 97 patients in the control group vs the cetuximab group with KRAS wild-type tumours, respectively). Overall survival differs by somatic mutation status irrespective of treatment received: BRAF mutant, 8·8 months (IQR 4·5–27·4); KRAS mutant, 14·4 months (8·5–24·0); all wild-type, 20·1 months (11·5–31·7). Interpretation This trial has not confirmed a benefit of addition of cetuximab to oxaliplatin-based chemotherapy in first-line treatment of patients with advanced colorectal cancer. Cetuximab increases response rate, with no evidence of benefit in progression-free or overall survival in KRAS wild-type patients or even in patients selected by additional mutational analysis of their tumours. The use of cetuximab in combination with oxaliplatin and capecitabine in first-line chemotherapy in patients with widespread metastases cannot be recommended. Funding Cancer Research UK, Cancer Research Wales, UK Medical Research Council, Merck KGgA. PMID:21641636
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Holmes, Tarquin
2017-06-01
Wild types in genetics are specialised strains of laboratory experimental organism which principally serve as standards against which variation is measured. As selectively inbred lineages highly isolated from ancestral wild populations, there appears to be little wild or typical about them. I will nonetheless argue that they have historically been successfully used as stand-ins for nature, allowing knowledge produced in the laboratory to be extrapolated to the natural world. In this paper, I will explore the 19th century origins of the wild type concept, the theoretical and experimental innovations which allowed concepts and organisms to move from wild nature to laboratory domestication c. 1900 (resulting in the production of standardised lab strains), and the conflict among early geneticists between interactionist and atomist accounts of wild type, which would eventually lead to the conceptual disintegration of wild types and the triumph of genocentrism and population genetics. I conclude by discussing how the strategy of using wild type strains to represent nature in the lab has nonetheless survived the downfall of the wild type concept and continues to provide, significant limitations acknowledged, an epistemically productive means of investigating heredity and evolutionary variation. Copyright © 2017 Elsevier Ltd. All rights reserved.
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MacAlpine, D M; Perlman, P S; Butow, R A
1998-06-09
Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (rho+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in rho+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (rho-). mtDNA recombination junctions are not observed in rho+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Deltamgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in rho+ mtDNA of Deltamgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by >/= 10-fold in wild-type rho+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of rho+ mtDNA.
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CMS-dependent prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer.
Smeby, J; Sveen, A; Merok, M A; Danielsen, S A; Eilertsen, I A; Guren, M G; Dienstmann, R; Nesbakken, A; Lothe, R A
2018-05-01
The prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer (CRC) varies with microsatellite instability (MSI) status. The gene expression-based consensus molecular subtypes (CMSs) of CRC define molecularly and clinically distinct subgroups, and represent a novel stratification framework in biomarker analysis. We investigated the prognostic value of these mutations within the CMS groups. Totally 1197 primary tumors from a Norwegian series of CRC stage I-IV were analyzed for MSI and mutation status in hotspots in KRAS (codons 12, 13 and 61) and BRAF (codon 600). A subset was analyzed for gene expression and confident CMS classification was obtained for 317 samples. This cohort was expanded with clinical and molecular data, including CMS classification, from 514 patients in the publically available dataset GSE39582. Gene expression signatures associated with KRAS and BRAFV600E mutations were used to evaluate differential impact of mutations on gene expression among the CMS groups. BRAFV600E and KRAS mutations were both associated with inferior 5-year overall survival (OS) exclusively in MSS tumors (BRAFV600E mutation versus KRAS/BRAF wild-type: Hazard ratio (HR) 2.85, P < 0.001; KRAS mutation versus KRAS/BRAF wild-type: HR 1.30, P = 0.013). BRAFV600E-mutated MSS tumors were strongly enriched and associated with metastatic disease in CMS1, leading to negative prognostic impact in this subtype (OS: BRAFV600E mutation versus wild-type: HR 7.73, P = 0.001). In contrast, the poor prognosis of KRAS mutations was limited to MSS tumors with CMS2/CMS3 epithelial-like gene expression profiles (OS: KRAS mutation versus wild-type: HR 1.51, P = 0.011). The subtype-specific prognostic associations were substantiated by differential effects of BRAFV600E and KRAS mutations on gene expression signatures according to the MSI status and CMS group. BRAFV600E mutations are enriched and associated with metastatic disease in CMS1 MSS tumors, leading to poor prognosis in this subtype. KRAS mutations are associated with adverse outcome in epithelial (CMS2/CMS3) MSS tumors.
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Georgopoulos, Angeliki; Aras, Omer; Noutsou, Marina; Tsai, Michael Y
2002-08-01
In type 2 diabetes, the threonine (Thr) for alanine (Ala) codon 54 polymorphism of the fatty acid binding protein 2 gene is associated with elevated fasting and postprandial triglycerides and dyslipidemia when compared with the wild type (Ala-54/Ala-54). To assess whether this is the case in patients with type 1 diabetes, who usually do not manifest the metabolic syndrome, we screened 181 patients with similar glycemic control as the type 2 patients. Thirty percent were heterozygous, and 9% were homozygous for the polymorphism. Mean (+/-SEM) fasting plasma triglyceride levels in patients with the wild type (n = 84), those heterozygous for Ala-54/Thr-54 (n = 44), and those homozygous for the Thr-54 (n = 13) were 1.0 +/- 0.07, 1.1 +/- 0.17, and 1.2 +/- 0.23 mmol/liter, respectively. In addition, there were no differences in total, low-density lipoprotein, high-density lipoprotein, and non-high density lipoprotein cholesterol among the three groups. After a fat load, the postprandial area under the curve of triglyceride in plasma, chylomicrons, and very low-density lipoprotein were similar between the wild type (n = 18) and the Thr-54 homozygotes (n = 12). In conclusion, in contrast to type 2, type 1 diabetes does not interact with the codon 54 polymorphism of the fatty acid binding protein 2 gene to cause hypertriglyceridemia/dyslipidemia. Insulin resistance could account possibly for this difference.
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Current Treatment Algorithms for Patients with Metastatic Non-Small Cell, Non-Squamous Lung Cancer
Melosky, Barbara
2017-01-01
The treatment paradigm for metastatic non-small cell, non-squamous lung cancer is continuously evolving due to new treatment options and our increasing knowledge of molecular signal pathways. As a result of treatments becoming more efficacious and more personalized, survival for selected groups of non-small cell lung cancer (NSCLC) patients is increasing. In this paper, three algorithms will be presented for treating patients with metastatic non-squamous, NSCLC. These include treatment algorithms for NSCLC patients whose tumors have EGFR mutations, ALK rearrangements, or wild-type/wild-type tumors. As the world of immunotherapy continues to evolve quickly, a future algorithm will also be presented. PMID:28373963
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Narendran, Gopalan; Kavitha, Dhanasekaran; Karunaianantham, Ramesh; Gil-Santana, Leonardo; Almeida-Junior, Jilson L.; Reddy, Sirasanambatti Devarajulu; Kumar, Marimuthu Makesh; Hemalatha, Haribabu; Jayanthi, Nagesh Nalini; Ravichandran, Narayanan; Krishnaraja, Raja; Prabhakar, Angamuthu; Manoharan, Tamizhselvan; Nithyananthan, Lokeswaran; Arjunan, Gunasundari; Natrajan, Mohan; Swaminathan, Soumya
2016-01-01
Background Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory phenomenon complicating HIV management in coincidental tuberculosis (TB) infection, upon immune reconstitution driven by antiretroviral therapy (ART). Leukotriene A4 hydroxylase (LTA4H), an enzyme which converts LTA4 to LTB4, regulates the balance between the anti-inflammatory lipoxins and pro-inflammatory LTB4, with direct implications in TB-driven inflammation. In humans, a single nucleotide polymorphism (SNP) in the LTA4H promoter which regulates its transcriptional activity (rs17525495) has been identified and described to impact clinical severity of TB presentation and response to corticosteroid therapy. Notably, the role of LTA4H on TB-IRIS has not been previously evaluated. Here, we performed an exploratory investigation testing the association of LTA4H polymorphism with respect to frequency of TB-IRIS occurrence and severity of TB-IRIS presentation in HIV-TB co-infected individuals. Methods Genotypic evaluation of the LTA4H enzyme from available samples was retrospectively correlated with clinical data captured in case sheets including IRIS details. The cohort included patients recruited from a prospective cohort study nested within a randomized clinical trial (NCT0933790) of ART-naïve HIV+ patients with newly diagnosed rifampicin sensitive pulmonary TB in South India. Frequency of the wild type genotype (CC), as well as of the mutant genotypes (CT or TT) in the IRIS and non-IRIS patients was estimated. Comparative analyses were performed between wild genotype (CC) and the mutant genotypes (CT or TT) and tested for association between the LTA4H polymorphisms and IRIS incidence and clinical severity. Results A total of 142 eligible ART-naïve patients were included in the analyses. Eighty-six individuals exhibited the wild genotype (CC) while 56 had mutant genotypes (43-CT and only 13-TT). Variant allele frequency was 0.23 and 0.26 in non-IRIS group and in IRIS group, respectively. Upon ART initiation, 51 patients developed IRIS while 91 did not. IRIS incidence was 34% and 37% in the wild (CC) and mutant type (CT/TT), respectively (p = 0.858) with a higher frequency of severe IRIS presentation in the mutant genotype group compared to the wild type genotype (p = 0.0006). A logistic regression model confirmed the association between the presence of CT/TT genotypes and occurrence of severe IRIS. Corticosteroid therapy successfully resolved IRIS in all cases irrespective of the LTA4H genotype. Conclusion A higher incidence of severe IRIS among patients with mutant LTA4H genotypes (CT and TT) was observed compared to the wild type, despite similar IRIS incidence and immune restoration in both groups. Steroids were effective in alleviating IRIS in all the genotypes. PMID:27643598
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Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice
DOE Office of Scientific and Technical Information (OSTI.GOV)
Baireddy, Praveena; Liu, Jing; Hinsdale, Myron
2011-11-15
Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemicalmore » changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and cannabinoid CB1 receptor knockout littermates responded similarly to the toxic effects of chlorpyrifos. Black-Right-Pointing-Pointer OP-induced changes in acetylcholine release appeared sensitive to modulation by CB1-mediated endocannabinoid signaling.« less
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Luanloet, Thikumporn; Sucharitakul, Jeerus; Chaiyen, Pimchai
2015-08-01
2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 is a flavin-dependent monooxygenase that catalyzes a hydroxylation and aromatic ring cleavage reaction. The functional roles of two residues, Tyr223 and Tyr82, located ~ 5 Å away from MHPC, were characterized using site-directed mutagenesis, along with ligand binding, product analysis and transient kinetic experiments. Mutation of Tyr223 resulted in enzyme variants that were impaired in their hydroxylation activity and had Kd values for substrate binding 5-10-fold greater than the wild-type enzyme. Because this residue is adjacent to the water molecule that is located next to the 3-hydroxy group of MHPC, the results indicate that the interaction between Tyr223, H2 O and the 3-hydroxyl group of MHPC are important for substrate binding and hydroxylation. By contrast, the Kd for substrate binding of Tyr82His and Tyr82Phe variants were similar to that of the wild-type enzyme. However, only ~ 40-50% of the substrate was hydroxylated in the reactions of both variants, whereas most of the substrate was hydroxylated in the wild-type enzyme reaction. In free solution, MHPC or 5-hydroxynicotinic acid exists in a mixture of monoanionic and tripolar ionic forms, whereas only the tripolar ionic form binds to the wild-type enzyme. The binding of tripolar ionic MHPC would allow efficient hydroxylation through an electrophilic aromatic substitution mechanism. For the Tyr82His and Tyr82Phe variants, both forms of substrates can bind to the enzymes, indicating that the mutation at Tyr82 abolished the selectivity of the enzyme towards the tripolar ionic form. Transient kinetic studies indicated that the hydroxylation rate constants of both Tyr82 variants are approximately two- to 2.5-fold higher than that of the wild-type enzyme. Altogether, our findings suggest that Tyr82 is important for the binding selectivity of MHPC oxygenase towards the tripolar ionic species, whereas the interaction between Tyr223 and the substrate is important for ensuring hydroxylation. These results highlight how the active site of a flavoenzyme is able to deal with the presence of multiple forms of a substrate in solution and ensure efficient hydroxylation. © 2015 FEBS.
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Lin, Hui-xing; Ma, Zhe; Yang, Xu-qiu; Fan, Hong-jie; Lu, Cheng-ping
2014-06-25
To develop a vaccine against Porcine circovirus type 2 (PCV2) and Streptococcus equi ssp. zooepidemicus (SEZ) co-infection, the genes of porcine IL-18, capsid protein (Cap) of PCV2 and M-like protein (SzP) of SEZ were inserted into the swinepox virus (SPV) genome by homologous recombination. The recombinant swinepox virus rSPV-ICS was verified by PCR and indirect immunofluorescence assays. To evaluate the immunogenicity of rSPV-ICS, 28 PCV2 and SEZ seronegative Bama minipigs were immunized with rSPV-ICS (n=8), commercial PCV2 vaccine and SEZ vaccine (n=8) or wild type SPV (n=8). The results showed that SzP-specific antibody and PCV2 neutralizing antibody of the rSPV-ICS immunized group increased significantly compared to the wild type SPV treated group after vaccination and increased continuously over time. The levels of IL-4 and IFN-γ in the rSPV-ICS immunized group were significantly higher than the other three groups, respectively. After been co-challenged with PCV2 and SEZ, 87.5% piglets in rSPV-ICS immunized group were survived. Significant reductions in gross lung lesion score, histopathological lung lesion score, and lymph node lesion score were noticed in the rSPV-ICS immunized group compared with the wtSPV treated group. The results suggested that the recombinant rSPV-ICS provided piglets with significant protection against PCV2-SEZ co-infection; thus, it offers proof-of-principle for the development of a vaccine for the prevention of these swine diseases. Copyright © 2014 Elsevier B.V. All rights reserved.
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Geredeli, Caglayan; Yasar, Nurgul
2018-03-27
The aim of this study was to investigate the efficacy and safety of first-line panitumumab plus folinic acid, 5-fluorouracil and irinotecan (FOLFIRI) in patients with wild-type KRAS and wild-type NRAS metastatic colorectal cancer (mCRC). Patients with wild-type KRAS and wild-type NRAS mCRC presenting to the medical oncology department of the Okmeydani Training and Research Hospital in Istanbul, Turkey, between April 2014 and January 2018 were enrolled in this study. A total of 64 patients (35 males and 29 females) with a median age of 59 (35-81) years old were enrolled. The median follow-up was 18.9 months, and the median progression-free survival was 13 months. The median overall survival (OS) was 26 months in the patients with wild-type KRAS and wild-type NRAS mCRC. It was 90.4% for the 6-month OS, 79.5% for the 1-year OS, 53.7% for the 2-year OS and 31.1% for the 3-year OS. The median OS of the patients who underwent metastasectomies was 40 [95% confidence interval (CI) = 19.9-60.1] months, and the median OS of the patients without metastasectomies was 22 (95% CI = 17.7-26.4) months. There was a statistically significant difference between these (P = 0.007). The first-line FOLFIRI plus panitumumab was associated with favourable efficacy in the patients with wild-type KRAS and wild-type NRAS mCRC, and it was well tolerated. The removal of the metastases that became resectable after chemotherapy further prolonged the patients' survival. Retrospectively registered: 33886.
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Georghiou, Sophia B.; Catanzaro, Donald; Rodrigues, Camilla; Crudu, Valeriu; Victor, Thomas C.; Garfein, Richard S.; Catanzaro, Antonino; Rodwell, Timothy C.
2016-01-01
Accurate identification of drug-resistant Mycobacterium tuberculosis is imperative for effective treatment and subsequent reduction in disease transmission. Line probe assays rapidly detect mutations associated with resistance and wild-type sequences associated with susceptibility. Examination of molecular-level performance is necessary for improved assay result interpretation and for continued diagnostic development. Using data collected from a large, multisite diagnostic study, probe hybridization results from line probe assays, MTBDRplus and MTBDRsl, were compared to those of sequencing, and the diagnostic performance of each individual mutation and wild-type probe was assessed. Line probe assay results classified as resistant due to the absence of wild-type probe hybridization were compared to those of sequencing to determine if novel mutations were inhibiting wild-type probe hybridization. The contribution of absent wild-type probe hybridization to the detection of drug resistance was assessed via comparison to a phenotypic reference standard. In our study, mutation probes demonstrated significantly higher specificities than wild-type probes and wild-type probes demonstrated marginally higher sensitivities than mutation probes, an ideal combination for detecting the presence of resistance conferring mutations while yielding the fewest number of false-positive results. The absence of wild-type probe hybridization without mutation probe hybridization was determined to be primarily the result of failure of mutation probe hybridization and not the result of novel or rare mutations. Compared to phenotypic culture-based drug susceptibility testing, the absence of wild-type probe hybridization without mutation probe hybridization significantly contributed to the detection of phenotypic rifampin and fluoroquinolone resistance with negligible increases in false-positive results. PMID:26763971
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Uehara, S
1982-01-01
During a short period, wild chimpanzees of group K in the Mahale Mountains employ a set of several techniques, including tool use, to feed on one species of termite (Pseudacanthotermes spiniger). They appear to use each technique appropriately according to phenological changes in the prey insect's activities. The chimpanzees also ingest small pieces of soil from the tower of P. spiniger's mound throughout the year. Geophagy presumably makes them visually and tactually aware of the phenological changes of the termite's reproductive cycle. Analyses of fecal samples from the chimpanzees indicate interannual fluctuations in the amount of termites ingested. On the other hand, the chimpanzees of group B, ranging to the north of group K, utilize a fishing technique to obtain another type of termite (Macrotermes?herus) on a large scale during the first half of the wet season. Fecal analysis data show that chimpanzees of group B consume far more termites than those of group K. The probability that the same or similar tool-using techniques as fishing may be employed in feeding on different types of insects by chimpanzees of different unit groups according to subtle local differences in the insect fauna of their home ranges is discussed.
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Campbell, Kevin; Gastier-Foster, Julie M; Mann, Meegan; Naranjo, Arlene H; Van Ryn, Collin; Bagatell, Rochelle; Matthay, Katherine K; London, Wendy B; Irwin, Meredith S; Shimada, Hiroyuki; Granger, M Meaghan; Hogarty, Michael D; Park, Julie R; DuBois, Steven G
2017-11-01
High-level MYCN amplification (MNA) is associated with poor outcome and unfavorable clinical and biological features in patients with neuroblastoma. To the authors' knowledge, less is known regarding these associations in patients with low-level MYCN copy number increases. In this retrospective study, the authors classified patients has having tumors with MYCN wild-type tumors, MYCN gain (2-4-fold increase in MYCN signal compared with the reference probe), or MNA (>4-fold increase). Tests of trend were used to investigate ordered associations between MYCN copy number category and features of interest. Log-rank tests and Cox models compared event-free survival and overall survival by subgroup. Among 4672 patients, 3694 (79.1%) had MYCN wild-type tumors, 133 (2.8%) had MYCN gain, and 845 (18.1%) had MNA. For each clinical/biological feature, the percentage of patients with an unfavorable feature was lowest in the MYCN wild-type category, intermediate in the MYCN gain category, and highest in the MNA category (P<.0001), except for 11q aberration, for which the highest rates were in the MYCN gain category. Patients with MYCN gain had inferior event-free survival and overall survival compared with those with MYCN wild-type. Among patients with high-risk disease, MYCN gain was associated with the lowest response rate after chemotherapy. Patients with non-stage 4 disease (according to the International Neuroblastoma Staging System) and patients with non-high-risk disease with MYCN gain had a significantly increased risk for death, a finding confirmed on multivariable testing. Increasing MYCN copy number is associated with an increasingly higher rate of unfavorable clinical/biological features, with 11q aberration being an exception. Patients with MYCN gain appear to have inferior outcomes, especially in otherwise more favorable groups. Cancer 2017;123:4224-4235. © 2017 American Cancer Society. © 2017 American Cancer Society.
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Michikawa, Mari; Ichinose, Hitomi; Momma, Mitsuru; Biely, Peter; Jongkees, Seino; Yoshida, Makoto; Kotake, Toshihisa; Tsumuraya, Yoichi; Withers, Stephen G; Fujimoto, Zui; Kaneko, Satoshi
2012-04-20
We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.
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Multigene signature for predicting prognosis of patients with 1p19q co-deletion diffuse glioma.
Hu, Xin; Martinez-Ledesma, Emmanuel; Zheng, Siyuan; Kim, Hoon; Barthel, Floris; Jiang, Tao; Hess, Kenneth R; Verhaak, Roel G W
2017-06-01
Co-deletion of 1p and 19q marks a diffuse glioma subtype associated with relatively favorable overall survival; however, heterogeneous clinical outcomes are observed within this category. We assembled gene expression profiles and sample annotation of 374 glioma patients carrying the 1p/19q co-deletion. We predicted 1p/19q status using gene expression when annotation was missing. A first cohort was randomly split into training (n = 170) and a validation dataset (n = 163). A second validation set consisted of 41 expression profiles. An elastic-net penalized Cox proportional hazards model was applied to build a classifier model through cross-validation within the training dataset. The selected 35-gene signature was used to identify high-risk and low-risk groups in the validation set, which showed significantly different overall survival (P = .00058, log-rank test). For time-to-death events, the high-risk group predicted by the gene signature yielded a hazard ratio of 1.78 (95% confidence interval, 1.02-3.11). The signature was also significantly associated with clinical outcome in the The Cancer Genome Atlas (CGA) IDH-mutant 1p/19q wild-type and IDH-wild-type glioma cohorts. Pathway analysis suggested that high risk was associated with increased acetylation activity and inflammatory response. Tumor purity was found to be significantly decreased in high-risk IDH-mutant with 1p/19q co-deletion gliomas and IDH-wild-type glioblastomas but not in IDH-wild-type lower grade or IDH-mutant, non-co-deleted gliomas. We identified a 35-gene signature that identifies high-risk and low-risk categories of 1p/19q positive glioma patients. We have demonstrated heterogeneity amongst a relatively new glioma subtype and provided a stepping stone towards risk stratification. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Michikawa, Mari; Ichinose, Hitomi; Momma, Mitsuru; Biely, Peter; Jongkees, Seino; Yoshida, Makoto; Kotake, Toshihisa; Tsumuraya, Yoichi; Withers, Stephen G.; Fujimoto, Zui; Kaneko, Satoshi
2012-01-01
We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-d-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)8-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu173 (acid base) and Glu287 (nucleophile), consistent with the retaining mechanism demonstrated by 1H NMR analysis. Glu45, Tyr243, Tyr292–Gly294, and Tyr334 form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln293 and Gly294 and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ∼200-fold lower (kcat/Km) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr334 in recognition of the C6 position of GlcA. The involvement of Glu45 in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (kcat/Km) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower. PMID:22367201
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Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus
DOE Office of Scientific and Technical Information (OSTI.GOV)
Suzuki, Kenichiro; Ito, Sohei; Shimizu-Ibuka, Akiko
2005-08-01
This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6{sub 2}22 and diffracted to a resolution of 2.4 Å. Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. Amore » crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6{sub 2}22, with unit-cell parameters a = b = 145.97, c = 118.03 Å.« less
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Perkins, David D.; Metzenberg, Robert L.; Raju, Namboori B.; Selker, Eric U.; Barry, Edward G.
1986-01-01
In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation x Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered. PMID:2947829
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Occurrence and distribution of Giardia species in wild rodents in Germany.
Helmy, Yosra A; Spierling, Nastasja G; Schmidt, Sabrina; Rosenfeld, Ulrike M; Reil, Daniela; Imholt, Christian; Jacob, Jens; Ulrich, Rainer G; Aebischer, Toni; Klotz, Christian
2018-03-27
Giardiasis is an important gastrointestinal parasitic disease in humans and other mammals caused by the protozoan Giardia duodenalis. This species complex is represented by genetically distinct groups (assemblages A-H) with varying zoonotic potential and host preferences. Wild rodents can harbor potentially zoonotic assemblages A and B, and the rodent-specific assemblage G. Other Giardia spp. found in these animals are Giardia muris and Giardia microti. For the latter, only limited information on genetic typing is available. It has been speculated that wild rodents might represent an important reservoir for parasites causing human giardiasis. The aim of this study was to investigate the occurrence and distribution of Giardia spp. and assemblage types in wild rodents from different study sites in Germany. Screening of 577 wild rodents of the genera Apodemus, Microtus and Myodes, sampled at eleven study sites in Germany, revealed a high overall Giardia prevalence. Giardia species determination at the SSU rDNA gene locus revealed that Apodemus mice, depending on species, were predominantly infected with one of two distinct G. muris sequence types. Giardia microti was the predominant parasite species found in voles of the genera Microtus and Myodes. Only a few animals were positive for potentially zoonotic G. duodenalis. Subtyping at the beta-giardin (bg) and glutamine dehydrogenase (gdh) genes strongly supported the existence of different phylogenetic subgroups of G. microti that are preferentially harbored by distinct host species. The present study highlights the preference of G. muris for Apodemus, and G. microti for Microtus and Myodes hosts and argues for a very low prevalence of zoonotic G. duodenalis assemblages in wild rodents in Germany. It also provides evidence that G. muris and G. microti subdivide into several phylogenetically distinguishable subgroups, each of which appears to be preferentially harbored by species of a particular rodent host genus. Finally, the study expands the database of sequences relevant for sequence typing of G. muris and G. microti isolates which will greatly help future analyses of these parasites' population structure.
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Randolph, Matthew E; Luo, Qingwei; Ho, Justin; Vest, Katherine E; Sokoloff, Alan J; Pavlath, Grace K
2014-01-01
The inability to swallow, or dysphagia, is a debilitating and life-threatening condition that arises with ageing or disease. Dysphagia results from neurological or muscular impairment of one or more pharyngeal muscles, which function together to ensure proper swallowing and prevent the aspiration of food or liquid into the lungs. Little is known about the effects of age or disease on pharyngeal muscles as a group. Here we show ageing affected pharyngeal muscle growth and atrophy in wild-type mice depending on the particular muscle analysed. Furthermore, wild-type mice also developed dysphagia with ageing. Additionally, we studied pharyngeal muscles in a mouse model for oculopharyngeal muscular dystrophy, a dysphagic disease caused by a polyalanine expansion in the RNA binding protein, PABPN1. We examined pharyngeal muscles of mice overexpressing either wild-type A10 or mutant A17 PABPN1. Overexpression of mutant A17 PABPN1 differentially affected growth of the palatopharyngeus muscle dependent on its location within the pharynx. Interestingly, overexpression of wild-type A10 PABPN1 was protective against age-related muscle atrophy in the laryngopharynx and prevented the development of age-related dysphagia. These results demonstrate that pharyngeal muscles are differentially affected by both ageing and muscular dystrophy in a region-dependent manner. These studies lay important groundwork for understanding the molecular and cellular mechanisms that regulate pharyngeal muscle growth and atrophy, which may lead to novel therapies for individuals with dysphagia. PMID:25326455
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Johnson, Larry; Atanasova, Kalina R.; Bui, Phuong Q.; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M.
2015-01-01
Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the proinflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. PMID:25828169
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Dugat, Thibaud; Zanella, Gina; Véran, Luc; Lesage, Céline; Girault, Guillaume; Durand, Benoît; Lagrée, Anne-Claire; Boulouis, Henri-Jean; Haddad, Nadia
2016-11-22
Anaplasma phagocytophilum is the causative agent of tick-borne fever, a disease with high economic impact for domestic ruminants in Europe. Epidemiological cycles of this species are complex, and involve different ecotypes circulating in various host species. To date, these epidemiological cycles are poorly understood, especially in Europe, as European reservoir hosts (i.e. vertebrate hosts enabling long-term maintenance of the bacterium in the ecosystem), of the bacterium have not yet been clearly identified. In this study, our objective was to explore the presence, the prevalence, and the genetic diversity of A. phagocytophilum in wild animals, in order to better understand their implications as reservoir hosts of this pathogen. The spleens of 101 wild animals were collected from central France and tested for the presence of A. phagocytophilum DNA by msp2 qPCR. Positive samples were then typed by multi-locus variable-number tandem repeat (VNTR) analysis (MLVA), and compared to 179 previously typed A. phagocytophilum samples. Anaplasma phagocytophilum DNA was detected in 82/101 (81.2%) animals including 48/49 red deer (98%), 20/21 roe deer (95.2%), 13/29 wild boars (44.8%), and 1/1 red fox. MLVA enabled the discrimination of two A. phagocytophilum groups: group A contained the majority of A. phagocytophilum from red deer and two thirds of those from cattle, while group B included a human strain and variants from diverse animal species, i.e. sheep, dogs, a horse, the majority of variants from roe deer, and the remaining variants from cattle and red deer. Our results suggest that red deer and roe deer are promising A. phagocytophilum reservoir host candidates. Moreover, we also showed that A. phagocytophilum potentially circulates in at least two epidemiological cycles in French cattle. The first cycle may involve red deer as reservoir hosts and cattle as accidental hosts for Group A strains, whereas the second cycle could involve roe deer as reservoir hosts and at least domestic ruminants, dogs, horses, and humans as accidental hosts for Group B strains.
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Wild rodents and shrews are natural hosts of Staphylococcus aureus.
Mrochen, Daniel M; Schulz, Daniel; Fischer, Stefan; Jeske, Kathrin; El Gohary, Heba; Reil, Daniela; Imholt, Christian; Trübe, Patricia; Suchomel, Josef; Tricaud, Emilie; Jacob, Jens; Heroldová, Marta; Bröker, Barbara M; Strommenger, Birgit; Walther, Birgit; Ulrich, Rainer G; Holtfreter, Silva
2017-09-22
Laboratory mice are the most commonly used animal model for Staphylococcus aureus infection studies. We have previously shown that laboratory mice from global vendors are frequently colonized with S. aureus. Laboratory mice originate from wild house mice. Hence, we investigated whether wild rodents, including house mice, as well as shrews are naturally colonized with S. aureus and whether S. aureus adapts to the wild animal host. 295 animals of ten different species were caught in different locations over four years (2012-2015) in Germany, France and the Czech Republic. 45 animals were positive for S. aureus (15.3%). Three animals were co-colonized with two different isolates, resulting in 48 S. aureus isolates in total. Positive animals were found in Germany and the Czech Republic in each studied year. The S. aureus isolates belonged to ten different spa types, which grouped into six lineages (clonal complex (CC) 49, CC88, CC130, CC1956, sequence type (ST) 890, ST3033). CC49 isolates were most abundant (17/48, 35.4%), followed by CC1956 (14/48, 29.2%) and ST890 (9/48, 18.8%). The wild animal isolates lacked certain properties that are common among human isolates, e.g., a phage-encoded immune evasion cluster, superantigen genes on mobile genetic elements and antibiotic resistance genes, which suggests long-term adaptation to the wild animal host. One CC130 isolate contained the mecC gene, implying wild rodents might be both reservoir and vector for methicillin-resistant S. aureus. In conclusion, we demonstrated that wild rodents and shrews are naturally colonized with S. aureus, and that those S. aureus isolates show signs of host adaptation. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.
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Variation in founder groups promotes establishment success in the wild
Forsman, Anders; Wennersten, Lena; Karlsson, Magnus; Caesar, Sofia
2012-01-01
Environmental changes currently pose severe threats to biodiversity, and reintroductions and translocations are increasingly used to protect declining populations and species from extinction. Theory predicts that establishment success should be higher for more variable groups of dissimilar individuals. To test this ‘diversity promotes establishment’ hypothesis, we introduced colour polymorphic pygmy grasshoppers (Tetrix subulata) to different sites in the wild. The number of descendants found at the release sites the subsequent year increased with increasing number of colour morphs in the founder group, and variation in founder groups also positively affected colour morph diversity in the established populations. Since colour morphs differ in morphology, physiology, behaviour, reproductive life history and types of niche used, these findings demonstrate that variation among individuals in functionally important traits promotes establishment success under natural conditions, and further indicate that founder diversity may contribute to evolutionary rescue and increased population persistence. PMID:22456885
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López-Pérez, Andrés M; Gage, Kenneth; Rubio, Andre V; Montenieri, John; Orozco, Libertad; Suzan, Gerardo
2018-06-01
Host identity, habitat type, season, and interspecific interactions were investigated as determinants of the community structure of fleas on wild carnivores in northwestern Mexico. A total of 540 fleas belonging to seven species was collected from 64 wild carnivores belonging to eight species. We found that the abundances of some flea species are explained by season and host identity. Pulex irritans and Echidnophaga gallinacea abundances were significantly higher in spring than in fall season. Flea communities on carnivore hosts revealed three clusters with a high degree of similarity within each group that was explained by the flea dominance of E. gallinacea, P. simulans, and P. irritans across host identity. Flea abundances did not differ statistically among habitat types. Finally, we found a negative correlation between the abundances of three flea species within wild carnivore hosts. Individual hosts with high loads of P. simulans males usually had significantly lower loads of P. irritans males or tend to have lower loads of E. gallinacea fleas and vice-versa. Additionally, the logistic regression model showed that the presence of P. simulans males is more likely to occur in wild carnivore hosts in which P. irritans males are absent and vice-versa. These results suggest that there is an apparent competitive exclusion among fleas on wild carnivores. The study of flea community structure on wild carnivores is important to identify the potential flea vectors for infectious diseases and provide information needed to design programs for human health and wildlife conservation. © 2018 The Society for Vector Ecology.
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Zhang, Chao; Li, Zhouquan; Zhang, Xiaohua; Yuan, Li; Dai, Heping; Xiao, Wei
2016-01-01
Transcriptomic profiles are generated by comparing wild-type and the yeast yap1 mutant to various chemicals in an attempt to establish a correlation between this gene mutation and chemical exposure. Test chemicals include ClonNAT as a non-genotoxic agent, methyl methanesulphonate (MMS) as an alkylating agent, tert-butyl hydroperoxide (t-BHP) as an oxidative agent and the mixture of t-BHP and MMS to reflect complex natural exposure. Differentially expressed genes (DEGs) were identified and specific DEGs were obtained by excluding overlapping DEGs with the control group. In the MMS exposure group, deoxyribonucleotide biosynthetic processes were upregulated, while oxidation-reduction processes were downregulated. In the t-BHP exposure group, metabolic processes were upregulated while peroxisome and ion transport pathways were downregulated. In the mixture exposure group, the proteasome pathway was upregulated, while the aerobic respiration was downregulated. Homologue analysis of DEGs related to human diseases showed that many of DEGs were linked to cancer, ageing and neuronal degeneration. These observations confirm that the yap1 mutant is more sensitive to chemicals than wild-type cells and that the susceptible individuals carrying the YAP1-like gene defect may enhance risk to chemical exposure. Hence, this study offers a novel approach to environmental risk assessment, based on the genetic backgrounds of susceptible individuals. Copyright © 2015 John Wiley & Sons, Ltd.
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Mitochondrial Group II Introns, Cytochrome c Oxidase, and Senescence in Podospora anserina†
Begel, Odile; Boulay, Jocelyne; Albert, Beatrice; Dufour, Eric; Sainsard-Chanet, Annie
1999-01-01
Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (α) has been thought to play a prominent role in this syndrome. Intron α is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the α sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron α. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron α is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron α plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, “immortality” can be acquired not by the absence of intron α but rather by the lack of active cytochrome c oxidase. PMID:10330149
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Chen, Xi; Sun, Weiwen; Pan, Ying; Yang, Quan; Cao, Kaiyi; Zhang, Jin; Zhang, Yizhi; Chen, Mincong; Chen, Feidi; Huang, Yueling; Dai, Lijun; Chen, Shengqiang
2013-10-01
To investigate whether lithium modifies open-field and elevated plus maze behavior, and brain phospho-glycogen synthase kinase 3 (P-GSK3beta) expression in Fmr1 knockout mice. One hundred and eighty FVB mice, including knockout and wild type, with an age of 30 days were used. An open-field and elevated plus maze was utilized to test behavior, while western blot was used to measure the P-GSK3beta expression. Six groups were formed: control (saline), lithium chloride 30, 60, 90, 120, and 200 mg/kg. The experiments were carried out in the Institute of Neuroscience, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China between January and June 2012. Lithium significantly decreased total distance, crossing, central area time, and center entry in the open-field test (p<0.05), and significantly reduced open-arm tracking, open-arm entry, and open-arm time in the elevated plus maze (p<0.05) in knockout mice. In wild type mice, significant changes were observed in both behavior tests in some treatment groups. Lithium ameliorated P-GSK3beta expression in the hippocampus of all the treatment groups in knockout mice (p<0.05). However, lithium did not modify either GSK3beta expression in tissues of knockout mice, or P-GSK3beta or GSK3beta expression in tissues of wild type mice. Lithium ameliorated open-field and elevated plus maze behaviors of Fmr1 knockout mice. This effect may be related to its enhancement of P-GSK3beta expression. Our findings suggest that lithium might have a therapeutic effect in fragile X syndrome.
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Liu, C Y; Li, Q; Zhou, X D
2016-03-29
To explore the inhibitory role of suppressor of cytokine signaling 1 (SOCS1) in lipopolysaccharide (LPS)-induced mucin5AC (MUC5AC) hypersecretion and the potential mechanism involved in this process. The human bronchial epithelial cells 16HBE were divided into 0, 0.5, 1, 6, 12 and 24 h groups according to the time of LPS challenge. In gain- and loss- of functions experiments, wild-type SOCS1 and SOCS1-targeted siRNA (SOCS1-siRNA) were synthesized to identify the function of SOCS1 in LPS-mediated MUC5AC hypersecretion, and named wild-type SOCS1 group and SOCS1-siRNA group, respectively, and the non-transfected group and non-targeted siRNA group were used as controls. In Filgotinib group, the specific inhibitor of Janus kinase 1 (JAK1), Filgotinib, was used to detect the role of JAK1/signal transducer and activator of transcription 1 (STAT1) signaling pathway in LPS challenge, and the aqueous physiological buffer group was used as the control. The production of MUC5AC protein was measured by enzyme linked immunosorbent assay (ELISA), and the amount of MUC5AC protein was normalized to the total protein in cell lysates and was expressed as μg/mg cell lysates. The proteins expressions of SOCS1, phosphorylation of JAK1 and STAT1 were measured by Western blot, and the total expression of its protein (for JAK1 and STAT1) or β-actin (for SOCS1) was used as the loading control. Compared to 0 h group, LPS induced a robust induction in MUC5AC expression, the expression levels of MUC5AC in 0, 0.5, 1, 6, 12 and 24 h groups were (2.86±0.20), (3.42±0.29), (3.43±0.12), (10.22±0.96), (14.56±1.12), (14.15±1.34) μg/mg, in association with a decrease of SOCS1 expression. And in 6 h group, the expressions of MUC5AC and SOCS1 were both medium up-regulated (all P<0.05). Consequently, the application of LPS for 6 h was selected as the optimal responses period in the ensuing experiments. Compared to the expression of MUC5AC protein in non-transfected group, high level of SOCS1 in wild-type SOCS1 group led to a reduced phosphorylation of JAK1/STAT1, as well as Filgotinib did, thereby suppressing excessive MUC5AC production in wild-type SOCS1 group and Filgotinib group [(4.04±0.65), (7.02±0.83) vs (10.37±1.00) μg/mg] (all P<0.05). Conversely, compared to the expression of MUC5AC in non-targeted siRNA group, down-regulation of SOCS1 in SOCS1-siRNA group promoted the phosphorylation of JAK1/STAT1, and then further increased MUC5AC production [(13.69±1.32) vs (11.01±1.41) μg/mg] (all P<0.05). These results show that SOCS1 suppresses LPS-promoted MUC5AC hypersecretion through the inhibition of JAK1/STAT1 signaling pathway.
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Moriwaki, Hiroshi; Koide, Remi; Yoshikawa, Ritsuko; Warabino, Yuya; Yamamoto, Hiroki
2013-04-01
The aim of this study is to investigate the potential of cell walls of wild-type and lipoteichoic acid-defective strains of Bacillus subtilis 168 to adsorb rare earth ions. Freeze-dried cell powders prepared from both strains were used for the evaluation of adsorption ability for the rare earth ions, namely, La(III), Eu(III), and Tm(III). The rare earth ions were efficiently adsorbed onto powders of both wild-type strain (WT powder) and lipoteichoic acid-defective strain (∆LTA powder) at pH 3. The maximum adsorption capacities for Tm(III) by WT and ∆LTA powders were 43 and 37 mg g(-1), respectively. Removal (in percent) of Tm(III), La(III), and Eu(III) from aqueous solution by WT powder was greater than by ∆LTA powder. These results indicate that rare earth ions are adsorbed to functional groups, such as phosphate and carboxyl groups, of lipoteichoic acid. We observed coagulated ∆LTA powder in the removal of rare earth ions (1-20 mg L(-1)) from aqueous solution. In contrast, sedimentation of WT powder did not occur under the same conditions. This unique feature of ∆LTA powder may be caused by the difference of the distribution between lipoteichoic acid and wall teichoic acid. It appears that ∆LTA powder is useful for removal of rare earth ions by adsorption, because aggregation allows for rapid separation of the adsorbent by filtration.
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Moleleki, Lucy Novungayo; Pretorius, Rudolph Gustav; Tanui, Collins Kipngetich; Mosina, Gabolwelwe; Theron, Jacques
2017-01-01
Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain. © 2016 BSPP and John Wiley & Sons Ltd.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Elling, R.A.; Fucini, R.V.; Romanowski, M.J.
Polo-like kinase 1 (Plk1) is a member of a family of serine/threonine kinases involved in the regulation of cell-cycle progression and cytokinesis and is an attractive target for the development of anticancer therapeutics. A zebrafish homolog of the human Plk1 (hPlk1) kinase domain (KD) was identified that can be expressed in large quantities in bacteria and crystallizes readily, whether in a wild-type form or as a variant containing the activating Thr196-->Asp substitution, in one space group and under similar conditions both in the absence and presence of active-site compounds. This construct was validated by testing a panel of hPlk1 inhibitorsmore » against human and zebrafish proteins and it was shown that the selected small molecules inhibited the homologs with a high degree of correlation. Crystal structures of ligand-free wild-type and activated zebrafish Plk1 (zPlk1) KDs revealed the organization of the secondary structural elements around the active site and demonstrated that the activation segment was disordered in the activated form of the domain but possessed a well defined secondary structure in the wild-type enzyme. The cocrystal structure of wild-type zPlk1 KD with ADP documented the hydrolysis of ATP and revealed the phosphorylation site. The cocrystal structure of the activated KD with wortmannin, a covalent inhibitor of Plk1 and PI3 kinases, showed the binding mode of the small molecule to the enzyme and may facilitate the design of more potent Plk1 inhibitors. The work presented in this study establishes the zPlk1 KD as a useful tool for rapid low- and high-throughput structure-based screening and drug discovery of compounds specific for this mitotic target.« less
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Vora, Kalpit A; Porter, Gene; Peng, Roche; Cui, Yan; Pryor, Kellyann; Eiermann, George; Zaller, Dennis M
2009-01-01
Background Current literature suggests that dipeptidyl peptidase IV (DPP-IV; CD26) plays an essential role in T-dependent immune responses, a role that could have important clinical consequences. To rigorously define the role of DPP-IV in the immune system, we evaluated genetic and pharmacological inhibition of the enzyme on T-dependent immune responses in vivo. Results The DPP-IV null animals mounted robust primary and secondary antibody responses to the T dependent antigens, 4-hydroxy-3-nitrophenylacetyl-ovalbumin (NP-Ova) and 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin (NP-CGG), which were comparable to wild type mice. Serum levels of antigen specific IgM, IgG1, IgG2a, IgG2b and IgG3 were similar between the two groups of animals. DPP-IV null animals mounted an efficient germinal center reaction by day 10 after antigen stimulation that was comparable to wild type mice. Moreover, the antibodies produced by DPP-IV null animals after repeated antigenic challenge were affinity matured. Similar observations were made using wild type animals treated with a highly selective DPP-IV inhibitor during the entire course of the experiments. T cell recall responses to ovalbumin and MOG peptide, evaluated by measuring proliferation and IL-2 release from cells isolated from draining lymph nodes, were equivalent in DPP-IV null and wild type animals. Furthermore, mice treated with DPP-IV inhibitor had intact T-cell recall responses to MOG peptide. In addition, female DPP-IV null and wild type mice treated with DPP-IV inhibitor exhibited normal and robust in vivo cytotoxic T cell responses after challenge with cells expressing the male H-Y minor histocompatibility antigen. Conclusion These data indicate Selective inhibition of DPP-IV does not impair T dependent immune responses to antigenic challenge. PMID:19358731
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Wilhelm, Annika; Aldridge, Victoria; Haldar, Debashis; Naylor, Amy J; Weston, Christopher J; Hedegaard, Ditte; Garg, Abhilok; Fear, Janine; Reynolds, Gary M; Croft, Adam P; Henderson, Neil C; Buckley, Christopher D; Newsome, Philip N
2016-07-01
CD248 (endosialin) is a stromal cell marker expressed on fibroblasts and pericytes. During liver injury, myofibroblasts are the main source of fibrotic matrix. To determine the role of CD248 in the development of liver fibrosis in the rodent and human setting. CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human and murine liver tissue and isolated hepatic stellate cells (HSCs). Hepatic fibrosis was induced in CD248(-/-) and wild-type controls with carbon tetrachloride (CCl4) treatment. Expression of CD248 was seen in normal liver of humans and mice but was significantly increased in liver injury using both immunostaining and gene expression assays. CD248 was co-expressed with a range of fibroblast/HSC markers including desmin, vimentin and α-smooth muscle actin (α-SMA) in murine and human liver sections. CD248 expression was restricted to isolated primary murine and human HSC. Collagen deposition and α-SMA expression, but not inflammation and neoangiogenesis, was reduced in CD248(-/-) mice compared with wild-type mice after CCl4 treatment. Isolated HSC from wild-type and CD248(-/-) mice expressed platelet-derived growth factor receptor α (PDGFR-α) and PDGFR-β at similar levels. As expected, PDGF-BB stimulation induced proliferation of wild-type HSC, whereas CD248(-/-) HSC did not demonstrate a proliferative response to PDGF-BB. Abrogated PDGF signalling in CD248(-/-) HSC was confirmed by significantly reduced c-fos expression in CD248(-/-) HSC compared with wild-type HSC. Our data show that deletion of CD248 reduces susceptibility to liver fibrosis via an effect on PDGF signalling, making it an attractive clinical target for the treatment of liver injury. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/
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Hansen, Laura M; Gupta, Divya; Joseph, Giji; Weiss, Daiana; Taylor, W Robert
2017-01-01
Diabetics often have poor perfusion in their limbs as a result of peripheral artery disease and an impaired ability to generate collateral vessels. The receptor for advanced glycation end products (RAGE) is one protein that is thought to play a detrimental role in collateral development in diabetics due to increased levels of advanced glycation end products (AGE), one of its ligands, in diabetes. Thus, the aim of this study was to investigate the role of RAGE in both diabetic and non-diabetic settings in a model of collateral formation in mice. Streptozotocin was used to induce diabetes in both wild type and RAGE knockout mice. Increased levels of the AGE, N ɛ -(carboxymethyl) lysine (CML), were confirmed via an ELISA. A hindlimb ischemia model, in which the femoral artery is ligated, was used to drive collateral growth and reperfusion was assessed using laser Doppler perfusion imaging and histological analysis of vessels in the muscle. Both of these measurements showed impaired collateral growth in diabetic compared with wild-type mice as well as improved collateral growth in both diabetic and non-diabetic RAGE knockout mice when compared their wild-type counterparts. Distance on a freely accessed running wheel, used as a measure of perfusion recovery, showed that wild-type diabetic mice had functionally impaired recovery compared with their wild-type counterparts. Immunohistochemistry and immunoblotting showed that HMGB-1 (high-mobility group box 1), another RAGE ligand, was increased in the ischemic leg compared with the non-ischemic leg in all mice. This increase in HMGB-1 may explain improvement in animals lacking RAGE and its subsequent signaling. In conclusion, this study shows that RAGE impairs collateral growth in a diabetic setting and also in a non-diabetic setting. This demonstrates the importance of RAGE and alternate RAGE ligands in the setting of collateral vessel growth.
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Avgoustidis, Dimitris; Nisyrios, Themistoklis; Nkenke, Emeka; Lijnen, Roger; Ragos, Vassilis; Perrea, Despina; Donta, Ismini; Vaena, Apostolia; Yapijakis, Christos; Vairaktaris, Eleftherios
2012-01-01
In an effort to assess the role of plasminogen activator inhibitor-1 (PAI-1) in oral squamous cancer development and progression, two different carcinogen treatment protocols were conducted. Protocol I included mice from a PAI-1 transgenic (Tg) breed (n=56) and their wild-type (WT) counterparts (n=56), divided into one control group and two main experimental groups, treated with 7,12-dimethylbenz[a]anthracene (DMBA) for 8 and 16 weeks, respectively. Protocol II included the same number and types of animals and groups, which were similarly treated with 4-Nitroquinoline 1-oxide (4-NQO) in drinking water. Two drugs that affect plasma PAI-1 levels, enalapril and pravastatin, were administered to certain subgroups of animals in both protocols. None of the animals developed macroscopically-visible oral cancer lesions. Eleven animals under Protocol I and 52 animals under Protocol II died. Skin lesions were noted only in DMBA-treated animals (n=9). Almost all animals administered with 4-NQO developed alopecia and lost weight, while two of them developed stomach tumours, and one female mouse developed a large ovarian cyst. Transgenic mice may respond differently when used in well-established carcinogen models and oral carcinogenesis is hard to achieve in these rodents.
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Altered Astrocyte-Neuron Interactions and Epileptogenesis in Tuberous Sclerosis Complex Disorder
2016-08-01
and physiological functions of wild - type and recombinant neurons, as well as the effects of Tsc1-deficient astrocytes on neuronal morphology and...intrinsic mTOR activation of synaptic activities on wild -type and recombinant neurons, as well as the effects of Tsc1- deficient astrocytes on neuronal...more dendritic spines than wild type, non-recombinant neurons. The latter show a similar spine density to that of pyramidal neurons in a TSC1 wild type
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2014-01-01
wild-type spores but ~15-fold higher deltaTrelease values; v ) germination kinetics of wild-type spores given a ? 30 sec 140 MPa HP pulse followed by...15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average Tlag values; and ( v ) the germination of wild-type...committed spores, as it does for nutrient-committed spores (14)? ( v ) Can these HP-com- mitted spores be isolated under conditions that do not allow
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Chou, Lee Yiung; Clarke, Charles M; Dykes, Gary A
2014-10-01
Nepenthes pitcher plants produce modified jug-shaped leaves to attract, trap and digest insect prey. We used 16S rDNA cloning and sequencing to compare bacterial communities in pitcher fluids of each of three species, namely Nepenthes ampullaria, Nepenthes gracilis and Nepenthes mirabilis, growing in the wild. In contrast to previous greenhouse-based studies, we found that both opened and unopened pitchers harbored bacterial DNA. Pitchers of N. mirabilis had higher bacterial diversity as compared to other Nepenthes species. The composition of the bacterial communities could be different between pitcher types for N. mirabilis (ANOSIM: R = 0.340, p < 0.05). Other Nepenthes species had similar bacterial composition between pitcher types. SIMPER showed that more than 50 % of the bacterial taxa identified from the open pitchers of N. mirabilis were not found in other groups. Our study suggests that bacteria in N. mirabilis are divided into native and nonnative groups.
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Origin and evolution of Andigena potatoes revealed by chloroplast and nuclear DNA markers.
Sukhotu, Thitaporn; Hosaka, Kazuyoshi
2006-06-01
Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are important, native-farmer-selected cultivars in the Andes, which form a primary gene pool for improving a worldwide grown potato (S. tuberosum subsp. tuberosum). To elucidate the origin of Andigena, 196 Andigena accessions were compared with 301 accessions of 33 closely related cultivated and wild species using several types of chloroplast DNA (ctDNA) markers and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Fourteen ctDNA types (haplotypes) and 115 RFLP bands were detected in Andigena, of which the main haplotypes and frequent RFLP bands were mostly shared with a cultivated diploid species, S. stenotomum Juz. et Buk. Principal component analysis of nDNA polymorphisms revealed a progressive and continuous variation from Peruvian wild species with C-type ctDNA to a group of wild species having S-type ctDNA in its variation range (S. bukasovii, S. canasense, S. candolleanum, and S. multidissectum), to cultivated diploid potatoes (S. phureja and S. stenotomum), and to cultivated tetraploid potatoes (Andigena and Chilean S. tuberosum subsp. tuberosum). These results suggest that the initial Andigena population arose with multiple origins exclusively from S. stenotomum. The overall evolutionary process toward the present-day Andigena was discussed.
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Stomatal control in tomato with ABA-deficient roots: response of grafted plants to soil drying.
Holbrook, N Michele; Shashidhar, V R; James, Richard A; Munns, Rana
2002-06-01
The hypothesis that ABA produced by roots in drying soil is responsible for stomatal closure was tested with grafted plants constructed from the ABA-deficient tomato mutants, sitiens and flacca and their near-isogenic wild-type parent. Three types of experiments were conducted. In the first type, reciprocal grafts were made between the wild type and sitiens or flacca. Stomatal conductance accorded with the genotype of the shoot, not the root. Stomates closed in all of the grafted plants in response to soil drying, regardless of the root genotype, i.e. regardless of the ability of the roots to produce ABA. In the second type of experiment, wild-type shoots were grafted onto a split-root system consisting of one wild-type root grafted to one mutant (flacca or sitiens) root. Water was withheld from one root system, while the other was watered well so that the shoots did not experience any decline in water potential or loss of turgor. Stomates closed to a similar extent when water was withheld from the mutant roots or the wild-type roots. In the third type of experiment, grafted plants with wild-type shoots and either wild-type or sitiens roots were established in pots that could be placed inside a pressure chamber, and the pressure increased as the soil dried so that the shoots remained fully turgid throughout. Stomates closed as the soil dried, regardless of whether the roots were wild type or sitiens. These experiments demonstrate that stomatal closure in response to soil drying can occur in the absence of leaf water deficit, and does not require ABA production by roots. A chemical signal from roots leading to a change in apoplastic ABA levels in leaves may be responsible for the stomatal closure.
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Vanilla, Shiny; Kotur, Pushpa F; Kutty, Moideen A; Vegi, Pradeep Kumar
2015-01-01
Introduction: Recurrent spontaneous abortion (RSA) is a multifactorial clinical obstetrics complication commonly occurring in pregnancy. Many research studies have noted the mutations such as C677T in N5, N10 - Methylenetetrahydrofolate reductase (MTHFR)gene which is regarded as RSA risk factor. This study was carried out to determine the occurrence of frequency of C677T of the MTHFR gene mutations with RSA. Aim: The purpose of present study is to determine the frequency of MTHFR C677T polymorphisms in couples with recurrent pregnancy loss and the impact of paternal polymorphisms of MTHFR C677T in recurrent pregnancy loss in population of couples living in Kolar district of Karnataka with RSA. Design: A total of 15 couples with a history of two or more unexplained RSA were enrolled as subjects in the study and a total of 15 couples with normal reproductive history, having two or more children and no history of miscarriages were enrolled as controls. Materials and Methods: DNA extraction from samples case and control group couples and its quantification by Agarose gel electrophoresis, assessment of DNA purity, MTHFR C 677T gene mutation detection by PCR-RFLP method. Statistical analysis: Carried out by web based online SPSS tool. Results: The frequency of C677T genotype showed homozygous wild type CC (80%), heterozygous CT type (13.3%) and homozygous mutation TT type (6.67%) observed in males. Similarly from female’s homozygous wild type CC (86.6%), heterozygous type (13.3%), and homozygous type mutations TT (0%) was recorded. In couple control groups, we observed homozygous wild type CC (86.6%), heterozygous CT type (13.3%) and homozygous type mutations TT type (0%). Conclusion: We noticed a high frequency of MTHFR specifically T allele associated with paternal side.Therefore, the present study indicated the impact of paternal gene polymorphism of MTHFR C677T on screening in couples with recurrent pregnancy loss. PMID:25859445
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Kattanek, Maria; Richardson, Kenneth C.; Hafez, Hafez Mohamed; Plendl, Johanna; Hünigen, Hana
2017-01-01
In this study the macroscopic and microscopic structure of the heart of a fast growing, meat-type turkey line (British United turkeys BUT Big 6) and a wild-type turkey line (Canadian Wild turkey) were compared. At 8 and 16 weeks of age, 10 birds of each genotype and sex were sampled. The body mass and heart mass of the meat-type turkey both increased at a faster rate than those of the wild-type turkey. However in both turkey lines, the relative heart mass decreased slightly with age, the decrease was statistically significant only in the male turkeys. Furthermore meat-type turkeys had a significantly (p < 0.01) lower relative heart mass and relative thickness of the left ventricle compared to the wild-type turkeys of the same age. The wild-type turkeys showed no significant change in the size of cardiomyocytes (cross sectional area and diameter) from 8 weeks to 16 weeks. In contrast, the size of cardiomyocytes increased significantly (p < 0.001) with age in the meat-type turkeys. The number of capillaries in the left ventricular wall increased significantly (p < 0.001) in wild-type turkeys from 2351 per mm2 at the age of 8 weeks to 2843 per mm2 at 16 weeks. However, in the meat-type turkeys there were no significant changes, capillary numbers being 2989 per mm2 at age 8 weeks and 2915 per mm2 at age 16 weeks. Correspondingly the area occupied by capillaries in the myocardium increased in wild-type turkeys from 8.59% at the age of 8 weeks to 9.15% at 16 weeks, whereas in meat-type turkeys this area decreased from 10.4% at 8 weeks to 9.95% at 16 weeks. Our results indicate a mismatch in development between body mass and heart mass and a compromised cardiac capillary density and architecture in the meat-type turkeys in comparison to the wild-type turkeys. PMID:28118415
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Jeong, Jipseol; Kang, Hyun-Mi; Lee, Eun-Kyoung; Song, Byung-Min; Kwon, Yong-Kuk; Kim, Hye-Ryoung; Choi, Kang-Seuk; Kim, Ji-Ye; Lee, Hyun-Jeong; Moon, Oun-Kyong; Jeong, Wooseog; Choi, Jida; Baek, Jong-Ho; Joo, Yi-Seok; Park, Yong Ho; Lee, Hee-Soo; Lee, Youn-Jeong
2014-10-10
Highly pathogenic H5N8 avian influenza viruses (HPAIVs) were introduced into South Korea during 2014, thereby caused outbreaks in wild birds and poultry farms. During the 2014 outbreak, H5N8 HPAIVs were isolated from 38 wild birds and 200 poultry farms (up to May 8, 2014). To better understand the introduction of these viruses and their relationships with wild birds and poultry farm, we analyzed the genetic sequences and available epidemiological data related to the viruses. Genetic analysis of 37 viruses isolated from wild birds and poultry farms showed that all of the isolates belonged to clade 2.3.4.6 of the hemagglutinin (HA) gene, but comprised two distinct groups. During the initial stage of the outbreak, identical isolates from each group were found in wild birds and poultry farms near Donglim Reservoir, which is a resting site for migratory birds, thereby indicating that two types of H5N8 HPAIVs were introduced into the lake at the same time. Interestingly, the one group of H5N8 HPAIV predominated around Donglim Reservoir, and the predominant virus was dispersed by wild birds among the migratory bird habitats in the western region of South Korea as time passed, and it was also detected in nearby poultry farms. Furthermore, compared with the results of the annual AIV surveillance of captured wild birds, which has been performed since 2008, more HPAIVs were isolated and H5 sero-prevalence was also detected during the 2014 outbreak. Overall, our results strongly suggest that migratory birds played a key role in the introduction and spread of viruses during the initial stage of the 2014 outbreak. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Ramadhani, Anggia N.; Puspitarini, Sapti; Sari, Anissa N.; Widodo
2017-11-01
Coronary artery disease (CAD) has emerged as a leading cause of death in Indonesia nowadays. WHO data in 2012 revealed that 37% of the Indonesian population died from this disease. CAD occurs because of endothelial dysfunction in the arteries. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a phospholipase A2 enzyme, encoded by the PLA2G7 gene. This protein is predicted to be involved in inflammatory phospholipid metabolism so it can be used as a biomarker of CAD in the early phase. Thus, the purpose of this research is to discover the difference in antibody production between wild-type and mutant V279F. The PAF-AH enzyme was isolated from mice lymphocyte cells in order to develop this enzyme as a biomarker of cardiovascular disease. PAF-AH migrates at 55kDa according to SDS-PAGE analysis. Flow cytometry analysis showed that mutant PAF-AH (V279F) is more antigenic than wild-type PAF-AH. The missense mutation of V279F PAF-AH means this enzyme cannot catabolize the acetyl group at the sn-2 position of PAF.
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Lee, Kah Meng; Chand, Kirat K; Hammond, Luke A; Lavidis, Nickolas A; Noakes, Peter G
2017-03-14
Laminin-α4 is involved in the alignment of active zones to postjunctional folds at the neuromuscular junction (NMJ). Prior study has implicated laminin-α4 in NMJ maintenance, with altered NMJ morphology observed in adult laminin-α4 deficient mice ( lama 4 -/- ). The present study further investigated the role of laminin-α4 in NMJ maintenance by functional characterization of transmission properties, morphological investigation of synaptic proteins including synaptic laminin-α4, and neuromotor behavioral testing. Results showed maintained perturbed transmission properties at lama 4 -/- NMJs from adult (3 months) through to aged (18-22 months). Hind-limb grip force demonstrated similar trends as transmission properties, with maintained weaker grip force across age groups in lama 4 -/- . Interestingly, both transmission properties and hind-limb grip force in aged wild-types resembled those observed in adult lama 4 -/- . Most significantly, altered expression of laminin-α4 was noted at the wild-type NMJs prior to the observed decline in transmission properties, suggesting that altered laminin-α4 expression precedes the decline of neurotransmission in aging wild-types. These findings significantly support the role of laminin-α4 in maintenance of the NMJ during aging.
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Assessment of Dental Fluorosis in Mmp20+/− Mice
Sharma, R.; Tye, C.E.; Arun, A.; MacDonald, D.; Chatterjee, A.; Abrazinski, T.; Everett, E.T.; Whitford, G.M.; Bartlett, J.D.
2011-01-01
The molecular mechanisms that underlie dental fluorosis are poorly understood. The retention of enamel proteins hallmarking fluorotic enamel may result from impaired hydrolysis and/or removal of enamel proteins. Previous studies have suggested that partial inhibition of Mmp20 expression is involved in the etiology of dental fluorosis. Here we ask if mice expressing only one functional Mmp20 allele are more susceptible to fluorosis. We demonstrate that Mmp20+/− mice express approximately half the amount of MMP20 as do wild-type mice. The Mmp20 heterozygous mice have normal-appearing enamel, with Vickers microhardness values similar to those of wild-type control enamel. Therefore, reduced MMP20 expression is not solely responsible for dental fluorosis. With 50-ppm-fluoride (F−) treatment ad libitum, the Mmp20+/− mice had F− tissue levels similar to those of Mmp20+/+ mice. No significant difference in enamel hardness was observed between the F−-treated heterozygous and wild-type mice. Interestingly, we did find a small but significant difference in quantitative fluorescence between these two groups, which may be attributable to slightly higher protein content in the Mmp20+/− mouse enamel. We conclude that MMP20 plays a nominal role in dental enamel fluorosis. PMID:21386097
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Overexpression of TGF-alpha increases lung tissue hysteresivity in transgenic mice.
Pillow, J J; Korfhagen, T R; Ikegami, M; Sly, P D
2001-12-01
Increased transforming growth factor (TGF)-alpha has been observed in neonatal chronic lung disease. Lungs of transgenic mice that overexpress TGF-alpha develop enlarged air spaces and pulmonary fibrosis compared with wild-type mice. We hypothesized that these pathological changes may alter the mechanical coupling of viscous and elastic forces within lung parenchyma. Respiratory impedance was measured in open-chested, tracheostomized adult wild-type and TGF-alpha mice by using the forced oscillation technique (0.25-19.63 Hz) delivered by flexiVent (Scireq, Montreal, PQ). Estimates of airway resistance (Raw), inertance (I), and the coefficients of tissue damping (G(L)) and tissue elastance (H(L)) were obtained by fitting a model to each impedance spectrum. Hysteresivity (eta) was calculated as G(L)/H(L). There was a significant increase in eta (P < 0.01) and a trend to a decrease in H(L) (P = 0.07) of TGF-alpha mice compared with the wild-type group. There was no significant change in Raw, I, or G(L). Structural abnormality present in the lungs of adult TGF-alpha mice alters viscoelastic coupling of the tissues, as evidenced by a change in eta.
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Wadsworth, Jonathan D. F.; Joiner, Susan; Linehan, Jacqueline M.; Desbruslais, Melanie; Fox, Katie; Cooper, Sharon; Cronier, Sabrina; Asante, Emmanuel A.; Mead, Simon; Brandner, Sebastian; Hill, Andrew F.; Collinge, John
2008-01-01
Kuru provides our principal experience of an epidemic human prion disease and primarily affected the Fore linguistic group of the Eastern Highlands of Papua New Guinea. Kuru was transmitted by the practice of consuming dead relatives as a mark of respect and mourning (transumption). To date, detailed information of the prion strain type propagated in kuru has been lacking. Here, we directly compare the transmission properties of kuru prions with sporadic, iatrogenic, and variant Creutzfeldt–Jakob disease (CJD) prions in Prnp-null transgenic mice expressing human prion protein and in wild-type mice. Molecular and neuropathological data from these transmissions show that kuru prions are distinct from variant CJD and have transmission properties equivalent to those of classical (sporadic) CJD prions. These findings are consistent with the hypothesis that kuru originated from chance consumption of an individual with sporadic CJD. PMID:18316717
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De novo establishment of wild-type song culture in the zebra finch.
Fehér, Olga; Wang, Haibin; Saar, Sigal; Mitra, Partha P; Tchernichovski, Ofer
2009-05-28
Culture is typically viewed as consisting of traits inherited epigenetically, through social learning. However, cultural diversity has species-typical constraints, presumably of genetic origin. A celebrated, if contentious, example is whether a universal grammar constrains syntactic diversity in human languages. Oscine songbirds exhibit song learning and provide biologically tractable models of culture: members of a species show individual variation in song and geographically separated groups have local song dialects. Different species exhibit distinct song cultures, suggestive of genetic constraints. Without such constraints, innovations and copying errors should cause unbounded variation over multiple generations or geographical distance, contrary to observations. Here we report an experiment designed to determine whether wild-type song culture might emerge over multiple generations in an isolated colony founded by isolates, and, if so, how this might happen and what type of social environment is required. Zebra finch isolates, unexposed to singing males during development, produce song with characteristics that differ from the wild-type song found in laboratory or natural colonies. In tutoring lineages starting from isolate founders, we quantified alterations in song across tutoring generations in two social environments: tutor-pupil pairs in sound-isolated chambers and an isolated semi-natural colony. In both settings, juveniles imitated the isolate tutors but changed certain characteristics of the songs. These alterations accumulated over learning generations. Consequently, songs evolved towards the wild-type in three to four generations. Thus, species-typical song culture can appear de novo. Our study has parallels with language change and evolution. In analogy to models in quantitative genetics, we model song culture as a multigenerational phenotype partly encoded genetically in an isolate founding population, influenced by environmental variables and taking multiple generations to emerge.
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Maggi, Maristella; Chiarelli, Laurent R; Valentini, Giovanna; Scotti, Claudia
2015-01-01
Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.
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Maggi, Maristella; Chiarelli, Laurent R.; Valentini, Giovanna; Scotti, Claudia
2015-01-01
Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy. PMID:25664771
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A 90-Day Feeding Study in Rats to Assess the Safety of Genetically Engineered Pork.
Xiao, Gao-Jun; Jiang, Sheng-Wang; Qian, Li-Li; Cai, Chun-Bo; Wang, Qing-Qing; Ma, De-Zun; Li, Biao; Xie, Shan-Shan; Cui, Wen-Tao; Li, Kui
2016-01-01
Our laboratory recently produced genetically engineered (GE) Meishan pigs containing a ZFN-edited myostatin loss-of-function mutant. These GE pigs develop and grow as normal as wild type pigs but produce pork with greater lean yield and lower fat mass. To assess any potential subchronic toxicity risks of this GE pork, a 90-day feeding study was conducted in Sprague-Dawley rats. Rats were randomly divided into five groups, and fed for 90 days with basic diet and basic diets formulated with low dose and high dose pork prepared from wild type pigs and GE pigs, respectively. Animal behaviors and clinical signs were monitored twice daily, and body weight and food consumption were measured and recorded weekly. At days 45 and 90, blood tests (lipid panel, electrolytes, parameters related to liver and kidney functions, and complete blood counts) were performed. Additionally, gross pathology and histopathological analyses were performed for major organs in each group. Data analysis shows that there were no significant differences in growth rate, food consumption, and blood test parameters between rat groups fed with GE pork and wild type pork. Although differences in some liver function parameters (such as aspartate aminotransferase, total proteins, albumin, and alkaline phosphatase) and white blood cell counts (such as lymphocyte percentage and monocyte percentage) were observed between rats fed with high dose GE pork and basic diet, all test results in rats fed with GE pork are in the normal range. Additionally, there are no apparent lesions noted in all organs isolated from rats in all five feeding groups on days 45 and 90. Overall, our results clearly indicate that food consumption of GE pork produced by ZFN-edited myostatin loss-of-function mutant pigs did not have any long-term adverse effects on the health status in rats.
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A 90-Day Feeding Study in Rats to Assess the Safety of Genetically Engineered Pork
Xiao, Gao-jun; Jiang, Sheng-Wang; Qian, Li-Li; Cai, Chun-Bo; Wang, Qing-qing; Ma, De-Zun; Li, Biao; Xie, Shan-shan; Cui, Wen-Tao; Li, Kui
2016-01-01
Our laboratory recently produced genetically engineered (GE) Meishan pigs containing a ZFN-edited myostatin loss-of-function mutant. These GE pigs develop and grow as normal as wild type pigs but produce pork with greater lean yield and lower fat mass. To assess any potential subchronic toxicity risks of this GE pork, a 90-day feeding study was conducted in Sprague-Dawley rats. Rats were randomly divided into five groups, and fed for 90 days with basic diet and basic diets formulated with low dose and high dose pork prepared from wild type pigs and GE pigs, respectively. Animal behaviors and clinical signs were monitored twice daily, and body weight and food consumption were measured and recorded weekly. At days 45 and 90, blood tests (lipid panel, electrolytes, parameters related to liver and kidney functions, and complete blood counts) were performed. Additionally, gross pathology and histopathological analyses were performed for major organs in each group. Data analysis shows that there were no significant differences in growth rate, food consumption, and blood test parameters between rat groups fed with GE pork and wild type pork. Although differences in some liver function parameters (such as aspartate aminotransferase, total proteins, albumin, and alkaline phosphatase) and white blood cell counts (such as lymphocyte percentage and monocyte percentage) were observed between rats fed with high dose GE pork and basic diet, all test results in rats fed with GE pork are in the normal range. Additionally, there are no apparent lesions noted in all organs isolated from rats in all five feeding groups on days 45 and 90. Overall, our results clearly indicate that food consumption of GE pork produced by ZFN-edited myostatin loss-of-function mutant pigs did not have any long-term adverse effects on the health status in rats. PMID:27812153
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Cusick, Matthew F; Libbey, Jane E; Cox Gill, Joan; Fujinami, Robert S; Eckels, David D
2013-01-01
Aim To determine whether modulation of T-cell responses by naturally occurring viral variants caused an increase in numbers of Tregs in HCV-infected patients. Patients, materials & methods Human peripheral blood mononuclear cells, having proliferative responses to a wild-type HCV-specific CD4+ T-cell epitope, were used to quantify, via proliferative assays, flow cytometry and class II tetramers, the effects of naturally occurring viral variants arising in the immunodominant epitope. Results In combination, the wild-type and variant peptides led to enhanced suppression of an anti-HCV T-cell response. The variant had a lower avidity for the wild-type-specific CD4+ T cell. Variant-stimulated CD4+ T cells had increased Foxp3, compared with wild-type-stimulated cells. Conclusion A stable viral variant from a chronic HCV subject was able to induce Tregs in multiple individuals that responded to the wild-type HCV-specific CD4+ T-cell epitope. PMID:24421862
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Ding, Xiao-Jie; Qin, Zhi-Yong; Hong, Christopher S.; Chen, Ling-Chao; Zhang, Xin; Zhao, Fang-Ping; Wang, Yin; Wang, Yang; Zhou, Liang-Fu; Zhuang, Zhengping; Ng, Ho-Keung; Yan, Hai; Yao, Yu; Mao, Ying
2015-01-01
IDH mutations frequently occur in WHO grade II and III diffuse gliomas and have favorable prognosis compared to wild-type tumors. However, whether IDH mutations in WHO grade II and II diffuse gliomas predict enhanced sensitivity to adjuvant radiation (RT) or chemotherapy (CHT) is still being debated. Recent studies have identified recurrent mutations in the promoter region of telomerase reverse transcriptase (TERT) in gliomas. We previously demonstrated that TERT promoter mutations may be promising biomarkers in glioma survival prognostication when combined with IDH mutations. This study analyzed IDH and TERT promoter mutations in 295 WHO grade II and III diffuse gliomas treated with or without adjuvant therapies to explore their impact on the sensitivity of tumors to genotoxic therapies. IDH mutations were found in 216 (73.2%) patients and TERT promoter mutations were found in 112 (38%) patients. In multivariate analysis, IDH mutations (p < 0.001) were independent prognostic factors for PFS and OS in patients receiving genotoxic therapies while TERT promoter mutations were not. In univariate analysis, IDH and TERT promoter mutations were not significant prognostic factors in patients who did not receive genotoxic therapies. Adjuvant RT and CHT were factors independently impacting PFS (RT p = 0.001, CHT p = 0.026) in IDH mutated WHO grade II and III diffuse gliomas but not in IDH wild-type group. Univariate and multivariate analyses demonstrated TERT promoter mutations further stratified IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to genotoxic therapies. Adjuvant RT and CHT were significant parameters influencing PFS in the IDH wt/TERT mut subgroup (RT p = 0.015, CHT p = 0.015) but not in the IDH wt/TERT wt subgroup. Our data demonstrated that IDH mutated WHO grade II and III diffuse gliomas had better PFS and OS than their IDH wild-type counterparts when genotoxic therapies were administered after surgery. Importantly, we also found that TERT promoter mutations further stratify IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to adjuvant therapies. Taken together, TERT promoter mutations may predict enhanced sensitivity to genotoxic therapies in IDH wild-type WHO grade II and III diffuse gliomas and may justify intensified treatment in this subgroup. PMID:26314843
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Seed fates in crop-wild hybrid sunflower: crop allele and maternal effects.
Pace, Brian A; Alexander, Helen M; Emry, Jason D; Mercer, Kristin L
2015-02-01
Domestication has resulted in selection upon seed traits found in wild populations, yet crop-wild hybrids retain some aspects of both parental phenotypes. Seed fates of germination, dormancy, and mortality can influence the success of crop allele introgression in crop-wild hybrid zones, especially if crop alleles or crop-imparted seed coverings result in out-of-season germination. We performed a seed burial experiment using crop, wild, and diverse hybrid sunflower (Helianthus annuus) cross types to test how a cross type's maternal parent and nuclear genetic composition might affect its fate under field conditions. We observed higher maladaptive fall germination in the crop- and F1- produced seeds than wild-produced seeds and, due to an interaction with percent crop alleles, fall germination was higher for cross types with more crop-like nuclear genetics. By spring, crop-produced cross types had the highest overwintering mortality, primarily due to higher fall germination. Early spring germination was identical across maternal types, but germination continued for F1-produced seeds. In conclusion, the more wild-like the maternal parent or the less proportion of the cross type's genome contributed by the crop, the greater likelihood a seed will remain ungerminated than die. Wild-like dormancy may facilitate introgression through future recruitment from the soil seed bank.
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Sebban, P; Maróti, P; Schiffer, M; Hanson, D K
1995-07-04
Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy. These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB. In the wild-type Rb. sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site. These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant. The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds. In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively. The pH dependences of K2, the QA-QB <--> QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)
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Reduced infarct size in neuroglobin-null mice after experimental stroke in vivo
2012-01-01
Background Neuroglobin is considered to be a novel important pharmacological target in combating stroke and neurodegenerative disorders, although the mechanism by which this protection is accomplished remains an enigma. We hypothesized that if neuroglobin is directly involved in neuroprotection, then permanent cerebral ischemia would lead to larger infarct volumes in neuroglobin-null mice than in wild-type mice. Methods Using neuroglobin-null mice, we estimated the infarct volume 24 hours after permanent middle cerebral artery occlusion using Cavalieri’s Principle, and compared the infarct volume in neuroglobin-null and wild-type mice. Neuroglobin antibody staining was used to examine neuroglobin expression in the infarct area of wild-type mice. Results Infarct volumes 24 hours after permanent middle cerebral artery occlusion were significantly smaller in neuroglobin-null mice than in wild-types (p < 0.01). Neuroglobin immunostaining of the penumbra area revealed no visible up-regulation of neuroglobin protein in ischemic wild-type mice when compared to uninjured wild-type mice. In uninjured wild-type mice, neuroglobin protein was seen throughout cortical layer II and sparsely in layer V. In contrast, no neuroglobin-immunoreactive neurons were observed in the aforementioned layers of the ischemia injured cortical area, or in the surrounding penumbra of ischemic wild-type mice. This suggests no selective sparing of neuroglobin expressing neurons in ischemia. Conclusions Neuroglobin-deficiency resulted in reduced tissue infarction, suggesting that, at least at endogenous expression levels, neuroglobin in itself is non-protective against ischemic injury. PMID:22901501
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Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina
2014-01-28
Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.
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Genetic studies of cell fusion induced by herpes simplex virus type 1
DOE Office of Scientific and Technical Information (OSTI.GOV)
Read, G.S.; Person, S.; Keller, P.M.
1980-07-01
Eight cell fusion-causing syn mutants were isolated from the KOS strain of herpes simplex virus type 1. Unlike the wild-type virus, the mutants produced plaques containing multinucleated cells, or syncytia. Fusion kinetics curves were established with a Coulter Counter assay for the mutants and wild-type virus in single infections of human embryonic lung (HEL) cells, for the mutants and wild-type virus in mixed infections (dominance test), and for pairs of mutants in mixed infection and proceeded with an exponential decrease in the number of small single cells. At some later time that was characteristic of the mutant, there was amore » significant reduction in the rate of fusion for all but possibly one of the mutants. Although the wild-type virus did not produce syncytial plaques, it did induce a small amount of fusion that stopped abruptly about 2 h after it started. These data are consistent with the hypothesis that both mutants and wild type induce an active fusion inducer and that the activity of this inducer is subsequently inhibited. The extent of fusion is apparently determined by the length of the interval during which the fusion inducer is active. That fusion is actively inhibited in wild-type infections is indicated by the observation that syn mutant-infected cells fused more readily with uninfected cells than with wild type-infected cells.« less
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NASA Technical Reports Server (NTRS)
Roten, L.; Nemoto, S.; Simsic, J.; Coker, M. L.; Rao, V.; Baicu, S.; Defreyte, G.; Soloway, P. J.; Zile, M. R.; Spinale, F. G.
2000-01-01
Alterations in the expression and activity of the matrix metalloproteinases (MMPs) and the tissue inhibitors of the MMPs (TIMPs) have been implicated in tissue remodeling in a number of disease states. One of the better characterized TIMPs, TIMP-1, has been shown to bind to active MMPs and to regulate the MMP activational process. The goal of this study was to determine whether deletion of the TIMP-1 gene in mice, which in turn would remove TIMP-1 expression in LV myocardium, would produce time-dependent effects on LV geometry and function. Age-matched sibling mice (129Sv) deficient in the TIMP-1 gene (TIMP-1 knock-out (TIMP-1 KO), n=10) and wild-type mice (n=10) underwent comparative echocardiographic studies at 1 and 4 months of age. LV catheterization studies were performed at 4 months and the LV harvested for histomorphometric studies. LV end-diastolic volume and mass increased (18+/-4 and 38+/-3%, respectively, P<0.05) at 4 months in the TIMP-1 KO group; a significant increase compared to wild-type controls (P<0.05). At 4 months, LV and end-diastolic wall stress was increased by over two-fold in the TIMP-1 KO compared to wild type (P<0.05). However, LV systolic pressure and ejection performance were unchanged in the two groups of mice. LV myocyte cross-sectional area was unchanged in the TIMP-1 KO mice compared to controls, but myocardial fibrillar collagen content was reduced. Changes in LV geometry occurred in TIMP-1 deficient mice and these results suggest that constitutive TIMP-1 expression participates in the maintenance of normal LV myocardial structure. Copyright 2000 Academic Press.
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Wang, Minxin; Zou, Xiaojing; Tian, Deying; Hu, Song; Jiang, Libin
2015-01-01
Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is an essential process for virus infection, such as HIV and hepatitis C, and plays a role in immune escape. However, the role of DC-SIGN in hepatitis B virus (HBV) infection is still unknown. The aim of this study was to investigate the role of DC-SIGN in mediating the maturation and activation of dendritic cells (DCs) when infected by HBV. Highly mannosylated HBV particles were obtained by treating HBV-producing HepG2.2.15 cells with the a-mannosidase I-inhibitor kifunensine. Highly mannosylated HBV or wild type HBV was added to infect the DCs of the DC-SIGN gene-silencing group and normal group, respectively. Then, the expression of CDla, CD80, CD83, CD86 and HLA-DR on DCs was detected by flow cytometry, the capacity of stimulating lymphocyte proliferation was tested by MTT assay, the level of IL-12p70 that was released by DCs was measured by enzyme-linked immunosorbent assay, and the expression of the proteins NF-κBp65 and p38 was detected by western blot. Both wild type and highly mannosylated HBV could promote DCs maturation and activation. However, the highly mannosylated HBV could promote DCs immune activation more strongly. The difference in the effect on DCs between the two types of HBV could be eliminated by DC-SIGN gene silencing. DC-SIGN can promote the maturation and activation of DCs when recognized HBV, but wild type HBV can escape recognition by DC-SIGN to a certain extent with the help of demannosylated modification, leading to defective DCs function and chronic HBV infection.
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Evaluation of MMX1902 as an Oral Treatment for Duchenne Muscular Dystrophy
2017-10-01
0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing...min for 60 minutes 2 times a week for 10 weeks. Four different SQ treatment groups (n = 9/group) were evaluated – wild-type controls and three groups...potential for MMX1902 treatment to stimulate and sustain regeneration even in the face of long- term, intensive exercise. 15. SUBJECT TERMS Duchenne
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NASA Astrophysics Data System (ADS)
Santos-Filho, Osvaldo Andrade; Hopfinger, Anton J.
2001-01-01
A set of 18 structurally diverse antifolates including pyrimethamine, cycloguanil, methotrexate, aminopterin and trimethoprim, and 13 pyrrolo[2,3-d]pyrimidines were studied using four-dimensional quantitative structure-activity relationship (4D-QSAR) analysis. The corresponding biological activities of these compounds include IC50 inhibition constants for both the wild type, and a specific mutant type of Plasmodium falciparum dihydrofolate reductase (DHFR). Two thousand conformations of each analog were sampled to generate a conformational ensemble profile (CEP) from a molecular dynamics simulation (MDS) of 100,000 conformer trajectory states. Each sampled conformation was placed in a 1 Å cubic grid cell lattice for each of five trial alignments. The frequency of occupation of each grid cell was computed for each of six types of pharmacophore groups of atoms of each compound. These grid cell occupancy descriptors (GCODs) were then used as a descriptor pool to construct 4D-QSAR models. Models for inhibition of both the `wild' type and the mutant enzyme were generated which provide detailed spatial pharmacophore requirements for inhibition in terms of atom types and their corresponding relative locations in space. The 4D-QSAR models indicate some structural features perhaps relevant to the mechanism of resistance of the Plasmodium falciparum DHFR to current antimalarials. One feature identified is a slightly different binding alignment of the ligands to the mutant form of the enzyme as compared to the wild type.
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2012-01-01
Background Gastrointestinal stromal tumors (GIST), one of the most common mesenchymal tumors of the gastrointestinal tract, prior to routine immunohistochemical staining and the introduction of tyrosine kinase inhibitors, were often mistaken for neoplasms of smooth muscle origin such as leiomyomas, leiomyosarcomas or leiomyoblastomas. Since the advent of imatinib, GIST has been further delineated into adult- (KIT or PDGFRα mutations) and pediatric- (typified by wild-type GIST/succinate dehydrogenase deficiencies) types. Using varying gender ratios at age of diagnosis we sought to elucidate prognostic factors for each sub-type and their impact on overall survival. Methods This is a long-term retrospective analysis of a large observational study of an international open cohort of patients from a GIST research and patient advocacy's lifetime registry. Demographic and disease-specific data were voluntarily supplied by its members from May 2000-October 2010; the primary outcome was overall survival. Associations between survival and prognostic factors were evaluated by univariate Cox proportional hazard analyses, with backward selection at P < 0.05 used to identify independent factors. Results Inflections in gender ratios by age at diagnosis in years delineated two distinct groups: above and below age 35 at diagnosis. Closer analysis confirmed the above 35 age group as previously reported for adult-type GIST, typified by mixed primary tumor sites and gender, KIT or PDGFRα mutations, and shorter survival times. The pediatric group (< age 18 at diagnosis) was also as previously reported with predominantly stomach tumors, females, wild-type GIST or SDH mutations, and extended survival. "Young adults" however formed a third group aged 18-35 at diagnosis, and were a clear mix of these two previously reported distinct sub-types. Conclusions Pediatric- and adult-type GIST have been previously characterized in clinical settings and these observations confirm significant prognostic factors for each from a diverse real-world cohort. Additionally, these findings suggest that extra diligence be taken with "young adults" (aged 18-35 at diagnosis) as pediatric-type GIST may present well beyond adolescence, particularly as these distinct sub-types have different causes, and consequently respond differently to treatments. PMID:22429770
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Call, Jerry; Walentas, Christopher D; Eickhoff, Jens C; Scherzer, Norman
2012-03-19
Gastrointestinal stromal tumors (GIST), one of the most common mesenchymal tumors of the gastrointestinal tract, prior to routine immunohistochemical staining and the introduction of tyrosine kinase inhibitors, were often mistaken for neoplasms of smooth muscle origin such as leiomyomas, leiomyosarcomas or leiomyoblastomas. Since the advent of imatinib, GIST has been further delineated into adult- (KIT or PDGFRα mutations) and pediatric- (typified by wild-type GIST/succinate dehydrogenase deficiencies) types. Using varying gender ratios at age of diagnosis we sought to elucidate prognostic factors for each sub-type and their impact on overall survival. This is a long-term retrospective analysis of a large observational study of an international open cohort of patients from a GIST research and patient advocacy's lifetime registry. Demographic and disease-specific data were voluntarily supplied by its members from May 2000-October 2010; the primary outcome was overall survival. Associations between survival and prognostic factors were evaluated by univariate Cox proportional hazard analyses, with backward selection at P < 0.05 used to identify independent factors. Inflections in gender ratios by age at diagnosis in years delineated two distinct groups: above and below age 35 at diagnosis. Closer analysis confirmed the above 35 age group as previously reported for adult-type GIST, typified by mixed primary tumor sites and gender, KIT or PDGFRα mutations, and shorter survival times. The pediatric group (< age 18 at diagnosis) was also as previously reported with predominantly stomach tumors, females, wild-type GIST or SDH mutations, and extended survival. "Young adults" however formed a third group aged 18-35 at diagnosis, and were a clear mix of these two previously reported distinct sub-types. Pediatric- and adult-type GIST have been previously characterized in clinical settings and these observations confirm significant prognostic factors for each from a diverse real-world cohort. Additionally, these findings suggest that extra diligence be taken with "young adults" (aged 18-35 at diagnosis) as pediatric-type GIST may present well beyond adolescence, particularly as these distinct sub-types have different causes, and consequently respond differently to treatments.
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Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei
2014-01-01
ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy. PMID:25031342
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Lee, Jeong Won; Lee, Jong Hoon; Shim, Byoung Yong; Kim, Sung Hwan; Chung, Mi-Joo; Kye, Bong-Hyeon; Kim, Hyung Jin; Cho, Hyeon Min; Jang, Hong Seok
2015-08-01
We evaluated the tumor response and survival according to the KRAS oncogene status in locally advanced rectal cancer. One hundred patients with locally advanced rectal cancer (cT3-4N0-2M0) received preoperative radiation of 50.4 Gy in 28 fractions with 5-fluorouracil and total mesorectal excision. Tumor DNA from each patient was obtained from pretreatment biopsy tissues. A Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation was found in 26 (26%) of the 100 patients. Downstaging (ypT0-2N0M0) rates after preoperative chemoradiotheray were not statistically different between the wild-type and mutant-type KRAS groups (30.8% vs 27.0%, P = 0.715, respectively). After a median follow-up time of 34 months, there was no statistically significant difference in the 3-year relapse-free survival (82.2% vs 82.6%, P = 0.512) and overall survival (94.7% vs 92.3%, P = 0.249) rates between wild-type and mutant-type KRAS groups, respectively. The KRAS mutation status does not influence the tumor response to the radiotherapy and survival in locally advanced rectal cancer patients who received preoperative chemoradiotherapy and curative surgery.
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Lee, Jeong Won; Lee, Jong Hoon; Shim, Byoung Yong; Kim, Sung Hwan; Chung, Mi-Joo; Kye, Bong-Hyeon; Kim, Hyung Jin; Cho, Hyeon Min; Jang, Hong Seok
2015-01-01
Abstract We evaluated the tumor response and survival according to the KRAS oncogene status in locally advanced rectal cancer. One hundred patients with locally advanced rectal cancer (cT3-4N0-2M0) received preoperative radiation of 50.4 Gy in 28 fractions with 5-fluorouracil and total mesorectal excision. Tumor DNA from each patient was obtained from pretreatment biopsy tissues. A Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation was found in 26 (26%) of the 100 patients. Downstaging (ypT0-2N0M0) rates after preoperative chemoradiotheray were not statistically different between the wild-type and mutant-type KRAS groups (30.8% vs 27.0%, P = 0.715, respectively). After a median follow-up time of 34 months, there was no statistically significant difference in the 3-year relapse-free survival (82.2% vs 82.6%, P = 0.512) and overall survival (94.7% vs 92.3%, P = 0.249) rates between wild-type and mutant-type KRAS groups, respectively. The KRAS mutation status does not influence the tumor response to the radiotherapy and survival in locally advanced rectal cancer patients who received preoperative chemoradiotherapy and curative surgery. PMID:26252300
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Choi, Jun-Ho; Kwak, Kyung-Won; Cho, Minhaeng
2013-12-12
The CO stretching mode of both wild-type and double mutant ( T67R / S92D ) MbCO (carbonmonoxymyoglobin) proteins is an ideal infrared (IR) probe for studying the local electrostatic environment inside the myoglobin heme pocket. Recently, to elucidate the conformational switching dynamics between two distinguishable states, extensive IR absorption, IR pump-probe, and two-dimensional (2D) IR spectroscopic studies for various mutant MbCO's have been performed by the Fayer group. They showed that the 2D IR spectroscopy of the double mutant, which has a peroxidase enzyme activity, reveals a rapid chemical exchange between two distinct states, whereas that of the wild-type does not. Despite the fact that a few simulation studies on these systems were already performed and reported, such complicated experimental results have not been fully reproduced nor described in terms of conformational state-to-state transition processes. Here, we first develop a distributed vibrational solvatochromic charge model for describing the CO stretch frequency shift reflecting local electric potential changes. Then, by carrying out molecular dynamic simulations of the two MbCO's and examining their CO frequency trajectories, it becomes possible to identify a proper reaction coordinate consisting of His64 imidazole ring rotation and its distance to the CO ligand. From the 2D surfaces of the resulting potential of mean forces, the spectroscopically distinguished A1 and A3 states of the wild-type as well as two more substates of the double mutant are identified and their vibrational frequencies and distributions are separately examined. Our simulated IR absorption and 2D IR spectra of the two MbCO's are directly compared with the previous experimental results reported by the Fayer group. The chemical exchange rate constants extracted from the two-state kinetic analyses of the simulated 2D IR spectra are in excellent agreement with the experimental values. On the basis of the quantitative agreement between the simulated spectra and experimental ones, we further examine the conformational differences in the heme pockets of the two proteins and show that the double mutation, T67R / S92D , suppresses the A1 population, restricts the imidazole ring rotation, and increases hydrogen-bond strength between the imidazole Nε-H and the oxygen atom of the CO ligand. It is believed that such delicate change of distal His64 imidazole ring dynamics induced by the double mutation may be responsible for its enhanced peroxidase catalytic activity as compared to the wild-type myoglobin.
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Perfluorohexane sulfonate (PFHxS) is one member of a group ofperfluoroakyl acids (PFAAs) presently recognized as widespread environmental contaminants. Like other PFAAs, PFHxS is also commonly found in human serum. Although PFHxS is presumed to be an activator of peroxisome proli...
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Frye, C A; Sumida, K; Lydon, J P; O'Malley, B W; Pfaff, D W
2006-05-01
Progesterone (P) and its 5alpha-reduced metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP), facilitate sexual behavior of rodents via agonist-like actions at intracellular progestin receptors (PRs) and membrane GABA(A)/benzodiazepine receptor complexes (GBRs), respectively. Given that ovarian secretion of progestins declines with aging, whether or not senescent mice are responsive to progestins was of interest. Homozygous PR knockout (PRKO) or wild-type mice that were between 10-12 (mid-aged) or 20-24 (aged) months of age were administered P or 3alpha,5alpha-THP, and the effect on lordosis were examined. Effects of a progestin-priming regimen that enhances PR-mediated (experiment 1) or more rapid, PR-independent effects of progestins (experiments 2 and 3) on sexual behavior were examined. Levels of P, 3alpha,5alpha-THP, and muscimol binding were examined in tissues from aged mice (experiment 4). Wild-type, but not PRKO, mice were responsive when primed with 17beta-estradiol (E(2); 0.5 microg) and administered P (500 microg, subcutaneously). Mid-aged wild-type mice demonstrated greater increases in lordosis 6 h later compared to their pre-P, baseline test than did aged wild-type mice (experiment 1). Lordosis of younger and older wild-type, but not PRKO, mice was significantly increased within 5 min of intravenous (IV) administration of P (100 ng), compared with E(2)-priming alone (experiment 2). However, wild-type and PRKO mice demonstrated significant increases in lordosis 5 min after IV administration of 3alpha,5alpha-THP, an effect which was more pronounced in mid-aged than in aged animals (100 ng-experiment 3). In tissues from aged wild-type and PRKO mice, levels of P, 3alpha,5alpha-THP, and muscimol binding were increased by P administration (experiment 4). PR binding was lower in the cortex of PRKO than that of wild-type mice. Mid-aged and aged PRKO and wild-type mice demonstrated rapid P or 3alpha,5alpha-THP-facilitated lordosis that may be, in part, independent of activity at PRs.
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Wu, Sanling; Wang, Ying-Ying; Ye, Chu-Yu; Bai, Xuefei; Li, Zefeng; Yan, Chenghai; Wang, Weidi; Wang, Ziqiang; Shu, Qingyao; Xie, Jiahua; Lee, Suk-Ha; Fan, Longjiang
2014-01-01
Semi-wild soybean is a unique type of soybean that retains both wild and domesticated characteristics, which provides an important intermediate type for understanding the evolution of the subgenus Soja population in the Glycine genus. In this study, a semi-wild soybean line (Maliaodou) and a wild line (Lanxi 1) collected from the lower Yangtze regions were deeply sequenced while nine other semi-wild lines were sequenced to a 3-fold genome coverage. Sequence analysis revealed that (1) no independent phylogenetic branch covering all 10 semi-wild lines was observed in the Soja phylogenetic tree; (2) besides two distinct subpopulations of wild and cultivated soybean in the Soja population structure, all semi-wild lines were mixed with some wild lines into a subpopulation rather than an independent one or an intermediate transition type of soybean domestication; (3) high heterozygous rates (0.19–0.49) were observed in several semi-wild lines; and (4) over 100 putative selective regions were identified by selective sweep analysis, including those related to the development of seed size. Our results suggested a hybridization origin for the semi-wild soybean, which makes a complex Soja population structure. PMID:25265539
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QUANTITATIVE ASSESSMENT OF INTEGRATED PHRENIC NERVE ACTIVITY
Nichols, Nicole L.; Mitchell, Gordon S.
2016-01-01
Integrated electrical activity in the phrenic nerve is commonly used to assess within-animal changes in phrenic motor output. Because of concerns regarding the consistency of nerve recordings, activity is most often expressed as a percent change from baseline values. However, absolute values of nerve activity are necessary to assess the impact of neural injury or disease on phrenic motor output. To date, no systematic evaluations of the repeatability/reliability have been made among animals when phrenic recordings are performed by an experienced investigator using standardized methods. We performed a meta-analysis of studies reporting integrated phrenic nerve activity in many rat groups by the same experienced investigator; comparisons were made during baseline and maximal chemoreceptor stimulation in 14 wild-type Harlan and 14 Taconic Sprague Dawley groups, and in 3 pre-symptomatic and 11 end-stage SOD1G93A Taconic rat groups (an ALS model). Meta-analysis results indicate: 1) consistent measurements of integrated phrenic activity in each sub-strain of wild-type rats; 2) with bilateral nerve recordings, left-to-right integrated phrenic activity ratios are ~1.0; and 3) consistently reduced activity in end-stage SOD1G93A rats. Thus, with appropriate precautions, integrated phrenic nerve activity enables robust, quantitative comparisons among nerves or experimental groups, including differences caused by neuromuscular disease. PMID:26724605
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Fructokinase activity mediates dehydration-induced renal injury.
Roncal Jimenez, Carlos A; Ishimoto, Takuji; Lanaspa, Miguel A; Rivard, Christopher J; Nakagawa, Takahiko; Ejaz, A Ahsan; Cicerchi, Christina; Inaba, Shinichiro; Le, MyPhuong; Miyazaki, Makoto; Glaser, Jason; Correa-Rotter, Ricardo; González, Marvin A; Aragón, Aurora; Wesseling, Catharina; Sánchez-Lozada, Laura G; Johnson, Richard J
2014-08-01
The epidemic of chronic kidney disease in Nicaragua (Mesoamerican nephropathy) has been linked with recurrent dehydration. Here we tested whether recurrent dehydration may cause renal injury by activation of the polyol pathway, resulting in the generation of endogenous fructose in the kidney that might subsequently induce renal injury via metabolism by fructokinase. Wild-type and fructokinase-deficient mice were subjected to recurrent heat-induced dehydration. One group of each genotype was provided water throughout the day and the other group was hydrated at night, after the dehydration. Both groups received the same total hydration in 24 h. Wild-type mice that received delayed hydration developed renal injury, with elevated serum creatinine, increased urinary NGAL, proximal tubular injury, and renal inflammation and fibrosis. This was associated with activation of the polyol pathway, with increased renal cortical sorbitol and fructose levels. Fructokinase-knockout mice with delayed hydration were protected from renal injury. Thus, recurrent dehydration can induce renal injury via a fructokinase-dependent mechanism, likely from the generation of endogenous fructose via the polyol pathway. Access to sufficient water during the dehydration period can protect mice from developing renal injury. These studies provide a potential mechanism for Mesoamerican nephropathy.
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Effect of glycogen synthase overexpression on insulin-stimulated muscle glucose uptake and storage.
Fogt, Donovan L; Pan, Shujia; Lee, Sukho; Ding, Zhenping; Scrimgeour, Angus; Lawrence, John C; Ivy, John L
2004-03-01
Insulin-stimulated muscle glucose uptake is inversely associated with the muscle glycogen concentration. To investigate whether this association is a cause and effect relationship, we compared insulin-stimulated muscle glucose uptake in noncontracted and postcontracted muscle of GSL3-transgenic and wild-type mice. GSL3-transgenic mice overexpress a constitutively active form of glycogen synthase, which results in an abundant storage of muscle glycogen. Muscle contraction was elicited by in situ electrical stimulation of the sciatic nerve. Right gastrocnemii from GSL3-transgenic and wild-type mice were subjected to 30 min of electrical stimulation followed by hindlimb perfusion of both hindlimbs. Thirty minutes of contraction significantly reduced muscle glycogen concentration in wild-type (49%) and transgenic (27%) mice, although transgenic mice retained 168.8 +/- 20.5 micromol/g glycogen compared with 17.7 +/- 2.6 micromol/g glycogen for wild-type mice. Muscle of transgenic and wild-type mice demonstrated similar pre- (3.6 +/- 0.3 and 3.9 +/- 0.6 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) and postcontraction (7.9 +/- 0.4 and 7.0 +/- 0.4 micromol.g(-1).h(-1) for transgenic and wild-type, respectively) insulin-stimulated glucose uptakes. However, the [14C]glucose incorporated into glycogen was greater in noncontracted (151%) and postcontracted (157%) transgenic muscle vs. muscle of corresponding wild-type mice. These results indicate that glycogen synthase activity is not rate limiting for insulin-stimulated glucose uptake in skeletal muscle and that the inverse relationship between muscle glycogen and insulin-stimulated glucose uptake is an association, not a cause and effect relationship.
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Ziv-Gal, A; Gao, L; Karman, B N; Flaws, J A
2015-03-01
The aryl hydrocarbon receptor (AHR) mediates the toxic effects of various endocrine disrupting chemicals. In female mice, global deletion of the Ahr (AhrKO) results in slow growth of ovarian antral follicles. No studies, however, have examined whether injection of the Ahr restores the phenotypes of cultured AhrKO ovarian antral follicles to wild-type levels. We developed a system to construct a recombinant adenovirus containing the Ahr to re-express the Ahr in AhrKO granulosa cells and whole antral follicles. We then compared follicle growth and levels of factors in the AHR signaling pathway (Ahr, Ahrr, Cyp1a1, and Cyp1b1) in wild-type, AhrKO, and Ahr re-expressed follicles. Further, we compared the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild-type, AhrKO, and Ahr re-expressed follicles. Ahr injection into AhrKO follicles partially restored their growth pattern to wild-type levels. Further, Ahr re-expressed follicles had significantly higher levels of Ahr, Ahrr, Cyp1a1, and Cyp1b1 compared to wild-type follicles. Upon TCDD treatment, only Cyp1a1 levels were significantly higher in Ahr re-expressed follicles compared to the levels in wild-type follicles. Our system of re-expression of the Ahr partially restores follicle growth and transcript levels of factors in the AHR signaling pathway to wild-type levels. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Li, Meng; Zhang, Xu; Zhu, Lingwei; Wang, Haifeng; Zhao, Na; Luo, Jing; Wang, Chengmin; Wang, Yutian; Liu, Yanhua; Zhou, Wei; Zhang, Bikai; Guo, Huancheng; He, Hongxuan
2017-07-01
Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that can induces gas gangrene or enteritis in poultry and humans and many other mammalian species. Here, we report an outbreak of C. perfringens type A and type C coinfection in wild boars ( Sus scrofa ). In February 2016, 10 dead wild boars, including two fresh carcasses, were found in Zhaosu County, Xinjiang Province, People's Republic of China. The two fresh carcasses were included in this study. Two strains of C. perfringens were isolated, identified, genotyped, and phylogenetically analyzed. Based on postmortem examination, bacterium isolation and identification, enterotoxin detection, and auxiliary tests, we made a diagnosis that the wild boar were killed by C. perfringens . Our findings provide the evidence that wild boar can be killed by C. perfringens intoxication. Wild boars are important reservoirs for many zoonotic agents. Therefore, more actions should be taken on the surveillance, prevention, and control of wild pig-borne diseases.
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Takenaka, Shotaro; Kawahara, Taihachi
2012-09-01
The sequence data from 5' UTR, intronic, coding and 3' UTR regions of Ppd-A1 and Ppd-B1 were investigated for a total of 158 accessions of emmer wheat landraces comprising 19 of wild emmer wheat (Triticum dicoccoides), 45 of hulled emmer wheat (T. dicoccum) and 94 of free-threshing (FT) emmer wheat (T. durum etc.). We detected some novel types of deletions in the coding regions from 22 hulled emmer accessions and 20 FT emmer accessions. Emmer wheat accessions with these deletions could produce predicted proteins likely to lack function. We also observed some novel mutations in Ppd-B1. Sixty-seven and forty-one haplotypes were found in Ppd-A1 and Ppd-B1, respectively. Some mutations found in this study have not been known, so they have potential for useful genetic resources for wheat breeding. On the basis of sequence data from the 5' UTR region, both Ppd-A1 and Ppd-B1 haplotypes were divided into two groups (Type AI/AII and Type BI/BII). Types AI and AII of Ppd-A1 suggested gene flow between wild and hulled emmer. On the other hand, Types BI and BII of Ppd-B1 suggested gene flow between wild and FT emmer. More than half of hulled emmer accessions were Type AII/BI but few FT emmer accessions were of this type. Therefore, over half of the hulled emmer did not contribute to evolution of FT emmer.
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Wang, Jin; Xiao, Qi-Zhi; Chen, You-Ming; Yi, Sheng; Liu, Dun; Liu, Yan-Hui; Zhang, Cui-Mei; Wei, Xiao-Feng; Zhou, Yu-Qiu; Zhong, Xing-Ming; Zhao, Cun-You; Xiong, Fu; Wei, Xiang-Cai; Xu, Xiang-Min
2014-12-01
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked incompletely dominant enzyme deficiency that results from G6PD gene mutations. Women heterozygous for G6PD mutations exhibit variation in the loss of enzyme activity but the cause of this phenotypic variation is unclear. We determined DNA methylation and X-inactivation patterns in 71 G6PD-deficient female heterozygotes and 68 G6PD non-deficient controls with the same missense mutations (G6PD Canton c.1376G>T or Kaiping c.1388G>A) to correlate determinants with variable phenotypes. Specific CpG methylations within the G6PD promoter were significantly higher in G6PD-deficient heterozygotes than in controls. Preferential X-inactivation of the G6PD wild-type allele was determined in heterozygotes. The incidence of preferential X-inactivation was 86.2% in the deficient heterozygote group and 31.7% in the non-deficient heterozygote group. A significant negative correlation was observed between X-inactivation ratios of the wild-type allele and G6PD/6-phosphogluconate dehydrogenase (6PGD) ratios in heterozygous G6PD Canton (r=-0.657, p<0.001) or Kaiping (r=-0.668, p<0.001). Multivariate logistic regression indicated that heterozygotes with hypermethylation of specific CpG sites in the G6PD promoter and preferential X-inactivation of the wild-type allele were at risk of enzyme deficiency. Copyright © 2014 Elsevier Inc. All rights reserved.
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Cha, Seung Bin; Lee, Won Jung; Shin, Min Kyoung; Jung, Myung Hwan; Shin, Seung Won; Yoo, An Na; Kim, Jong Wan; Yoo, Han Sang
2013-06-27
Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further.
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2013-01-01
Background Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Results Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. Conclusions In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further. PMID:23802650
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Sumby, Paul; Barbian, Kent D; Gardner, Donald J; Whitney, Adeline R; Welty, Diane M; Long, R Daniel; Bailey, John R; Parnell, Michael J; Hoe, Nancy P; Adams, Gerald G; Deleo, Frank R; Musser, James M
2005-02-01
Many pathogenic bacteria produce extracellular DNase, but the benefit of this enzymatic activity is not understood. For example, all strains of the human bacterial pathogen group A Streptococcus (GAS) produce at least one extracellular DNase, and most strains make several distinct enzymes. Despite six decades of study, it is not known whether production of DNase by GAS enhances virulence. To test the hypothesis that extracellular DNase is required for normal progression of GAS infection, we generated seven isogenic mutant strains in which the three chromosomal- and prophage-encoded DNases made by a contemporary serotype M1 GAS strain were inactivated. Compared to the wild-type parental strain, the isogenic triple-mutant strain was significantly less virulent in two mouse models of invasive infection. The triple-mutant strain was cleared from the skin injection site significantly faster than the wild-type strain. Preferential clearance of the mutant strain was related to the differential extracellular killing of the mutant and wild-type strains, possibly through degradation of neutrophil extracellular traps, innate immune structures composed of chromatin and granule proteins. The triple-mutant strain was also significantly compromised in its ability to cause experimental pharyngeal disease in cynomolgus macaques. Comparative analysis of the seven DNase mutant strains strongly suggested that the prophage-encoded SdaD2 enzyme is the major DNase that contributes to virulence in this clone. We conclude that extracellular DNase activity made by GAS contributes to disease progression, thereby resolving a long-standing question in bacterial pathogenesis research.
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Benschop, Jackie; Biggs, Patrick J.; Marshall, Jonathan C.; Hayman, David T.S.; Carter, Philip E.; Midwinter, Anne C.; Mather, Alison E.; French, Nigel P.
2017-01-01
During 1998–2012, an extended outbreak of Salmonella enterica serovar Typhimurium definitive type 160 (DT160) affected >3,000 humans and killed wild birds in New Zealand. However, the relationship between DT160 within these 2 host groups and the origin of the outbreak are unknown. Whole-genome sequencing was used to compare 109 Salmonella Typhimurium DT160 isolates from sources throughout New Zealand. We provide evidence that DT160 was introduced into New Zealand around 1997 and rapidly propagated throughout the country, becoming more genetically diverse over time. The genetic heterogeneity was evenly distributed across multiple predicted functional protein groups, and we found no evidence of host group differentiation between isolates collected from human, poultry, bovid, and wild bird sources, indicating ongoing transmission between these host groups. Our findings demonstrate how a comparative genomic approach can be used to gain insight into outbreaks, disease transmission, and the evolution of a multihost pathogen after a probable point-source introduction. PMID:28516864
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Sharma, Vijay K; Dean-Nystrom, Evelyn A; Casey, Thomas A
2011-07-12
Escherichia coli O157:H7 colonizes cattle intestines by using the locus of enterocyte effacement (LEE)-encoded proteins. The induction of systemic immune response against LEE-encoded proteins, therefore, will prove effective in reducing E. coli O157:H7 colonization in cattle. The previous studies have demonstrated that a hha (encodes for a hemolysin expression modulating protein) deletion enhances expression of LEE-encoded proteins and a sepB (encodes an ATPase required for the secretion of LEE-encoded proteins) deletion results in intracellular accumulation of LEE proteins. In this study, we demonstrate the efficacy of the hha and hha sepB deletion mutants as bacterins for reducing fecal shedding of E. coli O157:H7 in experimentally inoculated weaned calves. The weaned calves were injected intramuscularly with the bacterins containing 10(9) heat-killed cells of the hha(+) wild-type or hha or hha sepB isogenic mutants, and boosted with the same doses 2- and 4-weeks later. The evaluation of the immune response two weeks after the last booster immunization revealed that the calves vaccinated with the hha mutant bacterin had higher antibody titers against LEE proteins compared to the titers for these antibodies in the calves vaccinated with the hha sepB mutant or hha(+) wild-type bacterins. Following oral inoculations with 10(10) CFU of the wild-type E. coli O157:H7, the greater numbers of calves in the group vaccinated with the hha or hha sepB mutant bacterins stopped shedding the inoculum strain within a few days after the inoculations compared to the group of calves vaccinated with the hha(+) wild-type bacterin or PBS sham vaccine. Thus, the use of bacterins prepared from the hha and hha sepB mutants for reducing colonization of E. coli O157:H7 in cattle could represent a potentially important pre-harvest strategy to enhance post-harvest safety of bovine food products, water and produce. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Estimated abundance of wild burros surveyed on Bureau of Land Management Lands in 2014
Griffin, Paul C.
2015-01-01
The Bureau of Land Management (BLM) requires accurate estimates of the numbers of wild horses (Equus ferus caballus) and burros (Equus asinus) living on the lands it manages. For over ten years, BLM in Arizona has used the simultaneous double-observer method of recording wild burros during aerial surveys and has reported population estimates for those surveys that come from two formulations of a Lincoln-Petersen type of analysis (Graham and Bell, 1989). In this report, I provide those same two types of burro population analysis for 2014 aerial survey data from six herd management areas (HMAs) in Arizona, California, Nevada, and Utah. I also provide burro population estimates based on a different form of simultaneous double-observer analysis, now in widespread use for wild horse surveys that takes into account the potential effects on detection probability of sighting covariates including group size, distance, vegetative cover, and other factors (Huggins, 1989, 1991). The true number of burros present in the six areas surveyed was not known, so population estimates made with these three types of analyses cannot be directly tested for accuracy in this report. I discuss theoretical reasons why the Huggins (1989, 1991) type of analysis should provide less biased estimates of population size than the Lincoln-Petersen analyses and why estimates from all forms of double-observer analyses are likely to be lower than the true number of animals present in the surveyed areas. I note reasons why I suggest using burro observations made at all available distances in analyses, not only those within 200 meters of the flight path. For all analytical methods, small sample sizes of observed groups can be problematic, but that sample size can be increased over time for Huggins (1989, 1991) analyses by pooling observations. I note ways by which burro population estimates could be tested for accuracy when there are radio-collared animals in the population or when there are simultaneous double-observer surveys before and after a burro gather and removal.
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Grewal, S I; Han, B; Johnstone, K
1995-01-01
Pseudomonas tolaasii, the causal agent of brown blotch disease of Agaricus bisporus, spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies. The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics. A genomic cosmid clone (pSISG29) from a wild-type P. tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans. Subcloning and saturation mutagenesis analysis with Tn5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form. Marker exchange of the Tn5lacZ-mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form. The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein. The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins. Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation. Analysis of pheN expression with a pheN::lacZ fusion demonstrated that expression is regulated by environmental factors. These results are related to a model for control for phenotypic variation in P. tolaasii. PMID:7642492
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Orthotopic transplantation of LH receptor knockout and wild-type ovaries.
Chudgar, Daksha; Lei, Zhenmin; Rao, Ch V
2005-10-07
Luteinizing hormone (LH) receptor knockout animals have an ovarian failure due to an arrest in folliculogenesis at the antral stage. As a result, the animals have an infertility phenotype. The present study was undertaken to determine whether this phenotype could be reversed by orthotopic transplantation of wild-type ovaries. The results revealed that transplanting wild-type ovaries into null animals did not result in resumption of estrus cycles. Although the number of different types of follicles increased, none progressed to ovulation. The serum hormone profiles improved, reflecting the ovarian changes. The wild-type animals with null ovaries also failed to cycle and their ovaries and serum hormone levels were more like null animals with their own ovaries. Although the lack of rescue of null ovaries placed into wild-type animals was predicted, the failure of wild-type ovaries placed in null animals was not, which could be due to chronic exposure of transplanted tissue to high circulating LH levels and also possibly due to altered internal milieu in null animals. These findings may have implications for potential future considerations of grafting normal donor ovaries into women who have an ovarian failure resulting from inactivating LH receptor mutations.
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Yamagata, A; Hirota, R; Kato, J; Kuroda, A; Ikeda, T; Takiguchi, N; Ohtake, H
2000-08-01
The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao1, hao2, and hao3) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao1::kan, hao2::kan, or hao3::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao1::kan and hao3::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.
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Yokoya, Kazutomo; Postel, Sarah; Fang, Rui
2017-01-01
Background Fungal endophytes are highly diverse ubiquitous asymptomatic microorganisms, some of which appear to be symbiotic. Depending on abiotic conditions and genotype of the plant, the diversity of endophytes may confer fitness benefits to plant communities. Methods We studied a crop wild relative (CWR) of strawberry, along environmental gradients with a view to understand the cultivable root-derived endophytic fungi that can be evaluated for promoting growth and tolerating stress in selected plant groups. The main objectives were to understand whether: (a) suboptimal soil types are drivers for fungal distribution and diversity; (b) high pH and poor nutrient availability lead to fungal-plant associations that help deliver fitness benefits; and (c) novel fungi can be identified for their use in improving plant growth, and alleviate stress in diverse crops. Results The study revealed that habitats with high pH and low nutrient availability have higher fungal diversity, with more rare fungi isolated from locations with chalky soil. Plants from location G were the healthiest even though soil from this location was the poorest in nutrients. Study of environmental gradients, especially extreme habitat types, may help understand the root zone fungal diversity of different functional classes. Two small in vitro pilot studies conducted with two isolates showed that endophytic fungi from suboptimal habitats can promote plant growth and fitness benefits in selected plant groups. Discussion Targeting native plants and crop wild relatives for research offers opportunities to unearth diverse functional groups of root-derived endophytic fungi that are beneficial for crops. PMID:28168102
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de Luis, Daniel Antonio; Aller, Rocío; Izaola, Olatz; Bachiller, R; Pacheco, D
2014-09-01
The role of GLP-1 R variants on body weight response after dietary intervention is unclear. Our aim was to evaluate the effect of this polymorphism on cardiovascular risk factors, adipokine levels and weight loss secondary to a high monounsaturated fat vs. a high polyunsaturated fat hypocaloric diets in obese subjects. 391 obese subjects were randomly allocated to one of these two diets for a period of 3 months; diet M (high monounsaturated fat hypocaloric diet) and diet P (high polyunsaturated (PUFAs) fat hypocaloric diet). Two hundred and twelve patients (54.2 %) had the genotype GG (wild group) and 179 (45.8 %) patients had the next genotypes; GA (146 patients, 37.3 %) or AA (33 patients, 8.7 %) (Mutant group). With both diets and in wild-type and mutant-type groups, BMI, weight, fat mass, waist circumference and systolic blood pressure decreased. Anthropometric parameters were higher in non-A-allele carriers than A-allele carriers. With both diets and in both genotypes, leptin, insulin levels and HOMA decreased. With the diet P and in wild genotype, total cholesterol and LDL-cholesterol levels decreased, too. Our data showed a lack of association of rs6923761 GLP-1 R polymorphism with weight loss after a high monounsaturated fat and a high polyunsaturated fat hypocaloric diets. Better anthropometric parameters in obese subjects with the mutant allele (A) of rs6923761 GLP-1 R polymorphism were observed. Insulin levels and HOMA decreased in non-A carriers.
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Yi, Li; Cheng, Shipeng; Xu, Hongli; Wang, Jianke; Cheng, Yuening; Yang, Shen; Luo, Bin
2012-01-01
A combined reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (CDV). A pair of primers (P1/P2) was used to detect both CDV wild-type strains and vaccines. Another pair (P3/P4) was used to detect only CDV wild-type strains. A 335bp fragment was amplified from the genomic RNA of the vaccine and wild-type strains. A 555bp fragment was amplified specifically from the genomic RNA of the wild-type strains. No amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. The combined RT-PCR method detected effectively and differentiated the CDV wild-type and vaccine strains by two separate RT-PCRs. The method can be used for clinical detection and epidemiological surveillance. The phylogenetic analysis of the hemagglutinin gene of the local wild-type CDV strains revealed that the seven local isolates all belonged to the Asia-1 lineage, and were clustered closely with one another at the same location. These results suggested that the CDV genotype Asia-1 is circulating currently in domestic dogs in China. Copyright © 2011 Elsevier B.V. All rights reserved.
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Highly Branched Phenotype of the Petunia dad1-1 Mutant Is Reversed by Grafting.
Napoli, C.
1996-01-01
The recessive dad1-1 allele conditions a highly branched growth habit resulting from a proliferation of first- and second-order branches. Unlike the wild-type parent, which has lateral branching delayed until the third or fourth leaf node distal to the cotyledons, dad1-1 initiates lateral branching from each cotyledon axil. In addition to initiating lateral branching sooner than the wild type, dad1-1 sustains branching through more nodes on the main shoot axis than the wild type. In keeping with a propensity for branching at basal nodes, dad1-1 produces second-order branches at the proximal-most nodes on first-order branches and small shoots from accessory buds at basal nodes on the main shoot axis. Additional traits associated with the mutation are late flowering, adventitious root formation, shortened internodes, and mild leaf chlorosis. Graft studies show that a dad1-1 scion, when grafted onto wild-type stock, is converted to a phenotype resembling the wild type. Furthermore, a small wild-type interstock fragment inserted between a mutant root stock and a mutant scion is sufficient to convert the dad1-1 scion from mutant to a near wild-type appearance. The recessive dad1-1 phenotype combines traits associated with cytokinin overexpression, auxin overexpression, and gibberellin limitation, which suggests a complex interaction of hormones in establishing the mutant phenotype. PMID:12226274
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Plumb, William; Townsend, Alexandra J; Rasool, Brwa; Alomrani, Sarah; Razak, Nurhayati; Karpinska, Barbara; Ruban, Alexander V; Foyer, Christine H
2018-05-03
The requirements of growth and photosynthesis for ascorbate were assessed under low (LL; 250 μmol m-2 s-1) or high (HL; 1600 μmol m-2 s-1) irradiance in wild type Arabidopsis thaliana and two ascorbate synthesis mutants (vtc2-1 and vtc2-4) that have 30% wild type ascorbate levels. The low ascorbate mutants had the same numbers of leaves but lower rosette area and biomass than the wild type under LL. Wild type plants experiencing HL had higher leaf ascorbate, anthocyanin and xanthophyll pigments than under LL. In contrast, leaf ascorbate levels were not increased under HL in the mutant lines. While the degree of oxidation measured using an in vivo redox reporter in the nuclei and cytosol of the leaf epidermal and stomatal cells was similar under both irradiances in all lines, anthocyanin levels were significantly lower than in the low ascorbate mutants than the wild type under HL. Differences in the photosynthetic responses of vtc2-1 and vtc2-4 mutants were observed. Unlike vtc2-1, the vtc2-4 mutants had wild type zeaxanthin contents. While both low ascorbate mutants had lower NPQ levels than the wild type under HL, qPd values were greater only in vtc2-1 leaves. Ascorbate is therefore essential for growth but not photoprotection.
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Riddick, Nadeene E.; Hermann, Emilia A.; Loftin, Lamorris M.; Elliott, Sarah T.; Wey, Winston C.; Cervasi, Barbara; Taaffe, Jessica; Engram, Jessica C.; Li, Bing; Else, James G.; Li, Yingying; Hahn, Beatrice H.; Derdeyn, Cynthia A.; Sodora, Donald L.; Apetrei, Cristian; Paiardini, Mirko; Silvestri, Guido; Collman, Ronald G.
2010-01-01
In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species. PMID:20865163
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Riddick, Nadeene E; Hermann, Emilia A; Loftin, Lamorris M; Elliott, Sarah T; Wey, Winston C; Cervasi, Barbara; Taaffe, Jessica; Engram, Jessica C; Li, Bing; Else, James G; Li, Yingying; Hahn, Beatrice H; Derdeyn, Cynthia A; Sodora, Donald L; Apetrei, Cristian; Paiardini, Mirko; Silvestri, Guido; Collman, Ronald G
2010-08-26
In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species.
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A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase
ERIC Educational Resources Information Center
Witherow, D. Scott
2016-01-01
This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…
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Ivanova, L A; Ronzhina, D A; Ivanov, L A; Stroukova, L V; Peuke, A D; Rennenberg, H
2009-07-01
Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best-characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild-type poplar hybrid Populus tremula x P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding gamma-glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild-type plants; soil contamination significantly decreased biomass accumulation in both wild-type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two-dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild-type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal-contaminated field sites. Further experiments are required to test these assumptions.
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The role of strigolactones in photomorphogenesis of pea is limited to adventitious rooting.
Urquhart, Shelley; Foo, Eloise; Reid, James B
2015-03-01
The recently discovered group of plant hormones, the strigolactones, have been implicated in regulating photomorphogenesis. We examined this extensively in our strigolactone synthesis and response mutants and could find no evidence to support a major role for strigolactone signaling in classic seedling photomorphogenesis (e.g. elongation and leaf expansion) in pea (Pisum sativum), consistent with two recent independent reports in Arabidopsis. However, we did find a novel effect of strigolactones on adventitious rooting in darkness. Strigolactone-deficient mutants, Psccd8 and Psccd7, produced significantly fewer adventitious roots than comparable wild-type seedlings when grown in the dark, but not when grown in the light. This observation in dark-grown plants did not appear to be due to indirect effects of other factors (e.g. humidity) as the constitutively de-etiolated mutant, lip1, also displayed reduced rooting in the dark. This role for strigolactones did not involve the MAX2 F-Box strigolactone response pathway as Psmax2 f-box mutants did not show a reduction in adventitious rooting in the dark compared with wild-type plants. The auxin-deficient mutant bushy also reduced adventitious rooting in the dark, as did decapitation of wild-type plants. Rooting was restored by the application of indole-3-acetic acid (IAA) to decapitated plants, suggesting a role for auxin in the rooting response. However, auxin measurements showed no accumulation of IAA in the epicotyls of wild-type plants compared with the strigolactone synthesis mutant Psccd8, suggesting that changes in the gross auxin level in the epicotyl are not mediating this response to strigolactone deficiency. © 2014 Scandinavian Plant Physiology Society.
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Fast, C; Goldmann, W; Berthon, P; Tauscher, K; Andréoletti, O; Lantier, I; Rossignol, C; Bossers, A; Jacobs, J G; Hunter, N; Groschup, M H; Lantier, F; Langeveld, J P M
2017-09-19
Breeding towards genetic resistance to prion disease is effective in eliminating scrapie. In sheep, classical forms of scrapie have been eradicated almost completely in several countries by breeding programs using a prion protein (PrP) gene (PRNP) amino acid polymorphism. For goats, field and experimental studies have provided evidence for several amino acid polymorphisms that are associated with resistance to scrapie, but only limited data are available concerning the susceptibility of caprine PRNP genotypes to BSE. In this study, goat kids representing five PRNP genotypes based on three polymorphisms (M142, Q211 and K222 and the wild type I142, R211 and Q222) were orally challenged with bovine or goat BSE. Wild type goats were killed with clinical signs between 24-28 months post inoculation (mpi) to both challenges, and goats with genotype R/Q211 succumbed between 29-36 mpi. I/M142 goats developed clinical signs at 44-45 mpi and M/M142 goats remained healthy until euthanasia at 48 mpi. None of the Q/K222 goats showed definite clinical signs. Taken together the highest attack ratios were seen in wild type and R/Q211 goats, and the lowest in I/M142, M/M142 and Q/K222. In all genotype groups, one or more goats remained healthy within the incubation period in both challenges and without detectable PrP deposition in the tissues. Our data show that both the K222 and M142 polymorphisms lengthen the incubation period significantly compared to wild type animals, but only K222 was associated with a significant increase in resistance to BSE infection after oral exposure to both BSE sources.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ponce, E.; Mear, J; Grabowski, G.A.
1994-09-01
Numerous mutations ({approximately}45) of the acid {beta}-glucosidase gene have been identified in patients with Gaucher disease. Many of these have been characterized by partial sequencing of cDNAs derived by RT-PCR or PCR of genomic DNA. In addition, genotype/phenotype correlations have been based on screening for known mutations. Thus, only a part of the gene is characterized in any population of affected patients. Several Gaucher disease alleles contain multiple, authentic point mutations that raises concern about conclusions based on only partial genetic characterization. Several wild-type cDNAs for acid {beta}-glucosidase have been sequenced. One contained a cloning artifact encoding R495H. We expressedmore » this cDNA and showed that the R495H enzyme had normal kinetic and stability properties. A disease-associated allele encoding R496H has been found by several groups. The close association and similarities of these two substitutions led us to question the disease casuality of the R496H allele. To evaluate this, we created and/or expressed cDNAs encoding R495, R496 (wild-type), (R495H, R496), (R495, R496H) and (R495H, R496H). The (wild-type) and (R495H, R496) enzymes had indistinguishable properties whereas the (R495, R496H) enzyme was essentially inactive. The introduction of both mutations (R495H, R496H) produced an enzyme whose activity was 25 to 50% of the wild-type. These results indicate that a pseudoreversion to a functional enzyme can occur by introducing a functionally neutral mutation together with a severe mutation. These results have major implications to structure/function and genotype/phenotype correlations in this disease.« less
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Both germ line and somatic genetics of the p53 pathway affect ovarian cancer incidence and survival.
Bartel, Frank; Jung, Juliane; Böhnke, Anja; Gradhand, Elise; Zeng, Katharina; Thomssen, Christoph; Hauptmann, Steffen
2008-01-01
Although p53 is one of the most studied genes/proteins in ovarian carcinomas, the predictive value of p53 alterations is still ambiguous. We performed analyses of the TP53 mutational status and its protein expression using immunohistochemistry. Moreover, the single nucleotide polymorphism SNP309 in the P2 promoter of the MDM2 gene was investigated. We correlated the results with age of onset and outcome from 107 patients with ovarian carcinoma. In our study, we identified a large group of patients with p53 overexpression despite having a wild-type gene (49% of all patients with wild-type TP53). This was associated with a significantly shortened overall survival time (P = 0.019). Patients with p53 alterations (especially those with overexpression of wild-type TP53) were also more refractory to chemotherapy compared with patients with normal p53 (P = 0.027). The G-allele of SNP309 is associated with an earlier age of onset in patients with estrogen receptor-overexpressing FIGO stage III disease (P = 0.048). In contrast, in patients with FIGO stage III disease, a weakened p53 pathway (either the G-allele of SNP309 or a TP53 mutation) was correlated with increased overall survival compared with patients whose tumors were wild-type for both TP53 and SNP309 (P = 0.0035). Our study provides evidence that both germ line and somatic alterations of the p53 pathway influence the incidence and survival of ovarian carcinoma, and it underscores the importance of assessing the functionality of p53 in order to predict the sensitivity of platinum-based chemotherapies and patient outcome.
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Zhou, Li; Wang, Na-Na; Gong, Si-Ying; Lu, Rui; Li, Yang; Li, Xue-Bao
2015-11-01
Soil salinity is one of the most serious threats in world agriculture, and often influences cotton growth and development, resulting in a significant loss in cotton crop yield. WRKY transcription factors are involved in plant response to high salinity stress, but little is known about the role of WRKY transcription factors in cotton so far. In this study, a member (GhWRKY34) of cotton WRKY family was functionally characterized. This protein containing a WRKY domain and a zinc-finger motif belongs to group III of cotton WRKY family. Subcellular localization assay indicated that GhWRKY34 is localized to the cell nucleus. Overexpression of GhWRKY34 in Arabidopsis enhanced the transgenic plant tolerance to salt stress. Several parameters (such as seed germination, green cotyledons, root length and chlorophyll content) in the GhWRKY34 transgenic lines were significantly higher than those in wild type under NaCl treatment. On the contrary, the GhWRKY34 transgenic plants exhibited a substantially lower ratio of Na(+)/K(+) in leaves and roots dealing with salt stress, compared with wild type. Growth status of the GhWRKY34 transgenic plants was much better than that of wild type under salt stress. Expressions of the stress-related genes were remarkably up-regulated in the transgenic plants under salt stress, compared with those in wild type. Based on the data presented in this study, we hypothesize that GhWRKY34 as a positive transcription regulator may function in plant response to high salinity stress through maintaining the Na(+)/K(+) homeostasis as well as activating the salt stress-related genes in cells. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
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White, Tomas L.; Lewis, Philip; Hayes, Sally; Fergusson, James; Bell, James; Farinha, Luis; White, Nick S.; Pereira, Lygia V.; Meek, Keith M.
2017-01-01
Purpose The presence of fibrillin-rich elastic fibers in the cornea has been overlooked in recent years. The aim of the current study was to elucidate their functional role using a mouse model for Marfan syndrome, defective in fibrillin-1, the major structural component of the microfibril bundles that constitute most of the elastic fibers. Methods Mouse corneas were obtained from animals with a heterozygous fibrillin-1 mutation (Fbn1+/−) and compared to wild type controls. Corneal thickness and radius of curvature were calculated using optical coherence tomography microscopy. Elastic microfibril bundles were quantified and visualized in three-dimensions using serial block face scanning electron microscopy. Transmission electron microscopy was used to analyze stromal ultrastructure and proteoglycan distribution. Center-to-center average interfibrillar spacing was determined using x-ray scattering. Results Fbn1+/− corneas were significantly thinner than wild types and displayed a higher radius of curvature. In the Fbn1+/− corneas, elastic microfibril bundles were significantly reduced in density and disorganized compared to wild-type controls, in addition to containing a higher average center-to-center collagen interfibrillar spacing in the center of the cornea. No other differences were detected in stromal ultrastructure or proteoglycan distribution between the two groups. Proteoglycan side chains appeared to colocalize with the microfibril bundles. Conclusions Elastic fibers have an important, multifunctional role in the cornea as highlighted by the differences observed between Fbn1+/− and wild type animals. We contend that the presence of normal quantities of structurally organized elastic fibers are required to maintain the correct geometry of the cornea, which is disrupted in Marfan syndrome. PMID:28395026
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Talfournier, F; Munro, A W; Basran, J; Sutcliffe, M J; Daff, S; Chapman, S K; Scrutton, N S
2001-06-08
The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is +153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) < -250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.
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Choudhary, Pooja; Pacholko, Anthony G; Palaschuk, Josh; Bekar, Lane K
2018-06-03
Alzheimer's disease (AD) is the sixth leading cause of death in the United States where it is estimated that one in three seniors dies with AD or another dementia. Are modern lifestyle habits a contributing factor? Increased carbohydrate (sugar) consumption, stress and disruption of sleep patterns are quickly becoming the norm rather than the exception. Interestingly, seven months on a non-invasive high sucrose diet (20% sucrose in drinking water) has been shown to induce behavioral, metabolic and pathological changes consistent with AD in wild-type mice. As chronic stress and depression are associated with loss of locus coeruleus (LC) noradrenergic neurons and projections (source of anti-inflammatory and trophic factor control), we assessed the ability for a selective LC neurotoxin (DSP4) to accelerate and aggravate a high-sucrose mediated AD-related phenotype in wild-type mice. Male C57/Bl6 mice were divided into four groups: 1) saline injected, 2) DSP4 injected, 3) high sucrose drinking water (20%) or 4) DSP4 injected and high sucrose drinking water. We demonstrate that high sucrose consumption and DSP4 treatment promote an early-stage AD-related phenotype after only 3-4 months, as evidenced by elevated fecal corticosterone, increased despair, spatial memory deficits, increased AChE activity, elevated NO production, decreased pGSK3β and increased pTau. Combined treatment appears to accelerate and aggravate pathological processes consistent with Alzheimer disease and dementia. Developing a simple model in wild-type mice will highlight environmental and lifestyle factors that need to be addressed to slow, prevent or even reverse the rising trend in dementia patient numbers and cost.
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Wild-Type Measles Viruses with Non-Standard Genome Lengths
Bankamp, Bettina; Liu, Chunyu; Rivailler, Pierre; Bera, Jayati; Shrivastava, Susmita; Kirkness, Ewen F.; Bellini, William J.; Rota, Paul A.
2014-01-01
The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible. PMID:24748123
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Schaevitz, Laura R; Picker, Jonathan D; Rana, Jasmine; Kolodny, Nancy H; Shane, Barry; Berger-Sweeney, Joanne E; Coyle, Joseph T
2012-06-01
Interactions between genetic and environmental risk factors underlie a number of neuropsychiatric disorders, including schizophrenia (SZ) and autism (AD). Due to the complexity and multitude of the genetic and environmental factors attributed to these disorders, recent research strategies focus on elucidating the common molecular pathways through which these multiple risk factors may function. In this study, we examine the combined effects of a haplo-insufficiency of glutamate carboxypeptidase II (GCPII) and dietary folic acid deficiency. In addition to serving as a neuropeptidase, GCPII catalyzes the absorption of folate. GCPII and folate depletion interact within the one-carbon metabolic pathway and/or of modulate the glutamatergic system. Four groups of mice were tested: wild-type, GCPII hypomorphs, and wild-types and GCPII hypomorphs both fed a folate deficient diet. Due to sex differences in the prevalence of SZ and AD, both male and female mice were assessed on a number of behavioral tasks including locomotor activity, rotorod, social interaction, prepulse inhibition, and spatial memory. Wild-type mice of both sexes fed a folic acid deficient diet showed motor coordination impairments and cognitive deficits, while social interactions were decreased only in males. GCPII mutant mice of both sexes also exhibited reduced social propensities. In contrast, all folate-depleted GCPII hypomorphs performed similarly to untreated wild-type mice, suggesting that reduced GCPII expression and folate deficiency are mutually protective. Analyses of folate and neurometabolite levels associated with glutamatergic function suggest several potential mechanisms through which GCPII and folate may be interacting to create this protective effect. Copyright © 2011 Wiley Periodicals, Inc.
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Fei, Dennis Liang; Motowski, Hayley; Chatrikhi, Rakesh; Gao, Shaojian; Kielkopf, Clara L.; Varmus, Harold
2016-01-01
We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele. PMID:27776121
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Zhang, Ning; Xie, Yong-Dun; Guo, Hui-Jun; Zhao, Lin-Shu; Xiong, Hong-Chun; Gu, Jia-Yu; Li, Jun-Hui; Kong, Fu-Quan; Sui, Li; Zhao, Zi-Wei; Zhao, Shi-Rong; Liu, Lu-Xiang
2016-10-01
Gibberellin (GA) is essential for determining plant height. Alteration of GA content or GA signaling results in a dwarf or slender phenotype. Here, we characterized a novel wheat mutant, quick development (qd), in which GA regulates stem elongation but does not affect mature plant height. qd and wild-type plants did not exhibit phenotypic differences at the seedling stage. From jointing to heading stage, qd plants were taller than wild-type plants due to elongated cells. However, wild-type and qd plants were the same height at heading. Unlike wild-type plants, qd plants were sensitive to exogenous GA due to mutation of Rht-B1. With continuous GA stimulation, qd seedlings and adult plants were taller than wild-type. Thus, the GA content of qd plants might differ from that of wild-type during the growth process. Analysis of GA biosynthetic gene expression verified this hypothesis and showed that TaKAO, which is involved in catalyzing the early steps of GA biosynthesis, was differentially expressed in qd plants compared with wild-type. The bioactive GA associated gene TaGA20ox was downregulated in qd plants during the late growth stages. Measurements of endogenous GA content were consistent with the gene-expression analysis results. Consistent with the GA content variation, the first three basal internodes were longer and the last two internodes were shorter in qd than in wild-type plants. The qd mutant might be useful in dissecting the mechanism by which GA regulates stem-growing process, and it may be serve as a GA responsive semi-dwarf germplasm in breeding programs. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
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Priengprom, Thongkoon; Ekalaksananan, Tipaya; Kongyingyoes, Bunkerd; Suebsasana, Supawadee; Aromdee, Chantana; Pientong, Chamsai
2015-03-11
An andrographolide analogue, 3, 19-isopropylideneandrographolide (IPAD), exerts an inhibitory effect on replication of wild-type herpes simplex virus serotype 1 (HSV-1). In this study, we examined the anti-viral activity of IPAD on HSV wild types (HSV-1 strain KOS and HSV-2 clinical isolate) and HSV-1 drug-resistant strains (DRs). Synergistic effects of IPAD with acyclovir (ACV) were also evaluated. MTT and cytopathic effect (CPE) reduction assays were performed to determine cytotoxicity and anti-viral activities, respectively. A combination assay was used to determine synergistic effects of IPAD and ACV. Presence of viral DNA and protein in experimental cells was investigated using the polymerase chain reaction and western blotting, respectively. A non-cytotoxic concentration of IPAD (20.50 μM) completely inhibited CPE formation induced by HSV wild types and HSV-1 DRs after viral entry into the cells. The anti-HSV activities included inhibition of viral DNA and protein synthesis. The minimum inhibitory concentrations of ACV for HSV wild types and HSV-1 DRs were 20.20 and 2,220.00 μM, respectively. Combination of ACV with IPAD showed synergistic effects in inhibition of CPE formation, viral DNA and protein synthesis by HSV wild types as well as HSV-1 DRs. For the synergistic effects on HSV wild types and HSV-1 DRs, the effective concentrations of ACV were reduced. These results showed the inhibitory potential of IPAD on HSV wild types and HSV-1 DRs and suggested that IPAD could be used in combination with ACV for treatment of HSV-1 DRs infections.
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Gause, K C; Homma, M K; Licciardi, K A; Seger, R; Ahn, N G; Peterson, M J; Krebs, E G; Meier, K E
1993-08-05
Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.
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Yu, Xia; Wang, Guirong; Chen, Suting; Wei, Guomei; Shang, Yuanyuan; Dong, Lingling; Schön, Thomas; Moradigaravand, Danesh; Peacock, Sharon J.
2016-01-01
Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective. PMID:27324769
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Activation Of Wild-Type Hras Suppresses The Earliest Stages Of Pancreatic Cancer.
Weyandt, Jamie
2015-08-01
The RAS family of small GTPases is comprised of HRAS, NRAS, and KRAS. KRAS is invariably oncogenically mutated in pancreatic cancers, which is known to induce this disease. Beyond oncogenic KRAS, redox-dependent reactions have been implicated in the activation of the remaining wild-type RAS proteins in pancreatic cancer cell lines. These results suggest a possible involvement of wild-type RAS proteins in pancreatic cancer. To evaluate the impact of genetically suppressing wild-type RAS expression on pancreatic cancer. Hras homozygous null mice (Hras -/- ) were crossed into a Pdx-Cre; LSL-Kras G12D/+ (KC) murine background in which oncogenic Kras is activated in the pancreas to promote preinvasive pancreatic cancer. Tumor burden was then measured at different stages of disease. HRas -/- ;KC mice exhibited more precancerous lesions in the pancreas and more off-target skin papillomas compared to their wild-type counterparts, suggesting that Hras suppresses early oncogenic Kras-driven tumorigenesis, possibly at the time of initiation. Loss of Hras also reduced the survival of mice engineered to develop aggressive pancreatic cancer by the additional disruption of one allele of the tumor suppressor p53 (Trp53 R172H/+ ). However, this survival advantage was lost when both alleles of Trp53 were mutated, suggesting that wild-type Hras inhibits tumorigenesis in a p53-dependent fashion. Loss of wild-type Hras promotes the earliest stages of pancreatic tumorigenesis, and moreover results in more rapid progression of the disease. As such, mechanisms leading to activation of wild-type Ras proteins, including but not limited to redox-dependent reactions, may influence the development of pancreatic cancer. Copyright © 2015. Published by Elsevier B.V.
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The complex history of the domestication of rice.
Sweeney, Megan; McCouch, Susan
2007-11-01
Rice has been found in archaeological sites dating to 8000 bc, although the date of rice domestication is a matter of continuing debate. Two species of domesticated rice, Oryza sativa (Asian) and Oryza glaberrima (African) are grown globally. Numerous traits separate wild and domesticated rices including changes in: pericarp colour, dormancy, shattering, panicle architecture, tiller number, mating type and number and size of seeds. Genetic studies using diverse methodologies have uncovered a deep population structure within domesticated rice. Two main groups, the indica and japonica subspecies, have been identified with several subpopulations existing within each group. The antiquity of the divide has been estimated at more than 100 000 years ago. This date far precedes domestication, supporting independent domestications of indica and japonica from pre-differentiated pools of the wild ancestor. Crosses between subspecies display sterility and segregate for domestication traits, indicating that different populations are fixed for different networks of alleles conditioning these traits. Numerous domestication QTLs have been identified in crosses between the subspecies and in crosses between wild and domesticated accessions of rice. Many of the QTLs cluster in the same genomic regions, suggesting that a single gene with pleiotropic effects or that closely linked clusters of genes underlie these QTL. Recently, several domestication loci have been cloned from rice, including the gene controlling pericarp colour and two loci for shattering. The distribution and evolutionary history of these genes gives insight into the domestication process and the relationship between the subspecies. The evolutionary history of rice is complex, but recent work has shed light on the genetics of the transition from wild (O. rufipogon and O. nivara) to domesticated (O. sativa) rice. The types of genes involved and the geographic and genetic distribution of alleles will allow scientists to better understand our ancestors and breed better rice for our descendents.
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Skyberg, Jerod A; Rollins, Maryclare F; Samuel, Joshua W; Sutherland, Marjorie D; Belisle, John T; Pascual, David W
2013-09-01
Francisella tularensis is a highly infectious intracellular bacterium that causes the zoonotic infection tularemia. While much literature exists on the host response to F. tularensis infection, the vast majority of work has been conducted using attenuated strains of Francisella that do not cause disease in humans. However, emerging data indicate that the protective immune response against attenuated F. tularensis versus F. tularensis type A differs. Several groups have recently reported that interleukin-17 (IL-17) confers protection against the live vaccine strain (LVS) of Francisella. While we too have found that IL-17Rα(-/-) mice are more susceptible to F. tularensis LVS infection, our studies, using a virulent type A strain of F. tularensis (SchuS4), indicate that IL-17Rα(-/-) mice display organ burdens and pulmonary gamma interferon (IFN-γ) responses similar to those of wild-type mice following infection. In addition, oral LVS vaccination conferred equivalent protection against pulmonary challenge with SchuS4 in both IL-17Rα(-/-) and wild-type mice. While IFN-γ was found to be critically important for survival in a convalescent model of SchuS4 infection, IL-17 neutralization from either wild-type or IFN-γ(-/-) mice had no effect on morbidity or mortality in this model. IL-17 protein levels were also higher in the lungs of mice infected with the LVS rather than F. tularensis type A, while IL-23p19 mRNA expression was found to be caspase-1 dependent in macrophages infected with LVS but not SchuS4. Collectively, these results demonstrate that IL-17 is dispensable for host immunity to type A F. tularensis infection, and that induced and protective immunity differs between attenuated and virulent strains of F. tularensis.
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Camacho, María; Burgos, Araceli; Chamber-Pérez, Manuel A
2003-04-01
Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.
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Kirdis, Ebru; Jonsson, Ing-Marie; Kubica, Malgorzata; Potempa, Jan; Josefsson, Elisabet; Masalha, Mahmud; Foster, Simon J; Tarkowski, Andrzej
2007-01-01
Staphylococcus aureus is the most common cause of joint infections. It also contributes to several other diseases such as pneumonia, osteomyelitis, endocarditis, and sepsis. Bearing in mind that S. aureus becomes rapidly resistant to new antibiotics, many studies survey the virulence factors, with the aim to find alternative prophylaxis/treatment regimens. One potential virulence factor is the bacterial ability to survive at different oxygen tensions. S. aureus expresses ribonucleotide reductases (RNRs), which help it to grow under both aerobic and anaerobic conditions, by reducing ribonucleotides to deoxyribonucleotides. In this study, we investigated the role of RNR class III, which is required for anaerobic growth, as a virulence determinant in the pathogenesis of staphylococcal arthritis. The wild-type S. aureus strain and its isogenic mutant nrdDG mutant were inoculated intravenously into mice. Mice inoculated with the wild-type strain displayed significantly more severe arthritis, with significantly more synovitis and destruction of the bone and cartilage versus mutant strain inoculated mice. Further, the persistence of bacteria in the kidneys was significantly more pronounced in the group inoculated with the wild-type strain. Together these results indicate that RNR class III is an important virulence factor for the establishment of septic arthritis.
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Will, Sabine Eva; Neumann-Schaal, Meina; Heydorn, Raymond Leopold; Bartling, Pascal; Petersen, Jörn; Schomburg, Dietmar
2017-01-01
Phaeobacter inhibens DSM 17395, a model organism for marine Roseobacter group, was studied for its response to its own antimicrobial compound tropodithietic acid (TDA). TDA biosynthesis is encoded on the largest extrachromosomal element of P. inhibens, the 262 kb plasmid, whose curation leads to an increased growth and biomass yield. In this study, the plasmid-cured strain was compared to the wild-type strain and to transposon mutants lacking single genes of the TDA biosynthesis. The data show that the growth inhibition of the wild-type strain can be mainly attributed to the TDA produced by P. inhibens itself. Oxygen uptake rates remained constant in all strains but the growth rate dropped in the wild-type which supports the recently proposed mode of TDA action. Metabolome analysis showed no metabolic alterations that could be attributed directly to TDA. Taken together, the growth of P. inhibens is limited by its own antibacterial compound due to a partial destruction of the proton gradient which leads to a higher energetic demand. The universal presence of TDA biosynthesis in genome-sequenced isolates of the genus Phaeobacter shows that there must be a high benefit of TDA for P. inhibens in its ecological niche despite the drawback on its metabolism.
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Organs on chip approach: a tool to evaluate cancer -immune cells interactions.
Biselli, Elena; Agliari, Elena; Barra, Adriano; Bertani, Francesca Romana; Gerardino, Annamaria; De Ninno, Adele; Mencattini, Arianna; Di Giuseppe, Davide; Mattei, Fabrizio; Schiavoni, Giovanna; Lucarini, Valeria; Vacchelli, Erika; Kroemer, Guido; Di Natale, Corrado; Martinelli, Eugenio; Businaro, Luca
2017-10-06
In this paper we discuss the applicability of numerical descriptors and statistical physics concepts to characterize complex biological systems observed at microscopic level through organ on chip approach. To this end, we employ data collected on a microfluidic platform in which leukocytes can move through suitably built channels toward their target. Leukocyte behavior is recorded by standard time lapse imaging. In particular, we analyze three groups of human peripheral blood mononuclear cells (PBMC): heterozygous mutants (in which only one copy of the FPR1 gene is normal), homozygous mutants (in which both alleles encoding FPR1 are loss-of-function variants) and cells from 'wild type' donors (with normal expression of FPR1). We characterize the migration of these cells providing a quantitative confirmation of the essential role of FPR1 in cancer chemotherapy response. Indeed wild type PBMC perform biased random walks toward chemotherapy-treated cancer cells establishing persistent interactions with them. Conversely, heterozygous mutants present a weaker bias in their motion and homozygous mutants perform rather uncorrelated random walks, both failing to engage with their targets. We next focus on wild type cells and study the interactions of leukocytes with cancerous cells developing a novel heuristic procedure, inspired by Lyapunov stability in dynamical systems.
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Guo, Yulong; Zhu, Qinlong; Zheng, Shangyong; Li, Mingyang
2007-06-01
A MADS box gene (GhMADS3) was cloned from cotton (Gossypium hirsutum L.) based on EST sequences. The predicted protein sequence of GhMADS3 showed 85%, 73%, and 62% identity with Theobroma cacao TcAG, Antirrhinum majus FAR, and Arabidopsis thaliana AG, respectively, and was grouped with AG homologues when the full length sequences excluding N-extensions were compared. GhMADS3 expressed in the wild type cotton flower primarily in stamens and carpels, which was comparable to AG in Arabidopsis. However, it was not expressed in floral buds of a homeotic cotton variant chv1. Ectopic expression of GhMADS3 in tobacco (Nicotiana tabacum L.) resulted in flowers with sepal-to-carpel and petal-to-stamen transformation. The carpelloid first whorl organs, with stigmatic tissue on their upper edges, had a white appearance when compared with the dark green color of the wild type sepals. At times, long filaments were observed at the fusion site of the first carpelloid oranges. The second whorl organs in staminoid were usually smaller than the wild type and the color was changed from pink to white. These results suggest that GhMADS3 has a homeotic role in flower development.
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Gene knockout of tau expression does not contribute to the pathogenesis of prion disease.
Lawson, Victoria A; Klemm, Helen M; Welton, Jeremy M; Masters, Colin L; Crouch, Peter; Cappai, Roberto; Ciccotosto, Giuseppe D
2011-11-01
Prion diseases or transmissible spongiform encephalopathies are a group of fatal and transmissible disorders affecting the central nervous system of humans and animals. The principal agent of prion disease transmission and pathogenesis is proposed to be an abnormal protease-resistant isoform of the normal cellular prion protein. The microtubule-associated protein tau is elevated in patients with Creutzfeldt-Jakob disease. To determine whether tau expression contributes to prion disease pathogenesis, tau knockout and control wild-type mice were infected with the M1000 strain of mouse-adapted human prions. Immunohistochemical analysis for total tau expression in prion-infected wild-type mice indicated tau aggregation in the cytoplasm of a subpopulation of neurons in regions associated with spongiform change. Western immunoblot analysis of brain homogenates revealed a decrease in total tau immunoreactivity and epitope-specific changes in tau phosphorylation. No significant difference in incubation period or other disease features were observed between tau knockout and wild-type mice with clinical prion disease. These results demonstrate that, in this model of prion disease, tau does not contribute to the pathogenesis of prion disease and that changes in the tau protein profile observed in mice with clinical prion disease occurs as a consequence of the prion-induced pathogenesis.
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Song, Xiaoyun; Liu, Xingcai; Ding, Xi
2017-04-01
The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first-line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small-molecule kinase inhibitors in EGFR-targeted chemotherapy. Previously, the reversible pan-kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR T790M ) over wild-type kinase (EGFR WT ), suggesting that the staurosporine scaffold is potentially to develop the wild-type sparing reversible inhibitors of EGFR T790M . Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold-based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico-in vitro analog-sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild-type sparing inhibitors UCN-01, UCN-02, AFN941, and SB-218078 with high or moderate selectivity of 30-, 45-, 5-, and 8-fold for EGFR T790M over EGFR WT , respectively, which are comparable with or even better than that of the parent compound staurosporine (24-fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN-02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding. A hydroxyl group of UCN-02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN-02 than staurosporine for mutant over wild-type kinase. Copyright © 2016 John Wiley & Sons, Ltd.
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Masugi-Tokita, M; Yoshida, T; Kageyama, S; Kawata, M; Kawauchi, A
2018-03-01
Metabotropic glutamate receptor subtype 7 (mGluR7) is one of the group III mGluRs, which are negatively coupled to adenylate cyclase via Gi/Go proteins and localised to presynaptic active zones of the mammalian central nervous system. We previously reported that mGluR7 is essential for intermale aggression and amygdala-dependent fear learning. To elucidate the role of mGluR7 in the neuroendocrine system, we performed biochemical analyses and found a significant reduction of testosterone levels in mGluR7 knockout (KO) mice. Testosterone replacement restored intermale aggressive behaviour in castrated wild-type mice to the level of gonadally intact wild-type mice. However, given the same dosage of testosterone replacement, mGluR7 KO mice showed almost no aggressive behaviour. These results indicate that reduction of plasma testosterone is unrelated to the deficit in intermale aggression in mGluR7 KO mice. Social investigating behaviour of intact mGluR7 KO mice also differed from that of wild-type mice; e.g. the KO mice showing less frequent anogenital sniffing and more frequent grooming behaviour. Testosterone replacement increased anogenital sniffing and grooming behaviour in castrated mGluR7 KO mice, while the differences were still present between castrated wild-type mice and KO mice after both underwent testosterone replacement. These results imply that reduction of plasma testosterone may partially inhibit social investigating behaviours in intact mGluR7 KO mice. Furthermore, castrated mGluR7 KO mice have smaller seminal vesicles than those of castrated wild-type mice, although seminal vesicle weights were normal in intact mice. These observations suggest that, besides testicular testosterone, some other hormone levels may be dysregulated in mGluR7 KO mice, and indicate a critical role of mGluR7 in the endocrine system. Taken together, our findings demonstrate that mGluR7 is essential for the regulation of the endocrine system, in addition to innate behaviours such as intermale aggression and fear response. © 2018 British Society for Neuroendocrinology.
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Molecular determinants for FMN-binding in Desulfovibrio gigas flavoredoxin.
Broco, Manuela; Soares, Cláudio M; Oliveira, Solange; Mayhew, Stephen G; Rodrigues-Pousada, Claudina
2007-09-18
Flavoredoxin participates in Desulfovibrio gigas thiosulfate reduction pathway. Its 3-dimensional model was generated allowing the oxidized riboflavin-5'-phosphate (FMN) site to be predicted. Residues likely to be involved in FMN-binding were identified (N29, W35, T56, K92, H131 and F164) and mutated to alanine. Fluorescence titration with apoprotein showed that FMN is strongly bound in the wild-type protein. Comparison of K(d) values for mutants suggests that interactions with the phosphate group of FMN, contribute more to binding than the interactions with the isoalloxazine ring. The redox potential of bound FMN determined for wild-type and mutants revealed shifts to less negative values. These findings were correlated with the protein structure in order to contribute to a better understanding of the structure-function relationships in flavoredoxin.
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CYP2E1-dependent elevation of serum cholesterol, triglycerides, and hepatic bile acids by isoniazid
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cheng, Jie; Krausz, Kristopher W.; Li, Feng
Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition–induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-typemore » mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria β-oxidation. -- Highlights: ► Isoniazid metabolites were elevated only in wild-type mice. ► Isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice. ► Isoniazid elevated serum cholesterol and triglycerides, and hepatic bile acids. ► Bile acid transporters were significantly decreased in isoniazid-treated mice.« less
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ERIC Educational Resources Information Center
Roy, Urmi
2016-01-01
This work presents a three-dimensional (3D) modeling exercise for undergraduate students in chemistry and health sciences disciplines, focusing on a protein-group linked to immune system regulation. Specifically, the exercise involves molecular modeling and structural analysis of tumor necrosis factor (TNF) proteins, both wild type and mutant. The…
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Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice.
Leon, L R; Kozak, W; Peschon, J; Kluger, M J
1997-02-01
We examined the effects of injections of systemic [lipopolysaccharide (LPS), 2.5 mg/kg or 50 pg/kg ip] or local (turpentine, 100 microl sc) inflammatory stimuli on fever, motor activity, body weight, and food intake in tumor necrosis factor (TNF) double receptor (TNFR)-knockout mice. A high dose of LPS resulted in exacerbated fevers in TNFR-knockout mice compared with wild-type mice for the early phase of fever (3-15 h); the late phase of fever (16-24 h) and fevers to a low dose of LPS were similar in both groups. Motor activity, body weight, and food intake were similarly reduced in both groups of mice after LPS administration. In response to turpentine, TNFR-knockout and wild-type mice developed virtually identical responses to all variables monitored. These results suggest that 1) TNF modulates fevers to LPS dose dependently, 2) TNF does not modulate fevers to a subcutaneous injection of turpentine, and 3) knockout mice may develop cytokine redundancy in the regulation of the acute phase response to intraperitoneally injected LPS or subcutaneously injected turpentine.
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Toyoda, M; Ihara, T; Nakano, T; Ito, M; Kamiya, H
1999-03-17
In response to two types of measles virus (MV) antigens, a vaccine strain CAM and a wild strain isolated in 1994, the expression of IL-2 receptor alpha (CD25)(+)CD45RO(+)CD4(+) T-lymphocytes (T-cell activation) was analyzed by flow cytometry. In 75 healthy subjects with measles hemagglutination inhibition tests > or =1:16, the percentage of T-cell activation was significantly increased compared with that in seronegative individuals (p) < 0.05). Moreover, the T-cell expression was not significantly different among the vaccinated (n = 38), the naturally infected (n = 28) and the subclinically infected (exposed with wild type without history of measles infection and HI titers > or =1:16) (n = 10) groups. T-cell activation stimulated with MV antigens and HI antibody titers persisted for almost 30 years in the vaccinated group. These results suggest that cell-mediated immunity persists for long periods after vaccination and does not be influenced by antigenic drift.
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Loke, K E; Messina, E J; Shesely, E G; Kaley, G; Hintze, T H
2001-01-01
Our aim was to investigate the potential therapeutic role of endothelial nitric oxide synthase (eNOS) in the modulation of cardiac O(2) consumption induced by the angiotensin converting enzyme (ACE) inhibitor ramiprilat and amlodipine. Three different groups of mice were used; wild type, wild type in the presence of N-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/l) or genetically altered mice lacking the eNOS gene (eNOS -/-). Myocardial O(2) consumption was measured using a Clark-type O(2) electrode in an air-tight stirred bath. Concentration-response curves to ramiprilat (RAM), amlodipine (AMLO), diltiazem (DIL), carbachol (CCL), substance P (SP) and S-nitroso-N-acetyl-penicillamine (SNAP) were performed. The rate of decrease in O(2) concentration was expressed as a percentage of the baseline. Baseline O(2) consumption was not different between the three groups of mice. In tissues from wild type mice, RAM (10(-5) mol/l), AMLO (10(-5) mol/l), DIL (10(-4) mol/l), CCL (10(-4) mol/l), SP (10(-7) mol/l) and SNAP (10(-4) mol/l) reduced myocardial O(2) consumption by -32+/-4, -27+/-10, -20+/-6, -25+/-2, -22+/-4 and -42+/-4%, respectively. The responses to RAM, AMLO, CCL and SP were absent in tissues taken from eNOS -/- mice (-7.1+/-4.3, -5.0+/-6.0, -5.2+/-5.1 and -0.4+/-0.2%, respectively). In addition, L-NAME significantly (P<0.05) inhibited the reduction in O(2) consumption induced by RAM (-9.8+/-4.4%), AMLO (-1.0+/-6.0%), CCL (-8.8+/-3.7%) and SP (-6.6+/-4.9%) in cardiac tissues from wild type mice. In contrast, NO-independent responses to the calcium channel antagonist, DIL, and responses to the NO donor, SNAP, were not affected in cardiac tissues taken from eNOS -/- (DIL: -20+/-4%; SNAP: -46+/-6%) or L-NAME-treated (DIL: -17+/-2%; SNAP: -33+/-5%) mice. These results suggest that endogenous endothelial NO synthase derived NO serves an important role in the regulation of myocardial O(2) consumption. This action may contribute to the therapeutic action of ACE inhibitors and amlodipine.
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Kim, Tae Won; Elme, Anneli; Park, Joon Oh; Udrea, Anghel Adrian; Kim, Sun Young; Ahn, Joong Bae; Valencia, Ricardo Villalobos; Krishnan, Srinivasan; Manojlovic, Nebojsa; Guan, Xuesong; Lofton-Day, Catherine; Jung, A Scott; Vrdoljak, Eduard
2018-03-21
Tumor rat sarcoma gene (RAS) status is a negative predictive biomarker for anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC). We analyzed outcomes according to RAS and v-Raf murine sarcoma viral oncogene homolog B (BRAF) mutational status, and evaluated early tumor shrinkage (ETS) and depth of response (DpR) for patients with wild type RAS. Patients with confirmed metastatic colon or rectum adenocarcinoma, wild type Kristen rat sarcoma gene tumor exon 2 status, clinical/radiologic disease progression or toxicity during irinotecan or oxaliplatin treatment, and no previous anti-EGFR therapy were randomized 1:1 to receive best supportive care (BSC) with or without panitumumab (6.0 mg/kg, intravenously, on day 1 of each 14-day cycle) in this open-label, multicenter, phase III study (20100007). RAS and BRAF mutation status were determined using Sanger sequencing. ETS was evaluated as maximum percentage change from baseline to week 8; DpR was calculated as the percentage change for tumor shrinkage at nadir versus baseline. Overall, 270 patients had RAS wild type mCRC (panitumumab with BSC, n = 142; BSC, n = 128). For patients with wild type RAS tumors, median overall survival (OS; hazard ratio [HR], 0.72; P = .015) and progression-free survival (PFS; HR, 0.45; P < .0001) were improved with panitumumab with BSC versus BSC. Similar improvements were seen for patients with wild type RAS, and wild type BRAF tumors (OS: HR, 0.75; P = .04; PFS: HR, 0.45; P < .0001). Median DpR was 16.9% for the evaluable panitumumab with BSC wild type RAS population. Overall, 69.5% experienced any type of tumor shrinkage at week 8; 38.2% experienced ≥ 20% shrinkage. Similar improvements in OS and PFS were seen with stratification according to ETS. This analysis showed that panitumumab improved outcomes in wild type RAS mCRC and indicated that ETS and DpR could be used as additional efficacy markers. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
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Aggressive behavior of the white-eye mutant crickets, Gryllus bimaculatus.
Sakura, Midori; Watanabe, T; Aonuma, H
2012-01-01
Aggressive behavior of white-eye mutant crickets was investigated and compared with that of wild-type crickets. In the dark, wild-type pairs performed long-lasting fights with significantly higher aggressive levels compared to those in the light. In contrast, fights between two white-eye mutants were not significantly different with those between two wild-type crickets both in duration and the aggressive levels. Ethograms of aggressive behavior showed that the mutants could show typical sequentially escalating fight with the same behavioral categories as the wild-type crickets. These results indicate that the white-eye mutants are able to express normal aggressive behavior.
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Tajima, Satoshi; Itoh, Yoko; Sugimoto, Takashi; Kato, Tatsuya; Park, Enoch Y
2009-10-01
The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.
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Seltmann, Anne; Majolo, Bonaventura
2013-01-01
Social animals have to coordinate activities and collective movements to benefit from the advantages of group living. Animals in large groups maintain cohesion by self-organization processes whereas in smaller groups consensus decisions can be reached. Where consensus decisions are relevant leadership may emerge. Variation in the organization of collective movements has been linked to variation in female social tolerance among macaque species ranging from despotic to egalitarian. Here we investigated the processes underlying group movements in a wild macaque species characterized by a degree of social tolerance intermediate to previously studied congeneric species. We focused on processes before, during and after the departure of the first individual. To this end, we observed one group of wild Barbary macaques (Macaca sylvanus) in the Middle Atlas, Morocco using all-occurrence behaviour sampling of 199 collective movements. We found that initiators of a collective movement usually chose the direction in which more individuals displayed pre-departure behavior. Dominant individuals contributed to group movements more than subordinates, especially juveniles, measured as frequencies of successful initiations and pre-departure behaviour. Joining was determined by affiliative relationships and the number of individuals that already joined the movement (mimetism). Thus, in our study group partially shared consensus decisions mediated by selective mimetism seemed to be prevalent, overall supporting the suggestion that a species’ social style affects the organization of group movements. As only the most tolerant species show equally shared consensus decisions whereas in others the decision is partially shared with a bias to dominant individuals the type of consensus decisions seems to follow a stepwise relation. Joining order may also follow a stepwise, however opposite, relationship, because dominance only determined joining in highly despotic, but not in intermediate and tolerant species. PMID:23805305
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Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B.
Fukasawa, Kayoko M; Hirose, Junzo; Hata, Toshiyuki; Ono, Yukio
2006-09-26
Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.
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Vanleene, Maximilien; Shefelbine, Sandra J.
2013-01-01
Osteogenesis imperfecta (OI) is characterized by extremely brittle bone. Currently, bisphosphonate drugs allow a decrease of fracture by inhibiting bone resorption and increasing bone mass but with possible long term side effects. Whole body mechanical vibrations (WBV) treatment may offer a promising route to stimulate bone formation in OI patients as it has exhibited health benefits on both muscle and bone mass in human and animal models. The present study has investigated the effects of WBV (45 Hz, 0.3 g, 15 minutes/days, 5 days/week) in young OI (oim) and wild type female mice from 3 to 8 weeks of age. Vibration therapy resulted in a significant increase in the cortical bone area and cortical thickness in the femur and tibia diaphysis of both vibrated oim and wild type mice compared to sham controls. Trabecular bone was not affected by vibration in the wild type mice; vibrated oim mice, however, exhibited significantly higher trabecular bone volume fraction in the proximal tibia. Femoral stiffness and yield load in three point bending were greater in the vibrated wild type mice than in sham controls, most likely attributed to the increase in femur cortical cross sectional area observed in the μCT morphology analyses. The vibrated oim mice showed a trend toward improved mechanical properties, but bending data had large standard deviations and there was no significant difference between vibrated and non-vibrated oim mice. No significant difference of the bone apposition was observed in the tibial metaphyseal trabecular bone for both the oim and wild type vibrated mice by histomorphometry analyses of calcein labels. At the mid diaphysis, the cortical bone apposition was not significantly influenced by the WBV treatment in both the endosteum and periosteum of the oim vibrated mice while a significant change is observed in the endosteum of the vibrated wild type mice. As only a weak impact in bone apposition between the vibrated and sham groups is observed in the histological sections, it is possible that WBV reduced bone resorption, resulting in a relative increase in cortical thickness. Whole body vibration appears as a potential effective and innocuous means for increasing bone formation and strength, which is particularly attractive for treating the growing skeleton of children suffering from brittle bone disease or low bone density pathologies without the long term disadvantages of current pharmacological therapies. PMID:23352925
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NASA Astrophysics Data System (ADS)
Goo, Yong Sook; Ye, Jang Hee; Lee, Seokyoung; Nam, Yoonkey; Ryu, Sang Baek; Kim, Kyung Hwan
2011-06-01
Retinal prostheses are being developed to restore vision for those with retinal diseases such as retinitis pigmentosa or age-related macular degeneration. Since neural prostheses depend upon electrical stimulation to control neural activity, optimal stimulation parameters for successful encoding of visual information are one of the most important requirements to enable visual perception. In this paper, we focused on retinal ganglion cell (RGC) responses to different stimulation parameters and compared threshold charge densities in wild-type and rd1 mice. For this purpose, we used in vitro retinal preparations of wild-type and rd1 mice. When the neural network was stimulated with voltage- and current-controlled pulses, RGCs from both wild-type and rd1 mice responded; however the temporal pattern of RGC response is very different. In wild-type RGCs, a single peak within 100 ms appears, while multiple peaks (approximately four peaks) with ~10 Hz rhythm within 400 ms appear in RGCs in the degenerated retina of rd1 mice. We find that an anodic phase-first biphasic voltage-controlled pulse is more efficient for stimulation than a biphasic current-controlled pulse based on lower threshold charge density. The threshold charge densities for activation of RGCs both with voltage- and current-controlled pulses are overall more elevated for the rd1 mouse than the wild-type mouse. Here, we propose the stimulus range for wild-type and rd1 retinas when the optimal modulation of a RGC response is possible.
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Soda, Hitoshi; Maeda, Hiromichi; Hasegawa, Junichi; Takahashi, Takao; Hazama, Shoichi; Fukunaga, Mutsumi; Kono, Emiko; Kotaka, Masahito; Sakamoto, Junichi; Nagata, Naoki; Oba, Koji; Mishima, Hideyuki
2015-10-14
The clinical benefit of cetuximab combined with oxaliplatin-based chemotherapy remains under debate. The aim of the present multicenter open-label Phase II study was to explore the efficacy and safety of biweekly administration of cetuximab and mFOLFOX-6 or XELOX as first-line chemotherapy in patients with metastatic colorectal cancer. Sixty-two patients with previously untreated KRAS/BRAF wild-type metastatic colorectal cancer were recruited to the study between April 2010 and May 2011. Patients received one of two treatment regimens, either cetuximab plus mFOLFOX-6 (FOLFOX + Cmab) or cetuximab plus biweekly XELOX (XELOX + Cmab), according to their own preference. Treatment was continued until disease progression or the appearance of intolerable toxicities. The primary endpoint was response rate; secondary endpoints were progression-free survival, overall survival, disease control rate, dose intensity, conversion rate to surgical resection, and safety. The response rates in the FOLFOX + Cmab (n = 37) and XELOX + Cmab (n = 25) groups were 64.9 % (24/37) and 72.0 % (18/25), respectively. The median PFS in the FOLFOX + Cmab and XELOX + Cmab groups was 13.1 months (95 % confidence interval [CI] 12.1-17.5) and 13.4 months (95 % CI 10.1-17.9), respectively. Neutropenia was the most frequent grade 3/4 adverse event in both groups (33.9 %), followed by anorexia, acneiform eruption, skin fissure and paronychia. A waterfall plot of tumor diameter showed prominent shrinkage of the tumors in 88.7 % of patients. The results of the present study indicate that biweekly cetuximab plus mFOLFOX-6/XELOX is an effective and tolerable treatment regimen. Biweekly administration of cetuximab requires only one hospital visit every 2 weeks, and may become a convenient treatment option for patients with KRAS/BRAF wild-type metastatic colorectal cancer. This study is registered with University Hospital Medical Information Network (UMIN 000003253 ). Registration date is 02/24/2010.
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Nfib hemizygous mice are protected from hyperoxic lung injury and death.
Kumar, Vasantha H S; Chaker El Khoury, Joseph; Gronostajski, Richard; Wang, Huamei; Nielsen, Lori; Ryan, Rita M
2017-08-01
Nuclear Factor I ( Nfi) genes encode transcription factors essential for the development of organ systems including the lung. Nfib null mice die at birth with immature lungs. Nfib hemizygous mice have reduced lung maturation with decreased survival. We therefore hypothesized that these mice would be more sensitive to lung injury and would have lower survival to hyperoxia. Adult Nfib hemizygous mice and their wild-type (Wt) littermates were exposed to 100% O 2 for 89, 80, 72 and 66 h for survival studies with lung outcome measurements at 66 h. Nfib hemizygous and Wt controls were also studied in RA at 66 h. Cell counts and cytokines were measured in bronchoalveolar lavage (BAL); lung sections examined by histopathology; lung angiogenic and oxidative stress gene expression assessed by real-time PCR Unexpectedly, Nfib hemizygous mice (0/14-0%) had significantly lower mortality compared to Wt mice (10/22-45%) at 80 h of hyperoxia ( P < 0.003). LD 50 was 80 h in the Wt group versus 89 h in the hemizygous group. There were no differences in BAL cell counts between the groups. Among the cytokines studied, MIP-2 was significantly lower in hemizygous mice exposed to hyperoxia. New vessel formation, edema, congestion, and alveolar hemorrhage were noted on histopathology at 72 and 80 h in wild-type mice. Nfib hemizygous lungs had significant downregulation of genes involved in redox signaling and inflammatory pathways. Adult Nfib hemizygous mice are relatively resistant to hyperoxia compared to wild-type littermates. Mechanisms contributing to this resistance are not clear; however, transcription factors such as Nfib may regulate cell survival and play a role in modulating postnatal lung development. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.
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Molecular Determinants of the Cellular Entry of Asymmetric Peptide Dendrimers and Role of Caveolae.
Rewatkar, Prarthana V; Parekh, Harendra S; Parat, Marie-Odile
2016-01-01
Caveolae are flask-shaped plasma membrane subdomains abundant in most cell types that participate in endocytosis. Caveola formation and functions require membrane proteins of the caveolin family, and cytoplasmic proteins of the cavin family. Cationic peptide dendrimers are non-vesicular chemical carriers that can transport pharmacological agents or genetic material across the plasma membrane. We prepared a panel of cationic dendrimers and investigated whether they require caveolae to enter into cells. Cell-based studies were performed using wild type or caveola-deficient i.e. caveolin-1 or PTRF gene-disrupted cells. There was a statistically significant difference in entry of cationic dendrimers between wild type and caveola-deficient cells. We further unveiled differences between dendrimers with varying charge density and head groups. Our results show, using a molecular approach, that (i) expression of caveola-forming proteins promotes cellular entry of cationic dendrimers and (ii) dendrimer structure can be modified to promote endocytosis in caveola-forming cells.
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Molecular Determinants of the Cellular Entry of Asymmetric Peptide Dendrimers and Role of Caveolae
Rewatkar, Prarthana V.; Parekh, Harendra S.; Parat, Marie-Odile
2016-01-01
Caveolae are flask-shaped plasma membrane subdomains abundant in most cell types that participate in endocytosis. Caveola formation and functions require membrane proteins of the caveolin family, and cytoplasmic proteins of the cavin family. Cationic peptide dendrimers are non-vesicular chemical carriers that can transport pharmacological agents or genetic material across the plasma membrane. We prepared a panel of cationic dendrimers and investigated whether they require caveolae to enter into cells. Cell-based studies were performed using wild type or caveola-deficient i.e. caveolin-1 or PTRF gene-disrupted cells. There was a statistically significant difference in entry of cationic dendrimers between wild type and caveola-deficient cells. We further unveiled differences between dendrimers with varying charge density and head groups. Our results show, using a molecular approach, that (i) expression of caveola-forming proteins promotes cellular entry of cationic dendrimers and (ii) dendrimer structure can be modified to promote endocytosis in caveola-forming cells. PMID:26788849
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Evolutionary history of the NAM-B1 gene in wild and domesticated tetraploid wheat.
Lundström, Maria; Leino, Matti W; Hagenblad, Jenny
2017-12-20
The NAM-B1 gene in wheat has for almost three decades been extensively studied and utilized in breeding programs because of its significant impact on grain protein and mineral content and pleiotropic effects on senescence rate and grain size. First detected in wild emmer wheat, the wild-type allele of the gene has been introgressed into durum and bread wheat. Later studies have, however, also found the presence of the wild-type allele in some domesticated subspecies. In this study we trace the evolutionary history of the NAM-B1 in tetraploid wheat species and evaluate it as a putative domestication gene. Genotyping of wild and landrace tetraploid accessions showed presence of only null alleles in durum. Domesticated emmer wheats contained both null alleles and the wild-type allele while wild emmers, with one exception, only carried the wild-type allele. One of the null alleles consists of a deletion that covers several 100 kb. The other null-allele, a one-basepair frame-shift insertion, likely arose among wild emmer. This allele was the target of a selective sweep, extending over several 100 kb. The NAM-B1 gene fulfils some criteria for being a domestication gene by encoding a trait of domestication relevance (seed size) and is here shown to have been under positive selection. The presence of both wild-type and null alleles in domesticated emmer does, however, suggest the gene to be a diversification gene in this species. Further studies of genotype-environment interactions are needed to find out under what conditions selection on different NAM-B1 alleles have been beneficial.
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Altered respiratory responses to hypoxia in mutant mice deficient in neuronal nitric oxide synthase
Kline, David D; Yang, Tianen; Huang, Paul L; Prabhakar, Nanduri R
1998-01-01
The role of endogenous nitric oxide (NO) generated by neuronal nitric oxide synthase (NOS-1) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS-1. Experiments were performed on awake and anaesthetized mutant and wild-type control mice. Respiratory responses to varying levels of inspired oxygen (100, 21 and 12 % O2) and carbon dioxide (3 and 5 % CO2 balanced oxygen) were analysed. In awake animals, respiration was monitored by body plethysmograph along with oxygen consumption (V̇O2), CO2 production (V̇CO2) and body temperature. In anaesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Cyclic 3′,5′-guanosine monophosphate (cGMP) levels in the brainstem were analysed by radioimmunoassay as an index of nitric oxide generation. Unanaesthetized mutant mice exhibited greater respiratory responses during 21 and 12 % O2 than the wild-type controls. Respiratory responses were associated with significant decreases in oxygen consumption in both groups of mice, and the magnitude of change was greater in mutant than wild-type mice. Changes in CO2 production and body temperature, however, were comparable between both groups of mice. Similar augmentation of respiratory responses during hypoxia was also observed in anaesthetized mutant mice. In addition, five of the fourteen mutant mice displayed periodic oscillations in respiration (brief episodes of increases in respiratory rate and tidal phrenic nerve activity) while breathing 21 and 12 % O2, but not during 100 % O2. The time interval between the episodes decreased by reducing inspired oxygen from 21 to 12 % O2. Changes in arterial blood pressure and arterial blood gases were comparable at any given level of inspired oxygen between both groups of mice, indicating that changes in these variables do not account for the differences in the response to hypoxia. Respiratory responses to brief hyperoxia (Dejours test) and to cyanide, a potent chemoreceptor stimulant, were more pronounced in mutant mice, suggesting augmented peripheral chemoreceptor sensitivity. cGMP levels were elevated in the brainstem during 21 and 12 % O2 in wild-type but not in mutant mice, indicating decreased formation of nitric oxide in mutant mice. The magnitude of respiratory responses to hypercapnia (3 and 5 % CO2 balanced oxygen) was comparable in both groups of mice in the awake and anaesthetized conditions. These observations suggest that the hypoxic responses were selectively augmented in mutant mice deficient in NOS-1. Peripheral as well as central mechanisms contributed to the altered responses to hypoxia. These results support the idea that nitric oxide generated by NOS-1 is an important physiological modulator of respiration during hypoxia. PMID:9679181
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Li, Mo-Lin; Li, Chuan-Gang; Shu, Xiao-Hong; Li, Ming-Xia; Jia, Yu-Jie; Qin, Zhi-Hai
2007-11-01
To investigate the immunological mechanism of anti-tumor effect of 5-FU by establishing lymphoma EL4 tumor-bearing mouse models in wild type C57BL/6 mice and nude C57BL/6 mice, respectively. The mouse lymphoma EL4 cells were inoculated subcutaneously into wild type C57BL/6 mice (immune-competent mice). Twelve days later, 5-FU of different doses was administered intraperitoneally to treat these wild type C57BL/6 tumor-bearing mice. The size of tumors in the wild type C57BL/6 mice was observed and recorded to explore the minimal dose of 5-FU that could cure the tumor-bearing mice. Then the same amount of EL4 tumor cells was inoculated subcutaneously into wild type C57BL/6 mice and nude C57BL/6 mice (T cell-deficient mice) simultaneously, which had the same genetic background of C57BL/6. Twelve days later, 5-FU of the minimal dose was given intraperitoneally to treat both the wild type and nude C57BL/6 tumor-bearing mice. The size of tumors in the two different types of mice was observed and recorded. A single dose of 5-FU (75 mg/kg) cured both the EL4 tumor-bearing wild type C57BL/6 mice and the EL4 tumor-bearing nude C57BL/6 mice in the first week. Two weeks after 5-FU treatment, all of the nude mice died of tumor relapse while most of the wild type C57BL/6 mice were fully recovered. A single dose of 5-FU has marked anti-tumor effects on lymphoma EL4 tumor-bearing C57BL/6 mice with or without T lymphocytes. The relapse of tumors after 5-FU treatment might be related to the function of T lymphocytes.
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Hammer, Ralf; Ritzmann, Mathias; Palzer, Andreas; Lang, Christiane; Hammer, Birgit; Pesch, Stefan; Ladinig, Andrea
2012-01-01
Samples were collected from 203 wild boars (Sus scrofa) hunted in Baden-Wurtemburg, Germany from November-January 2008 and 2009. Samples from the lung and tonsil were analyzed by quantitative polymerase chain reaction (qPCR) for porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European type) and type 2 (American type). A qPCR to detect porcine circovirus type 2 (PCV2)-specific genome was performed on tissue homogenates including lung, tonsils, and inguinal lymph nodes. Serum samples were tested for antibodies against PRRSV and PCV2 by enzyme-linked immunosorbent assay (ELISA). No PRRSV was detected in any of the 203 samples and one sample had detectable antibodies against PRRSV. We detected PCV2 in organ materials from 103 wild boars with a prevalence of 50.7%. The number of wild boars positive for PCV2 by PCR varied according to the population density of wild boars among woodlands. More positive samples were detected in woodlands with a high density of wild boars. We found no correlation between the number of PCV2-positive wild boars and the density of domestic pigs in the surrounding area. The number of wild boars positive for antibodies against PCV2 by the INGEZIM Circovirus IgG/IgM test kit was low (53 sera positive for IgG- and three sera positive for IgM-antibodies) in comparison to the higher positive results from the INGEZIM CIRCO IgG test kit (102 positive and 12 inconclusive results).
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Singh, Balwant; Singh, Nisha; Mishra, Shefali; Tripathi, Kabita; Singh, Bikram P; Rai, Vandna; Singh, Ashok K; Singh, Nagendra K
2018-01-01
Wild relatives of crops possess adaptive mutations for agronomically important traits, which could play significant role in crop improvement for sustainable agriculture. However, global climate change and human activities pose serious threats to the natural habitats leading to erosion of genetic diversity of wild rice populations. The purpose of this study was to explore and characterize India's huge untapped wild rice diversity in Oryza rufipogon Griff. species complex from a wide range of ecological niches. We made strategic expeditions around diversity hot spots in 64 districts of nine different agro-climatic zones of the country and collected 418 wild rice accessions. Significant variation was observed among the accessions for 46 morphological descriptors, allowing classification into O. nivara, O. rufipogon , and O. sativa f. spontanea morpho-taxonomic groups. Genome-specific pSINE1 markers confirmed all the accessions having AA genome, which were further classified using ecotype-specific pSINE1 markers into annual, perennial, intermediate, and an unknown type. Principal component analysis revealed continuous variation for the morphological traits in each ecotype group. Genetic diversity analysis based on multi-allelic SSR markers clustered these accessions into three major groups and analysis of molecular variance for nine agro-climatic zones showed that 68% of the genetic variation was inherent amongst individuals while only 11% of the variation separated the zones, though there was significant correlation between genetic and spatial distances of the accessions. Model based population structure analysis using genome wide bi-allelic SNP markers revealed three sub-populations designated 'Pro-Indica,' 'Pro-Aus,' and 'Mid-Gangetic,' which showed poor correspondence with the morpho - taxonomic classification or pSINE1 ecotypes. There was Pan-India distribution of the 'Pro-Indica' and 'Pro-Aus' sub-populations across agro-climatic zones, indicating a more fundamental grouping based on the ancestry closely related to 'Indica' and 'Aus' groups of rice cultivars. The Pro-Indica population has substantial presence in the Eastern Himalayan Region and Lower Gangetic Plains, whereas 'Pro-Aus' sub-population was predominant in the Upper Gangetic Plains, Western Himalayan Region, Gujarat Plains and Hills, and Western Coastal Plains. In contrast 'Mid-Gangetic' population was largely concentrated in the Mid Gangetic Plains. The information presented here will be useful in the utilization of wild rice resources for varietal improvement.
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Sata, F; Sapone, A; Elizondo, G; Stocker, P; Miller, V P; Zheng, W; Raunio, H; Crespi, C L; Gonzalez, F J
2000-01-01
To determine the existence of mutant and variant CgammaP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression. A bacterial artificial chromosome that contains the complete CgammaP3A4 gene was isolated and the exons and surrounding introns were directly sequenced to develop primers to polymerase chain reaction (PCR) amplify and sequence the gene from lymphocyte DNA. DNA samples from Chinese, black, and white subjects were screened. Mutating the affected amino acid in the wild-type cDNA and expressing the variant enzyme with use of the baculovirus system was used to functionally evaluate the variant allele having a missense mutation. To investigate the existence of mutant and variant CgammaP3A4 alleles in humans, all 13 exons and the 5'-flanking region of the human CgammaP3A4 gene in three racial groups were sequenced and four alleles were identified. An A-->G point mutation in the 5'-flanking region of the human CgammaP3A4 gene, designated CgammaP3A4*1B, was found in the three different racial groups. The frequency of this allele in a white population was 4.2%, whereas it was 66.7% in black subjects. The CgammaP3A4*1B allele was not found in Chinese subjects. A second variant allele, designated CgammaP3A4*2, having a Ser222Pro change, was found at a frequency of 2.7% in the white population and was absent in the black subjects and Chinese subjects analyzed. Baculovirus-directed cDNA expression revealed that the CYP3A4*2 P450 had a lower intrinsic clearance for the CYP3A4 substrate nifedipine compared with the wild-type enzyme but was not significantly different from the wild-type enzyme for testosterone 6beta-hydroxylation. Another rare allele, designated CgammaP3A4*3, was found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450. These are the first examples of potential function polymorphisms resulting from missense mutations in the CgammaP3A4 gene. The CgammaP3A4*2 allele was found to encode a P450 with substrate-dependent altered kinetics compared with the wild-type P450.
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Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J.; Critser, John; Arwert, Fre; Haneline, Laura S.; Clapp, D. Wade
2006-01-01
Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and Fancg–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients. PMID:16946306
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Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J; Critser, John; Arwert, Fre; Haneline, Laura S; Clapp, D Wade
2006-12-15
Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.
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Guo, Rongxian; Jiao, Yang; Li, Zhuoyang; Zhu, Shanshan; Fei, Xiao; Geng, Shizhong; Pan, Zhiming; Chen, Xiang; Li, Qiuchun; Jiao, Xinan
2017-01-01
Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum ( Salmonella Pullorum) is highly adapted to chickens causing an acute systemic disease that results in high mortality. Vaccination represents one approach for promoting animal health, food safety and reducing environmental persistence in Salmonella control. An important consideration is that Salmonella vaccination in poultry should not interfere with the salmonellosis monitoring program. This is the basis of the DIVA (Differentiation of Infected and Vaccinated Animals) program. In order to achieve this goal, waaL mutant was developed on a spiC mutant that was developed previously. The safety, efficacy, and DIVA features of this vaccine candidate ( Salmonella Pullorum Δ spiC Δ waaL ) were evaluated in broilers. Our results show that the truncated LPS in the vaccine strain has a differentiating use as both a bacteriological marker (rough phenotype) and also as a serological marker facilitating the differentiation between infected and vaccinated chickens. The rough mutant showed adequate safety being avirulent in the host chicks and showed increased sensitivity to environmental stresses. Single intramuscular immunization of day-old broiler chicks with the mutant confers ideal protection against lethal wild type challenge by significantly stimulating both humoral and cellular immune responses as well as reducing the colonization of the challenge strain. Significantly lower mean pathology scores were observed in the vaccination group compared to the control group. Additionally, the mutant strain generated cross-protection against challenge with the wild type Salmonella Gallinarum thereby improving survival and with the wild type Salmonella Enteritidis thereby reducing colonization. These results suggest that the double-mutant strain may be a safe, effective, and cross-protective vaccine against Salmonella infection in chicks while conforming to the requirements of the DIVA program.
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Bouvier d'Yvoire, Madeleine; Bouchabke-Coussa, Oumaya; Voorend, Wannes; Antelme, Sébastien; Cézard, Laurent; Legée, Frédéric; Lebris, Philippe; Legay, Sylvain; Whitehead, Caragh; McQueen-Mason, Simon J; Gomez, Leonardo D; Jouanin, Lise; Lapierre, Catherine; Sibout, Richard
2013-02-01
Brachypodium distachyon (Brachypodium) has been proposed as a model for grasses, but there is limited knowledge regarding its lignins and no data on lignin-related mutants. The cinnamyl alcohol dehydrogenase (CAD) genes involved in lignification are promising targets to improve the cellulose-to-ethanol conversion process. Down-regulation of CAD often induces a reddish coloration of lignified tissues. Based on this observation, we screened a chemically induced population of Brachypodium mutants (Bd21-3 background) for red culm coloration. We identified two mutants (Bd4179 and Bd7591), with mutations in the BdCAD1 gene. The mature stems of these mutants displayed reduced CAD activity and lower lignin content. Their lignins were enriched in 8-O-4- and 4-O-5-coupled sinapaldehyde units, as well as resistant inter-unit bonds and free phenolic groups. By contrast, there was no increase in coniferaldehyde end groups. Moreover, the amount of sinapic acid ester-linked to cell walls was measured for the first time in a lignin-related CAD grass mutant. Functional complementation of the Bd4179 mutant with the wild-type BdCAD1 allele restored the wild-type phenotype and lignification. Saccharification assays revealed that Bd4179 and Bd7591 lines were more susceptible to enzymatic hydrolysis than wild-type plants. Here, we have demonstrated that BdCAD1 is involved in lignification of Brachypodium. We have shown that a single nucleotide change in BdCAD1 reduces the lignin level and increases the degree of branching of lignins through incorporation of sinapaldehyde. These changes make saccharification of cells walls pre-treated with alkaline easier without compromising plant growth. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.
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Quantitative assessment of integrated phrenic nerve activity.
Nichols, Nicole L; Mitchell, Gordon S
2016-06-01
Integrated electrical activity in the phrenic nerve is commonly used to assess within-animal changes in phrenic motor output. Because of concerns regarding the consistency of nerve recordings, activity is most often expressed as a percent change from baseline values. However, absolute values of nerve activity are necessary to assess the impact of neural injury or disease on phrenic motor output. To date, no systematic evaluations of the repeatability/reliability have been made among animals when phrenic recordings are performed by an experienced investigator using standardized methods. We performed a meta-analysis of studies reporting integrated phrenic nerve activity in many rat groups by the same experienced investigator; comparisons were made during baseline and maximal chemoreceptor stimulation in 14 wild-type Harlan and 14 Taconic Sprague Dawley groups, and in 3 pre-symptomatic and 11 end-stage SOD1(G93A) Taconic rat groups (an ALS model). Meta-analysis results indicate: (1) consistent measurements of integrated phrenic activity in each sub-strain of wild-type rats; (2) with bilateral nerve recordings, left-to-right integrated phrenic activity ratios are ∼1.0; and (3) consistently reduced activity in end-stage SOD1(G93A) rats. Thus, with appropriate precautions, integrated phrenic nerve activity enables robust, quantitative comparisons among nerves or experimental groups, including differences caused by neuromuscular disease. Copyright © 2015 Elsevier B.V. All rights reserved.
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Sun, Mingbo; Li, Changgui; Xu, Wenbo; Liao, Guoyang; Li, Rongcheng; Zhou, Jian; Li, Yanping; Cai, Wei; Yan, Dongmei; Che, Yanchun; Ying, Zhifang; Wang, Jianfeng; Yang, Huijuan; Ma, Yan; Ma, Lei; Ji, Guang; Shi, Li; Jiang, Shude; Li, Qihan
2017-05-15
A Sabin strain-based inactivated poliomyelitis vaccine (Sabin-IPV) is the rational option for completely eradicating poliovirus transmission. The neutralizing capacity of Sabin-IPV immune serum to different strains of poliovirus is a key indicator of the clinical protective efficacy of this vaccine. Sera collected from 500 infants enrolled in a randomized, blinded, positive control, phase 2 clinical trial were randomly divided into 5 groups: Groups A, B, and C received high, medium, and low doses, respectively, of Sabin-IPV, while groups D and E received trivalent oral polio vaccine and Salk strain-based IPV, respectively, all on the same schedule. Immune sera were collected after the third dose of primary immunization, and tested in cross-neutralization assays against 19 poliovirus strains of all 3 types. All immune sera from all 5 groups interacted with the 19 poliovirus strains with various titers and in a dose-dependent manner. One type 2 immunodeficiency-associated vaccine-derived poliovirus strain was not recognized by these immune sera. Sabin-IPV vaccine can induce protective antibodies against currently circulating and reference wild poliovirus strains and most vaccine-derived poliovirus strains, with rare exceptions. NCT01056705. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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De novo establishment of wild-type song culture in the zebra finch
Feher, Olga; Wang, Haibin; Saar, Sigal; Mitra, Partha P.; Tchernichovski, Ofer
2009-01-01
What sort of culture would evolve in an island colony of naive founders? This question cannot be studied experimentally in humans. We performed the analogous experiment using socially learned birdsong. Culture is typically viewed as consisting of traits inherited epigenetically, via social learning. However, cultural diversity has species-typical constraints1, presumably of genetic origin. A celebrated, if contentious, example is whether a universal grammar constrains syntactic diversity in human languages2. Oscine songbirds exhibit song learning and provide biologically tractable models of culture: members of a species show individual variation in song3 and geographically separated groups have local song dialects 4,5. Different species exhibit distinct song cultures6,7, suggestive of genetic constraints8,9. Absent such constraints, innovations and copying errors should cause unbounded variation over multiple generations or geographical distance, contrary to observations9. We asked if wild-type song culture might emerge over multiple generations in an isolated colony founded by isolates, and if so, how this might happen and what type of social environment is required10. Zebra finch isolates, unexposed to singing males during development, produce song with characteristics that differ from the wild-type song found in laboratory11 or natural colonies. In tutoring lineages starting from isolate founders, we quantified alterations in song across tutoring generations in two social environments: tutor-pupil pairs in sound-isolated chambers and an isolated semi-natural colony. In both settings, juveniles imitated the isolate tutors, but changed certain characteristics of the songs. These alterations accumulated over learning generations. Consequently, songs evolved toward the wild-type in 3–4 generations. Thus, species-typical song culture can appear de novo. Our study has parallels with language change and evolution12,13. In analogy to models in quantitative genetics14,15, we model song culture as a multi-generational phenotype, partly encoded genetically in an isolate founding population, influenced by environmental variables, and taking multiple generations to emerge. PMID:19412161
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Barnabe, Christian; Buitrago, Rosio; Bremond, Philippe; Aliaga, Claudia; Salas, Renata; Vidaurre, Pablo; Herrera, Claudia; Cerqueira, Frédérique; Bosseno, Marie-France; Waleckx, Etienne; Breniere, Simone Frédérique
2013-01-01
Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into six discrete typing units (DTUs; TcI–TcVI) of which TcI is ubiquitous and genetically highly variable. While clonality is the dominant mode of propagation, recombinant events play a significant evolutive role. Recently, foci of wild Triatoma infestans have been described in Bolivia, mainly infected by TcI. Hence, for the first time, we evaluated the level of genetic exchange within TcI natural potentially panmictic populations (single DTU, host, area and sampling time). Seventy-nine TcI stocks from wild T. infestans, belonging to six populations were characterized at eight microsatellite loci. For each population, Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), and presence of repeated multilocus genotypes (MLG) were analyzed by using a total of seven statistics, to test the null hypothesis of panmixia (H0). For three populations, none of the seven statistics allowed to rejecting H0; for another one the low size did not allow us to conclude, and for the two others the tests have given contradictory results. Interestingly, apparent panmixia was only observed in very restricted areas, and was not observed when grouping populations distant of only two kilometers or more. Nevertheless it is worth stressing that for the statistic tests of "HWE", in order to minimize the type I error (i. e. incorrect rejection of a true H0), we used the Bonferroni correction (BC) known to considerably increase the type II error ( i. e. failure to reject a false H0). For the other tests (LD and MLG), we did not use BC and the risk of type II error in these cases was acceptable. Thus, these results should be considered as a good indicator of the existence of panmixia in wild environment but this must be confirmed on larger samples to reduce the risk of type II error. PMID:24312410
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Zhao, Ruozhi; Le, Khuong; Moghadasian, Mohammed H; Shen, Garry X
2013-08-01
Cardiovascular disease is the predominant cause of death in diabetic patients. Fibroblasts are one of the major types of cells in the heart or vascular wall. Increased levels of glycated low-density lipoprotein (glyLDL) were detected in diabetic patients. Previous studies in our group demonstrated that oxidized LDL increased the amounts of NADPH oxidase (NOX), plasminogen activator inhibitor-1 (PAI-1), and heat shock factor-1 (HSF1) in fibroblasts. This study examined the expression of NOX, PAI-1, and HSF1 in glyLDL-treated wild-type or HSF1-deficient mouse embryo fibroblasts (MEFs) and in leptin receptor-knockout (db/db) diabetic mice. Treatment with physiologically relevant levels of glyLDL increased superoxide and H2O2 release and the levels of NOX4 and p22phox (an essential component of multiple NOX complexes) in wild-type or HSF1-deficient MEFs. The levels of HSF1 and PAI-1 were increased by glyLDL in wild-type MEFs, but not in HSF1-deficient MEFs. Diphenyleneiodonium (a nonspecific NOX inhibitor) or small interfering RNA for p22phox prevented glyLDL-induced increases in the levels of NOX4, HSF1, or PAI-1 in MEFs. The amounts of NOX4, HSF1, and PAI-1 were elevated in hearts of db/db diabetic mice compared to wild-type mice. The results suggest that glyLDL increased the abundance of NOX4 or p22phox via an HSF1-independent pathway, but that of PAI-1 via an HSF1-dependent manner. NOX4 plays a crucial role in glyLDL-induced expression of HSF1 and PAI-1 in mouse fibroblasts. Increased expression of NOX4, HSF1, and PAI-1 was detected in cardiovascular tissue of diabetic mice. Copyright © 2013 Elsevier Inc. All rights reserved.
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Sigurdardóttir, Anna Gudný; Arnórsdóttir, Jóhanna; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Suhre, Karsten; Kristjánsson, Magnús M
2009-03-01
Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin I, have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin I. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR, Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by approximately 3 degrees C, both in T(50%) and T(m). Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity (k(cat)) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15DDeltaC) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation. Removal of the C-terminal arm improved the thermal stability (T(m) approximately +1.5 degrees C) of the truncated enzyme (VPRDeltaC) as compared to the wild type VPR. Introduction of the N15D substitution into VPRDeltaC improved the thermal stability further by about 3 degrees C, or to about the same extent as in the wild type. However, contrary to what was observed for the wild type, the introduction of the putative salt bridge did not affect the catalytic properties (k(cat)) of the C-terminal truncated enzyme.
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Pringle, Märit; Landén, Annica; Unnerstad, Helle Ericsson; Molander, Benedicta; Bengtsson, Björn
2012-09-21
The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed. The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum. The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78T (ATCC® 27164T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml. The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli.
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2012-01-01
Background The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed. Methods The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum. Results The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78T (ATCC® 27164T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml. Conclusions The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli. PMID:22998753
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Ohri, S K; Parratt, R; White, T; Becket, J; Brannan, J J; Hunt, B J; Taylor, K M
2001-05-01
A recombinant human serine protease inhibitor known as Kunitz protease inhibitor (KPI) wild type has functional similarities to the bovine Kunitz inhibitor, aprotinin, and had shown a potential to reduce bleeding in an ovine model of cardiopulmonary bypass (CPB). The aim of this study was to assess KPI-185, a modification of KPI-wild type that differs from KPI-wild type in two amino acid residues and which enhances anti-kallikrein activity in a further double-blind, randomized study in an ovine model of CPB, and to compare with our previous study of KPI-wild type and aprotinin in the same ovine model. Post-operative drain losses and subjective assessment of wound 'dryness' showed no significant differences between KPI-185 and KPI-wild type, despite the significant enhancement of kallikrein inhibition using KPI-185 seen in serial kallikrein inhibition assays. These preliminary findings support the hypothesis that kallikrein inhibition is not the major mechanism by which Kunitz inhibitors such as aprotinin reduce perioperative bleeding.
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Park, Miseon; Mitchell, Wilfrid J.
2016-01-01
Trehalose has been shown to protect bacterial cells from environmental stress. Its uptake and osmoprotective effect in Clostridium perfringens were investigated by comparing wild type C. perfringens ATCC 13124 with a fluoroquinolone- (gatifloxacin-) resistant mutant. In a chemically defined medium, trehalose and sucrose supported the growth of the wild type but not that of the mutant. Microarray data and qRT-PCR showed that putative genes for the phosphorylation and transport of sucrose and trehalose (via phosphoenolpyruvate-dependent phosphotransferase systems, PTS) and some regulatory genes were downregulated in the mutant. The wild type had greater tolerance than the mutant to salts and low pH; trehalose and sucrose further enhanced the osmotolerance of the wild type to NaCl. Expression of the trehalose-specific PTS was lower in the fluoroquinolone-resistant mutant. Protection of C. perfringens from environmental stress could therefore be correlated with the ability to take up trehalose. PMID:28058047
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Glucocorticoid Receptor Polymorphisms and Outcomes in Pediatric Septic Shock.
Cvijanovich, Natalie Z; Anas, Nick; Allen, Geoffrey L; Thomas, Neal J; Bigham, Michael T; Weiss, Scott L; Fitzgerald, Julie; Checchia, Paul A; Meyer, Keith; Quasney, Michael; Gedeit, Rainer; Freishtat, Robert J; Nowak, Jeffrey; Raj, Shekhar S; Gertz, Shira; Grunwell, Jocelyn R; Opoka, Amy; Wong, Hector R
2017-04-01
Polymorphisms of the glucocorticoid receptor gene are associated with outcome and corticosteroid responsiveness among patients with inflammatory disorders. We conducted a candidate gene association study to test the hypothesis that these polymorphisms are associated with outcome and corticosteroid responsiveness among children with septic shock. We genotyped 482 children with septic shock for the presence of two glucocorticoid receptor polymorphisms (rs56149945 and rs41423247) associated with increased sensitivity and one glucocorticoid receptor polymorphism (rs6198) associated with decreased sensitivity to corticosteroids. The primary outcome variable was complicated course, defined as 28-day mortality or the persistence of two or more organ failures 7 days after a septic shock diagnosis. We used logistic regression to test for an association between corticosteroid exposure and outcome, within genotype group, and adjusted for illness severity. Multiple PICUs in the United States. Standard care. There were no differences in outcome when comparing the various genotype groups. Among patients homozygous for the wild-type glucocorticoid receptor allele, corticosteroids were independently associated with increased odds of complicated course (odds ratio, 2.30; 95% CI, 1.01-5.21; p = 0.047). Based on these glucocorticoid receptor polymorphisms, we could not detect a beneficial effect of corticosteroids among any genotype group. Among children homozygous for the wild-type allele, corticosteroids were independently associated with increased odds of poor outcome.
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Mesenchymal Stem Cells Improve Motor Functions and Decrease Neurodegeneration in Ataxic Mice
Jones, Jonathan; Estirado, Alicia; Redondo, Carolina; Pacheco-Torres, Jesus; Sirerol-Piquer, Maria-Salomé; Garcia-Verdugo, José M; Martinez, Salvador
2015-01-01
The main objective of this work is to demonstrate the feasibility of using bone marrow-derived stem cells in treating a neurodegenerative disorder such as Friedreich's ataxia. In this disease, the dorsal root ganglia of the spinal cord are the first to degenerate. Two groups of mice were injected intrathecally with mesenchymal stem cells isolated from either wild-type or Fxntm1Mkn/Tg(FXN)YG8Pook (YG8) mice. As a result, both groups presented improved motor skills compared to nontreated mice. Also, frataxin expression was increased in the dorsal root ganglia of the treated groups, along with lower expression of the apoptotic markers analyzed. Furthermore, the injected stem cells expressed the trophic factors NT3, NT4, and BDNF, which bind to sensory neurons of the dorsal root ganglia and increase their survival. The expression of antioxidant enzymes indicated that the stem cell-treated mice presented higher levels of catalase and GPX-1, which are downregulated in the YG8 mice. There were no significant differences in the use of stem cells isolated from wild-type and YG8 mice. In conclusion, bone marrow mesenchymal stem cell transplantation, both autologous and allogeneic, is a feasible therapeutic option to consider in delaying the neurodegeneration observed in the dorsal root ganglia of Friedreich's ataxia patients. PMID:25070719
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USDA-ARS?s Scientific Manuscript database
Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while th...
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Higgins, LeeAnn; Markowski, Todd; Brambl, Robert
2016-01-01
A moderate heat shock induces Neurospora crassa to synthesize large quantities of heat shock proteins that are protective against higher, otherwise lethal temperatures. However, wild type cells do not survive when carbohydrate deprivation is added to heat shock. In contrast, a mutant strain defective in a stress-activated protein kinase does survive the combined stresses. In order to understand the basis for this difference in survival, we have determined the relative levels of detected proteins in the mutant and wild type strain during dual stress, and we have identified gene transcripts in both strains whose quantities change in response to heat shock or dual stress. These data and supportive experimental evidence point to reasons for survival of the mutant strain. By using alternative respiratory mechanisms, these cells experience less of the oxidative stress that proves damaging to wild type cells. Of central importance, mutant cells recycle limited resources during dual stress by undergoing autophagy, a process that we find utilized by both wild type and mutant cells during heat shock. Evidence points to inappropriate activation of TORC1, the central metabolic regulator, in wild type cells during dual stress, based upon behavior of an additional signaling mutant and inhibitor studies. PMID:27870869
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NASA Technical Reports Server (NTRS)
Wagner, T. A.; Cove, D. J.; Sack, F. D.
1997-01-01
Wild-type Ceratodon purpureus (Hedw.) Brid. protonemata grow up in the dark by negative gravitropism. When upright wild-type protonemata are reoriented 90 degrees, they temporarily grow down soon after reorientation ("initial reversal") and also prior to cytokinesis ("mitotic reversal"). A positively gravitropic mutant designated wrong- way response (wwr-1) has been isolated by screening ultraviolet light-mutagenized Ceratodon protonemata. Protonemata of wwr-l reoriented from the vertical to the horizontal grow down with kinetics comparable to those of the wild-type. Protonemata of wwr-1 also show initial and mitotic reversals where they temporarily grow up. Thus, the direction of gravitropism, initial reversal, and mitotic reversal are coordinated though each are opposite in wwr-1 compared to the wild-type. Normal plastid zonation is still maintained in dark-grown wwr-1 apical cells, but the plastids are more numerous and plastid sedimentation is more pronounced. In addition, wwr-1 apical cells are wider and the tips greener than in the wild-type. These data suggest that a functional WWR gene product is not necessary for the establishment of some gravitropic polarity, for gravitropism, or for the coordination of the reversals. Thus, the WWR protein may normally transduce information about cell orientation.
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Gamma ray-induced small plaque mutants of western equine encephalitis virus
DOE Office of Scientific and Technical Information (OSTI.GOV)
Simizu, B.; Yamazaki, S.; Suzuki, K.
1973-12-01
Small plaque mutants of Western equine encephalitis virus were obtained from the surviving fractions of wild-type virus which was irradiated with gamma rays. The frequency with which small plaque mutants appeared in the surviving fraction increased with the radiation dose. These mutants were not more resistant to radiation than wild-type virus. The growth rate of a mutant, S127, was lower than that of wild-type. Clonally purified mutant virions presented two peaks in a velocity sedimentation profile; peak 1 corresponded to the peak of wild type and peak 2 moved faster than peak 1. Virions of both peaks were infectious andmore » consistently formed small plaques in chicken embryo cells. Virions reisolated from either peak and grown in chicken embryo cells also revealed two peaks in sedimentation analysis. In the electron microscope examination peak 2 proved to consist of giant form particles, each of which contained more than one nucleoid surrounded with a common envelope. Despite this remarkable morphological difference, densities of the wild-type and S127 mutant virions were similar in cesium chloride gradients. The RNAs and proteins of mutant virions could not be distinguished from those of wild types on the basis of size or change. (auth)« less
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de Mooij, Tim; Schediwy, Kira; Wijffels, René H; Janssen, Marcel
2016-12-20
Under high light conditions, microalgae are oversaturated with light which significantly reduces the light use efficiency. Microalgae with a reduced pigment content, antenna size mutants, have been proposed as a potential solution to increase the light use efficiency. The goal of this study was to investigate the competition between antenna size mutants and wild type microalgae in mass cultures. Using a kinetic model and literature-derived experimental data from wild type Chlorella sorokiniana, the productivity and competition of wild type cells and antenna size mutants were simulated. Cultivation was simulated in an outdoor microalgal raceway pond production system which was assumed to be limited by light only. Light conditions were based on a Mediterranean location (Tunisia) and a more temperate location (the Netherlands). Several wild type contamination levels were simulated in each mutant culture separately to predict the effect on the productivity over the cultivation time of a hypothetical summer season of 100days. The simulations demonstrate a good potential of antenna size reduction to increase the biomass productivity of microalgal cultures. However, it was also found that after a contamination with wild type cells the mutant cultures will be rapidly overgrown resulting in productivity loss. Copyright © 2016 Elsevier B.V. All rights reserved.
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Baccari, Clelia; Killiny, Nabil; Ionescu, Michael; Almeida, Rodrigo P P; Lindow, Steven E
2014-01-01
The hypothesis that a wild-type strain of Xylella fastidiosa would restore the ability of rpfF mutants blocked in diffusible signal factor production to be transmitted to new grape plants by the sharpshooter vector Graphocephala atropunctata was tested. While the rpfF mutant was very poorly transmitted by vectors irrespective of whether they had also fed on plants infected with the wild-type strain, wild-type strains were not efficiently transmitted if vectors had fed on plants infected with the rpfF mutant. About 100-fewer cells of a wild-type strain attached to wings of a vector when suspended in xylem sap from plants infected with an rpfF mutant than in sap from uninfected grapes. The frequency of transmission of cells suspended in sap from plants that were infected by the rpfF mutant was also reduced over threefold. Wild-type cells suspended in a culture supernatant of an rpfF mutant also exhibited 10-fold less adherence to wings than when suspended in uninoculated culture media. A factor released into the xylem by rpfF mutants, and to a lesser extent by the wild-type strain, thus inhibits their attachment to, and thus transmission by, sharpshooter vectors and may also enable them to move more readily through host plants.
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van der Hoeven, Ransome; Forst, Steven
2009-09-01
The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded beta-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the DeltaopnS strain. Coinjection of the wild-type and DeltaopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or DeltaopnS strain were colonized by the wild-type strain. In addition, the DeltaopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The DeltaopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment.
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Takeuchi, Tadayoshi; Tanaka, Keisuke; Nakajima, Hidemitsu; Matsui, Minoru; Azuma, Yasu-Taka
2007-01-01
The involvement of muscarinic receptors in neurogenic responses of the ileum was studied in wild-type and muscarinic-receptor (M-receptor) knockout (KO) mice. Electrical field stimulation to the wild-type mouse ileum induced a biphasic response, a phasic and sustained contraction that was abolished by tetrodotoxin. The sustained contraction was prolonged for an extended period after the termination of electrical field stimulation. The phasic contraction was completely inhibited by atropine. In contrast, the sustained contraction was enhanced by atropine. Ileal strips prepared from M2-receptor KO mice exhibited a phasic contraction similar to that seen in wild-type mice and a sustained contraction that was larger than that in wild-type mice. In M3-receptor KO mice, the phasic contraction was smaller than that observed in wild-type mice. Acetylcholine exogenously administrated induced concentration-dependent contractions in strips isolated from wild-type, M2- and M3-receptor KO mice. However, contractions in M3-receptor KO mice shifted to the right. The sustained contraction was inhibited by capsaicin and neurokinin NK2 receptor antagonist, suggesting that it is mediated by substance P (SP). SP-induced contraction of M2-receptor KO mice did not differ from that of wild-type mice. SP immunoreactivity was located in enteric neurons, colocalized with M2 receptor immunoreactivity. These results suggest that atropine-sensitive phasic contraction is mainly mediated via the M3 receptor, and SP-mediated sustained contraction is negatively regulated by the M2 receptor at a presynaptic level.
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Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max
2014-01-01
During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase–polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains. PMID:25424931
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Matson, David O; Vesikari, Timo; Dennehy, Penelope; Dallas, Michael D; Goveia, Michelle G; Itzler, Robbin F; Ciarlet, Max
2014-01-01
During the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST), the period between the administration of dose 1 through 13 days after the administration of dose 3, there were more wild-type rotavirus gastroenteritis (RVGE) cases among vaccine recipients compared with placebo recipients using the protocol-specified microbiological plaque assay in the clinical-efficacy cohort, a subset of subjects where vaccine efficacy against RVGE of any severity was assessed. In this study, a rotavirus genome segment 6-based reverse transcriptase-polymerase chain reaction assay was applied post hoc to clarify the accuracy of type categorization of all these RVGE cases in vaccine recipients during the vaccination phase of REST. The assay characterized 147 (90%) of 163 re-assayed RVGE cases or rotavirus-associated health care contacts as type-determinable: either wild-type or vaccine-type rotavirus strains. In the clinical-efficacy cohort (N = 5673), 19 (18.8%) of 101 samples from RVGE cases contained wild-type rotavirus, 70 (69.3%) vaccine virus, and 12 (11.9%) were indeterminable. In the large-scale cohort (N = 68,038), 10 (34.5%) of 29 samples from RVGE-related health care contacts contained wild-type rotavirus strains, 15 (51.7%) vaccine-type rotavirus strains, and 4 (13.8%) were indeterminable. Of the 33 samples from RVGE cases in placebo recipients, all were confirmed to contain wild-type rotaviruses. Altogether, this post-hoc re-evaluation showed that the majority (75%) of type-determinable RVGE cases or health care contacts that occurred during the vaccination phase of REST in vaccine recipients were associated with vaccine-type rotavirus strains rather than wild-type rotavirus strains.
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Revisiting PC1/3 Mutants: Dominant-Negative Effect of Endoplasmic Reticulum-Retained Mutants.
Blanco, Elias H; Ramos-Molina, Bruno; Lindberg, Iris
2015-10-01
Prohormone convertase 1/3 (PC1/3), encoded by the gene PCSK1, is critical for peptide hormone synthesis. An increasing number of studies have shown that inactivating mutations in PCSK1 are correlated with endocrine pathologies ranging from intestinal dysfunction to morbid obesity, whereas the common nonsynonymous polymorphisms rs6232 (N221D) and rs6234-rs6235 (Q665E-S690T) are highly associated with obesity risk. In this report, we revisited the biochemical and cellular properties of PC1/3 variants in the context of a wild-type PC1/3 background instead of the S357G hypermorph background used for all previous studies. In the wild-type background the PC1/3 N221D variant exhibited 30% lower enzymatic activity in a fluorogenic assay than wild-type PC1/3; this inhibition was greater than that detected in an equivalent experiment using the PC1/3 S357G background. A PC1/3 variant with the linked carboxyl-terminal polymorphisms Q665E-S690T did not show this difference. We also analyzed the biochemical properties of 2 PC1/3 mutants, G209R and G593R, which are retained in the endoplasmic reticulum (ER), and studied their effects on wild-type PC1/3. The expression of ER-retained mutants induced ER stress markers and also resulted in dominant-negative blockade of wild-type PC1/3 prodomain cleavage and decreased expression of wild-type PC1/3, suggesting facilitation of the entry of wild-type protein to a degradative proteasomal pathway. Dominant-negative effects of PC1/3 mutations on the expression and maturation of wild-type protein, with consequential effects on PC1/3 availability, add a new element which must be considered in population and clinical studies of this gene.
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2010-01-01
A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance. PMID:20433759
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Li, Mo-lin; Li, Chuan-gang; Shu, Xiao-hong; Jia, Yu-jie; Qin, Zhi-hai
2006-03-01
To establish mouse lymphoma EL4 tumor-bearing mouse models in wild type C57BL/6 mice and nude C57BL/6 mice respectively, and to further investigate the immunological mechanisms of anti-tumor effect of melphalan. Mouse lymphoma EL4 cells were inoculated subcutaneously into wild type C57BL/6 mice (immune-competent mice). Twelve days later, melphalan of different doses were administered intraperitoneally to treat these wild type C57BL/6 tuomr-bearing mice. Tumor sizes were observed and recorded subsequently to find out the minimal dose of melphalan that could cure the tuomr-bearing mice. Then the same amount of EL4 tumor cells were inoculated subcutaneously into wild type C57BL/6 mice and nude C57BL/6 mice (T cell-deficient mice) simultaneously, which had the same genetic background of C57BL/6. Twelve days later, melphalan of the minimal dose was given intraperitoneally to treat both the wild type and nude C57BL/6 tuomr-bearing mice. Tumor sizes were observed and recorded in these two different types of mice subsequently. A single dose of melphalan (7.5 mg/kg) could cure EL4 tumor-bearing wild type C57BL/6 mice, but could not induce tumor regression in EL4 tumor-bearing nude C57BL/6 mice. A single dose of melphalan has obvious anti-tumor effect on mouse lymphoma EL4 tumor-bearing wild type C57BL/6mice, which requires the involvement of T lymphocytes in the host probably related to their killing functions.
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Mohanty, Madhu C; Deshpande, Jagadish M
2013-01-01
Polioviruses are the causative agent of paralytic poliomyelitis. Attenuated polioviruses (Sabin oral poliovirus vaccine strains) do not replicate efficiently in neurons as compared to the wild type polioviruses and therefore do not cause disease. This study was aimed to investigate the differential host immune response to wild type 1 poliovirus (wild PV) and Sabin attenuated type 1 poliovirus (Sabin PV) in cultured human neuronal cells. By using flow cytometry and real time PCR methods we examined host innate immune responses and compared the role of toll like receptors (TLRs) and cytoplasmic RNA helicases in cultured human neuronal cells (SK-N-SH) infected with Sabin PV and wild PV. Human neuronal cells expressed very low levels of TLRs constitutively. Sabin PV infection induced significantly higher expression of TLR3, TLR7 and melanoma differentiation-associated protein-5 (MDA-5) m-RNA in neuronal cells at the beginning of infection (up to 4 h) as compared to wild PV. Further, Sabin PV also induced the expression of interferon α/β at early time point of infection. The induced expression of IFN α/β gene by Sabin PV in neuronal cells could be suppressed by inhibiting TLR7. Neuronal cell innate immune response to Sabin and wild polioviruses differ significantly for TLR3, TLR7, MDA5 and type 1 interferons. Effects of TLR7 activation and interferon production and Sabin virus replication in neuronal cells need to be actively investigated in future studies.
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Tregnaghi, Miguel W; Abate, Héctor J; Valencia, Alejandra; Lopez, Pio; Da Silveira, Themis Reverbel; Rivera, Luis; Rivera Medina, Doris Maribel; Saez-Llorens, Xavier; Gonzalez Ayala, Silvia Elena; De León, Tirza; Van Doorn, Leen-Jan; Pilar Rubio, Maria Del; Suryakiran, Pemmaraju Venkata; Casellas, Javier M; Ortega-Barria, Eduardo; Smolenov, Igor V; Han, Htay-Htay
2011-06-01
The efficacy of a rotavirus vaccine against severe rotavirus gastroenteritis when coadministered with routine Expanded Program on Immunization (EPI) vaccines including oral polio vaccine (OPV) was evaluated in this study. Double-blind, randomized (2:1), placebo-controlled study conducted across 6 Latin American countries. Healthy infants (N = 6568) 6 to 12 weeks of age received 2 doses of RIX4414 vaccine or placebo following a 0, 1- to 2-month schedule. Routine vaccines including OPV were coadministered according to local EPI schedule. Vaccine efficacy (VE) against severe rotavirus gastroenteritis caused by circulating wild-type rotavirus from 2 weeks post-Dose 2 until 1 year of age was calculated with 95% confidence interval [CI]. Safety was assessed during the entire study period. Immunogenicity of RIX4414 and OPV was also assessed. During the efficacy follow-up period (mean duration = 7.4 months), 7 and 19 cases of severe rotavirus gastroenteritis were reported in the vaccine and placebo groups, respectively, with a VE of 81.6% (95% CI: 54.4-93.5). VE against severe rotavirus gastroenteritis caused by G1 type was 100% (95% CI: <0-100) and 80.6% (95% CI: 51.4-93.2) against the pooled non-G1 rotavirus types, respectively. There was no difference (P = 0.514) in the incidence of serious adverse events reported in the 2 groups. Antirotavirus IgA seropositivity rate at 1 to 2 months post-Dose 2 was 61.4% (95% CI: 53.7-68.6) in the RIX4414 group; similar seroprotection rates (≥96.0%) against the 3 antipoliovirus types was observed 1 month post-Dose 3 of OPV in both groups. RIX4414 was highly efficacious against severe rotavirus gastroenteritis caused by the circulating wild-type rotavirus (G1 and non-G1) when coadministered with routine EPI vaccines including OPV.
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Klomps, Lawrence V; Zomorodi, Naseem; Kim, H Mike
2017-12-01
Rotator cuff muscle fatty degeneration after a chronic tendon tear is an irreversible pathologic change associated with poor clinical outcomes of tendon repair, and its exact pathogenesis remains unknown. We sought to investigate the role of transplanted bone marrow cells in the development of fatty degeneration, specifically in adipocyte accumulation, using a mouse model. Fourteen mice were divided into 2 bone marrow chimeric animal groups: bone marrow transplantation (BMT) group and reverse BMT group. For the BMT group, C57BL/6J wild-type mice underwent whole body irradiation followed by BMT into the retro-orbital sinus from green fluorescent protein (GFP)-transgenic donor mice. For the reverse BMT group, GFP-transgenic mice received BMT from C57BL/6J wild-type donor mice after irradiation. The supraspinatus tendon, infraspinatus tendon, and suprascapular nerve were surgically transected 3 weeks after transplantation. The rotator cuff muscles were harvested 13 weeks after transplantation for histologic analysis and GFP immunohistochemistry. On histologic examination, both groups showed substantial fatty degeneration, fibrosis, and atrophy of the cuff muscles. The BMT group showed no noticeable GFP immunostaining, whereas the reverse BMT group showed significantly stronger GFP staining in most adipocytes (P < .001). However, both groups also showed that a small number of adipocytes originated from transplanted bone marrow cells. A small number of myocytes showed a large cytoplasmic lipid vacuole resembling adipocytes. This study's findings suggest that most adipocytes in fatty degeneration of the rotator cuff muscles originate from sources other than bone marrow-derived stem cells, and there may be more than 1 source for the adipocytes. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.
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2013-01-01
The E46K genetic missense mutation of the wild-type α-synuclein protein was recently identified in a family of Spanish origin with hereditary Parkinson’s disease. Detailed understanding of the structures of the monomeric E46K mutant-type α-synuclein protein as well as the impact of the E46K missense mutation on the conformations and free energy landscapes of the wild-type α-synuclein are required for gaining insights into the pathogenic mechanism of Parkinson’s disease. In this study, we use extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations to assess the secondary and tertiary structural properties as well as the conformational preferences of the monomeric wild-type and E46K mutant-type α-synuclein proteins in an aqueous solution environment. We also present the residual secondary structure component conversion stabilities with dynamics using a theoretical strategy, which we most recently developed. To the best of our knowledge, this study presents the first detailed comparison of the structural and thermodynamic properties of the wild-type and E46K mutant-type α-synuclein proteins in an aqueous solution environment at the atomic level with dynamics. We find that the E46K mutation results not only in local but also in long-range changes in the structural properties of the wild-type α-synuclein protein. The mutation site shows a significant decrease in helical content as well as a large increase in β-sheet structure formation upon E46K mutation. In addition, the β-sheet content of the C-terminal region increases significantly in the E46K mutant-type αS in comparison to the wild-type αS. Our theoretical strategy developed to assess the thermodynamic preference of secondary structure transitions indicates that this shift in secondary structure is the result of a decrease in the thermodynamic preference of turn to helix conversions while the coil to β-sheet preference increases for these residues. Long-range intramolecular protein interactions of the C-terminal with the N-terminal and NAC regions increase upon E46K mutation, resulting in more compact structures for the E46K mutant-type rather than wild-type αS. However, the E46K mutant-type αS structures are less stable than the wild-type αS. Overall, our results show that the E46K mutant-type αS has a higher propensity to aggregate than the wild-type αS and that the N-terminal and C-terminal regions are reactive toward fibrillization and aggregation upon E46K mutation and we explain the associated reasons based on the structural properties herein. Small molecules or drugs that can block the specific residues forming abundant β-sheet structure, which we report here, might help to reduce the reactivity of these intrinsically disordered fibrillogenic proteins toward aggregation and their toxicity. PMID:23374074
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Boklund, A; Goldbach, S G; Uttenthal, A; Alban, L
2008-07-15
Denmark has no free-range wild-boar population. However, Danish wildlife organizations have suggested that wild boar should be reintroduced into the wild to broaden national biodiversity. Danish pig farmers fear that this would lead to a higher risk of introduction of classical swine fever virus (CSFV), which could have enormous consequences in terms of loss of pork exports. We conducted a risk assessment to address the additional risk of introducing and spreading CSFV due to the reintroduction of wild boar. In this paper, we present the part of the risk assessment that deals with the spread of CSFV between the hypothetical wild-boar population and the domestic population. Furthermore, the economic impact is assessed taking the perspective of the Danish national budget and the Danish pig industry. We used InterSpreadPlus to model the differential classical swine fever (CSF) risk due to wild boar. Nine scenarios were run to elucidate the effect of: (a) presence of wild boar (yes/no), (b) locations for the index case (domestic pig herd/wild-boar group), (c) type of control strategy for wild boar (hunting/vaccination) and (d) presence of free-range domestic pigs. The presence of free-range wild boar was simulated in two large forests using data from wildlife studies and Danish habitat data. For each scenario, we estimated (1) the control costs borne by the veterinary authorities, (2) the control-related costs to farmers and (3) the loss of exports associated with an epidemic. Our simulations predict that CSFV will be transmitted from the domestic pig population to wild boar if the infected domestic pig herd is located close to an area with wild boar (<5 km). If an outbreak begins in the wild-boar population, the epidemic will last longer and will occasionally lead to several epidemics because of periodic transfer of virus from groups of infected wild boar to domestic pig herds. The size and duration of the epidemic will be reduced if there are no free-range domestic pig herds in the area with CSF-infected wild boar. The economic calculations showed that the total national costs for Denmark (i.e. the direct costs to the national budget and the costs to the pig industry) related to an outbreak of CSF in Denmark will be highly driven by the reactions of the export markets and in particular of the non-EU markets. Unfortunately, there is a substantial amount of uncertainty surrounding this issue. If hunting is used as a control measure, the average expenses related to a CSF outbreak will be 40% higher if wild boar are present compared with not present. However, a vaccination strategy for wild boar will double the total costs compared with a hunting strategy.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Phillips, J.; Warby, C; Whitby, F
2009-01-01
Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connectedmore » by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.« less
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Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sprong, Debbie; Janssen, Hilde L.; Vens, Conchita
2006-02-01
Purpose: To determine the role of DNA repair in hypoxic radioresistance. Methods and Materials: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. Results: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficientmore » line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. Conclusion: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity.« less
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Rcan2 and estradiol independently regulate body weight in female mice
Ding, Ling-Cui; Gong, Qian-Qian; Li, Shi-Wei; Fu, Xiao-Long; Jin, Ye-Cheng; Zhang, Jian; Gao, Jian-Gang; Sun, Xiao-Yang
2017-01-01
Rcan2 increases food intake and plays an important role in the development of age- and diet- induced obesity in male mice. However, in females, wild-type mice grow almost at a similar rate as Rcan2−/− mice on normal chow diet from 6 weeks of age. Here we showed that the ability of Rcan2 to promote weight gain was attenuated by energy expenditure mediated by 17β-estradiol in female mice. Using ovariectomy-operated models, we found that 17β-estradiol deprivation did not alter food intake, but induced more weight gain in wild-type mice than Rcan2−/− mice. If wild-type mice ingested equally as Rcan2−/− mice, in the same ovarian state they exhibited similar weight changes, but the mice in ovariectomized groups were significantly heavier than the ovarian-intact mice, suggesting that body weight is not only regulated by Rcan2, but also by 17β-estradiol. Furthermore, we demonstrated that Rcan2 and 17β-estradiol independently regulated body weight even on high-fat diets. Therefore, our findings indicate that Rcan2 and 17β-estradiol regulate body weight through different mechanisms. Rcan2 increases food intake, whereas 17β-estradiol promotes energy expenditure. These findings provide novel insights into the sexual dimorphism of body weight regulation. PMID:28624805
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The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages
Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara
2013-01-01
S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches. PMID:23593170
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Apoptotic cell death in erythrocytes of p53-deficient medaka (Oryzias latipes) after γ-irradiation.
Sayed, Alaa El-Din Hamid; Watanabe-Asaka, Tomomi; Oda, Shoji; Mitani, Hiroshi
2016-10-01
Previous studies have examined the effects of γ-irradiation (γ-IR) on wild-type and p53 mutant Medaka (Oryzias latipes) 24 hours after irradiation and in the present work, apoptosis and alterations in erythrocytes of 4, 8 and 24 h and 14 days after gamma-ray irradiation were reported as genotoxic biomarkers of γ-irradiation. Sexually mature wild-type, WT (Hd-rR) and p53(-/-) adult female medaka (O. latipes) were exposed to 4 Gy dose of γ-IR and sampling were collected after 4, 8 and 24 h and 14 days. Apoptosis and morphological alterations were observed from 4 h after irradiation and remarkably increased 8 h after irradiation in the wild-type. Apoptotic cell death has been observed 8 h after irradiation most prominently but subtle in p53 mutant medaka. All these phenotypes were recovered 14 days after irradiation in both strains. Although no micronuclei were seen in any group, nuclear abnormalities were observed in red blood cells. Both apoptosis and morphological alterations in erythrocytes were decreased after 24 and 14 days after γ-irradiation. We conclude that apoptosis and malformations caused by 4 Gy γ-irradiation in the erythrocytes of medaka fish occurs from 4-24 h and the initial response until 8 h was p53-dependent.
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The β-hemolysin and intracellular survival of Streptococcus agalactiae in human macrophages.
Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara
2013-01-01
S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.
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Sadoyama, Shinko; Sekine, Akimasa; Satoh, Hiroaki; Iwasawa, Tae; Kato, Terufumi; Ikeda, Satoshi; Sata, Masafumi; Baba, Tomohisa; Tabata, Erina; Minami, Yuko; Nemoto, Kenji; Hayashihara, Kenji; Saito, Takefumi; Okudela, Koji; Ohashi, Kenichi; Tajiri, Michihiko; Ogura, Takashi
2018-01-01
The aim of this study was to clarify the incidence and disease behavior of brain metastases (BM) without extracranial disease (ie, isolated BM) as the first relapse after curative surgery in non-small-cell lung cancer (NSCLC) patients, analyzed according to epidermal growth factor receptor (EGFR) mutation status. A review of the medical charts of consecutive NSCLC patients diagnosed between 2005 and 2016 with BM as the first relapse after curative surgery was performed. Among 1191 patients evaluated for EGFR mutation status, 28 patients who met the inclusion criteria were divided into 2 groups: EGFR mutation group (16 patients) and wild type group (12 patients). At BM diagnosis, the EGFR-mutation group tended to have more commonly isolated BM compared with that in the wild type group (11 of 16 vs. 3 of 12; P = .054). In the EGFR mutation group, the patients with isolated BM showed longer overall survival than those with non-isolated BM (39.6 vs. 18.7 months; P = .038). Notably, isolated BM in the EGFR mutation group was neurologically asymptomatic in 10 of the 11 patients. With regard to upfront treatment for isolated BM in the EGFR mutation group, 10 of 11 patients were treated with only cranial radiotherapy without EGFR tyrosine kinase inhibitors, but two-thirds of the patients (7 of 11; 64%) developed extracranial disease during the study period. In curatively resected NSCLC patients with EGFR mutation, isolated BM would be correlated with better prognosis, but regarded as a precursor to systemic disease. Because isolated BM can be neurologically asymptomatic, it would be important to periodically perform cranial evaluation to detect isolated BM. Copyright © 2017 Elsevier Inc. All rights reserved.
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Ayeh, Kwadwo Owusu; Lee, YeonKyeong; Ambrose, Mike J; Hvoslef-Eide, Anne Kathrine
2009-06-23
In pea seeds (Pisum sativum L.), the Def locus defines an abscission event where the seed separates from the funicle through the intervening hilum region at maturity. A spontaneous mutation at this locus results in the seed failing to abscise from the funicle as occurs in wild type peas. In this work, structural differences between wild type peas that developed a distinct abscission zone (AZ) between the funicle and the seed coat and non-abscission def mutant were characterized. A clear abscission event was observed in wild type pea seeds that were associated with a distinct double palisade layers at the junction between the seed coat and funicle. Generally, mature seeds fully developed an AZ, which was not present in young wild type seeds. The AZ was formed exactly below the counter palisade layer. In contrast, the palisade layers at the junction of the seed coat and funicle were completely absent in the def mutant pea seeds and the cells in this region were seen to be extensions of surrounding parenchymatous cells. The Def wild type developed a distinct AZ associated with palisade layer and counterpalisade layer at the junction of the seed coat and funicle while the def mutant pea seed showed non-abscission and an absence of the double palisade layers in the same region. We conclude that the presence of the double palisade layer in the hilum of the wild type pea seeds plays an important structural role in AZ formation by delimiting the specific region between the seed coat and the funicle and may play a structural role in the AZ formation and subsequent detachment of the seed from the funicle.
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Sullivan, James A.; Gray, John C.
2000-01-01
The pea lip1 (light-independent photomorphogenesis1) mutant shows many of the characteristics of light-grown development when grown in continuous darkness. To investigate the identity of LIP1, cDNAs encoding the pea homolog of COP1, a repressor of photomorphogenesis identified in Arabidopsis, were isolated from wild-type and lip1 pea seedlings. lip1 seedlings contained a wild-type COP1 transcript as well as a larger COP1′ transcript that contained an internal in-frame duplication of 894 bp. The COP1′ transcript segregated with the lip1 phenotype in F2 seedlings and could be translated in vitro to produce a protein of ∼100 kD. The COP1 gene in lip1 peas contained a 7.5-kb duplication, consisting of exons 1 to 7 of the wild-type sequence, located 2.5 kb upstream of a region of genomic DNA identical to the wild-type COP1 DNA sequence. Transcription and splicing of the mutant COP1 gene was predicted to produce the COP1′ transcript, whereas transcription from an internal promoter in the 2.5-kb region of DNA located between the duplicated regions of COP1 would produce the wild-type COP1 transcript. The presence of small quantities of wild-type COP1 transcripts may reduce the severity of the phenotype produced by the mutated COP1′ protein. The genomic DNA sequences of the COP1 gene from wild-type and lip1 peas and the cDNA sequences of COP1 and COP1′ transcripts have been submitted to the EMBL database under the EMBL accession numbers AJ276591, AJ276592, AJ289773, and AJ289774, respectively. PMID:11041887
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van der Hoeven, Ransome; Forst, Steven
2009-01-01
The gammaproteobacterium Xenorhabdus nematophila engages in a mutualistic association with an entomopathogenic nematode and also functions as a pathogen toward different insect hosts. We studied the role of the growth-phase-regulated outer membrane protein OpnS in host interactions. OpnS was shown to be a 16-stranded β-barrel porin. opnS was expressed during growth in insect hemolymph and expression was elevated as the cell density increased. When wild-type and opnS deletion strains were coinjected into insects, the wild-type strain was predominantly recovered from the insect cadaver. Similarly, an opnS-complemented strain outcompeted the ΔopnS strain. Coinjection of the wild-type and ΔopnS strains together with uncolonized nematodes into insects resulted in nematode progeny that were almost exclusively colonized with the wild-type strain. Likewise, nematode progeny recovered after coinjection of a mixture of nematodes carrying either the wild-type or ΔopnS strain were colonized by the wild-type strain. In addition, the ΔopnS strain displayed a competitive growth defect when grown together with the wild-type strain in insect hemolymph but not in defined culture medium. The ΔopnS strain displayed increased sensitivity to antimicrobial compounds, suggesting that deletion of OpnS affected the integrity of the outer membrane. These findings show that the OpnS porin confers a competitive advantage for the growth and/or the survival of X. nematophila in the insect host and provides a new model for studying the biological relevance of differential regulation of porins in a natural host environment. PMID:19465651
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Perception of sweet taste is important for voluntary alcohol consumption in mice.
Blednov, Y A; Walker, D; Martinez, M; Levine, M; Damak, S; Margolskee, R F
2008-02-01
To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: alpha-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild-type mice, whereas Tas1r3 null mice were not different from wild type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion (CTA) to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in CTA to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol.
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Sharma, Reetu; Sastry, G Narahari
2015-01-01
Thermus thermophilius isopropylmalate dehydrogenase catalyzes oxidative decarboxylation and dehydrogenation of isopropylmalate. Substitution of leucine to alanine at position 172 enhances the thermal stability among the known point mutants. Exploring the dynamic properties of non-covalent interactions such as saltbridges, hydrogen bonds and hydrophobic interactions to explain thermal stability of a protein is interesting in its own right. In this study dynamic changes in the non-covalent interactions are studied to decipher the deterministic features of thermal stability of a protein considering a case study of a point mutant in Thermus thermophilus isopropylmalate dehydrogenase. A total of four molecular dynamic simulations of 0.2 μs were carried out on wild type and mutant's functional dimers at 300 K and 337 K. Higher thermal stability of the mutant as compared to wild type is revealed by root mean square deviation, root mean square fluctuations and Cα-Cα distance with an increase in temperature from 300 K to 337 K. Most of the regions of wild type fluctuate higher than the corresponding regions of mutant with an increase in temperature. Cα-Cα distance analysis suggests that long distance networks are significantly affected in wild type as compared to the mutant. Short lived contacts are higher in wild type, while long lived contacts are lost at 337 K. The mutant forms less hydrogen bonds with water as compared to wild type at 337 K. In contrast to wild type, the mutant shows significant increase in unique saltbridges, hydrogen bonds and hydrophobic contacts at 337 K. The current study indicates that there is a strong inter-dependence of thermal stability on the way in which non-covalent interactions reorganize, and it is rewarding to explore this connection in single mutant studies.
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Meshach Paul, D; Chadah, Tania; Senthilkumar, B; Sethumadhavan, Rao; Rajasekaran, R
2017-11-03
The major candidate for multiple sulfatase deficiency is a defective formylglycine-generating enzyme (FGE). Though adequately produced, mutations in FGE stall the activation of sulfatases and prevent their activity. Missense mutations, viz. E130D, S155P, A177P, W179S, C218Y, R224W, N259I, P266L, A279V, C336R, R345C, A348P, R349Q and R349W associated with multiple sulfatase deficiency are yet to be computationally studied. Aforementioned mutants were initially screened through ws-SNPs&GO 3D program. Mutant R345C acquired the highest score, and hence was studied in detail. Discrete molecular dynamics explored structural distortions due to amino acid substitution. Therein, comparative analyses of wild type and mutant were carried out. Changes in structural contours were observed between wild type and mutant. Mutant had low conformational fluctuation, high atomic mobility and more compactness than wild type. Moreover, free energy landscape showed mutant to vary in terms of its conformational space as compared to wild type. Subsequently, wild type and mutant were subjected to single-model analyses. Mutant had lesser intra molecular interactions than wild type suggesting variations pertaining to its secondary structure. Furthermore, simulated thermal denaturation showed dissimilar pattern of hydrogen bond dilution. Effects of these variations were observed as changes in elements of secondary structure. Docking studies of mutant revealed less favourable binding energy towards its substrate as compared to wild type. Therefore, theoretical explanations for structural distortions of mutant R345C leading to multiple sulfatase deficiency were revealed. The protocol of the study could be useful to examine the effectiveness of pharmacological chaperones prior to experimental studies.
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Watanabe, Toru; Bartrand, Timothy A; Omura, Tatsuo; Haas, Charles N
2012-03-01
Reported data sets on infection of volunteers challenged with wild-type influenza A virus at graded doses are few. Alternatively, we aimed at developing a dose-response assessment for this virus based on the data sets for its live attenuated reassortants. Eleven data sets for live attenuated reassortants that were fit to beta-Poisson and exponential dose-response models. Dose-response relationships for those reassortants were characterized by pooling analysis of the data sets with respect to virus subtype (H1N1 or H3N2), attenuation method (cold-adapted or avian-human gene reassortment), and human age (adults or children). Furthermore, by comparing the above data sets to a limited number of reported data sets for wild-type virus, we quantified the degree of attenuation of wild-type virus with gene reassortment and estimated its infectivity. As a result, dose-response relationships of all reassortants were best described by a beta-Poisson model. Virus subtype and human age were significant factors determining the dose-response relationship, whereas attenuation method affected only the relationship of H1N1 virus infection to adults. The data sets for H3N2 wild-type virus could be pooled with those for its reassortants on the assumption that the gene reassortment attenuates wild-type virus by at least 63 times and most likely 1,070 times. Considering this most likely degree of attenuation, 10% infectious dose of H3N2 wild-type virus for adults was estimated at 18 TCID50 (95% CI = 8.8-35 TCID50). The infectivity of wild-type H1N1 virus remains unknown as the data set pooling was unsuccessful. © 2011 Society for Risk Analysis.
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Contribution of Secretory Antibodies to Intestinal Mucosal Immunity against Helicobacter pylori
Wijburg, Odilia L. C.; Pedersen, John S.; Walduck, Anna K.; Kwok, Terry; Strugnell, Richard A.; Robins-Browne, Roy M.
2013-01-01
The natural immune response to Helicobacter pylori neither clears infection nor prevents reinfection. However, the ability of secretory antibodies to influence the course of H. pylori infection has not been determined. We compared the natural progression of H. pylori infection in wild-type C57BL/6 mice with that in mice lacking the polymeric immunoglobulin receptor (pIgR) that is essential for the secretion of polymeric antibody across mucosal surfaces. H. pylori SS1-infected wild-type and pIgR knockout (KO) mice were sampled longitudinally for gastrointestinal bacterial load, antibody response, and histological changes. The gastric bacterial loads of wild-type and pIgR KO mice remained constant and comparable at up to 3 months postinfection (mpi) despite SS1-reactive secretory IgA in the intestinal contents of wild-type mice at that time. Conversely, abundant duodenal colonization of pIgR KO animals contrasted with the near-total eradication of H. pylori from the intestine of wild-type animals by 3 mpi. H. pylori was cultured only from the duodenum of those animals in which colonization in the distal gastric antrum was of sufficient density for immunohistological detection. By 6 mpi, the gastric load of H. pylori in wild-type mice was significantly lower than in pIgR KO animals. While there was no corresponding difference between the two mouse strains in gastric pathology results at 6 mpi, reductions in gastric bacterial load correlated with increased gastric inflammation together with an intestinal secretory antibody response in wild-type mice. Together, these results suggest that naturally produced secretory antibodies can modulate the progress of H. pylori infection, particularly in the duodenum. PMID:23918779
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Singh, Dharmendra K.; Maximova, Siela N.; Jensen, Philip J.; Lehman, Brian L.; Ngugi, Henry K.; McNellis, Timothy W.
2010-01-01
The fibrillins are a large family of chloroplast proteins that have been linked with stress tolerance and disease resistance. FIBRILLIN4 (FIB4) is found associated with the photosystem II light-harvesting complex, thylakoids, and plastoglobules, which are chloroplast compartments rich in lipophilic antioxidants. For this study, FIB4 expression was knocked down in apple (Malus 3 domestica) using RNA interference. Plastoglobule osmiophilicity was decreased in fib4 knockdown (fib4 KD) tree chloroplasts compared with the wild type, while total plastoglobule number was unchanged. Compared with the wild type, net photosynthetic CO2 fixation in fib4 KD trees was decreased at high light intensity but was increased at low light intensity. Furthermore, fib4 KD trees produced more anthocyanins than the wild type when transferred from low to high light intensity, indicating greater sensitivity to high light stress. Relative to the wild type, fib4 KD apples were more sensitive to methyl viologen and had higher superoxide levels during methyl viologen treatment. Arabidopsis (Arabidopsis thaliana) fib4 mutants and fib4 KD apples were more susceptible than their wild-type counterparts to the bacterial pathogens Pseudomonas syringae pathovar tomato and Erwinia amylovora, respectively, and were more sensitive to ozone-induced tissue damage. Following ozone stress, plastoglobule osmiophilicity decreased in wild-type apple and remained low in fib4 KD trees; total plastoglobule number increased in fib4 KD apples but not in the wild type. These results indicate that FIB4 is required for plastoglobule development and resistance to multiple stresses. This study suggests that FIB4 is involved in regulating plastoglobule content and that defective regulation of plastoglobule content leads to broad stress sensitivity and altered photosynthetic activity. PMID:20813909
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Juréen, P; Angeby, K; Sturegård, E; Chryssanthou, E; Giske, C G; Werngren, J; Nordvall, M; Johansson, A; Kahlmeter, G; Hoffner, S; Schön, T
2010-05-01
The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.
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Juréen, P.; Ängeby, K.; Sturegård, E.; Chryssanthou, E.; Giske, C. G.; Werngren, J.; Nordvall, M.; Johansson, A.; Kahlmeter, G.; Hoffner, S.; Schön, T.
2010-01-01
The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis. PMID:20237102
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PERCEPTION OF SWEET TASTE IS IMPORTANT FOR VOLUNTARY ALCOHOL CONSUMPTION IN MICE
Blednov, Y.A.; Walker, D.; Martinez, M.; Levine, M.; Damak, S.; Margolskee, R.F.
2012-01-01
To directly evaluate the association between taste perception and alcohol intake, we used three different mutant mice, each lacking a gene expressed in taste buds and critical to taste transduction: α-gustducin (Gnat3), Tas1r3 or Trpm5. Null mutant mice lacking any of these three genes showed lower preference score for alcohol and consumed less alcohol in a two-bottle choice test, as compared with wild-type littermates. These null mice also showed lower preference score for saccharin solutions than did wild-type littermates. In contrast, avoidance of quinine solutions was less in Gnat3 or Trpm5 knockout mice than in wild type mice, whereas Tas1r3 null mice were not different from wild-type in their response to quinine solutions. There were no differences in null vs. wild-type mice in their consumption of sodium chloride solutions. To determine the cause for reduction of ethanol intake, we studied other ethanol-induced behaviors known to be related to alcohol consumption. There were no differences between null and wild-type mice in ethanol-induced loss of righting reflex, severity of acute ethanol withdrawal or conditioned place preference for ethanol. Weaker conditioned taste aversion to alcohol in null mice may have been caused by weaker rewarding value of the conditioned stimulus (saccharin). When saccharin was replaced by sodium chloride, no differences in conditioned taste aversion to alcohol between knockout and wild-type mice were seen. Thus, deletion of any one of three different genes involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in pharmacological actions of ethanol. PMID:17376151
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Barazani, Oz; von Dahl, Caroline C.; Baldwin, Ian T.
2007-01-01
Sebacina vermifera, a growth-promoting endophytic fungus, significantly increases Nicotiana attenuata's growth but impairs both its herbivore resistance and its accumulation of the costly, jasmonic acid (JA)-regulated defense protein, trypsin proteinase inhibitor (TPI). To determine if the fungi's growth-promoting effects can be attributed to lower TPI-related defense costs, we inoculated transformed N. attenuata plants silenced in their ability to synthesize JA, JA-isoleucine, and TPI by antisense (lipoxygenase 3 [as-lox3] and Thr deaminase [as-td]) and inverted repeat (ir-tpi) expression, and found that inoculation promoted plant growth as in untransformed wild-type plants. Moreover, herbivore-elicited increases in JA and JA-isoleucine concentrations did not differ between inoculated and uninoculated wild-type plants. However, inoculation significantly reduced the morphological effect of 1-aminocyclopropane-1-carboxylic acid on wild-type seedlings in a triple response assay, suggesting that ethylene signaling was impaired. Furthermore, S. vermifera failed to promote the growth of N. attenuata plants transformed to silence ethylene production (1-aminocyclopropane-1-carboxylic acid oxidase [ir-aco]). Inoculating wild-type plants with S. vermifera decreased the ethylene burst elicited by applying Manduca sexta oral secretions to mechanical wounds. Accordingly, oral secretion-elicited transcript levels of the ethylene synthesis genes NaACS3, NaACO1, and NaACO3 in inoculated plants were significantly lower compared to these levels in uninoculated wild-type plants. Inoculation accelerated germination in wild-type seeds; however, uninoculated wild-type seeds germinated as rapidly as inoculated seeds in the presence of the ethylene scrubber KMnO4. In contrast, neither inoculation nor KMnO4 exposure influenced the germination of ir-aco seeds. We conclude that S. vermifera increases plant growth by impairing ethylene production independently of JA signaling and TPI production. PMID:17416638
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Miller, Kyle J; Cao, Wei; Ibrahim, Mohamed M; Levinson, Howard
2017-04-01
Hemostasis, the initial phase of wound healing, sets the stage for tissue repair. Microporous polysaccharide hemosphere powder (MPH) is an FDA-approved hemostatic agent that may impact the wound-healing process. This study examined the role of MPH in murine wild-type and diabetic (db/db) wound-healing models and a foreign body response scarring model. The powder was topically applied to excisional wounds in wild-type C57BL/6 mice and db/db mice. The effect of MPH on scarring was evaluated by applying it to the expanded polytetrafluoroethylene tube implantation model. In wild-type mice, topically applied MPH increased epithelial thickness. Levels of α-smooth muscle actin (α-SMA) were decreased in MPH-treated wild-type wounds, whereas Rho-associated protein kinase 2 (ROCK2) and transforming growth factor β levels were increased. In db/db mice, topical wound MPH application decreased epithelial thickness and delayed wound closure. The db/db wounds displayed an increased collagen index. The ROCK2 was increased in a similar manner to wild-type mice, whereas α-SMA and transforming growth factor β levels were decreased. The MPH-treated expanded polytetrafluoroethylene tube mice showed increased α-SMA levels and depressed ROCK2 levels. There were no changes in histologic parameters of the foreign body response. The results suggest that MPH does not adversely impact wound healing in wild-type mice, both topically and around implants, but prolongs time to closure and diminishes thickness in db/db wounds. The MPH application alters contractile proteins in all wound models. These changes could have downstream effects on the wound healing process, and further investigation into the use of MPH in altered or impaired states of wound healing is warranted.
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The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females
Sakai, Takaomi; Kasuya, Junko; Kitamoto, Toshihiro; Aigaki, Toshiro
2009-01-01
Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless (pain) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. Here we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNAi in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. On the other hand, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity. PMID:19531155
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The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females.
Sakai, T; Kasuya, J; Kitamoto, T; Aigaki, T
2009-07-01
Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless (pain) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. In this study, we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNA interference (RNAi) in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. However, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity.
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Two alternative juvenile life history types for fall Chinook salmon in the Snake River basin
Connor, W.P.; Sneva, J.G.; Tiffan, K.F.; Steinhorst, R.K.; Ross, D.
2005-01-01
Fall Chinook salmon Oncorhynchus tshawytscha in the Snake River basin were listed under the Endangered Species Act in 1992. At the time of listing, it was assumed that fall Chinook salmon juveniles in the Snake River basin adhered strictly to an ocean-type life history characterized by saltwater entry at age 0 and first-year wintering in the ocean. Research showed, however, that some fall Chinook salmon juveniles in the Snake River basin spent their first winter in a reservoir and resumed seaward movement the following spring at age 1 (hereafter, reservoir-type juveniles). We collected wild and hatchery ocean-type fall Chinook salmon juveniles in 1997 and wild and hatchery reservoir-type juveniles in 1998 to assess the condition of the reservoir-type juveniles at the onset of seaward movement. The ocean-type juveniles averaged 112-139 mm fork length, and the reservoir-type juveniles averaged 222-224 mm fork length. The large size of the reservoir-type juveniles suggested a high potential for survival to salt water and subsequent return to freshwater. Scale pattern analyses of the fall Chinook salmon spawners we collected during 1998-2003 supported this point. Of the spawners sampled, an overall average of 41% of the wild fish and 51% of the hatchery fish had been reservoir-type juveniles. Males that had been reservoir-type juveniles often returned as small "minijacks" (wild, 16% of total; hatchery, 40% of total), but 84% of the wild males, 60% of the hatchery males, and 100% of the wild and hatchery females that had been reservoir-type juveniles returned at ages and fork lengths commonly observed in populations of Chinook salmon. We conclude that fall Chinook salmon in the Snake River basin exhibit two alternative juvenile life histories, namely ocean-type and reservoir-type. ?? Copyright by the American Fisheries Society 2005.
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Kost, Matthew A; Alexander, Helen M; Jason Emry, D; Mercer, Kristin L
2015-01-01
Hybridization produces strong evolutionary forces. In hybrid zones, selection can differentially occur on traits and selection intensities may differ among hybrid generations. Understanding these dynamics in crop–wild hybrid zones can clarify crop-like traits likely to introgress into wild populations and the particular hybrid generations through which introgression proceeds. In a field experiment with four crop–wild hybrid Helianthus annuus (sunflower) cross types, we measured growth and life history traits and performed phenotypic selection analysis on early season traits to ascertain the likelihood, and routes, of crop allele introgression into wild sunflower populations. All cross types overwintered, emerged in the spring, and survived until flowering, indicating no early life history barriers to crop allele introgression. While selection indirectly favored earlier seedling emergence and taller early season seedlings, direct selection only favored greater early season leaf length. Further, there was cross type variation in the intensity of selection operating on leaf length. Thus, introgression of multiple early season crop-like traits, due to direct selection for greater early season leaf length, should not be impeded by any cross type and may proceed at different rates among generations. In sum, alleles underlying early season sunflower crop-like traits are likely to introgress into wild sunflower populations. PMID:26029263
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Kost, Matthew A; Alexander, Helen M; Jason Emry, D; Mercer, Kristin L
2015-06-01
Hybridization produces strong evolutionary forces. In hybrid zones, selection can differentially occur on traits and selection intensities may differ among hybrid generations. Understanding these dynamics in crop-wild hybrid zones can clarify crop-like traits likely to introgress into wild populations and the particular hybrid generations through which introgression proceeds. In a field experiment with four crop-wild hybrid Helianthus annuus (sunflower) cross types, we measured growth and life history traits and performed phenotypic selection analysis on early season traits to ascertain the likelihood, and routes, of crop allele introgression into wild sunflower populations. All cross types overwintered, emerged in the spring, and survived until flowering, indicating no early life history barriers to crop allele introgression. While selection indirectly favored earlier seedling emergence and taller early season seedlings, direct selection only favored greater early season leaf length. Further, there was cross type variation in the intensity of selection operating on leaf length. Thus, introgression of multiple early season crop-like traits, due to direct selection for greater early season leaf length, should not be impeded by any cross type and may proceed at different rates among generations. In sum, alleles underlying early season sunflower crop-like traits are likely to introgress into wild sunflower populations.
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Deshpande, J M; Nadkarni, S S; Siddiqui, Z A
2003-12-01
Significant progress has been made towards eradication of poliomyelitis in India. Surveillance for acute flaccid paralysis (AFP) has reached high standards. Among the 3 types of polioviruses, type 2 had been eliminated in India and eradicated globally as of October 1999. However, we isolated wild poliovirus type 2 from a small number of polio cases in northern India in 2000 and again during December 2002 to February 2003. Using molecular tools the origin, of the wild type 2 poliovirus was investigated. Polioviruses isolated from stool samples collected from patients with AFP were differentiated as wild virus or Sabin vaccine-like by ELISA and probe hybridization assays. Complete VP1 gene nucleotide sequences of the wild type 2 poliovirus isolates were determined by reverse transcriptase polymerase chain reaction (RT-PCR), followed by cycle sequencing. VP1 nucleotide sequences were compared with those of wild type 2 polioviruses that were indigenous in India in the past as well as prototype/laboratory strains and the GenBank database. Wild poliovirus type 2 was detected in stool samples from 6 patients with AFP in western Uttar Pradesh and 1 in Gujarat. In addition, the virus was isolated from one healthy contact child and from environmental sewage sample in Moradabad where three of these patients were reported. These isolates were identified as genetically closely related to laboratory reference strain MEF-1. Molecular characterization of the isolates confirmed that there was no evidence of extensive person-to-person transmission of the virus in the community. Laboratory reference strain (MEF-1) of poliovirus type 2 caused paralytic poliomyelitis in 10 patients in September 2000 and November 2002 to February 2003. The origin of the virus was some laboratory as yet not identified. This episode highlights the urgent need for stringent containment of wild poliovirus containing materials in the laboratories across the country in order to prevent recurrence of such incidents.
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Schwarz, Sandra; West, T. Eoin; Boyer, Frédéric; Chiang, Wen-Chi; Carl, Mike A.; Hood, Rachel D.; Rohmer, Laurence; Tolker-Nielsen, Tim; Skerrett, Shawn J.; Mougous, Joseph D.
2010-01-01
Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans—leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections. PMID:20865170
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Genetics of Ustilago violacea. I. Carotenoid mutants and carotenogenesis
DOE Office of Scientific and Technical Information (OSTI.GOV)
Garber, E.D.; Baird, M.L.; Chapman, D.J.
1975-12-01
Wild-type strains of Ustilago violacea produce pink colonies on laboratory medium and yield white, orange, pumpkin, and yellow colonies after uv mutagenesis. The wild-type strains contain neurosporene and lycopene; one orange mutant, $gamma$-carotene; and one yellow mutant, $beta$-carotene. One white mutant had no detectable carotenoids. Diploid colonies heterozygous for wild type and orange, pumpkin, yellow, or white are phenotypically wild type. Diploid colonies heterozygous for yellow and orange are also phenotypically wild type. Diploid colonies heterozygous for white and orange; white and yellow; and white, yellow, and orange are phenotypically light orange, light yellow, and orange- yellow, respectively. The whitemore » mutants give a circular complementation map; the color mutants fit a linear complementation map. We propose a multienzyme of four identical dehydrogenases and one or two identical cyclases for carotenogenesis in this species. The white and color mutants represent structural mutations altering the conformation of the dehydrogenase or cyclase, respectively. Furthermore, cyclases may or may not aggregate in association with the dehydrogenase aggregate to form the multienzyme aggregate responsible for the color mutants. (auth)« less
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Kiss, John Z.; Sack, Fred D.
1990-01-01
Gravitropism in dark-grown hypocotyls of the wild type was compared with a starch-deficient Nicotiana sylvestris mutant (NS 458) to test the effects of starch deficiency on gravity sensing. In a time course of curvature measured using infrared video, the response of the mutant was greatly reduced compared to the wild type; 72 hours after reorientation, curvature was about 10° for NS 458 and about 70° for wild type. In dishes maintained in a vertical orientation, wild-type hypocotyls were predominantly vertical, whereas NS 458 hypocotyls were severely disoriented with about 5 times more orientational variability than wild type. Since the growth rates were equal for both genotypes and phototropic curvature was only slightly inhibited in NS 458, the mutation probably affects gravity perception rather than differential growth. Our data suggest that starch deficiency reduces gravitropic sensitivity more in dark-grown hypocotyls than in dark- or light-grown roots in this mutant and support the hypothesis that amyloplasts function as statoliths in shoots as well as roots. Images Figure 2 Figure 3 Figure 4 PMID:11537476
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NASA Technical Reports Server (NTRS)
Kiss, J. Z.; Sack, F. D.
1990-01-01
Gravitropism in dark-grown hypocotyls of the wild type was compared with a starch-deficient Nicotiana sylvestris mutant (NS 458) to test the effects of starch deficiency on gravity sensing. In a time course of curvature measured using infrared video, the response of the mutant was greatly reduced compared to the wild type; 72 hours after reorientation, curvature was about 10 degrees for NS 458 and about 70 degrees for wild type. In dishes maintained in a vertical orientation, wild-type hypocotyls were predominantly vertical, whereas NS 458 hypocotyls were severely disoriented with about 5 times more orientational variability than wild type. Since the growth rates were equal for both genotypes and phototropic curvature was only slightly inhibited in NS 458, the mutation probably affects gravity perception rather than differential growth. Our data suggest that starch deficiency reduces gravitropic sensitivity more in dark-grown hypocotyls than in dark- or light-grown roots in this mutant and support the hypothesis that amyloplasts function as statoliths in shoots as well as roots.
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Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice.
Tsokas, Panayiotis; Hsieh, Changchi; Yao, Yudong; Lesburguères, Edith; Wallace, Emma Jane Claire; Tcherepanov, Andrew; Jothianandan, Desingarao; Hartley, Benjamin Rush; Pan, Ling; Rivard, Bruno; Farese, Robert V; Sajan, Mini P; Bergold, Peter John; Hernández, Alejandro Iván; Cottrell, James E; Shouval, Harel Z; Fenton, André Antonio; Sacktor, Todd Charlton
2016-05-17
PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice.
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Huang, Jie-Hong; Kortstee, Anne; Dees, Dianka C T; Trindade, Luisa M; Schols, Henk A; Gruppen, Harry
2016-08-01
Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyzes the conversion of UDP-glucose to UDP-galactose. Cell wall materials from the cv. Kardal (wild-type, background) and two UGE transgenic lines (UGE 45-1 and UGE 51-16) were isolated and fractionated. The galactose (Gal) content (mg/100g tuber) from UGE 45-1 transgenic line was 38% higher than that of wild-type, and resulted in longer pectin side chains. The Gal content present in UGE 51-16 was 17% lower than that of wild-type, although most pectin populations maintained the same level of Gal. Both UGE transgenic lines showed unexpectedly a decrease in acetylation and an increase in methyl-esterification of pectin. Both UGE transgenic lines showed similar proportions of homogalacturonan and rhamnogalacturonan I within pectin backbone as the wild-type, except for the calcium-bound pectin fraction exhibiting relatively less rhamnogalacturonan I. Next to pectin modification, xyloglucan populations from both transgenic lines were altered resulting in different XSGG and XXGG proportion in comparison to wild-type. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E.; Jaspers, Nicolaas G. J.; Kaptein, Robert; Hoeijmakers, Jan H. J.; Boelens, Rolf
2015-01-01
The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe231, Leu231 lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. PMID:26085086
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Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution
NASA Technical Reports Server (NTRS)
Tsang, Joyce; Joyce, Gerald F.
1996-01-01
In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.
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Chen, Guangli; Gong, Shusheng; Chen, Pei; Luo, Linghui
2014-09-01
To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer. Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry. Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phβApr-neol cells (P < 0.01). These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.
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Why genetic modification of lignin leads to low-recalcitrance biomass
Carmona, Christopher; Langan, Paul; Smith, Jeremy C.; ...
2014-11-11
Genetic modification of plants via down-regulation of cinnamyl alcohol dehydrogenase leads to incorporation of aldehyde groups in the lignin polymer. Moreover, the resulting lignocellulosic biomass has increased bioethanol yield. However, a molecular-scale explanation of this finding is currently lacking. We perform molecular dynamics simulation of the copolymer with hemicellulose of wild type and the genetically modified lignin, in aqueous solution. We find that the non-covalent association with hemicellulose of lignin containing aldehyde groups is reduced compared to the wild-type. This phase separation may increase the cell wall porosity in the mutant plants, thus explaining their easier deconstruction to biofuels. Themore » thermodynamic origin of the reduced lignin-hemicellulose association is found to be a more favorable self-interaction energy and less favorable interaction with hemicellulose for the mutant lignin. Furthermore, reduced hydration water density fluctuations are found for the mutant lignin, implying a more hydrophobic lignin surface. Our results provide a detailed description of how aldehyde incorporation makes lignin more hydrophobic and reduces its association with hemicellulose, thus suggesting that increased lignin hydrophobicity may be an optimal characteristic required for improved biofuel production.« less
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Graham, Kirsty E; Furuichi, Takeshi; Byrne, Richard W
2017-03-01
In animal communication, signallers and recipients are typically different: each signal is given by one subset of individuals (members of the same age, sex, or social rank) and directed towards another. However, there is scope for signaller-recipient interchangeability in systems where most signals are potentially relevant to all age-sex groups, such as great ape gestural communication. In this study of wild bonobos (Pan paniscus), we aimed to discover whether their gestural communication is indeed a mutually understood communicative repertoire, in which all individuals can act as both signallers and recipients. While past studies have only examined the expressed repertoire, the set of gesture types that a signaller deploys, we also examined the understood repertoire, the set of gestures to which a recipient reacts in a way that satisfies the signaller. We found that most of the gestural repertoire was both expressed and understood by all age and sex groups, with few exceptions, suggesting that during their lifetimes all individuals may use and understand all gesture types. Indeed, as the number of overall gesture instances increased, so did the proportion of individuals estimated to both express and understand a gesture type. We compared the community repertoire of bonobos to that of chimpanzees, finding an 88 % overlap. Observed differences are consistent with sampling effects generated by the species' different social systems, and it is thus possible that the repertoire of gesture types available to Pan is determined biologically.
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Li, Li; Liu, Kai; Wei, Sheng-Li; Cheng, Xiao-Li; Liu, Juan; Ren, Guang-Xi; Wang, Wen-Quan
2014-04-01
This study was conducted to investigate the wild and cultivated resource situation of Rheum tanguticum in main production area of China, estimate its reserves, and put forward the feasible approach for the sustainable utilization of R. tanguticum. On the basis of the literature data about R. tanguticum, conbined with interview, investigation and sampling investigation, the total reserve of resources is estimated using the route-quadrat method and the vegetation and soil-type map area method proposed by our research group. The results indicate that there is no obvious change between the present distribution ranges of the wild R. tanguticum and its historical records, but its population density has changed clearly. The reserve of the wild R. tanguticum has seriously declined in lots of place, even faced the exhaustion in some regions. According to the investigation, the resource reserve of the wild R. tanguticum is no more than 5 000 t, and the cultivated is about 1 607 t. The resource reserve of the wild R. tanguticum is nearly depleted, and this suggests that the wild R. tanguticum should be enrolled in the protection plant list, and the cultivated will become the main resource of Rhubarb in the future. So it is extremely neccessary to collect and protect the germplasm resource of R. tanguticum, establish the germplasm nursery and repository, and conduct breeding research on those bases.
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de Luis, Daniel Antonio; Izaola, Olatz; Primo, David; Aller, Rocio
2017-11-14
Several adiponectin gene (ADIPOQ) single nucleotide polymorphisms (SNPS) have been related with adiponectin levels and risk for obesity. Our aim was to analyze the effects of rs1501299 ADIPOQ gene polymorphism on total adiponectin levels, insulin resistance and weight loss after a Mediterranean hypocaloric diet in obese subjects. A Caucasian population of 82 obese patients was analyzed, before and after 3 months on a Mediterranean hypocaloric diet. Before and after 3 months on a hypocaloric diet, an anthropometric evaluation, an assessment of nutritional intake and a biochemical analysis were performed. After dietary treatment and in wild type group, weight, BMI, fat mass, leptin levels, systolic blood pressure and waist circumference decreases were similar to the mutant type group. In wild type group, the decrease in total cholesterol was -28.1±15.3 mg/dl (mutant group: -12.6±16.7 mg/dl:p=0.009), LDL- cholesterol was -31.8±20.5 mg/dl (-12.2±11.5 mg/dl:p=0.006), fasting glucose plasma -4.8±2.5 mg/dL (-0.5±0.1 mg/dL:p=0.02), insulin -3.6±1.5 mUI/L (+0.6±1.1 mUI/L:p=0.02) and HOMA-IR -1.2±0.9 (-0.1±1.1:p=0.03). The present study suggests that T allele of ADIPO (rs1501299) could be a predictor of a lack of response of HOMA-IR, insulin, fasting glucose and LDL cholesterol secondary to a Mediterranean hypocaloric diet in obese subjects. Copyright © 2017 Elsevier B.V. All rights reserved.
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de Luis, Daniel Antonio; Romero, Enrique; Izaola, Olatz; Primo, David; Aller, Rocío
2017-01-01
The role of brain-derived neurotrophic factor (BDNF) variants on change in body weight and cardiovascular risk factors after weight loss remains unclear in obese patients. Our aim was to analyze the effects of the rs10767664 BDNF gene polymorphism on body weight, cardiovascular risk factors, and serum adipokine levels after a high monounsaturated fatty acids (MUFAs) hypocaloric diet (diet M) versus a high polyunsaturated fatty acids (PUFAs) hypocaloric diet (diet P). A Caucasian population of 361 obese patients was enrolled. Subjects who met the inclusion criteria were randomly allocated to one of two diets for a period of 3 months. Two hundred and sixteen subjects (59.8%) had the genotype AA (wild-type group), and 145 (40.2%) patients had the genotypes AT (122 patients, 33.8%) or TT (23 patients, 6.4%) (mutant-type group). After weight loss with diet P and diet M and in both genotype groups, body mass index, weight, fat mass, waist circumference, systolic blood pressure, serum leptin levels, low-density lipoprotein cholesterol, and total cholesterol decreased in a significant way. Secondary to weight loss with diet M and only in the wild-type group, insulin levels (-2.1 ± 2.0 vs. -0.7 ± 2.9 IU/L, p < 0.05) and homeostatic model assessment of insulin resistance (-0.7 ± 0.9 vs. -0.3 ± 1.0 U, p < 0.05) decreased. Our data show that the rs10767664 variant of the BDNF gene modifies insulin resistance and insulin levels after weight loss with a hypocaloric diet enriched with MUFAs. © 2018 S. Karger AG, Basel.
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Ribeiro, Márcio Garcia; Takai, Shinji; Guazzelli, Alessandro; Lara, Gustavo Henrique Batista; da Silva, Aristeu Vieira; Fernandes, Marta Catarina; Condas, Larissa Anuska Zeni; Siqueira, Amanda Keller; Salerno, Tatiana
2011-12-01
The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15-17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil. Copyright © 2010 Elsevier Ltd. All rights reserved.
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Poythress, J C; Affolter, James M
2018-04-14
Because of concerns over recent declines in overall biodiversity in suburban areas, homeowners are attempting to improve the ecological functioning of their landscapes by incorporating native plants. Native plants are important for supporting native herbivorous insects, but it is unknown whether the native plants that are commercially available, typically cultivated varieties (cultivars) of a single genotype, are equally effective as food sources as the local, wild-type plants. We compared the hemipteran communities feeding on cultivars and wild-propagated plants for four species of native perennials commonly used as ornamentals. Of 65 hemipteran species collected, 35 exhibited a preference for some plant species over others, indicating a high degree of host-plant specialization. Moreover, the insect community associated with cultivars was distinct from the insect community associated with wild-type plants for each plant species, with three to four insect species accounting for most of the observed difference. Total insect abundance and insect biomass differed between cultivars and wild-propagated plants, but the direction of the difference changed over time and was not consistent among plant species. Species richness and a diversity index (the Q statistic) did not differ between cultivars and wild-type plants. These data suggest that abundance and diversity of hemipteran insects does not depend on the source of the plant material per se, but rather on the particular characteristics of cultivars that distinguish them from the wild type.
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Conversion between parallel and antiparallel β -sheets in wild-type and Iowa mutant Aβ40 fibrils
NASA Astrophysics Data System (ADS)
Xi, Wenhui; Hansmann, Ulrich H. E.
2018-01-01
Using a variant of Hamilton-replica-exchange, we study for wild type and Iowa mutant Aβ40 the conversion between fibrils with antiparallel β-sheets and such with parallel β-sheets. We show that wild type and mutant form distinct salt bridges that in turn stabilize different fibril organizations. The conversion between the two fibril forms leads to the release of small aggregates that in the Iowa mutant may shift the equilibrium from fibrils to more toxic oligomers.
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Characterization of the Origin of DNA Replication of the Coxiella burnetii Chromosome
1990-01-26
chromosomal DNAs (FIG. IB): the 19.4-kb EcoR I fragment of Salmonella typhimurium DNA (lane 4),9 the 17.5-kb Sal I fragment of Enterobacter aerogenes ...IacZYA-argF) U 1694680d IacZAM15 Salmonella typhimurium Wild type WVUd Kiebsiella pneumoniae Wild type WVUd Enterobacter aero genes Wild type WVUd... aerogenes and K. pneumoniae were digested with appropriate restriction enzymes. The restriction fragments were separated on a 0.9% agarose gel, transferred to
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Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains.
Médici, K C; Barry, A F; Alfieri, A F; Alfieri, A A
2010-01-01
Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species.
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Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.
Martini, Sabrina V; Silva, Adriana L; Ferreira, Debora; Rabelo, Rafael; Ornellas, Felipe M; Gomes, Karina; Rocco, Patricia R M; Petrs-Silva, Hilda; Morales, Marcelo M
2016-01-01
Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy. © 2016 The Author(s) Published by S. Karger AG, Basel.
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Steele, Andrew Duncan; Neuzil, Kathleen M; Cunliffe, Nigel A; Madhi, Shabir A; Bos, Pieter; Ngwira, Bagrey; Witte, Desiree; Todd, Stacy; Louw, Cheryl; Kirsten, Mari; Aspinall, Sanet; Van Doorn, Leen Jan; Bouckenooghe, Alain; Suryakiran, Pemmaraju V; Han, Htay Htay
2012-09-13
Rotaviruses are the most important cause of severe acute gastroenteritis worldwide in children <5 years of age. The human, G1P[8] rotavirus vaccine Rotarix™ significantly reduced severe rotavirus gastroenteritis episodes in a Phase III clinical trial conducted in infants in South Africa and Malawi. This paper examines rotavirus vaccine efficacy in preventing severe rotavirus gastroenteritis, during infancy, caused by the various G and P rotavirus types encountered during the first rotavirus-season. Healthy infants aged 5-10 weeks were enrolled and randomized into three groups to receive either two (10 and 14 weeks) or three doses of Rotarix™ (together forming the pooled Rotarix™ group) or three doses of placebo at a 6,10,14-week schedule. Weekly home visits were conducted to identify gastroenteritis episodes. Rotaviruses were detected by ELISA and genotyped by RT-PCR and nucleotide sequencing. The percentage of infants with severe rotavirus gastroenteritis caused by the circulating G and P types from 2 weeks post-last dose until one year of age and the corresponding vaccine efficacy was calculated with 95% CI. Overall, 4939 infants were vaccinated and 4417 (pooled Rotarix™ = 2974; placebo = 1443) were included in the per protocol efficacy cohort. G1 wild-type was detected in 23 (1.6%) severe rotavirus gastroenteritis episodes from the placebo group. This was followed in order of detection by G12 (15 [1%] in placebo) and G8 types (15 [1%] in placebo). Vaccine efficacy against G1 wild-type, G12 and G8 types were 64.1% (95% CI: 29.9%; 82%), 51.5% (95% CI:-6.5%; 77.9%) and 64.4% (95% CI: 17.1%; 85.2%), respectively. Genotype P[8] was the predominant circulating P type and was detected in 38 (2.6%) severe rotavirus gastroenteritis cases in placebo group. The remaining circulating P types comprised of P[4] (20 [1.4%] in placebo) and P[6] (13 [0.9%] in placebo). Vaccine efficacy against P[8] was 59.1% (95% CI: 32.8%; 75.3%), P[4] was 70.9% (95% CI: 37.5%; 87.0%) and P[6] was 55.2% (95% CI: -6.5%; 81.3%) Rotarix™ vaccine demonstrated efficacy against severe gastroenteritis caused by diverse circulating rotavirus types. These data add to a growing body of evidence supporting heterotypic protection provided by Rotarix™. NCT00241644.
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Kaneko, Mei; Takanashi, Sayaka; Thongprachum, Aksara; Hanaoka, Nozomu; Fujimoto, Tsuguto; Nagasawa, Koo; Kimura, Hirokazu; Okitsu, Shoko; Mizuguchi, Masashi; Ushijima, Hiroshi
2017-01-01
Two live attenuated oral rotavirus vaccines, Rotarix and RotaTeq, have been introduced as voluntary vaccination in Japan since 2011 and 2012, respectively. Effectiveness of the vaccines has been confirmed, whereas concerns such as shedding of the vaccine strains and gastroenteritis cases caused by vaccine strains are not well assessed. We aimed to identify the vaccine strains in children with acute gastroenteritis (AGE) to investigate the prevalence of AGE caused by vaccination or horizontal transmission of vaccine strains. A total of 1,824 stool samples were collected from children with AGE at six outpatient clinics in 2012-2015. Among all, 372 group A rotavirus (RVA) positive samples were screened for vaccine components by real-time RT-PCR which were designed to differentiate vaccine strains from rotavirus wild-type strains with high specificity. For samples possessing both vaccine and wild-type strains, analyses by next-generation sequencing (NGS) were conducted to characterize viruses existed in the intestine. As a result, Rotarix-derived strains were identified in 6 of 372 (1.6%) RVA positive samples whereas no RotaTeq strain was detected. Among six samples, four possessed Rotarix-derived strains while two possessed both Rotarix-derived strains and wild-type strains. In addition, other pathogens such as norovirus, enterovirus and E.coli were detected in four samples. The contribution of these vaccine strains to each patient's symptoms was unclear as all of the cases were vaccinated 2-14 days before sample collection. Proportion of average coverage for each segmented gene by NGS strongly suggested the concurrent infection of the vaccine-derived strain and the wild-type strain rather than reassortment of these two strains in one sample. This is the first study to report the prevalence of vaccine-derived strains in patients with RVA AGE in Japan as 1.6% without evidence of horizontal transmission. The results emphasized the importance of continuous monitoring on vaccine strains and their clinical impacts on children.
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Ha, Ji-Hee; Kim, Jung-Eun; Kim, Yong-Sung
2016-01-01
The monospecific and bivalent characteristics of naturally occurring immunoglobulin G (IgG) antibodies depend on homodimerization of the fragment crystallizable (Fc) regions of two identical heavy chains (HCs) and the subsequent assembly of two identical light chains (LCs) via disulfide linkages between each HC and LC. Immunoglobulin Fc heterodimers have been engineered through modifications to the CH3 domain interface, with different mutations on each domain such that the engineered Fc fragments, carrying the CH3 variant pair, preferentially form heterodimers rather than homodimers. Many research groups have adopted different strategies to generate Fc heterodimers, with the goal of high heterodimerization yield, while retaining biophysical and biological properties of the wild-type Fc. Based on their ability to enforce heterodimerization between the two different HCs, the established Fc heterodimers have been extensively exploited as a scaffold to generate bispecific antibodies (bsAbs) in full-length IgG and IgG-like formats. These have many of the favorable properties of natural IgG antibodies, such as high stability, long serum half-life, low immunogenicity, and immune effector functions. As of July 2016, more than seven heterodimeric Fc-based IgG-format bsAbs are being evaluated in clinical trials. In addition to bsAbs, heterodimeric Fc technology is very promising for the generation of Fc-fused proteins and peptides, as well as cytokines (immunocytokines), which can present the fusion partners in the natural monomeric or heterodimeric form rather than the artificial homodimeric form with wild-type Fc. Here, we present relevant concepts and strategies for the generation of heterodimeric Fc proteins, and their application in the development of bsAbs in diverse formats for optimal biological activity. In addition, we describe wild-type Fc-fused monomeric and heterodimeric proteins, along with the difficulties associated with their preparations, and discuss the use of heterodimeric Fc as an alternative scaffold of wild-type Fc for naturally monomeric or heterodimeric proteins, to create Fc-fusion proteins with novel therapeutic modality.
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Aroca, Angeles; Schneider, Markus; Scheibe, Renate; Gotor, Cecilia; Romero, Luis C
2017-06-01
Hydrogen sulfide is an important signaling molecule comparable with nitric oxide and hydrogen peroxide in plants. The underlying mechanism of its action is unknown, although it has been proposed to be S-sulfhydration. This post-translational modification converts the thiol groups of cysteines within proteins to persulfides, resulting in functional changes of the proteins. In Arabidopsis thaliana, S-sulfhydrated proteins have been identified, including the cytosolic isoforms of glyceraldehyde-3-phosphate dehydrogenase GapC1 and GapC2. In this work, we studied the regulation of sulfide on the subcellular localization of these proteins using two different approaches. We generated GapC1-green fluorescent protein (GFP) and GapC2-GFP transgenic plants in both the wild type and the des1 mutant defective in the l-cysteine desulfhydrase DES1, responsible for the generation of sulfide in the cytosol. The GFP signal was detected in the cytoplasm and the nucleus of epidermal cells, although with reduced nuclear localization in des1 compared with the wild type, and exogenous sulfide treatment resulted in similar signals in nuclei in both backgrounds. The second approach consisted of the immunoblot analysis of the GapC endogenous proteins in enriched nuclear and cytosolic protein extracts, and similar results were obtained. A significant reduction in the total amount of GapC in des1 in comparison with the wild type was determined and exogenous sulfide significantly increased the protein levels in the nuclei in both plants, with a stronger response in the wild type. Moreover, the presence of an S-sulfhydrated cysteine residue on GapC1 was demonstrated by mass spectrometry. We conclude that sulfide enhances the nuclear localization of glyceraldehyde-3-phosphate dehydrogenase. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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Gully, Neville; Bright, Richard; Marino, Victor; Marchant, Ceilidh; Cantley, Melissa; Haynes, David; Butler, Catherine; Dashper, Stuart; Reynolds, Eric; Bartold, Mark
2014-01-01
Objectives To investigate the suggested role of Porphyromonas gingivalis peptidylarginine deiminase (PAD) in the relationship between the aetiology of periodontal disease and experimentally induced arthritis and the possible association between these two conditions. Methods A genetically modified PAD-deficient strain of P. gingivalis W50 was produced. The effect of this strain, compared to the wild type, in an established murine model for experimental periodontitis and experimental arthritis was assessed. Experimental periodontitis was induced following oral inoculation with the PAD-deficient and wild type strains of P. gingivalis. Experimental arthritis was induced via the collagen antibody induction process and was monitored by assessment of paw swelling and micro-CT analysis of the radio-carpal joints. Experimental periodontitis was monitored by micro CT scans of the mandible and histological assessment of the periodontal tissues around the mandibular molars. Serum levels of anti-citrullinated protein antibodies (ACPA) and P. gingivalis were assessed by ELISA. Results The development of experimental periodontitis was significantly reduced in the presence of the PAD-deficient P. gingivalis strain. When experimental arthritis was induced in the presence of the PAD-deficient strain there was less paw swelling, less erosive bone damage to the joints and reduced serum ACPA levels when compared to the wild type P. gingivalis inoculated group. Conclusion This study has demonstrated that a PAD-deficient strain of P. gingivalis was associated with significantly reduced periodontal inflammation. In addition the extent of experimental arthritis was significantly reduced in animals exposed to prior induction of periodontal disease through oral inoculation of the PAD-deficient strain versus the wild type. This adds further evidence to the potential role for P. gingivalis and its PAD in the pathogenesis of periodontitis and exacerbation of arthritis. Further studies are now needed to elucidate the mechanisms which drive these processes. PMID:24959715
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Giuntini, Serena; Reason, Donald C; Granoff, Dan M
2012-01-01
Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.
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Nrf2-Dependent Induction of NQO1 in Mouse Aortic Endothelial Cells Overexpressing Catalase
Lin, Xinghua; Yang, Hong; Zhou, LiChun; Guo, ZhongMao
2011-01-01
Overexpression of catalase has been shown to accelerate benzo(a)pyrene (BaP) detoxification in mouse aortic endothelial cells (MAECs ). NAD(P)H:quinone oxidoreductase1 (NQO1) is an enzyme that catalyzes BaP-quinone detoxification. Aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor-2 (Nrf2) are transcription factors that control NQO1 expression. Here, we investigated the effect of catalase overexpression on NQO1, Nrf2 and AhR expressions. The levels of NQO1 mRNA and protein were comparable in MAECs isolated from wild-type and transgenic mice that overexpress human catalase (hCatTg). BaP treatment increased NQO1 mRNA and protein levels in both groups, with a significantly greater induction in hCatTg MAECs than in wild-type cells. BaP-induced NQO1 promoter activity was dramatically higher in hCatTg MAECs than in wild-type cells. Our data also showed that the basal level of AhR and the BaP-induced level of Nrf2 were significantly higher in hCatTg MAECs than in wild-type cells. Inhibition of specificity protein-1 (Sp1) binding to the AhR promoter region by mithramycin A reversed the enhanced effect of catalase overexpression on AhR expression. Knockdown of AhR by RNA interference diminished BaP-induced expression of Nrf2 and NQO1. Knockdown of Nrf2 significantly decreased NQO1 mRNA and protein levels in cells with or without BaP treatment. NQO1 promoter activity was abrogated by mutation of the Nrf2-binding site in this promoter. In contrast, mutation of the AhR-binding site in NQO1 promoter did not affect the promoter activity. These results suggest that catalase overexpression upregulates BaP-induced NQO1 expression via enhancing the Sp1-AhR-Nrf2 signaling cascade. PMID:21569840
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Tran, Anh N.; Lai, Albert; Li, Sichen; Pope, Whitney B.; Teixeira, Stephanie; Harris, Robert J.; Woodworth, Davis C.; Nghiemphu, Phioanh L.; Cloughesy, Timothy F.; Ellingson, Benjamin M.
2014-01-01
Background Isocitrate dehydrogenase 1 (IDH1) mutations have been linked to favorable outcomes in patients with glioblastoma multiforme (GBM). Recent in vitro experiments suggest that IDH1 mutation sensitizes tumors to radiation damage. We hypothesized that radiographic treatment response would be significantly different between IDH1 mutant versus wild-type GBMs after radiotherapy (RT) and concurrent temozolomide (TMZ). Methods A total of 39 newly diagnosed GBM patients with known IDH1 mutational status (10 IDH1 mutants), who followed standard therapy and had regular post-contrast T1W (T1+C) and T2W/ fluid-attenuated inversion recovery (FLAIR) images in the 6-month period after starting RT, were enrolled. The volume of contrast-enhancing and FLAIR hyperintensity were calculated from each scan. Linear and polynomial regression techniques were used to estimate the rate of change and temporal patterns in tumor volumes. Results IDH1 mutant GBMs demonstrated a favorable response to RT/TMZ in the study period, as demonstrated by 10 of 10 mutants showing radiographic response (decreasing VT1+C), compared with 13 of 29 wild-types (P < .001). During the study period, VT1+C and VFLAIR changed at −3.6% per week and +0.6% per week in IDH1 mutant tumors, respectively, as compared with +0.8% per week and +5.2% per week in IDH1 wild-type tumors (P = .0076 and P = .0118, respectively). Amongst the radiographic responders, IDH1 mutant GBMs still demonstrated significant progression-free and overall survival benefit. Aggregated tumor kinetics by group showed significant lower rate in IDH1 mutant GBMs in specific periods: >105 days for VFLAIR and 95–120 and >150 days for VT1+C from starting RT/TMZ. Conclusions The current study supports the hypothesis that IDH1 mutant GBMs are more sensitive to radiochemotherapy than IDH1 wild-type GBMs. PMID:24305712
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Pillidge, Katharine; Porter, Ashley J; Young, Jared W; Stanford, S Clare
2016-01-01
The underlying cause(s) of abnormalities expressed by patients with attention deficit hyperactivity disorder (ADHD) have yet to be delineated. One factor that has been associated with increased vulnerability to ADHD is polymorphism(s) of TACR1, which is the human equivalent of the rodent NK1 (substance P-preferring) receptor gene (Nk1r). We have reported previously that genetically altered mice, lacking functional NK1R (NK1R–/–), express locomotor hyperactivity, which was blunted by the first-line treatment for ADHD, methylphenidate. Here, we compared the effects of this psychostimulant (3, 10 and 30 mg/kg, intraperitoneally) on the behaviour of NK1R-/- mice and their wild types in the 5-Choice Continuous Performance Test, which emulates procedures used to study attention and response control in ADHD patients. Methylphenidate increased total trials (a measure of ‘productivity’) completed by wild types, but not by NK1R-/- mice. Conversely, this drug reduced perseveration by NK1R-/- mice, but not by wild types. Other drug-induced changes in key behaviours were not genotype dependent, especially at the highest dose: for example, % omissions (an index of inattentiveness) was increased, whereas % false alarms and % premature responses (measures of impulsivity) declined in both genotypes, indicating reduced overall response. These findings are discussed in the context of the efficacy of methylphenidate in the treatment of ADHD. Moreover, they lead to several testable proposals. First, methylphenidate does not improve attention in a subgroup of ADHD patients with a functional deficit of TACR1. Second, these patients do not express excessive false alarms when compared with other groups of subjects, but they do express excessive perseveration, which would be ameliorated by methylphenidate. PMID:27097734
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Nikodinovic-Runic, Jasmina; Coulombel, Lydie; Francuski, Djordje; Sharma, Narain D; Boyd, Derek R; Ferrall, Rory Moore O; O'Connor, Kevin E
2013-06-01
Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min(-1) g cell dry weight(-1) resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite (S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[b]thiophene and 2-methylbenzo[b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group.
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Kaneko, Mei; Thongprachum, Aksara; Hanaoka, Nozomu; Fujimoto, Tsuguto; Nagasawa, Koo; Kimura, Hirokazu; Okitsu, Shoko; Mizuguchi, Masashi; Ushijima, Hiroshi
2017-01-01
Two live attenuated oral rotavirus vaccines, Rotarix and RotaTeq, have been introduced as voluntary vaccination in Japan since 2011 and 2012, respectively. Effectiveness of the vaccines has been confirmed, whereas concerns such as shedding of the vaccine strains and gastroenteritis cases caused by vaccine strains are not well assessed. We aimed to identify the vaccine strains in children with acute gastroenteritis (AGE) to investigate the prevalence of AGE caused by vaccination or horizontal transmission of vaccine strains. A total of 1,824 stool samples were collected from children with AGE at six outpatient clinics in 2012–2015. Among all, 372 group A rotavirus (RVA) positive samples were screened for vaccine components by real-time RT-PCR which were designed to differentiate vaccine strains from rotavirus wild-type strains with high specificity. For samples possessing both vaccine and wild-type strains, analyses by next-generation sequencing (NGS) were conducted to characterize viruses existed in the intestine. As a result, Rotarix-derived strains were identified in 6 of 372 (1.6%) RVA positive samples whereas no RotaTeq strain was detected. Among six samples, four possessed Rotarix-derived strains while two possessed both Rotarix-derived strains and wild-type strains. In addition, other pathogens such as norovirus, enterovirus and E.coli were detected in four samples. The contribution of these vaccine strains to each patient’s symptoms was unclear as all of the cases were vaccinated 2–14 days before sample collection. Proportion of average coverage for each segmented gene by NGS strongly suggested the concurrent infection of the vaccine-derived strain and the wild-type strain rather than reassortment of these two strains in one sample. This is the first study to report the prevalence of vaccine-derived strains in patients with RVA AGE in Japan as 1.6% without evidence of horizontal transmission. The results emphasized the importance of continuous monitoring on vaccine strains and their clinical impacts on children. PMID:28902863
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Xu, Songtao; Zhang, Yan; Zhu, Zhen; Liu, Chunyu; Mao, Naiying; Ji, Yixin; Wang, Huiling; Jiang, Xiaohong; Li, Chongshan; Tang, Wei; Feng, Daxing; Wang, Changyin; Zheng, Lei; Lei, Yue; Ling, Hua; Zhao, Chunfang; Ma, Yan; He, Jilan; Wang, Yan; Li, Ping; Guan, Ronghui; Zhou, Shujie; Zhou, Jianhui; Wang, Shuang; Zhang, Hong; Zheng, Huanying; Liu, Leng; Ma, Hemuti; Guan, Jing; Lu, Peishan; Feng, Yan; Zhang, Yanjun; Zhou, Shunde; Xiong, Ying; Ba, Zhuoma; Chen, Hui; Yang, Xiuhui; Bo, Fang; Ma, Yujie; Liang, Yong; Lei, Yake; Gu, Suyi; Liu, Wei; Chen, Meng; Featherstone, David; Jee, Youngmee; Bellini, William J; Rota, Paul A; Xu, Wenbo
2013-01-01
China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993-2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10(-3) substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure. Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.
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Burkitt, Michael D; Hanedi, Abdalla F; Duckworth, Carrie A; Williams, Jonathan M; Tang, Joseph M; O'Reilly, Lorraine A; Putoczki, Tracy L; Gerondakis, Steve; Dimaline, Rod; Caamano, Jorge H; Pritchard, D Mark
2015-07-01
NF-κB signalling is an important factor in the development of inflammation-associated cancers. Mouse models of Helicobacter-induced gastric cancer and colitis-associated colorectal cancer have demonstrated that classical NF-κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF-κB proteins, including NF-κB1/p50, NF-κB2/p52, and c-Rel, differentially regulate the development of gastric pre-neoplasia. To investigate the effect of NF-κB subunit loss on colitis-associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1(-/-), Nfkb2(-/-), and c-Rel(-/-) mice. Animals lacking the c-Rel subunit were more susceptible to colitis-associated cancer than wild-type mice, developing 3.5 times more colonic polyps per animal than wild-type mice. Nfkb2(-/-) mice were resistant to colitis-associated cancer, developing fewer polyps per colon than wild-type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2(-/-) mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c-Rel(-/-) mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild-type counterparts. These observations demonstrate different functions of specific NF-κB subunits in this model of colitis-associated carcinogenesis. NF-κB2/p52 is necessary for the development of colitis, whilst c-Rel-mediated signalling regulates colonic epithelial cell turnover following DNA damage. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Ranchon, Isabelle; LaVail, Matthew M; Kotake, Yashige; Anderson, Robert E
2003-07-09
Phenyl-N-tert-butylnitrone (PBN) protects rat retinas against light damage. Because the degenerative process involved in light damage and inherited retinal degeneration both lead to a common final cell death, apoptosis, we used transgenic rats with a P23H or S334ter rhodopsin mutation to test the effects of PBN on retinal degeneration and light damage and the susceptibility of the transgenic rats to light damage. In the first study, 3-week-old mutant and wild-type rats were given no drug, 0.25% PBN in drinking water, or 0.25% PBN in drinking water plus three daily intraperitoneal injections of PBN (100 mg/kg, i.p., every 8 hr). Electroretinograms were recorded at postnatal day 49, after which the rats were killed for morphometric analysis. There was no photoreceptor rescue by PBN in P23H or S334ter rats, as evidenced by equivalent loss of function and photoreceptor cells in the three treatment groups. In the second study, P23H, S334ter, and wild-type rats were exposed for 24 hr to 2700 lux light. The rats were untreated or treated with PBN (50 mg/kg per injection, every 6 hr, starting before exposure). ERGs were recorded before and 1 d after exposure. Animals were killed 6 d later for morphometric analysis. PBN protected wild-type and P23H but not S334ter retinas from light damage. S334ter retinas were relatively less susceptible to light damage than P23H and wild-type rats. The results suggest that the initiating event(s) that causes photoreceptor cell death in the mutated rats is different from that which occurs in light damage, although both ultimately undergo an apoptotic cell death.
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Dominguez, Jessica A; Samocha, Alexandr J; Liang, Zhe; Burd, Eileen M; Farris, Alton B; Coopersmith, Craig M
2013-10-01
Nuclear factor-κB is a critical regulator of cell-survival genes and the host inflammatory response. The purpose of this study was to investigate the role of enterocyte-specific NF-kB in sepsis through selective ablation of IkB kinase. Prospective, randomized controlled study. Animal laboratories in university medical centers. Mice lacking functional NF-kB in their intestinal epithelium (Vil-Cre/Ikkβ) and wild-type mice were subjected to sham laparotomy or cecal ligation and puncture. Animals were killed at 24 hours or followed 7 days for survival. Septic wild-type mice had decreased villus length compared with sham mice, whereas villus atrophy was further exacerbated in septic Vil-Cre/Ikkβ mice. Sepsis induced an increase in intestinal epithelial apoptosis compared with sham mice, which was further exacerbated in Vil-Cre/Ikkβ mice. Sepsis induced intestinal hyperpermeability in wild-type mice compared with sham mice, which was further exacerbated in septic Vil-Cre/Ikkβ mice. This was associated with increased intestinal expression of claudin-2 in septic wild-type mice, which was further increased in septic Vil-Cre/Ikkβ mice. Both, pro-inflammatory and anti-inflammatory cytokines were increased in serum following cecal ligation and puncture, and interleukin 10 and monocyte chemoattractant protein-1 levels were higher in septic Vil-Cre/Ikkβ mice than in septic wild-type mice. All septic mice were bacteremic, but no differences in bacterial load were identified between wild-type and Vil-Cre/Ikkβ mice. To determine the functional significance of these results, animals were followed for survival. Septic wild-type mice had lower mortality than septic Vil-Cre/Ikkβ mice (47% vs 80%, p<0.05). Antitumor necrosis factor administration decreased intestinal apoptosis, permeability, and mortality in wild-type septic mice, and a similar improvement in intestinal integrity and survival were seen when antitumor necrosis factor was given to Vil-Cre/Ikkβ mice. Enterocyte-specific NF-kB has a beneficial role in sepsis by partially preventing sepsis-induced increases in apoptosis and permeability, which are associated with worsening mortality.
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Hoon Park, Ji; Kumar, Naresh; Hoon Park, Dae; Yusupov, Maksudbek; Neyts, Erik C.; Verlackt, Christof C. W.; Bogaerts, Annemie; Ho Kang, Min; Sup Uhm, Han; Ha Choi, Eun; Attri, Pankaj
2015-01-01
Bacteria can be inactivated through various physical and chemical means, and these have always been the focus of extensive research. To further improve the methodology for these ends, two types of plasma systems were investigated: nano-second pulsed plasma (NPP) as liquid discharge plasma and an Argon gas-feeding dielectric barrier discharge (Ar-DBD) as a form of surface plasma. To understand the sterilizing action of these two different plasma sources, we performed experiments with Staphylococcus aureus (S. aureus) bacteria (wild type) and multidrug resistant bacteria (Penicillum-resistant, Methicillin-resistant and Gentamicin-resistant). We observed that both plasma sources can inactivate both the wild type and multidrug-resistant bacteria to a good extent. Moreover, we observed a change in the surface morphology, gene expression and β-lactamase activity. Furthermore, we used X-ray photoelectron spectroscopy to investigate the variation in functional groups (C-H/C-C, C-OH and C=O) of the peptidoglycan (PG) resulting from exposure to plasma species. To obtain atomic scale insight in the plasma-cell interactions and support our experimental observations, we have performed molecular dynamics simulations to study the effects of plasma species, such as OH, H2O2, O, O3, as well as O2 and H2O, on the dissociation/formation of above mentioned functional groups in PG. PMID:26351132
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Andrianarivelo, M R; Rabarijaona, L; Boisier, P; Chezzi, C; Zeller, H G
1999-01-01
From July 1995 to December 1996, 3185 stool specimens from healthy children aged 6-59 months attending 6 dispensaries in the Antananarivo area were examined for poliovirus. The children had been routinely immunized according to the Expanded Programme on Immunization (EPI) schedule and received the last dose of oral polio vaccine (OPV) more than 1 month before stool collection. 99.4% of the children were immunized with at least 3 doses of OPV. HEp-2 cell culture revealed virus infections in 192 stools (6.0%), including 9 poliovirus (0.3%) and 183 nonpolio enterovirus isolates (5.7%). Infections occurred throughout the year, but incidence was higher during the hot and rainy season (P=0.01). Using a neutralization test with monoclonal antibodies and PCR-RFLP in two genomic regions coding for the VP1 capsid and RNA polymerase, 4 wild polioviruses (3 type 1 and 1 type 3) and 5 vaccine-related polioviruses (2 Sabin 1-like variants, 1 Sabin 2-like and 2 Sabin 3-like) strains were identified. The wild polioviruses were isolated at the beginning and the end of the dry season. Similar RFLP patterns were observed for the 3 wild type 1 polioviruses. Comparison of partial genomic sequences in the VP1/2 A region of 1 of the wild type 1 isolates with 2 wild type strains isolated in Antananarivo in 1992 and 1993 showed a divergence of at least 10% between the strains, suggesting at least two different pathways of transmission during this period. Our findings demonstrate that immunization with 3 doses of OPV did not prevent intestinal carriage of wild poliovirus strains, and that there is a risk of wild poliovirus transmission to susceptible children in the area. Multiple strategies are required to improve immunization coverage in Madagascar.
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Toll-like receptors 2 and 4 contribute to sepsis-induced depletion of spleen dendritic cells.
Pène, Frédéric; Courtine, Emilie; Ouaaz, Fatah; Zuber, Benjamin; Sauneuf, Bertrand; Sirgo, Gonzalo; Rousseau, Christophe; Toubiana, Julie; Balloy, Viviane; Chignard, Michel; Mira, Jean-Paul; Chiche, Jean-Daniel
2009-12-01
Depletion of dendritic cells (DC) in secondary lymphoid organs is a hallmark of sepsis-induced immune dysfunction. In this setting, we investigated if Toll-like receptor (TLR)-dependent signaling might modulate the maturation process and the survival of DC. Using a model of sublethal polymicrobial sepsis induced by cecal ligation and puncture, we investigated the quantitative and functional features of spleen DC in wild-type, TLR2(-/-), TLR4(-/-), and TLR2(-/-) TLR4(-/-) mice. By 24 h, a decrease in the relative percentage of CD11c(high) spleen DC occurred in wild-type mice but was prevented in TLR2(-/-), TLR4(-/-), and TLR2(-/-) TLR4(-/-) mice. In wild-type mice, sepsis dramatically affected both CD11c(+) CD8alpha(+) and CD11c(+) CD8alpha(-) subsets. In all three types of knockout mice studied, the CD11c(+) CD8alpha(+) subset followed a depletion pattern similar to that for wild-type mice. In contrast, the loss of CD11c(+) CD8alpha(-) cells was attenuated in TLR2(-/-) and TLR4(-/-) mice and completely prevented in TLR2(-/-) TLR4(-/-) mice. Accordingly, apoptosis of spleen DC was increased in septic wild-type mice and inhibited in knockout mice. In addition we characterized the functional features of spleen DC obtained from septic mice. As shown by increased expression of major histocompatibility complex class II and CD86, polymicrobial sepsis induced maturation of DC, with subsequent increased capacity to prime T lymphocytes, similarly in wild-type and knockout mice. In response to CpG DNA stimulation, production of interleukin-12 was equally impaired in DC obtained from wild-type and knockout septic mice. In conclusion, although dispensable for the DC maturation process, TLR2 and TLR4 are involved in the mechanisms leading to depletion of spleen DC following polymicrobial sepsis.
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Singh, Balwant; Singh, Nisha; Mishra, Shefali; Tripathi, Kabita; Singh, Bikram P.; Rai, Vandna; Singh, Ashok K.; Singh, Nagendra K.
2018-01-01
Wild relatives of crops possess adaptive mutations for agronomically important traits, which could play significant role in crop improvement for sustainable agriculture. However, global climate change and human activities pose serious threats to the natural habitats leading to erosion of genetic diversity of wild rice populations. The purpose of this study was to explore and characterize India’s huge untapped wild rice diversity in Oryza rufipogon Griff. species complex from a wide range of ecological niches. We made strategic expeditions around diversity hot spots in 64 districts of nine different agro-climatic zones of the country and collected 418 wild rice accessions. Significant variation was observed among the accessions for 46 morphological descriptors, allowing classification into O. nivara, O. rufipogon, and O. sativa f. spontanea morpho-taxonomic groups. Genome-specific pSINE1 markers confirmed all the accessions having AA genome, which were further classified using ecotype-specific pSINE1 markers into annual, perennial, intermediate, and an unknown type. Principal component analysis revealed continuous variation for the morphological traits in each ecotype group. Genetic diversity analysis based on multi-allelic SSR markers clustered these accessions into three major groups and analysis of molecular variance for nine agro-climatic zones showed that 68% of the genetic variation was inherent amongst individuals while only 11% of the variation separated the zones, though there was significant correlation between genetic and spatial distances of the accessions. Model based population structure analysis using genome wide bi-allelic SNP markers revealed three sub-populations designated ‘Pro-Indica,’ ‘Pro-Aus,’ and ‘Mid-Gangetic,’ which showed poor correspondence with the morpho-taxonomic classification or pSINE1 ecotypes. There was Pan-India distribution of the ‘Pro-Indica’ and ‘Pro-Aus’ sub-populations across agro-climatic zones, indicating a more fundamental grouping based on the ancestry closely related to ‘Indica’ and ‘Aus’ groups of rice cultivars. The Pro-Indica population has substantial presence in the Eastern Himalayan Region and Lower Gangetic Plains, whereas ‘Pro-Aus’ sub-population was predominant in the Upper Gangetic Plains, Western Himalayan Region, Gujarat Plains and Hills, and Western Coastal Plains. In contrast ‘Mid-Gangetic’ population was largely concentrated in the Mid Gangetic Plains. The information presented here will be useful in the utilization of wild rice resources for varietal improvement. PMID:29467785
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Wells, Randall S.; McHugh, Katherine A.; Douglas, David C.; Shippee, Steve; McCabe, Elizabeth Berens; Barros, Nélio B.; Phillips, Goldie T.
2014-01-01
Free-ranging bottlenose dolphins (Tursiops truncatus) living in Sarasota Bay, Florida appear to have a lower risk of developing insulin resistance and metabolic syndrome compared to a group of dolphins managed under human care. Similar to humans, differences in diet and activity cycles between these groups may explain why Sarasota dolphins have lower insulin, glucose, and lipids. To identify potential protective factors against metabolic syndrome, existing and new data were incorporated to describe feeding and activity patterns of the Sarasota Bay wild dolphin community. Sarasota dolphins eat a wide variety of live fish and spend 10–20% of daylight hours foraging and feeding. Feeding occurs throughout the day, with the dolphins eating small proportions of their total daily intake in brief bouts. The natural pattern of wild dolphins is to feed as necessary and possible at any time of the day or night. Wild dolphins rarely eat dead fish or consume large amounts of prey in concentrated time periods. Wild dolphins are active throughout the day and night; they may engage in bouts of each key activity category at any time during daytime. Dive patterns of radio-tagged dolphins varied only slightly with time of day. Travel rates may be slightly lower at night, suggesting a diurnal rhythm, albeit not one involving complete, extended rest. In comparison, the managed dolphins are older; often fed a smaller variety of frozen-thawed fish types; fed fish species not in their natural diet; feedings and engaged activities are often during the day; and they are fed larger but fewer meals. In summary, potential protective factors against metabolic syndrome in dolphins may include young age, activity, and small meals fed throughout the day and night, and specific fish nutrients. These protective factors against insulin resistance and type 2 diabetes are similar to those reported in humans. Further studies may benefit humans and dolphins.
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Wells, Randall S.; McHugh, Katherine A.; Douglas, David C.; Shippee, Steve; McCabe, Elizabeth Berens; Barros, Nélio B.; Phillips, Goldie T.
2013-01-01
Free-ranging bottlenose dolphins (Tursiops truncatus) living in Sarasota Bay, Florida appear to have a lower risk of developing insulin resistance and metabolic syndrome compared to a group of dolphins managed under human care. Similar to humans, differences in diet and activity cycles between these groups may explain why Sarasota dolphins have lower insulin, glucose, and lipids. To identify potential protective factors against metabolic syndrome, existing and new data were incorporated to describe feeding and activity patterns of the Sarasota Bay wild dolphin community. Sarasota dolphins eat a wide variety of live fish and spend 10–20% of daylight hours foraging and feeding. Feeding occurs throughout the day, with the dolphins eating small proportions of their total daily intake in brief bouts. The natural pattern of wild dolphins is to feed as necessary and possible at any time of the day or night. Wild dolphins rarely eat dead fish or consume large amounts of prey in concentrated time periods. Wild dolphins are active throughout the day and night; they may engage in bouts of each key activity category at any time during daytime. Dive patterns of radio-tagged dolphins varied only slightly with time of day. Travel rates may be slightly lower at night, suggesting a diurnal rhythm, albeit not one involving complete, extended rest. In comparison, the managed dolphins are older; often fed a smaller variety of frozen-thawed fish types; fed fish species not in their natural diet; feedings and engaged activities are often during the day; and they are fed larger but fewer meals. In summary, potential protective factors against metabolic syndrome in dolphins may include young age, activity, and small meals fed throughout the day and night, and specific fish nutrients. These protective factors against insulin resistance and type 2 diabetes are similar to those reported in humans. Further studies may benefit humans and dolphins. PMID:24133483
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Vaid, Mudit; Singh, Tripti; Prasad, Ram; Elmets, Craig A.; Xu, Hui; Katiyar, Santosh K.
2013-01-01
Ultraviolet (UV) radiation-induced immunosuppression has been implicated in skin carcinogenesis. Grape seed proanthocyanidins (GSPs) have anti-skin carcinogenic effects in mice and GSPs-fed mice exhibit a reduction in UV-induced suppression of allergic contact hypersensitivity (CHS), a prototypic T cell-mediated response. Here, we report that dietary GSPs did not inhibit UVB-induced suppression of CHS in xeroderma pigmentosum complementation group A (XPA)-deficient mice, which lack nucleotide excision repair mechanisms. GSPs enhanced repair of UVB-induced DNA damage (cyclobutane pyrimidine dimers) in wild-type, but not XPA-deficient, dendritic cells (DCs). Co-culture of CD4+ T cells with DCs from UVB-irradiated wild-type mice resulted in suppression of T-cell proliferation and secretion of Th-1 type cytokines that was ameliorated when the DCs were obtained from GSPs-fed mice; whereas, DCs obtained from GSPs-fed XPA-KO mice failed to restore T-cell proliferation. In adoptive transfer experiments, donor DCs were positively selected from the draining lymph nodes of UVB-exposed donor mice that were sensitized to 2,4, dinitrofluorobenzene were transferred into naïve recipient mice and the CHS response assessed. Naïve recipients that received DCs from UVB-exposed wild-type donors that had been fed GSPs exhibited a full CHS response, whereas no significant CHS was observed in mice that received DCs from XPA-KO mice fed GSPs. These results suggest that GSPs prevent UVB-induced immunosuppression through DNA repair-dependent functional activation of dendritic cells in mice. PMID:23321928
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Giebink, G S; Wright, P F
1983-01-01
We have previously shown that chinchillas infected with a multiply passaged laboratory strain of influenza A/NWS/33 (H1N1) develop negative middle-ear pressure; polymorphonuclear leukocyte oxidative, bactericidal, and chemotactic dysfunction; and increased susceptibility to pneumococcal otitis media. Because influenza A virus strains show different virulence in humans, three such strains were compared in the chinchilla model. Negative middle-ear pressure and tympanic membrane inflammation developed significantly more often in chinchillas infected with wild-type H3N2 virus than with either wild-type H1N1 virus or an attenuated, cold-adapted H3N2 vaccine strain, CR29. Marked depression in polymorphonuclear leukocyte chemiluminescent activity also developed significantly more often in H3N2 infected animals than in H1N1- or CR29-infected animals. Intranasal challenge of influenza virus-infected animals with type 7 Streptococcus pneumoniae resulted in a significantly greater occurrence of pneumococcal otitis media in H3N2-infected animals than in H1N1-, CR29-, or non-influenza-infected control animals. Clearance of pneumococci from nasal washings of animals infected with wild-type H3N2 was significantly delayed in comparison with the other groups. Thus, the previously demonstrated increased susceptibility to otitis media among children infected with H3N2 influenza virus may relate to the capacity of this strain to induce negative middle-ear pressure, polymorphonuclear leukocyte dysfunction, and alteration in the mucosal clearance of pneumococci. PMID:6885170
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Mercer, Kristin L.; Emry, D. Jason; Snow, Allison A.; Kost, Matthew A.; Pace, Brian A.; Alexander, Helen M.
2014-01-01
Understanding the likelihood and extent of introgression of novel alleles in hybrid zones requires comparison of lifetime fitness of parents and hybrid progeny. However, fitness differences among cross types can vary depending on biotic conditions, thereby influencing introgression patterns. Based on past work, we predicted that increased competition would enhance introgression between cultivated and wild sunflower (Helianthus annuus) by reducing fitness advantages of wild plants. To test this prediction, we established a factorial field experiment in Kansas, USA where we monitored the fitness of four cross types (Wild, F1, F2, and BCw hybrids) under different levels of interspecific and intraspecific competition. Intraspecific manipulations consisted both of density of competitors and of frequency of crop-wild hybrids. We recorded emergence of overwintered seeds, survival to reproduction, and numbers of seeds produced per reproductive plant. We also calculated two compound fitness measures: seeds produced per emerged seedling and seeds produced per planted seed. Cross type and intraspecific competition affected emergence and survival to reproduction, respectively. Further, cross type interacted with competitive treatments to influence all other fitness traits. More intense competition treatments, especially related to density of intraspecific competitors, repeatedly reduced the fitness advantage of wild plants when considering seeds produced per reproductive plant and per emerged seedling, and F2 plants often became indistinguishable from the wilds. Wild fitness remained superior when seedling emergence was also considered as part of fitness, but the fitness of F2 hybrids relative to wild plants more than quadrupled with the addition of interspecific competitors and high densities of intraspecific competitors. Meanwhile, contrary to prediction, lower hybrid frequency reduced wild fitness advantage. These results emphasize the importance of taking a full life cycle perspective. Additionally, due to effects of exogenous selection, a given hybrid generation may be especially well-suited to hastening introgression under particular environmental conditions. PMID:25295859
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Mercer, Kristin L; Emry, D Jason; Snow, Allison A; Kost, Matthew A; Pace, Brian A; Alexander, Helen M
2014-01-01
Understanding the likelihood and extent of introgression of novel alleles in hybrid zones requires comparison of lifetime fitness of parents and hybrid progeny. However, fitness differences among cross types can vary depending on biotic conditions, thereby influencing introgression patterns. Based on past work, we predicted that increased competition would enhance introgression between cultivated and wild sunflower (Helianthus annuus) by reducing fitness advantages of wild plants. To test this prediction, we established a factorial field experiment in Kansas, USA where we monitored the fitness of four cross types (Wild, F1, F2, and BCw hybrids) under different levels of interspecific and intraspecific competition. Intraspecific manipulations consisted both of density of competitors and of frequency of crop-wild hybrids. We recorded emergence of overwintered seeds, survival to reproduction, and numbers of seeds produced per reproductive plant. We also calculated two compound fitness measures: seeds produced per emerged seedling and seeds produced per planted seed. Cross type and intraspecific competition affected emergence and survival to reproduction, respectively. Further, cross type interacted with competitive treatments to influence all other fitness traits. More intense competition treatments, especially related to density of intraspecific competitors, repeatedly reduced the fitness advantage of wild plants when considering seeds produced per reproductive plant and per emerged seedling, and F2 plants often became indistinguishable from the wilds. Wild fitness remained superior when seedling emergence was also considered as part of fitness, but the fitness of F2 hybrids relative to wild plants more than quadrupled with the addition of interspecific competitors and high densities of intraspecific competitors. Meanwhile, contrary to prediction, lower hybrid frequency reduced wild fitness advantage. These results emphasize the importance of taking a full life cycle perspective. Additionally, due to effects of exogenous selection, a given hybrid generation may be especially well-suited to hastening introgression under particular environmental conditions.
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Curry, Jennifer Wendy; Hohl, Rodrigo; Noakes, Timothy David; Kohn, Tertius Abraham
2012-01-01
SUMMARY Some wild antelopes are fast sprinters and more resistant to fatigue than others. This study therefore investigated two wild antelope species to better understand their reported performance capability. Muscle samples collected post mortem from the vastus lateralis and longissimus lumborum of fallow deer (Dama dama) and springbok (Antidorcas marsupialis) were analysed for myosin heavy chain isoform content, citrate synthase, 3-hydroxyacyl CoA dehydrogenase, phosphofructokinase, lactate dehydrogenase and creatine kinase activities. Cross-sectional areas, fibre type and oxidative capacities of each fibre type were determined in the vastus lateralis only. The predominant fibre type in both muscle groups and species were type IIX (>50%), with springbok having more type IIX fibres than fallow deer (P<0.05). Overall cross-sectional area was not different between the two species. The metabolic pathway analyses showed high glycolytic and oxidative capacities for both species, but springbok had significantly higher CS activities than fallow deer. Large variation and overlap in oxidative capacities existed within and between the fibre types. Some type IIX fibres presented with oxidative capacities similar to those from type I and IIA fibres. The data suggest that springbok and fallow deer are able sprint at >90 and 46 km h−1, respectively, partly from having large type IIX fibre contents and high glycolytic capacities. The high oxidative capacities also suggest that these animals may be able to withstand fatigue for long periods of time. PMID:22899533
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Tiwari, Sameeksha; Awasthi, Manika; Singh, Swati; Pandey, Veda P; Dwivedi, Upendra N
2017-10-23
Protein-protein interactions (PPI) are a new emerging class of novel therapeutic targets. In order to probe these interactions, computational tools provide a convenient and quick method towards the development of therapeutics. Keeping this in view the present study was initiated to analyse interaction of tumour suppressor protein p53 (TP53) and breast cancer associated protein (BRCA1) as promising target against breast cancer. Using computational approaches such as protein-protein docking, hot spot analyses, molecular docking and molecular dynamics simulation (MDS), stepwise analyses of the interactions of the wild type and mutant TP53 with that of wild type BRCA1 and their modulation by alkaloids were done. Protein-protein docking method was used to generate both wild type and mutant complexes of TP53-BRCA1. Subsequently, the complexes were docked using sixteen different alkaloids, fulfilling ADMET and Lipinski's rule of five criteria, and were compared with that of a well-known inhibitor of PPI, namely nutlin. The alkaloid dicentrine was found to be the best docked alkaloid among all the docked alklaloids as well as that of nutlin. Furthermore, MDS analyses of both wild type and mutant complexes with the best docked alkaloid i.e. dicentrine, revealed higher stability of mutant complex than that of the wild one, in terms of average RMSD, RMSF and binding free energy, corroborating the results of docking. Results suggested more pronounced interaction of BRCA1 with mutant TP53 leading to increased expression of mutated TP53 thus showing a dominant negative gain of function and hampering wild type TP53 function leading to tumour progression.
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Role of Mrx Fimbriae of Xenorhabdus nematophila in Competitive Colonization of the Nematode Host ▿
Snyder, Holly; He, Hongjun; Owen, Heather; Hanna, Chris; Forst, Steven
2011-01-01
Xenorhabdus nematophila engages in mutualistic associations with the infective juvenile (IJ) stage of specific entomopathogenic nematodes. Mannose-resistant (Mrx) chaperone-usher-type fimbriae are produced when the bacteria are grown on nutrient broth agar (NB agar). The role of Mrx fimbriae in the colonization of the nematode host has remained unresolved. We show that X. nematophila grown on LB agar produced flagella rather than fimbriae. IJs propagated on X. nematophila grown on LB agar were colonized to the same extent as those propagated on NB agar. Further, progeny IJs were normally colonized by mrx mutant strains that lacked fimbriae both when bacteria were grown on NB agar and when coinjected into the insect host with aposymbiotic nematodes. The mrx strains were not competitively defective for colonization when grown in the presence of wild-type cells on NB agar. In addition, a phenotypic variant strain that lacked fimbriae colonized as well as the wild-type strain. In contrast, the mrx strains displayed a competitive colonization defect in vivo. IJ progeny obtained from insects injected with comixtures of nematodes carrying either the wild-type or the mrx strain were colonized almost exclusively with the wild-type strain. Likewise, when insects were coinjected with aposymbiotic IJs together with a comixture of the wild-type and mrx strains, the resulting IJ progeny were predominantly colonized with the wild-type strain. These results revealed that Mrx fimbriae confer a competitive advantage during colonization in vivo and provide new insights into the role of chaperone-usher fimbriae in the life cycle of X. nematophila. PMID:21856828
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Yan, Da-Wei; Wang, Jing; Yuan, Ting-Ting; Hong, Li-Wei; Gao, Xiang; Lu, Ying-Tang
2013-01-01
Aux/IAAs interact with auxin response factors (ARFs) to repress their transcriptional activity in the auxin signaling pathway. Previous studies have focused on gain-of-function mutations of domain II and little is known about whether the expression level of wild-type Aux/IAAs can modulate auxin homeostasis. Here we examined the perturbation of auxin homeostasis by ectopic expression of wild-type IAA15. Root gravitropism and stem cell differentiation were also analyzed. The transgenic lines were less sensitive to exogenous auxin and exhibited low-auxin phenotypes including failures in gravity response and defects in stem cell differentiation. Overexpression lines also showed an increase in auxin concentration and reduced polar auxin transport. These results demonstrate that an alteration in the expression of wild-type IAA15 can disrupt auxin homeostasis.
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Roper, Jatin; Sinnamon, Mark J.; Coffee, Erin M.; Belmont, Peter; Keung, Lily; Georgeon-Richard, Larissa; Wang, Wei Vivian; Faber, Anthony C.; Yun, Jihye; Yilmaz, Omer H.; Bronson, Roderick T.; Martin, Eric S.; Tsichlis, Philip N.; Hung, Kenneth E.
2014-01-01
PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer. PMID:24576621
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Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D
2016-01-01
Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8-100% sensitivity, 100% specificity, 86-89% efficiency and a limit of detection of 12-400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82-90% efficiency and limit of detection of 120-4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.
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Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.
2016-01-01
Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains. PMID:26839745
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Watanabe, Kazuki; Sakai, Takaomi
2016-01-01
In the fruitfly Drosophila melanogaster, females take the initiative to mate successfully because they decide whether to mate or not. However, little is known about the molecular and neuronal mechanisms regulating sexual receptivity in virgin females. Genetic tools available in Drosophila are useful for identifying molecules and neural circuits involved in the regulation of sexual receptivity. We previously demonstrated that insulin-producing cells (IPCs) in the female brain are critical to the regulation of female sexual receptivity. Ablation and inactivation of IPCs enhance female sexual receptivity, suggesting that neurosecretion from IPCs inhibits female sexual receptivity. IPCs produce and release insulin-like peptides (Ilps) that modulate various biological processes such as metabolism, growth, lifespan and behaviors. Here, we report a novel role of the Ilps in sexual behavior in Drosophila virgin females. Compared with wild-type females, females with knockout mutations of Ilps showed a high mating success rate toward wild-type males, whereas wild-type males courted wild-type and Ilp-knockout females to the same extent. Wild-type receptive females retard their movement during male courtship and this reduced female mobility allows males to copulate. Thus, it was anticipated that knockout mutations of Ilps would reduce general locomotion. However, the locomotor activity in Ilp-knockout females was significantly higher than that in wild-type females. Thus, our findings indicate that the high mating success rate in Ilp-knockout females is caused by their enhanced sexual receptivity, but not by improvement of their sex appeal or by general sluggishness.
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Rao, Shailaja Kishan; Ross, Jordan M; Harrison, Fiona E; Bernardo, Alexandra; Reiserer, Randall S; Reiserer, Ronald S; Mobley, James A; McDonald, Michael P
2015-06-01
Physical exercise may provide protection against the cognitive decline and neuropathology associated with Alzheimer's disease, although the mechanisms are not clear. In the present study, APP/PSEN1 double-transgenic and wild-type mice were allowed unlimited voluntary exercise for 7months. Consistent with previous reports, wheel-running improved cognition in the double-transgenic mice. Interestingly, the average daily distance run was strongly correlated with spatial memory in the water maze in wild-type mice (r(2)=.959), but uncorrelated in transgenics (r(2)=.013). Proteomics analysis showed that sedentary transgenic mice differed significantly from sedentary wild-types with respect to proteins involved in synaptic transmission, cytoskeletal regulation, and neurogenesis. When given an opportunity to exercise, the transgenics' deficiencies in cytoskeletal regulation and neurogenesis largely normalized, but abnormal synaptic proteins did not change. In contrast, exercise enhanced proteins associated with cytoskeletal regulation, oxidative phosphorylation, and synaptic transmission in wild-type mice. Soluble and insoluble Aβ40 and Aβ42 levels were significantly decreased in both cortex and hippocampus of active transgenics, suggesting that this may have played a role in the cognitive improvement in APP/PSEN1 mice. β-secretase was significantly reduced in active APP/PSEN1 mice compared to sedentary controls, suggesting a mechanism for reduced Aβ. Taken together, these data illustrate that exercise improves memory in wild-type and APP-overexpressing mice in fundamentally different ways. Copyright © 2015 Elsevier Inc. All rights reserved.
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Microbiota-induced obesity requires farnesoid X receptor
Parséus, Ava; Sommer, Nina; Sommer, Felix; Caesar, Robert; Molinaro, Antonio; Ståhlman, Marcus; Greiner, Thomas U; Perkins, Rosie; Bäckhed, Fredrik
2017-01-01
Objective The gut microbiota has been implicated as an environmental factor that modulates obesity, and recent evidence suggests that microbiota-mediated changes in bile acid profiles and signalling through the bile acid nuclear receptor farnesoid X receptor (FXR) contribute to impaired host metabolism. Here we investigated if the gut microbiota modulates obesity and associated phenotypes through FXR. Design We fed germ-free (GF) and conventionally raised (CONV-R) wild-type and Fxr−/− mice a high-fat diet (HFD) for 10 weeks. We monitored weight gain and glucose metabolism and analysed the gut microbiota and bile acid composition, beta-cell mass, accumulation of macrophages in adipose tissue, liver steatosis, and expression of target genes in adipose tissue and liver. We also transferred the microbiota of wild-type and Fxr-deficient mice to GF wild-type mice. Results The gut microbiota promoted weight gain and hepatic steatosis in an FXR-dependent manner, and the bile acid profiles and composition of faecal microbiota differed between Fxr−/− and wild-type mice. The obese phenotype in colonised wild-type mice was associated with increased beta-cell mass, increased adipose inflammation, increased steatosis and expression of genes involved in lipid uptake. By transferring the caecal microbiota from HFD-fed Fxr−/− and wild-type mice into GF mice, we showed that the obesity phenotype was transferable. Conclusions Our results indicate that the gut microbiota promotes diet-induced obesity and associated phenotypes through FXR, and that FXR may contribute to increased adiposity by altering the microbiota composition. PMID:26740296
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pigk Mutation underlies macho behavior and affects Rohon-Beard cell excitability
Carmean, V.; Yonkers, M. A.; Tellez, M. B.; Willer, J. R.; Willer, G. B.; Gregg, R. G.; Geisler, R.; Neuhauss, S. C.
2015-01-01
The study of touch-evoked behavior allows investigation of both the cells and circuits that generate a response to tactile stimulation. We investigate a touch-insensitive zebrafish mutant, macho (maco), previously shown to have reduced sodium current amplitude and lack of action potential firing in sensory neurons. In the genomes of mutant but not wild-type embryos, we identify a mutation in the pigk gene. The encoded protein, PigK, functions in attachment of glycophosphatidylinositol anchors to precursor proteins. In wild-type embryos, pigk mRNA is present at times when mutant embryos display behavioral phenotypes. Consistent with the predicted loss of function induced by the mutation, knock-down of PigK phenocopies maco touch insensitivity and leads to reduced sodium current (INa) amplitudes in sensory neurons. We further test whether the genetic defect in pigk underlies the maco phenotype by overexpressing wild-type pigk in mutant embryos. We find that ubiquitous expression of wild-type pigk rescues the touch response in maco mutants. In addition, for maco mutants, expression of wild-type pigk restricted to sensory neurons rescues sodium current amplitudes and action potential firing in sensory neurons. However, expression of wild-type pigk limited to sensory cells of mutant embryos does not allow rescue of the behavioral touch response. Our results demonstrate an essential role for pigk in generation of the touch response beyond that required for maintenance of proper INa density and action potential firing in sensory neurons. PMID:26133798
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Kimura, Yoshio; Tanaka, Chihiro; Sasaki, Katsuho; Sasaki, Masashi
2017-01-01
Diadenosine polyphosphates (ApnA) are thought to act as signalling molecules regulating stress responses and biofilm formation in prokaryotes. However, ApnA function in Myxococcus xanthus remains unknown. Here, we investigated the role of ApnA in M. xanthus, using the wild-type and ApnA hydrolase (apaH) mutant strains exposed to various stress conditions. In both wild-type and apaH mutant cells cultured on starvation medium (CF agar), the levels of intracellular diadenosine tetraphosphate (Ap4A) and pentaphosphate (Ap5A) increased several fold during the first 16 h of development and decreased gradually thereafter. The levels of Ap4A and Ap5A in the apaH mutant were about 5- and 11-fold higher than those in the wild-type strain at 16 h, respectively. ApnA hydrolase activity of the wild-type strain increased 1.5-fold during the first 8 h of development, and it then gradually decreased. The apaH mutant formed spores 1-2 days after the wild-type strain did, and the yield of viable spores was 5.5 % of that in the wild-type strain 5 days after inoculation onto CF agar. These results suggest the possibility that high intracellular levels of Ap4A and/or Ap5A may inhibit M. xanthus sporulation at the early stage of development and that the bacteria reduce intracellular Ap4A and Ap5A accumulation through ApnA hydrolase activity.
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Vaidya, Sunil R; Dvivedi, Garima M; Jadhav, Santoshkumar M
2016-01-01
The reports from the countries where mumps vaccine is given as routine immunization suggest differences in mumps virus neutralizing antibody titres when tested with vaccine and wild type viruses. Such reports are unavailable from countries like India where mumps vaccine is not included in routine immunization. We, therefore, undertook this study to understand the cross-neutralization activity of Indian mumps viruses. By using commercial mumps IgG enzyme immunoassay (EIA) and a rapid focus reduction neutralization test (FRNT), a panel of serum samples was tested. The panel consisted of 14 acute and 14 convalescent serum samples collected during a mumps outbreak and 18 archived serum samples. Two wild types (genotypes C and G) and Leningrad-Zagreb vaccine strain (genotype N) were used for the challenge experiments and FRNT titres were determined and further compared. The HN protein sequence of three mumps viruses was analyzed for the presence of key epitopes. All serum samples effectively neutralized mumps virus wild types and a vaccine strain. However, significantly lower FRNT titres were noted to wild types than to vaccine strain (P<0.05). The comparison between EIA and FRNT results revealed 95.6 per cent agreement. No amino acid changes were seen in the epitopes in the Indian wild type strains. All potential N-linked glycosylation sites were observed in Indian strains. Good cross-neutralization activity was observed for three mumps virus strains, however, higher level of FRNT titres was detected for mumps virus vaccine strain compared to Indian wild type isolates.
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Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo.
Myer, A; Wagner, D S; Vivian, J L; Olson, E N; Klein, W H
1997-05-15
Skeletal muscle is formed via a complex series of events during embryogenesis. These events include commitment of mesodermal precursor cells, cell migration, cell-cell recognition, fusion of myoblasts, activation of structural genes, and maturation. In mice lacking the bHLH transcription factor myogenin, myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This indicates that myogenin is critical for the fusion process and subsequent differentiation events of myogenesis. To further define the nature of the myogenic defects in myogenin-null mice, we investigated whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos demonstrated that myogenin-null myoblasts readily fused in the presence of wild-type myoblasts. However, chimeric myofibers did not express wild-type levels of muscle-specific gene products, and myofibers with a high percentage of mutant nuclei appeared abnormal, suggesting that the wild-type nuclei could not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific subset of genes with diverse function. Thus, myogenin appears to control not only transcription of muscle structural genes but also the extracellular environment in which myoblast fusion takes place. We propose that myogenin regulates the expression of one or more extracellular or cell surface proteins required to initiate the muscle differentiation program.
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Cdx mutant axial progenitor cells are rescued by grafting to a wild type environment.
Bialecka, Monika; Wilson, Valerie; Deschamps, Jacqueline
2010-11-01
Cdx transcription factors are required for axial extension. Cdx genes are expressed in the posterior growth zone, a region that supplies new cells for axial elongation. Cdx2(+/-)Cdx4(-/-) (Cdx2/4) mutant embryos show abnormalities in axis elongation from E8.5, culminating in axial truncation at E10.5. These data raised the possibility that the long-term axial progenitors of Cdx mutants are intrinsically impaired in their ability to contribute to posterior growth. We investigated whether we could identify cell-autonomous defects of the axial progenitor cells by grafting mutant cells into a wild type growth zone environment. We compared the contribution of GFP labeled mutant and wild type progenitors grafted to unlabeled wild type recipients subsequently cultured over the period during which Cdx2/4 defects emerge. Descendants of grafted cells were scored for their contribution to differentiated tissues in the elongating axis and to the posterior growth zone. No difference between the contribution of descendants from wild type and mutant grafted progenitors was detected, indicating that rescue of the Cdx mutant progenitors by the wild type recipient growth zone is provided non-cell autonomously. Recently, we showed that premature axial termination of Cdx mutants can be partly rescued by stimulating canonical Wnt signaling in the posterior growth zone. Taken together with the data shown here, this suggests that Cdx genes function to maintain a signaling-dependent niche for the posterior axial progenitors. Copyright © 2010 Elsevier Inc. All rights reserved.
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2013-01-01
The genetic missense A30P mutation of the wild-type α-synuclein protein results in the replacement of the 30th amino acid residue from alanine (Ala) to proline (Pro) and was initially found in the members of a German family who developed Parkinson’s disease. Even though the structures of these proteins have been measured before, detailed understanding about the structures and their relationships with free energy landscapes is lacking, which is of interest to provide insights into the pathogenic mechanism of Parkinson’s disease. We report the secondary and tertiary structures and conformational free energy landscapes of the wild-type and A30P mutant-type α-synuclein proteins in an aqueous solution environment via extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations. In addition, we present the residual secondary structure component transition stabilities at the atomic level with dynamics in terms of free energy change calculations using a new strategy that we reported most recently. Our studies yield new interesting results; for instance, we find that the A30P mutation has local as well as long-range effects on the structural properties of the wild-type α-synuclein protein. The helical content at Ala18-Gly31 is less prominent in comparison to the wild-type α-synuclein protein. The β-sheet structure abundance decreases in the N-terminal region upon A30P mutation of the wild-type α-synuclein, whereas the NAC and C-terminal regions possess larger tendencies for β-sheet structure formation. Long-range intramolecular protein interactions are less abundant upon A30P mutation, especially between the NAC and C-terminal regions, which is linked to the less compact and less stable structures of the A30P mutant-type rather than the wild-type α-synuclein protein. Results including the usage of our new strategy for secondary structure transition stabilities show that the A30P mutant-type α-synuclein tendency toward aggregation is higher than the wild-type α-synuclein but we also find that the C-terminal and NAC regions of the A30P mutant-type α-synuclein are reactive toward fibrillzation and aggregation based on atomic level studies with dynamics in an aqueous solution environment. Therefore, we propose that small molecules or drugs blocking the specific residues, which we report herein, located in the NAC- and C-terminal regions of the A30P mutant-type α-synuclein protein might help to reduce the toxicity of the A30P mutant-type α-synuclein protein. PMID:23374072
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Wise-Scira, Olivia; Aloglu, Ahmet Kemal; Dunn, Aquila; Sakallioglu, Isin Tuna; Coskuner, Orkid
2013-03-20
The genetic missense A30P mutation of the wild-type α-synuclein protein results in the replacement of the 30th amino acid residue from alanine (Ala) to proline (Pro) and was initially found in the members of a German family who developed Parkinson's disease. Even though the structures of these proteins have been measured before, detailed understanding about the structures and their relationships with free energy landscapes is lacking, which is of interest to provide insights into the pathogenic mechanism of Parkinson's disease. We report the secondary and tertiary structures and conformational free energy landscapes of the wild-type and A30P mutant-type α-synuclein proteins in an aqueous solution environment via extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations. In addition, we present the residual secondary structure component transition stabilities at the atomic level with dynamics in terms of free energy change calculations using a new strategy that we reported most recently. Our studies yield new interesting results; for instance, we find that the A30P mutation has local as well as long-range effects on the structural properties of the wild-type α-synuclein protein. The helical content at Ala18-Gly31 is less prominent in comparison to the wild-type α-synuclein protein. The β-sheet structure abundance decreases in the N-terminal region upon A30P mutation of the wild-type α-synuclein, whereas the NAC and C-terminal regions possess larger tendencies for β-sheet structure formation. Long-range intramolecular protein interactions are less abundant upon A30P mutation, especially between the NAC and C-terminal regions, which is linked to the less compact and less stable structures of the A30P mutant-type rather than the wild-type α-synuclein protein. Results including the usage of our new strategy for secondary structure transition stabilities show that the A30P mutant-type α-synuclein tendency toward aggregation is higher than the wild-type α-synuclein but we also find that the C-terminal and NAC regions of the A30P mutant-type α-synuclein are reactive toward fibrillzation and aggregation based on atomic level studies with dynamics in an aqueous solution environment. Therefore, we propose that small molecules or drugs blocking the specific residues, which we report herein, located in the NAC- and C-terminal regions of the A30P mutant-type α-synuclein protein might help to reduce the toxicity of the A30P mutant-type α-synuclein protein.
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Mundell, S J; Matharu, A-L; Nisar, S; Palmer, T M; Benovic, J L; Kelly, E
2010-02-01
We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.
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Prevalence and genotype identification of Toxoplasma gondii in wild animals from southwestern Spain.
Calero-Bernal, Rafael; Saugar, José M; Frontera, Eva; Pérez-Martín, Juan E; Habela, Miguel A; Serrano, Francisco J; Reina, David; Fuentes, Isabel
2015-01-01
We used PCR to detect Toxoplasma gondii in the principal game species in southwestern Spain. We detected T. gondii in 32.2% of animals tested. Prevalences varied from 14.7% in wild boar (Sus scrofa) to 51.2% in red fox (Vulpes vulpes). The most prevalent genotype was type II (50.0%), followed by type III (20.6%) and type I (5.9%). Mixed infections (11.8%) were detected in wild boar (types I+III) and red fox (types II+III). Polymorphic strains (11.8%) were detected in several species. The high prevalence and the genetic variability shown could have implications for infection of farm animals and humans.
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Ding, Jianqiang; Yannam, Govardhana R; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I; Wong, Ronald J; Avsar, Yesim; Guha, Chandan; Perlmutter, David H; Fox, Ira J; Roy-Chowdhury, Jayanta
2011-05-01
α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.
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Peptidomics of Cpefat/fat mouse brain regions: Implications for neuropeptide processing
Zhang, Xin; Che, Fa-Yun; Berezniuk, Iryna; Sonmez, Kemal; Toll, Lawrence; Fricker, Lloyd D.
2009-01-01
SUMMARY Quantitative peptidomics was used to compare levels of peptides in wild type and Cpefat/fat mice, which lack carboxypeptidase E (CPE) activity due to a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in wild type mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpefat/fat mouse brain, relative to wild type mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpefat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpefat/fat mouse brain at levels similar to those in wild type mouse brain. Many peptides were greatly elevated in the Cpefat/fat mice; these peptide processing intermediates with C-terminal Lys and/or Arg were generally not detectable in wild type mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides. PMID:19014391
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Yow, Geok-Yong; Uo, Takuma; Yoshimura, Tohru; Esaki, Nobuyoshi
2006-03-01
Saccharomyces cerevisiae is sensitive to D-amino acids: those corresponding to almost all proteinous L-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that D-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of D-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to D-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to D-amino acids than the wild type. We further confirmed that, upon cultivation with D-phenylalanine, N-acetyl-D-phenylalanine was accumulated in the culture but not in the wild type and hpa3Delta cells overproducing DNT cells. Thus, D-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.
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Avery, Simon V.; Malkapuram, Srividya; Mateus, Carolina; Babb, Kimberly S.
2000-01-01
Saccharomyces cerevisiae, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S. cerevisiae hypersensitive to oxytetracycline (OTC): a sod1Δ mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 μg ml−1, whereas concentrations of up to 1,000 μg ml−1 had no effect on the growth of the wild type. OTC resistance was restored in the sod1Δ mutant by complementation with wild-type SOD1. The effect of OTC appeared to be cytotoxic and was not evident in a ctt1Δ (cytosolic catalase) mutant or in the presence of tetracycline. SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and sod1Δ strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1Δ mutant, but not the wild type, to OTC. We propose that Sod1p protects S. cerevisiae against a mode of OTC action that is dependent on oxidative damage. PMID:10613865
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Compensation for PKMζ in long-term potentiation and spatial long-term memory in mutant mice
Tsokas, Panayiotis; Hsieh, Changchi; Yao, Yudong; Lesburguères, Edith; Wallace, Emma Jane Claire; Tcherepanov, Andrew; Jothianandan, Desingarao; Hartley, Benjamin Rush; Pan, Ling; Rivard, Bruno; Farese, Robert V; Sajan, Mini P; Bergold, Peter John; Hernández, Alejandro Iván; Cottrell, James E; Shouval, Harel Z; Fenton, André Antonio; Sacktor, Todd Charlton
2016-01-01
PKMζ is a persistently active PKC isoform proposed to maintain late-LTP and long-term memory. But late-LTP and memory are maintained without PKMζ in PKMζ-null mice. Two hypotheses can account for these findings. First, PKMζ is unimportant for LTP or memory. Second, PKMζ is essential for late-LTP and long-term memory in wild-type mice, and PKMζ-null mice recruit compensatory mechanisms. We find that whereas PKMζ persistently increases in LTP maintenance in wild-type mice, PKCι/λ, a gene-product closely related to PKMζ, persistently increases in LTP maintenance in PKMζ-null mice. Using a pharmacogenetic approach, we find PKMζ-antisense in hippocampus blocks late-LTP and spatial long-term memory in wild-type mice, but not in PKMζ-null mice without the target mRNA. Conversely, a PKCι/λ-antagonist disrupts late-LTP and spatial memory in PKMζ-null mice but not in wild-type mice. Thus, whereas PKMζ is essential for wild-type LTP and long-term memory, persistent PKCι/λ activation compensates for PKMζ loss in PKMζ-null mice. DOI: http://dx.doi.org/10.7554/eLife.14846.001 PMID:27187150
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ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS
The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...
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2013-01-01
The A53T genetic missense mutation of the wild-type α-synuclein (αS) protein was initially identified in Greek and Italian families with familial Parkinson’s disease. Detailed understanding of the structures and the changes induced in the wild-type αS structure by the A53T mutation, as well as establishing the direct relationships between the rapid conformational changes and free energy landscapes of these intrinsically disordered fibrillogenic proteins, helps to enhance our fundamental knowledge and to gain insights into the pathogenic mechanism of Parkinson’s disease. We employed extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations to determine the secondary and tertiary structural properties as well as the conformational free energy surfaces of the wild-type and A53T mutant-type αS proteins in an aqueous solution medium using both implicit and explicit water models. The confined aqueous volume effect in the simulations of disordered proteins using an explicit model for water is addressed for a model disordered protein. We also assessed the stabilities of the residual secondary structure component interconversions in αS based on free energy calculations at the atomic level with dynamics using our recently developed theoretical strategy. To the best of our knowledge, this study presents the first detailed comparison of the structural properties linked directly to the conformational free energy landscapes of the monomeric wild-type and A53T mutant-type α-synuclein proteins in an aqueous solution environment. Results demonstrate that the β-sheet structure is significantly more altered than the helical structure upon A53T mutation of the monomeric wild-type αS protein in aqueous solution. The β-sheet content close to the mutation site in the N-terminal region is more abundant while the non-amyloid-β component (NAC) and C-terminal regions show a decrease in β-sheet abundance upon A53T mutation. Obtained results utilizing our new theoretical strategy show that the residual secondary structure conversion stabilities resulting in α-helix formation are not significantly affected by the mutation. Interestingly, the residual secondary structure conversion stabilities show that secondary structure conversions resulting in β-sheet formation are influenced by the A53T mutation and the most stable residual transition yielding β-sheet occurs directly from the coil structure. Long-range interactions detected between the NAC region and the N- or C-terminal regions of the wild-type αS disappear upon A53T mutation. The A53T mutant-type αS structures are thermodynamically more stable than those of the wild-type αS protein structures in aqueous solution. Overall, the higher propensity of the A53T mutant-type αS protein to aggregate in comparison to the wild-type αS protein is related to the increased β-sheet formation and lack of strong intramolecular long-range interactions in the N-terminal region in comparison to its wild-type form. The specific residual secondary structure component stabilities reported herein provide information helpful for designing and synthesizing small organic molecules that can block the β-sheet forming residues, which are reactive toward aggregation. PMID:23607785
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Coskuner, Orkid; Wise-Scira, Olivia
2013-07-17
The A53T genetic missense mutation of the wild-type α-synuclein (αS) protein was initially identified in Greek and Italian families with familial Parkinson's disease. Detailed understanding of the structures and the changes induced in the wild-type αS structure by the A53T mutation, as well as establishing the direct relationships between the rapid conformational changes and free energy landscapes of these intrinsically disordered fibrillogenic proteins, helps to enhance our fundamental knowledge and to gain insights into the pathogenic mechanism of Parkinson's disease. We employed extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations to determine the secondary and tertiary structural properties as well as the conformational free energy surfaces of the wild-type and A53T mutant-type αS proteins in an aqueous solution medium using both implicit and explicit water models. The confined aqueous volume effect in the simulations of disordered proteins using an explicit model for water is addressed for a model disordered protein. We also assessed the stabilities of the residual secondary structure component interconversions in αS based on free energy calculations at the atomic level with dynamics using our recently developed theoretical strategy. To the best of our knowledge, this study presents the first detailed comparison of the structural properties linked directly to the conformational free energy landscapes of the monomeric wild-type and A53T mutant-type α-synuclein proteins in an aqueous solution environment. Results demonstrate that the β-sheet structure is significantly more altered than the helical structure upon A53T mutation of the monomeric wild-type αS protein in aqueous solution. The β-sheet content close to the mutation site in the N-terminal region is more abundant while the non-amyloid-β component (NAC) and C-terminal regions show a decrease in β-sheet abundance upon A53T mutation. Obtained results utilizing our new theoretical strategy show that the residual secondary structure conversion stabilities resulting in α-helix formation are not significantly affected by the mutation. Interestingly, the residual secondary structure conversion stabilities show that secondary structure conversions resulting in β-sheet formation are influenced by the A53T mutation and the most stable residual transition yielding β-sheet occurs directly from the coil structure. Long-range interactions detected between the NAC region and the N- or C-terminal regions of the wild-type αS disappear upon A53T mutation. The A53T mutant-type αS structures are thermodynamically more stable than those of the wild-type αS protein structures in aqueous solution. Overall, the higher propensity of the A53T mutant-type αS protein to aggregate in comparison to the wild-type αS protein is related to the increased β-sheet formation and lack of strong intramolecular long-range interactions in the N-terminal region in comparison to its wild-type form. The specific residual secondary structure component stabilities reported herein provide information helpful for designing and synthesizing small organic molecules that can block the β-sheet forming residues, which are reactive toward aggregation.
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Effect of Environmental Enrichment on Singly- and Group-Housed Squirrel Monkeys
NASA Technical Reports Server (NTRS)
Spring, Sarah E.; Clifford, James O.; Tomko, David L.; Hargens, Alan R. (Technical Monitor)
1996-01-01
Nonhuman primates display an interest in novel places, habituate to new situations, and spend most of their daily activity in the wild in large groups engaging in feeding behaviors. Captivity changes these behaviors, and disrupts normal social hierarchies. In captivity, animals may exhibit stereotypical behaviors which are thought to indicate decreased psychological well-being (PWB). If an animal's behaviors can be made to approach those seen in the wild, and stereotypical behaviors are minimal it is assumed that PWB is adequate. Environmental enrichment (EE) devices have been used to address the Animal Welfare Act's requirement that the PWB of captive nonhuman primates be considered. The purpose of the present study was to examine whether various EE devices improve the PWB of captive squirrel monkeys. The present study used behavioral observation to quantify the effectiveness of several EE devices in reducing stereotypical behaviors in squirrel monkeys housed singly or in groups. Results showed that the EE devices used did not affect the expression of normal or stereotypical behaviors, but that the type of housing did.
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Thioredoxin-1 attenuates sepsis-induced cardiomyopathy after cecal ligation and puncture in mice.
Wilson, Rickesha L; Selvaraju, Vaithinathan; Lakshmanan, Rajesh; Thirunavukkarasu, Mahesh; Campbell, Jacob; McFadden, David W; Maulik, Nilanjana
2017-12-01
Sepsis is a leading cause of mortality among patients in intensive care units across the USA. Thioredoxin-1 (Trx-1) is an essential 12 kDa cytosolic protein that, apart from maintaining the cellular redox state, possesses multifunctional properties. In this study, we explored the possibility of controlling adverse myocardial depression by overexpression of Trx-1 in a mouse model of severe sepsis. Adult C57BL/6J and Trx-1 Tg/+ mice were divided into wild-type sham (WTS), wild-type cecal ligation and puncture (WTCLP), Trx-1 Tg/+ sham (Trx-1 Tg/+ S), and Trx-1 Tg/+ CLP groups. Cardiac function was evaluated before surgery, 6 and 24 hours after CLP surgery. Immunohistochemical and Western blot analysis were performed after 24 hours in heart tissue sections. Echocardiography analysis showed preserved cardiac function in the Trx-1 Tg/+ CLP group compared with the WTCLP group. Similarly, Western blot analysis revealed increased expression of Trx-1, heme oxygenase-1 (HO-1), survivin (an inhibitor of apoptosis [IAP] protein family), and decreased expression of thioredoxin-interacting protein (TXNIP), caspase-3, and 3- nitrotyrosine in the Trx-1 Tg/+ CLP group compared with the WTCLP group. Immunohistochemical analysis showed reduced 4-hydroxynonenal, apoptosis, and vascular leakage in the cardiac tissue of Trx-1 Tg/+ CLP mice compared with mice in the WTCLP group. Our results indicate that overexpression of Trx-1 attenuates cardiac dysfunction during CLP. The mechanism of action may involve reduction of oxidative stress, apoptosis, and vascular permeability through activation of Trx-1/HO-1 and anti-apoptotic protein survivin. Copyright © 2017 Elsevier Inc. All rights reserved.
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Omega-3 Fatty Acids Supplementation: Therapeutic Potential in a Mouse Model of Stargardt Disease.
Prokopiou, Ekatherine; Kolovos, Panagiotis; Kalogerou, Maria; Neokleous, Anastasia; Nicolaou, Orthodoxia; Sokratous, Kleitos; Kyriacou, Kyriacos; Georgiou, Tassos
2018-06-01
To evaluate the therapeutic effects of omega-3 (ω3) fatty acids on retinal degeneration in the ABCA4-/- model of Stargardt disease when the blood level of arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio is between 1 and 1.5. Eight-month-old mice were allocated to three groups: wild type (129S1), ABCA4-/- untreated, and ABCA4-/- ω3 treated. ω3 treatment lasted 3 months and comprised daily gavage administration of EPA and docosahexaenoic acid (DHA). Blood and retinal fatty acid analysis was performed using gas chromatography to adjust the blood AA/EPA ∼1 to 1.5. Eyecups were histologically examined using transmission electron microscopy and confocal microscopy to evaluate lipofuscin granules and the photoreceptor layer. Retinal N-retinylidene-N-retinylethanolamine (A2E), a major component of retinal pigment epithelium lipofuscin, was quantified using liquid chromatography and tandem mass spectrometry, in addition to retinal proteomic analysis to determine changes in inflammatory proteins. EPA levels increased and AA levels decreased in the blood and retinas of the treatment group. Significantly less A2E and lipofuscin granules were observed in the treatment group. The thickness of the outer nuclear layer was significantly greater in the treatment group (75.66 ± 4.80 μm) than in the wild-type (61.40 ± 1.84 μm) or untreated ABCA4-/- (56.50 ± 3.24 μm) groups. Proteomic analysis indicated lower levels of complement component 3 (C3) in the treatment group, indicative of lower complement-induced inflammatory response. Three months of ω3 supplementation (AA/EPA ∼1-1.5) reduces A2E levels, lipofuscin granules, and C3 levels in the ABCA4-/- mouse model of Stargardt disease, consistent with slowing of the disease.
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Yamada, Hanano; Vreysen, Marc J B; Gilles, Jeremie R L; Munhenga, Givemore; Damiens, David D
2014-08-13
To enable the release of only sterile male Anopheles arabiensis mosquitoes for the sterile insect technique, the genetic background of a wild-type strain was modified to create a genetic sexing strain ANO IPCL1 that was based on a dieldrin resistance mutation. Secondly, the eggs of ANO IPCL1 require treatment with dieldrin to allow complete elimination of female L1 larvae from the production line. Finally, male mosquito pupae need to be treated with an irradiation dose of 75 Gy for sterilization. The effects of these treatments on the competitiveness of male An. arabiensis were studied. The competitiveness of ANO IPCL1 males that were treated either with irradiation or both dieldrin and irradiation, was compared with that of the wild-type strain (An. arabiensis Dongola) at a 1:1 ratio in 5.36 m3 semi-field cages located in a climate-controlled greenhouse. In addition, three irradiated: untreated male ratios were tested in semi-field cages (1:1, 5:1 and 10:1) and their competition for virgin wild-type females was assessed. The ANO IPCL1 males were equally competitive as the wild-type males in this semi-field setting. The ANO IPCL1 males irradiated at 75 Gy were approximately half as competitive as the unirradiated wild-type males. ANO IPCL1 males that had been treated with dieldrin as eggs, and irradiated with 75 Gy as pupae were slightly more competitive than males that were only irradiated. Ratios of 1:1, 5:1 and 10:1 irradiated ANO IPCL1 males: untreated wild-type males resulted in 31, 66 and 81% induced sterility in the female cage population, respectively. An irradiation dose of 75 Gy reduced the competitiveness of male ANO IPCL1 significantly and will need to be compensated by releasing higher numbers of sterile males in the field. However, the dieldrin treatment used to eliminate females appears to have an unexpected radioprotectant effect, however the mechanism is not understood. A sterile to wild-type ratio of 10:1 effectively reduced the population's fertility under the experimental field cage conditions, but further studies in the field will be needed to confirm the efficiency of sterile ANO IPCL1 males when competing against wild males for wild females.
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Munhenga, Givemore; Brooke, Basil D; Gilles, Jeremie R L; Slabbert, Kobus; Kemp, Alan; Dandalo, Leonard C; Wood, Oliver R; Lobb, Leanne N; Govender, Danny; Renke, Marius; Koekemoer, Lizette L
2016-03-02
Anopheles arabiensis Patton is primarily responsible for malaria transmission in South Africa after successful suppression of other major vector species using indoor spraying of residual insecticides. Control of An. arabiensis using current insecticide based approaches is proving difficult owing to the development of insecticide resistance, and variable feeding and resting behaviours. The use of the sterile insect technique as an area-wide integrated pest management system to supplement the control of An. arabiensis was proposed for South Africa and is currently under investigation. The success of this technique is dependent on the ability of laboratory-reared sterile males to compete with wild males for mates. As part of the research and development of the SIT technique for use against An. arabiensis in South Africa, radio-sensitivity and mating competitiveness of a local An. arabiensis sexing strain were assessed. The optimal irradiation dose inducing male sterility without compromising mating vigour was tested using Cobalt 60 irradiation doses ranging from 70-100 Gy. Relative mating competitiveness of sterile laboratory-reared males (GAMA strain) compared to fertile wild-type males (AMAL strain) for virgin wild-type females (AMAL) was investigated under laboratory and semi-field conditions using large outdoor cages. Three different sterile male to fertile male to wild-type female ratios were evaluated [1:1:1, 5:1:1 and 10:1:1 (sterile males: fertile, wild-type males: fertile, wild-type females)]. Irradiation at the doses tested did not affect adult emergence but had a moderate effect on adult survivorship and mating vigour. A dose of 75 Gy was selected for the competitiveness assays. Mating competitiveness experiments showed that irradiated GAMA male mosquitoes are a third as competitive as their fertile AMAL counterparts under semi-field conditions. However, they were not as competitive under laboratory conditions. An inundative ratio of 10:1 induced the highest sterility in the representative wild-type population, with potential to effectively suppress reproduction. Laboratory-reared and sterilised GAMA male An. arabiensis at a release ratio of 3:1 (3 sterile males to 1 wild, fertile male) can successfully compete for insemination of wild-type females. These results will be used to inform subsequent small-scale pilot field releases in South Africa.
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Olaya-Abril, Alfonso; Luque-Almagro, Víctor M; Manso, Isabel; Gates, Andrew J; Moreno-Vivián, Conrado; Richardson, David J
2017-01-01
Abstract Paracoccus denitrificans PD1222 accumulates short-length polyhydroxyalkanoates, poly(3-hydroxybutyrate), under nitrogen-deficient conditions. Polyhydroxybutyrate metabolism requires the 3-ketoacyl-CoA thiolase PhaA, the acetoacetyl-CoA dehydrogenase/reductase PhaB and the synthase PhaC for polymerization. Additionally, P. denitrificans PD1222 grows aerobically with nitrate as sole nitrogen source. Nitrate assimilation is controlled negatively by ammonium through the two-component NtrBC system. NtrB is a sensor kinase that autophosphorylates a histidine residue under low-nitrogen concentrations and, in turn, transfers a phosphoryl group to an aspartate residue of the response regulator NtrC protein, which acts as a transcriptional activator of the P. denitrificans PD1222 nasABGHC genes. The P. denitrificans PD1222 NtrB mutant was unable to use nitrate efficiently as nitrogen source when compared to the wild-type strain, and it also overproduced poly(3-hydroxybutyrate). Acetyl-CoA concentration in the P. denitrificans PD1222 NtrB mutant strain was higher than in the wild-type strain. The expression of the phaC gene was also increased in the NtrB mutant when compared to the wild-type strain. These results suggest that accumulation of poly(3-hydroxybutyrate) in the NtrB mutant strain of PD1222 responds to the high levels of acetyl-CoA that accumulate in the cytoplasm as consequence of its inability to efficiently use nitrate as nitrogen source. PMID:29228177
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The role of carbonic anhydrase VI in bitter taste perception: evidence from the Car6−/− mouse model
2014-01-01
Background Carbonic anhydrase VI (CA VI) is a secretory isozyme of the α-CA gene family. It is highly expressed in the salivary and mammary glands and secreted into saliva and milk. Although CA VI was first described as a gustatory protein, its exact functional roles have remained enigmatic. Interestingly, polymorphism of the CA6 gene was recently linked to bitter taste perception in humans. In this study, we compared the preference of Car6−/− and wild-type mice for different taste modalities in an IntelliCage monitoring environment. Morphologies of taste buds, tongue papillae, and von Ebner’s glands were evaluated by light microscopy. Cell proliferation and rate of apoptosis in tongue specimens were examined by Ki67 immunostaining and fluorescent DNA fragmentation staining, respectively. Results The behavioral follow up of the mice in an IntelliCage system revealed that Car6−/− mice preferred 3 μM quinine (bitter) solution, whereas wild type mice preferred water. When the quinine concentration increased, both groups preferentially selected water. Histological analysis, Ki67 immunostaining and detection of apoptosis did not reveal any significant changes between tongue specimens of the knockout and wild type mice. Conclusions Our knockout mouse model confirms that CA VI is involved in bitter taste perception. CA VI may be one of the factors which contribute to avoidance of bitter, potentially harmful, substances. PMID:25134447
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Kalfusová, Alena; Kodet, Roman
2017-01-01
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Most of them arise due to activating mutations in KIT (75 - 85 %) or PDGFRA (less than 10 %) genes. Identification of the activating mutations in KIT and PDGFRA genes, which code for receptor tyrosine kinases (RTKs), has improved the outcome of targeted therapy of metastatic, unresectable or recurrent GISTs. Primary and/or secondary resistance represents a significant problem in the targeted therapy by Imatinib mesylate (IM) in patients with GIST. An important mechanism of the secondary resistance is the evolvement of secondary mutations. Except for primary and secondary resistance, there is another problem of disease progression - a failure of tumor cells eradication even in the long term therapy of tyrosine kinase inhibitors. GISTs without mutations in KIT/PDGFRA genes constitute 10 - 15% GISTs in adults, and a majority (85 %) of pediatric GISTs. KIT/PDGFRA wild-type GISTs represent a heterogeneous group of tumors with several molecular-genetics and/or morphologic differences. KIT/PDGFRA wild-type GISTs are different in their molecular features, for example in mutations in the BRAF, KRAS, NF1 genes or defects of succinate dehydrogenase (SDH) subunits. KIT/PDGFRA wild-type GISTs are generally less sensitive to targeted therapy by tyrosine kinase inhibitors in comparison with KIT/PDGFRA mutated GISTs. Inhibitors of BRAF, PI3K (mTOR) or inhibitors of IGF1R and VEGFR receptors provide alternative therapeutic strategies.
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Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae.
Vambutas, A; Ackerman, S H; Tzagoloff, A
1991-11-01
C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo.
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Kim, Hye Ryun; Kim, Jae-Ho; Bae, Dong-Hoon; Ahn, Byung-Hak
2010-12-01
Korean traditional rice wines yakju and takju are generally brewed with nuruk as the source of the saccharogenic enzymes by natural fermentation. To improve the quality of Korean rice wine, the microorganisms in the nuruk need to be studied. The objective of this research was to improve the quality of Korean wine with the wild-type yeast strains isolated from the fermentation starter, nuruk. Only strain YA-6 showed high activity in 20% ethanol. Precipitation of Y89-5-3 was similar to that of very flocculent yeast (〉80%) at 75.95%. Using 18S rRNA sequencing, all 10 strains were identified as Saccharomyces cerevisiae. Volatile compounds present in yakju were analyzed by gas chromatography-mass selective detector. The principal component analysis (PCA) of the volatile compounds grouped long-chain esters on the right side of the first principal component, PC1; these compounds were found in yakju that was made with strains YA-6, Y89-5-3, Y89-5- 2, Y90-9, and Y89-1-1. On the other side of PC1 were short-chain esters; these compounds were found in wines that were brewed with strains Y183-2, Y268-3, Y54-3, Y98-4, and Y88-4. Overall, the results indicated that using different wild-type yeast strains in the fermentation process significantly affects the chemical characteristics of the glutinous rice wine.
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Chicken IgY Fc expressed by Eimeria mitis enhances the immunogenicity of E. mitis.
Qin, Mei; Tang, Xinming; Yin, Guangwen; Liu, Xianyong; Suo, Jingxia; Tao, Geru; Ei-Ashram, Saeed; Li, Yuan; Suo, Xun
2016-03-21
Eimeria species are obligate intracellular apicomplexan parasites, causing great economic losses in the poultry industry. Currently wild-and attenuated- type anticoccidial vaccines are used to control coccidiosis. However, their use in fast growing broilers is limited by vaccination side effects caused by medium and/or low immunogenic Eimeria spp. There is, therefore, a need for a vaccine with high immunogenicity for broilers. The avian yolk sac IgY Fc is the avian counterpart of the mammalian IgG Fc, which enhances immunogenicity of Fc-fusion proteins. Here, we developed a stable transgenic Eimeria mitis expressing IgY Fc (Emi.chFc) and investigated whether the avian IgY Fc fragment enhances the immunogenicity of E. mitis. Two-week-old broilers were immunized with either Emi.chFc or wild type Eimeria and challenged with wild type E. mitis to analyze the protective properties of transgenic Emi.chFc. Chickens immunized with Emi.chFc had significantly lower oocyst output, in comparison with PBS, mock control (transgenic E. mitis expressing HA1 from H9N2 avian influenza virus) and wildtype E. mitis immunized groups after challenge, indicating that IgY Fc enhanced the immunogenicity of E. mitis. Our findings suggest that IgY Fc-expressing Eimeria may be a better coccidiosis vaccine, and transgenic Eimeria expressing Fc-fused exogenous antigens may be used as a novel vaccine-delivery vehicle against a wide variety of pathogens.
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de Souza, João Maria Gomes Alencar; Molina, Wagner Franco; de Almeida, Lúcia Maria; de Gouveia, Milson Bezerra; de Macêdo, Francisco Pepino; Laumann, Raul Alberto; Paranhos, Beatriz Aguiar Jordão
2015-01-01
The sterile insect technique (SIT) is widely utilized in the biological control of fruit flies of the family Tephritidae, particularly against the Mediterranean fruit fly. This study investigated the interaction between mating success and morphometric variation in the wings and the production of acoustic signals among three male groups of Ceratitis capitata (Wiedemann): (1) wild males, (2) irradiated with Co-60 (steriles), and (3) irradiated (steriles) and treated with ginger oil. The canonical variate analysis discriminated two groups (males irradiated and males wild), based on the morphological shape of the wings. Among males that emit buzz signals, wild males obtained copulation more frequently than males in Groups 2 and 3. The individuals of Group 3 achieved more matings than those in Group 2. Wild males displayed lower pulse duration, higher intervals between pulses, and higher dominant frequency. Regarding the reproductive success, the morphological differences in the wings' shape between accepted and nonaccepted males are higher in wild males than in the irradiated ones. The present results can be useful in programs using the sterile insect technique for biological control of C. capitata. PMID:26075293
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Cong, Yu; McArthur, Monica A.; Cohen, Melanie; Jahrling, Peter B.; Janosko, Krisztina B.; Josleyn, Nicole; Kang, Kai; Zhang, Tengfei; Holbrook, Michael R.
2016-01-01
Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease. PMID:27191161
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Cong, Yu; McArthur, Monica A; Cohen, Melanie; Jahrling, Peter B; Janosko, Krisztina B; Josleyn, Nicole; Kang, Kai; Zhang, Tengfei; Holbrook, Michael R
2016-05-01
Humans infected with yellow fever virus (YFV), a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM) and dendritic cells (MoDC) from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease.
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Bernal, Alexandra; Simón, Oihane; Williams, Trevor; Muñoz, Delia
2013-01-01
A Chrysodeixis chalcites single-nucleocapsid nucleopolyhedrovirus wild-type isolate from the Canary Islands, Spain, named ChchSNPV-TF1 (ChchTF1-wt), appears to have great potential as the basis for a biological insecticide for control of the pest. An improved understanding of the genotypic structure of this wild-type strain population should facilitate the selection of genotypes for inclusion in a bioinsecticidal product. Eight genetically distinct genotypes were cloned in vitro: ChchTF1-A to ChchTF1-H. Quantitative real-time PCR (qPCR) analysis confirmed that ChchTF1-A accounted for 36% of the genotypes in the wild-type population. In bioassays, ChchTF1-wt occlusion bodies (OBs) were significantly more pathogenic than any of the component single-genotype OBs, indicating that genotype interactions were likely responsible for the pathogenicity phenotype of wild-type OBs. However, the wild-type population was slower killing and produced higher OB yields than any of the single genotypes alone. These results strongly suggested that the ChchTF1-wt population is structured to maximize its transmission efficiency. Experimental OB mixtures and cooccluded genotype mixtures containing the most abundant and the rarest genotypes, at frequencies similar to those at which they were isolated, revealed a mutualistic interaction that restored the pathogenicity of OBs. In OB and cooccluded mixtures containing only the most abundant genotypes, ChchTF1-ABC, OB pathogenicity was even greater than that of wild-type OBs. The ChchTF1-ABC cooccluded mixture killed larvae 33 h faster than the wild-type population and remained genotypically and biologically stable throughout five successive passages in vivo. In conclusion, the ChchTF1-ABC mixture shows great potential as the active ingredient of a bioinsecticide to control C. chalcites in the Canary Islands. PMID:24096419
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Biko, Lydia; Hose, Dorothea; Hofmann, Lukas; Sommer, Claudia
2016-01-01
Background Fabry disease is an X-linked lysosomal storage disorder due to impaired activity of alpha-galactosidase A with intracellular accumulation of globotriaosylceramide. Associated small fiber pathology leads to characteristic pain in Fabry disease. We systematically assessed sensory system, physical activity, metabolic parameters, and morphology of male and female mice with alpha-galactosidase A deficiency (Fabry ko) from 2 to 27 months of age and compared results with those of age- and gender-matched wild-type littermates of C57Bl/6J background. Results From the age of two months, male and female Fabry mice showed mechanical hypersensitivity (p < 0.001 each) compared to wild-type littermates. Young Fabry ko mice of both genders were hypersensitive to heat stimulation (p < 0.01) and developed heat hyposensitivity with aging (p < 0.05), while cold hyposensitivity was present constantly in young (p < 0.01) and old (p < 0.05) Fabry ko mice compared to wild-type littermates. Stride angle increased only in male Fabry ko mice with aging (p < 0.01) in comparison to wild-type littermates. Except for young female mice, male (p < 0.05) and female (p < 0.01) Fabry ko mice had a higher body weight than wild-type littermates. Old male Fabry ko mice were physically less active than their wild-type littermates (p < 0.05), had lower chow intake (p < 0.001), and lost more weight (p < 0.001) in a one-week treadmill experiment than wild-type littermates. Also, Fabry ko mice showed spontaneous pain protective behavior and developed orofacial dysmorphism resembling patients with Fabry disease. Conclusions Mice with alpha-galactosidase A deficiency show age-dependent and distinct deficits of the sensory system. alpha-galactosidase A-deficient mice seem to model human Fabry disease and may be helpful when studying the pathophysiology of Fabry-associated pain. PMID:27145802
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Park, Won; Feng, Yufeng; Kim, Hyojin; Suh, Mi Chung; Ahn, Sung-Ju
2015-09-01
Under heavy-metal stress, CsHMA3 overexpressing transgenic Camelina plants displayed not only a better quality, but also a higher quantity of unsaturated fatty acids in their seeds compared with wild type. Camelina sativa L. belongs to the Brassicaceae family and is frequently used as a natural vegetable oil source, as its seeds contain a high content of fatty acids. In this study, we observed that, when subjected to heavy metals (Cd, Co, Zn and Pb), the seeds of CsHMA3 (Heavy-Metal P1B-ATPase 3) transgenic lines retained their original golden yellow color and smooth outline, unlike wild-type seeds. Furthermore, we investigated the fatty acids content and composition of wild type and CsHMA3 transgenic lines after heavy metal treatments compared to the control. The results showed higher total fatty acid amounts in seeds of CsHMA3 transgenic lines compared with those in wild-type seeds under heavy-metal stresses. In addition, the compositions of unsaturated fatty acids-especially 18:1 (oleic acid), 18:2 (linoleic acid; only in case of Co treatment), 18:3 (linolenic acid) and 20:1 (eicosenoic acid)-in CsHMA3 overexpressing transgenic lines treated with heavy metals were higher than those of wild-type seeds under the same conditions. Furthermore, reactive oxygen species (ROS) contents in wild-type leaves and roots when treated with heavy metal were higher than in CsHMA3 overexpressing transgenic lines. These results indicate that overexpression of CsHMA3 affects fatty acid composition and content-factors that are responsible for the fuel properties of biodiesel-and can alleviate ROS accumulation caused by heavy-metal stresses in Camelina. Due to these factors, we propose that CsHMA3 transgenic Camelina can be used for phytoremediation of metal-contaminated soil as well as for oil production.
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Kim, Ju Sun; Kim, Jung Eun; Kim, Kyung; Lee, Jeeyun; Park, Joon Oh; Lim, Ho Yeong; Park, Young Suk; Kang, Won Ki; Kim, Seung Tae
2017-01-01
Background: Anti-EGFR therapies have been recommended for advanced colorectal cancer (CRC) with wild-type RAS and PIK3CA mutation. However, PIK3CA mutations are a poor prognostic marker and a negative predictor of response to anti-EGFR therapies in RAS wild-type CRC. Therefore, new and advanced treatment strategies are needed for personalized medical treatment of patients with wild-type RAS and PIK3CA mutation. Methods: Patient-derived tumor cells were collected from the ascites of a refractory colon cancer patient with wild-type RAS and PIK3CA mutation. We performed a cell viability assay for cetuximab, AZD5363 (AKT inhibitor), and everolimus (mTOR inhibitor) using PDCs. We also evaluated combinations of cetuximab plus AZD5363 or everolimus in a cell viability assay. Results: Based on cellular proliferation by MTT assay, tumor cells were significantly inhibited by 1uM cetuximab (control vs. cetuximab, mean growth = 100.0% vs 58.07%, p = 0.0103), 1uM AZD5363 (control vs. AZD5363, mean growth = 100.0% vs 58.22%, p = 0.0123), and 1uM everolimus (control vs. everolimus, mean growth = 100.0% vs 52.17%, p = 0.0011). Tumor cell growth was more profoundly reduced by combinations of cetuximab plus AZD5363 (control vs. cetuximab plus AZD5363, mean growth = 100.0% vs 25.00%, p < 0.0001) or everolimus (control vs. cetuximab+everolimus, mean growth = 100.0% vs 28.24%, p < 0.0001). Conclusions: Taken together, these results indicate that RAS wild-type and PIK3CA mutant PDCs originating from CRC are considerably inhibited by treatment with cetuximab plus AZD5363 or everolimus, with downregulation of the AKT and ERK pathways. These combinations may be considered as new options for advanced CRC patients with wild-type RAS and PIK3CA mutation in the context of clinical trials.
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Role of carotenoids in first positive phototropism of etiolated Arabidopsis thaliana seedlings
DOE Office of Scientific and Technical Information (OSTI.GOV)
Orbovic, V.; Poff, K.L.
1991-05-01
A mutant of Arabidopsis thaliana, chosen for is pale cotyledon phenotype in dark grown material, has been obtained from Klaus Apel (ETH-Zentrum, Zurich, Switzerland). Fluence response curves for this putative carotenoidless mutant and its wild-type parent have been measured. The shape of the fluence response curve for the pale mutant is similar to that of its wild-type parent. However, the amplitude of curvature by the mutant is considerably lower than curvature of the wild-type. If the amplitude of the curvature is enhanced with a red light pre-irradiation, peaks of the two photoreceptor pigments, P{sub I} and P{sub II} can bemore » seen in both the pale mutant and its wild-type parent. Based on these data, the authors conclude that neither photoreceptor pigment is altered in the pale mutant.« less
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Dong, X. Y.; Li, W. H.; Zhu, J. L.; Liu, W. J.; Zhao, M. Q.; Luo, Y. W.; Chen, J. D.
2015-01-01
Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance. PMID:27175171
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Efficacy of an inactivated, recombinant bovine herpesvirus type 5 (BoHV-5) vaccine.
Campos, F S; Dezen, D; Antunes, D A; Santos, H F; Arantes, T S; Cenci, A; Gomes, F; Lima, F E S; Brito, W M E D; Filho, H C K; Batista, H B C R; Spilki, F R; Franco, A C; Rijsewijk, F A M; Roehe, P M
2011-02-24
Bovine herpesvirus type 5 (BoHV-5) is the causative agent of bovine herpetic encephalitis. In countries where BoHV-5 is prevalent, attempts to vaccinate cattle to prevent clinical signs from BoHV-5-induced disease have relied essentially on vaccination with BoHV-1 vaccines. However, such practice has been shown not to confer full protection to BoHV-5 challenge. In the present study, an inactivated, oil adjuvanted vaccine prepared with a recombinant BoHV-5 from which the genes coding for glycoprotein I (gI), glycoprotein E (gE) and membrane protein US9 were deleted (BoHV-5 gI/gE/US9(-)), was evaluated in cattle in a vaccination/challenge experiment. The vaccine was prepared from a virus suspension containing a pre-inactivation antigenic mass equivalent to 10(7.69) TCID(50)/dose. Three mL of the inactivated vaccine were administered subcutaneously to eight calves serologically negative for BoHV-5 (vaccinated group). Four other calves were mock-vaccinated with an equivalent preparation without viral antigens (control group). Both groups were boostered 28 days later. Neither clinical signs of disease nor adverse effects were observed during or after vaccination. A specific serological response, revealed by the development of neutralizing antibodies, was detected in all vaccinated animals after the first dose of vaccine, whereas control animals remained seronegative. Calves were subsequently challenged on day 77 post-vaccination (pv) with 10(9.25) TCID(50) of the wild-type BoHV-5 (parental strain EVI 88/95). After challenge, vaccinated cattle displayed mild signs of respiratory disease, whereas the control group developed respiratory disease and severe encephalitis, which led to culling of 2/4 calves. Searches for viral DNA in the central nervous system (CNS) of vaccinated calves indicated that wild-type BoHV-5 did not replicate, whereas in CNS tissues of calves on the control group, viral DNA was widely distributed. BoHV-5 shedding in nasal secretions was significantly lower in vaccinated calves than in the control group on days 2, 3, 4 and 6 post-challenge (pc). In addition, the duration of virus shedding was significantly shorter in the vaccinated (7 days) than in controls (12 days). Attempts to reactivate latent infection by administration of dexamethasone at 147 days pv led to recrudescence of mild signs of respiratory disease in both vaccinated and control groups. Infectious virus shedding in nasal secretions was detected at reactivation and was significantly lower in vaccinated cattle than in controls on days 11-13 post-reactivation (pr). It is concluded that the inactivated vaccine prepared with the BoHV-5 gI/gE/US9(-) recombinant was capable of conferring protection to encephalitis when vaccinated cattle were challenged with a large infectious dose of the parental wild type BoHV-5. However, it did not avoid the establishment of latency nor impeded dexamethasone-induced reactivation of the virus, despite a significant reduction in virus shedding after challenge and at reactivation on vaccinated calves. Copyright © 2010 Elsevier B.V. All rights reserved.
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Ruaño, Gualberto; Villagra, David; Rahim, Umme Salma; Windemuth, Andreas; Kocherla, Mohan; Bower, Bruce; Szarek, Bonnie L; Goethe, John W
2008-11-01
This study compared the types and carrier prevalences of clinically significant DNA polymorphisms in the cytochrome P450 (CYP450) genes CYP2C9, CYP2C19 and CYP2D6 in major depressive disorder patients with a control group of nonpsychiatrically ill, medical outpatients. We conducted a case-control study using 73 psychiatric outpatients diagnosed with depression and referred to a tertiary center, The Institute of Living (Hartford, CT, USA), for treatment resistance or intolerable side-effects to psychotropic drugs. The controls were 120 cardiovascular patients from Hartford Hospital being treated for dyslipidemia but otherwise healthy and not psychiatrically ill. DNA typing to detect polymorphisms in the genes CYP2C9, CYP2C19 and CYP2D6 was accomplished with the Tag-It™ mutation detection assay and the Luminex xMAP ® system. The percentage of individuals in psychiatric versus control groups with two wild-type alleles for CYP2C9, CYP2C19 and CYP2D6 genes, were 50 versus 74% (p < 0.001), 71 versus 73% (not statistically significant) and 36 versus 43% (trend, p < 0.2), respectively. Within the psychiatric population, 57% of individuals were carriers of non-wild-type alleles for 2-3 genes, compared with 36% in the control population (p < 0.0001). The balance, 43% in the psychiatric population and 64% in the control, were carriers of non-wild-type alleles for none or one gene. These findings reveal that clinically relevant CYP2C9 polymorphisms occur more frequently in depressed psychiatric patients than in nonpsychiatric controls. The same trend was found for polymorphisms in the CYP2D6 gene. We found a significant cumulative metabolic deficiency in the psychiatric population for combinations of the CYP2C9, CYP2C19 and CYP2D6 genes. The significant enrichment of CYP2C9-deficient alleles in the psychiatric population validates a previously reported association of this gene with the risk for depression disorders. The high prevalence of carriers with deficient and null alleles suggests that CYP450 DNA typing may play a role in the management of psychiatric patients at tertiary care institutions.
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Wild chimpanzees' use of single and combined vocal and gestural signals.
Hobaiter, C; Byrne, R W; Zuberbühler, K
2017-01-01
We describe the individual and combined use of vocalizations and gestures in wild chimpanzees. The rate of gesturing peaked in infancy and, with the exception of the alpha male, decreased again in older age groups, while vocal signals showed the opposite pattern. Although gesture-vocal combinations were relatively rare, they were consistently found in all age groups, especially during affiliative and agonistic interactions. Within behavioural contexts rank (excluding alpha-rank) had no effect on the rate of male chimpanzees' use of vocal or gestural signals and only a small effect on their use of combination signals. The alpha male was an outlier, however, both as a prolific user of gestures and recipient of high levels of vocal and gesture-vocal signals. Persistence in signal use varied with signal type: chimpanzees persisted in use of gestures and gesture-vocal combinations after failure, but where their vocal signals failed they tended to add gestural signals to produce gesture-vocal combinations. Overall, chimpanzees employed signals with a sensitivity to the public/private nature of information, by adjusting their use of signal types according to social context and by taking into account potential out-of-sight audiences. We discuss these findings in relation to the various socio-ecological challenges that chimpanzees are exposed to in their natural forest habitats and the current discussion of multimodal communication in great apes. All animal communication combines different types of signals, including vocalizations, facial expressions, and gestures. However, the study of primate communication has typically focused on the use of signal types in isolation. As a result, we know little on how primates use the full repertoire of signals available to them. Here we present a systematic study on the individual and combined use of gestures and vocalizations in wild chimpanzees. We find that gesturing peaks in infancy and decreases in older age, while vocal signals show the opposite distribution, and patterns of persistence after failure suggest that gestural and vocal signals may encode different types of information. Overall, chimpanzees employed signals with a sensitivity to the public/private nature of information, by adjusting their use of signal types according to social context and by taking into account potential out-of-sight audiences.
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Brown, André E X; Yemini, Eviatar I; Grundy, Laura J; Jucikas, Tadas; Schafer, William R
2013-01-08
Visible phenotypes based on locomotion and posture have played a critical role in understanding the molecular basis of behavior and development in Caenorhabditis elegans and other model organisms. However, it is not known whether these human-defined features capture the most important aspects of behavior for phenotypic comparison or whether they are sufficient to discover new behaviors. Here we show that four basic shapes, or eigenworms, previously described for wild-type worms, also capture mutant shapes, and that this representation can be used to build a dictionary of repetitive behavioral motifs in an unbiased way. By measuring the distance between each individual's behavior and the elements in the motif dictionary, we create a fingerprint that can be used to compare mutants to wild type and to each other. This analysis has revealed phenotypes not previously detected by real-time observation and has allowed clustering of mutants into related groups. Behavioral motifs provide a compact and intuitive representation of behavioral phenotypes.
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Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models.
Ng, Samuel Y; Yoshida, Noriaki; Christie, Amanda L; Ghandi, Mahmoud; Dharia, Neekesh V; Dempster, Joshua; Murakami, Mark; Shigemori, Kay; Morrow, Sara N; Van Scoyk, Alexandria; Cordero, Nicolas A; Stevenson, Kristen E; Puligandla, Maneka; Haas, Brian; Lo, Christopher; Meyers, Robin; Gao, Galen; Cherniack, Andrew; Louissaint, Abner; Nardi, Valentina; Thorner, Aaron R; Long, Henry; Qiu, Xintao; Morgan, Elizabeth A; Dorfman, David M; Fiore, Danilo; Jang, Julie; Epstein, Alan L; Dogan, Ahmet; Zhang, Yanming; Horwitz, Steven M; Jacobsen, Eric D; Santiago, Solimar; Ren, Jian-Guo; Guerlavais, Vincent; Annis, D Allen; Aivado, Manuel; Saleh, Mansoor N; Mehta, Amitkumar; Tsherniak, Aviad; Root, David; Vazquez, Francisca; Hahn, William C; Inghirami, Giorgio; Aster, Jon C; Weinstock, David M; Koch, Raphael
2018-05-22
T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.
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CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation
Forzati, Floriana; Federico, Antonella; Pallante, Pierlorenzo; Colamaio, Marianna; Esposito, Francesco; Sepe, Romina; Gargiulo, Sara; Luciano, Antonio; Arra, Claudio; Palma, Giuseppe; Bon, Giulia; Bucher, Stefania; Falcioni, Rita; Brunetti, Arturo; Battista, Sabrina; Fedele, Monica; Fusco, Alfredo
2014-01-01
ABSTRACT We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation. PMID:25190058
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In vitro evolution of a ribozyme that contains 5-bromouridine
NASA Technical Reports Server (NTRS)
Dai, X.; Joyce, G. F.; Bada, J. L. (Principal Investigator)
2000-01-01
The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.
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Hunter, Margaret E.; Nico, Leo G.
2015-01-01
Invasive Asian Black Carp (Mylopharyngodon piceus) have been present in USA aquaculture facilities since the 1980s and wild Black Carp have been found in the Mississippi River Basin since the early 1990s. This study characterizes the genetic diversity and relatedness of the Basin’s Black Carp and clarifies the introduction history. Analyses focused on three mitochondrial markers (control region, cytochrome-b, and 16S) and seven nuclear microsatellite markers (nDNA), using aquaculture and wild-caught samples collected in the upper and lower Mississippi Basin. Of the three mitochondrial haplotypes, two were shared between the aquaculture and wild populations, while a third was only present in upper Mississippi wild-caught specimens. Due to the presence of diploid and triploid fish, microsatellite markers were scored as pseudodominant and revealed low polymorphism (NA = 4.6, NA Ave = 1.5). Nuclear Bayesian clustering analyses identified two genetically distinct groups and four subclusters, each primarily composed of a unique haplotype. Samples from three aquaculture farms were assigned to group 1, while a fourth farm included samples from both groups 1 and 2. Wild-caught fish from the upper Basin were predominantly group 1, whereas wild samples from the lower Mississippi were assigned to both genetic groups. The presence of divergent haplotypes and distinct nDNA groups, along with geographic distribution patterns, indicate that wild populations in the basin likely resulted from multiple introductions. Genetic similarities between wild and captive populations support claims that aquaculture is the introduction source, but a shortage of samples and a history of repeated transfers among facilities obscure the precise pathway.
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Resistin deficiency in mice has no effect on pulmonary responses induced by acute ozone exposure
Razvi, Shehla S.; Richards, Jeremy B.; Malik, Farhan; Cromar, Kevin R.; Price, Roger E.; Bell, Cynthia S.; Weng, Tingting; Atkins, Constance L.; Spencer, Chantal Y.; Cockerill, Katherine J.; Alexander, Amy L.; Blackburn, Michael R.; Alcorn, Joseph L.; Haque, Ikram U.
2015-01-01
Acute exposure to ozone (O3), an air pollutant, causes pulmonary inflammation, airway epithelial desquamation, and airway hyperresponsiveness (AHR). Pro-inflammatory cytokines—including IL-6 and ligands of chemokine (C-X-C motif) receptor 2 [keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-2], TNF receptor 1 and 2 (TNF), and type I IL-1 receptor (IL-1α and IL-1β)—promote these sequelae. Human resistin, a pleiotropic hormone and cytokine, induces expression of IL-1α, IL-1β, IL-6, IL-8 (the human ortholog of murine KC and MIP-2), and TNF. Functional differences exist between human and murine resistin; yet given the aforementioned observations, we hypothesized that murine resistin promotes O3-induced lung pathology by inducing expression of the same inflammatory cytokines as human resistin. Consequently, we examined indexes of O3-induced lung pathology in wild-type and resistin-deficient mice following acute exposure to either filtered room air or O3. In wild-type mice, O3 increased bronchoalveolar lavage fluid (BALF) resistin. Furthermore, O3 increased lung tissue or BALF IL-1α, IL-6, KC, TNF, macrophages, neutrophils, and epithelial cells in wild-type and resistin-deficient mice. With the exception of KC, which was significantly greater in resistin-deficient compared with wild-type mice, no genotype-related differences in the other indexes existed following O3 exposure. O3 caused AHR to acetyl-β-methylcholine chloride (methacholine) in wild-type and resistin-deficient mice. However, genotype-related differences in airway responsiveness to methacholine were nonexistent subsequent to O3 exposure. Taken together, these data demonstrate that murine resistin is increased in the lungs of wild-type mice following acute O3 exposure but does not promote O3-induced lung pathology. PMID:26386120
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Taguchi, Ayumi; Kawana, Kei; Tomio, Kensuke; Yamashita, Aki; Isobe, Yosuke; Nagasaka, Kazunori; Koga, Kaori; Inoue, Tomoko; Nishida, Haruka; Kojima, Satoko; Adachi, Katsuyuki; Matsumoto, Yoko; Arimoto, Takahide; Wada-Hiraike, Osamu; Oda, Katsutoshi; Kang, Jing X; Arai, Hiroyuki; Arita, Makoto; Osuga, Yutaka; Fujii, Tomoyuki
2014-01-01
Cancer associated fibroblasts (CAFs) are responsible for tumor growth, angiogenesis, invasion, and metastasis. Matrix metalloproteinase (MMP)-9 secreted from cancer stroma populated by CAFs is a prerequisite for cancer angiogenesis and metastasis. Omega-3 polyunsaturated fatty acids (omega-3 PUFA) have been reported to have anti-tumor effects on diverse types of malignancies. Fat-1 mice, which can convert omega-6 to omega-3 PUFA independent of diet, are useful to investigate the functions of endogenous omega-3 PUFA. To examine the effect of omega-3 PUFA on tumorigenesis, TC-1 cells, a murine epithelial cell line immortalized by human papillomavirus (HPV) oncogenes, were injected subcutaneously into fat-1 or wild type mice. Tumor growth and angiogenesis of the TC-1 tumor were significantly suppressed in fat-1 compared to wild type mice. cDNA microarray of the tumors derived from fat-1 and wild type mice revealed that MMP-9 is downregulated in fat-1 mice. Immunohistochemical study demonstrated immunoreactivity for MMP-9 in the tumor stromal fibroblasts was diffusely positive in wild type whereas focal in fat-1 mice. MMP-9 was expressed in primary cultured fibroblasts isolated from fat-1 and wild type mice but was not expressed in TC-1 cells. Co-culture of fibroblasts with TC-1 cells enhanced the expression and the proteinase activity of MMP-9, although the protease activity of MMP-9 in fat-1-derived fibroblasts was lower than that in wild type fibroblasts. Our data suggests that omega-3 PUFAs suppress MMP-9 induction and tumor angiogenesis. These findings may provide insight into mechanisms by which omega-3 PUFAs exert anti-tumor effects by modulating tumor microenvironment.
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Project Wild and the Dominant Western Paradigm: A Content Analysis Utilizing Deep Ecology.
ERIC Educational Resources Information Center
Ingraham, Blake
Environmental educators utilize activity guides as a primary method of diffusing environmental education material into educational settings. The most popular environmental education activity guide in use today is Project WILD. Project WILD has come under fire by various groups, especially animal rights groups. Accordingly, a content analysis study…
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Jain, Rishabh; Agarwal, Ankit; Kierski, Patricia R.; Schurr, Michael J.; Murphy, Christopher J.; McAnulty, Jonathan F.; Abbott, Nicholas L.
2012-01-01
The development of versatile methods that provide spatial and temporal control over the presentation of physical and biochemical cues on wound beds can lead to new therapeutic approaches that expedite wound healing by favorably influencing cellular behaviors. Towards that goal, we report that native chemical functional groups presented by wound beds can be utilized for direct covalent attachment of polymeric microbeads. Specifically, we demonstrated the covalent attachment of maleimide-functionalized and catechol-functionalized microbeads, made of either polystyrene (non-degradable) or poly(lactic-co-glycolic acid) ((PLGA), degradable), to sulfhydryl and amine groups present on porcine dermis used here as an ex vivo model wound bed. A pronounced increase (10–70 fold) in the density and persistence of the covalently reactive microbeads was observed relative to microbeads that adsorb via non-covalent interactions. Complementary characterization of the surface chemistry of the ex vivo wound beds using Raman microspectroscopy provides support for our conclusion that the increased adherence of the maleimide-functionalized beads results from their covalent bond formation with sulfhydryl groups on the wound bed. The attachment of maleimide-functionalized microbeads to wounds created in live wild-type and diabetic mice led to observations of differential immobilization of microbeads on them and were consistent with anticipated differences in the presentation of sulfhydryl groups on the two different wound types. Finally, the incorporation of maleimide-functionalized microbeads in wounds created in wild-type mice did not impair the rate of wound closure relative to an untreated wound. Overall, the results presented in this paper enable a general and facile approach to the engineering of wound beds in which microbeads are covalently immobilized to wound beds. Such immobilized microbeads could be used in future studies to release bioactive factors (e.g., antimicrobial agents or growth factors) and/or introduce topographical cues that promote cell behaviors underlying healing and wound closure. PMID:23088838
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Thust, S C; Hassanein, S; Bisdas, S; Rees, J H; Hyare, H; Maynard, J A; Brandner, S; Tur, C; Jäger, H R; Yousry, T A; Mancini, L
2018-03-23
To investigate if quantitative apparent diffusion coefficient (ADC) measurements can predict genetic subtypes of non-gadolinium-enhancing gliomas, comparing whole tumour against single slice analysis. Volumetric T2-derived masks of 44 gliomas were co-registered to ADC maps with ADC mean (ADC mean ) calculated. For the slice analysis, two observers placed regions of interest in the largest tumour cross-section. The ratio (ADC ratio ) between ADC mean in the tumour and normal appearing white matter was calculated for both methods. Isocitrate dehydrogenase (IDH) wild-type gliomas showed the lowest ADC values throughout (p < 0.001). ADC mean in the IDH-mutant 1p19q intact group was significantly higher than in the IDH-mutant 1p19q co-deleted group (p < 0.01). A volumetric ADC mean threshold of 1201 × 10 -6 mm 2 /s identified IDH wild-type with a sensitivity of 83% and a specificity of 86%; a volumetric ADC ratio cut-off value of 1.65 provided a sensitivity of 80% and a specificity of 92% (area under the curve (AUC) 0.9-0.94). A slice ADC ratio threshold for observer 1 (observer 2) of 1.76 (1.83) provided a sensitivity of 80% (86%), specificity of 91% (100%) and AUC of 0.95 (0.96). The intraclass correlation coefficient was excellent (0.98). ADC measurements can support the distinction of glioma subtypes. Volumetric and two-dimensional measurements yielded similar results in this study. • Diffusion-weighted MRI aids the identification of non-gadolinium-enhancing malignant gliomas • ADC measurements may permit non-gadolinium-enhancing glioma molecular subtyping • IDH wild-type gliomas have lower ADC values than IDH-mutant tumours • Single cross-section and volumetric ADC measurements yielded comparable results in this study.
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Maize pollen coat xylanase facilitates pollen tube penetration into silk during sexual reproduction.
Suen, Der Fen; Huang, Anthony H C
2007-01-05
Cell wall hydrolases are well documented to be present on pollen, but their roles on the stigma during sexual reproduction have not been previously demonstrated. We explored the function of the tapetum-synthesized xylanase, ZmXYN1, on maize (Zea mays L.) pollen. Transgenic lines (xyl-less) containing little or no xylanase in the pollen coat were generated with use of an antisense construct of the xylanase gene-coding region driven by the XYN1 gene promoter. Xyl-less and wild-type plants had similar vegetative growth. Electron microscopy revealed no appreciable morphological difference in anther cells and pollen between xyl-less lines and the wild type, whereas immunofluorescence microscopy and biochemical analyses indicated an absence of xylanase on xyl-less pollen. Xyl-less pollen germinated as efficiently as wild-type pollen in vitro in a liquid medium but less so on gel media of increasing solidity or on silk, which is indicative of partial impaired water uptake. Once germinated in vitro or on silk, the xyl-less and wild-type pollen tubes elongated at comparable rates. Tubes of germinated xyl-less pollen on silk did not penetrate into the silk as efficiently as tubes of wild-type pollen, and this lower efficiency could be overcome by the addition of xylanase to the silk. For wild-type pollen, coat xylanase activity on oat spelled xylan in vitro and tube penetration into silk were inhibited by xylose but not glucose. The overall findings indicate that maize pollen coat xylanase facilitates pollen tube penetration into silk via enzymatic xylan hydrolysis.
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NASA Astrophysics Data System (ADS)
Ren, Zhao-Yu; Xu, Xiao-Ming; Wang, Shui-Cai; Xin, Yue-Yong; He, Jun-Fang; Hou, Xun
2003-10-01
A high yielding rice variety mutant (Oryza sativa L., Zhenhui 249) with low chlorophyll b (Chl b) has been discovered in natural fields. It has a quality character controlled by a pair of recessive genes (nuclear gene). The partial loss of Chl b in content affects the efficiency of light harvest in a light harvest complex (LHC), thus producing the difference of the exciting energy transfer and the efficiency of photochemistry conversion between the mutant and wild-type rice in photosynthetic unit. The efficiency of utilizing light energy is higher in the mutant than that in the wild-type rice relatively. For further discussion of the above-mentioned difference and learning about the mechanism of the increase in the photochemical efficiency of the mutant, the pico-second resolution fluorescence spectrum measurement with delay-frame-scanning single photon counting technique is adopted. Thylakoid membranes of the mutant and the wild-type rice are excited by an Ar+ laser with a pulse width of 120 ps, repetition rate of 4 MHz and wavelength of 514 nm. Compared with the time and spectrum property of exciting fluorescence, conclusions of those ultrafast dynamic experiments are: 1) The speeds of the exciting energy transferred in photo-system I are faster than that in photo-system II in both samples. 2) The speeds of the exciting energy transfer of mutant sample are faster than those of the wild-type. This might be one of the major reasons why the efficiency of photosynthesis is higher in mutant than that in the wild-type rice.
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Davidson, F F; Loewen, P C; Khorana, H G
1994-01-01
A disulfide bond that is evidently conserved in the guanine nucleotide-binding protein-coupled receptors is present in rhodopsin between Cys-110 and Cys-187. We have replaced these two cysteine residues by alanine residues and now report on the properties of the resulting rhodopsin mutants. The mutant protein C110A/C187A expressed in COS cells resembles wild-type rhodopsin in the ground state. It folds correctly to bind 11-cis-retinal and form the characteristic rhodopsin chromophore. It is inert to hydroxylamine in the dark, and its stability to dark thermal decay is reduced, relative to that of the wild type, by a delta delta G not equal to of only -2.9 kcal/mol. Further, the affinities of the mutant and wild-type rhodopsins to the antirhodopsin antibody rho4D2 are similar, both in the dark and in light. However, the metarhodopsin II (MII) and MIII photointermediates of the mutant are less stable than those formed by the wild-type rhodopsin. Although the initial rates of transducin activation are the same for both mutant and wild-type MII intermediates at 4 degrees C, at 15 degrees C the MII photointermediate in the mutant decays more than 20 times faster than in wild type. We conclude that the disulfide bond between Cys-110 and Cys-187 is a key component in determining the stability of the MII structure and its coupling to transducin activation. PMID:8171030
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Vaidya, Sunil R.; Dvivedi, Garima M.; Jadhav, Santoshkumar M.
2016-01-01
Background & objectives: The reports from the countries where mumps vaccine is given as routine immunization suggest differences in mumps virus neutralizing antibody titres when tested with vaccine and wild type viruses. Such reports are unavailable from countries like India where mumps vaccine is not included in routine immunization. We, therefore, undertook this study to understand the cross-neutralization activity of Indian mumps viruses. Methods: By using commercial mumps IgG enzyme immunoassay (EIA) and a rapid focus reduction neutralization test (FRNT), a panel of serum samples was tested. The panel consisted of 14 acute and 14 convalescent serum samples collected during a mumps outbreak and 18 archived serum samples. Two wild types (genotypes C and G) and Leningrad-Zagreb vaccine strain (genotype N) were used for the challenge experiments and FRNT titres were determined and further compared. The HN protein sequence of three mumps viruses was analyzed for the presence of key epitopes. Results: All serum samples effectively neutralized mumps virus wild types and a vaccine strain. However, significantly lower FRNT titres were noted to wild types than to vaccine strain (P<0.05). The comparison between EIA and FRNT results revealed 95.6 per cent agreement. No amino acid changes were seen in the epitopes in the Indian wild type strains. All potential N-linked glycosylation sites were observed in Indian strains. Interpretation & conclusions: Good cross-neutralization activity was observed for three mumps virus strains, however, higher level of FRNT titres was detected for mumps virus vaccine strain compared to Indian wild type isolates. PMID:26997012
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Hocher, Berthold; Haumann, Hannah; Rahnenführer, Jan; Reichetzeder, Christoph; Kalk, Philipp; Pfab, Thiemo; Tsuprykov, Oleg; Winter, Stefan; Hofmann, Ute; Li, Jian; Püschel, Gerhard P.; Lang, Florian; Schuppan, Detlef; Schwab, Matthias; Schaeffeler, Elke
2016-01-01
ABSTRACT Maternal environmental factors can impact on the phenotype of the offspring via the induction of epigenetic adaptive mechanisms. The advanced fetal programming hypothesis proposes that maternal genetic variants may influence the offspring's phenotype indirectly via epigenetic modification, despite the absence of a primary genetic defect. To test this hypothesis, heterozygous female eNOS knockout mice and wild type mice were bred with male wild type mice. We then assessed the impact of maternal eNOS deficiency on the liver phenotype of wild type offspring. Birth weight of male wild type offspring born to female heterozygous eNOS knockout mice was reduced compared to offspring of wild type mice. Moreover, the offspring displayed a sex specific liver phenotype, with an increased liver weight, due to steatosis. This was accompanied by sex specific differences in expression and DNA methylation of distinct genes. Liver global DNA methylation was significantly enhanced in both male and female offspring. Also, hepatic parameters of carbohydrate metabolism were reduced in male and female offspring. In addition, male mice displayed reductions in various amino acids in the liver. Maternal genetic alterations, such as partial deletion of the eNOS gene, can affect liver metabolism of wild type offspring without transmission of the intrinsic defect. This occurs in a sex specific way, with more detrimental effects in females. This finding demonstrates that a maternal genetic defect can epigenetically alter the phenotype of the offspring, without inheritance of the defect itself. Importantly, these acquired epigenetic phenotypic changes can persist into adulthood. PMID:27175980
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Piña-Vázquez, Denia M; Mayoral-Peña, Zyanya; Gómez-Sánchez, Maricela; Salazar-Olivo, Luis A; Arellano-Carbajal, Fausto
2017-04-18
Psidium guajava and Tagetes erecta have been used traditionally to treat gastrointestinal parasites, but their active metabolites and mechanisms of action remain largely unknown. To evaluate the anthelmintic potential of Psidium guajava and Tagetes erecta extracts on Levamisole-sensitive and Levamisole-resistant strains of the model nematode Caenorhabditis elegans. Aqueous extracts of Psidium guajava (PGE) and Tagetes erecta (TEE) were assayed on locomotion and egg-laying behaviors of the wild-type (N2) and Levamisole-resistant (CB193) strains of Caenorhabditis elegans. Both extracts paralyzed wild-type and Levamisole-resistant nematodes in a dose-dependent manner. In wild-type worms, TEE 25mg/mL induced a 75% paralysis after 8h of treatment and PGE 25mg/mL induced a 100% paralysis after 4h of treatment. PGE exerted a similar paralyzing effect on N2 wild-type and CB193 Levamisole-resistant worms, while TEE only partially paralyzed CB193 worms. TEE 25mg/mL decreased N2 egg-laying by 65% with respect to the untreated control, while PGE did it by 40%. Psidium guajava leaves and Tagetes erecta flower-heads possess hydrosoluble compounds that block the motility of Caenorhabditis elegans by a mechanism different to that of the anthelmintic drug Levamisole. Effects are also observable on oviposition, which was diminished in the wild-type worms. The strong anthelmintic effects in crude extracts of these plants warrants future work to identify their active compounds and to elucidate their molecular mechanisms of action. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.
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Richman, D D; Murphy, B R; Belshe, R B; Rusten, H M; Chanock, R M; Blacklow, N R; Parrino, T A; Rose, F B; Levine, M M; Caplan, E
1977-08-01
The two temperature-sensitive (ts) lesions present in influenza A/Hong Kong/68-ts-1[E] (H3N2 68) virus were transferred via genetic reassortment to influenza A/Georgia/74 (H3N2 74) wild-type virus. A recombinant clone possessing both ts lesions and the shutoff temperature of 38 C of the Hong Kong/68 ts donor and the two surface antigens of the Georgia/74 wild-type virus was administered to 32 seronegative adult volunteers. Thirty-one volunteers were infected, of whom only five experienced mild afebrile upper respiratory tract illness. The wild-type recipient virus was a cloned population that induced illness in five of six infected volunteers. Therfore, the attenuation exhibited by the Georgia/74-ts-1[E] virus could reasonably be assumed to be due to the acquisition of the two ts-1[E] lesions by the Georgia/74 wild-type virus. The serum and nasal wash antibody responses of the ts-1[E] vaccinees were equivalent to those of the volunteers who received wild-type virus. The two ts lesions present in the Hong Kong/68-ts-1[E] virus have now been transferred three times to a wild-type virus bearing a new hemagglutinin, and in each instance the new ts recombination exhibited a similar, satisfactory level of attenuation and antigenicity for adults. It seems likely that the transfer of the ts-1[E] lesions to any new influenza virus will regularly result in attenuation of a recombinat virus possessing the new surface antigens.
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Miller, J B; Amy, N K
1983-01-01
We examined molybdenum cofactor activity in chlorate-resistant (chl) and nitrate reductase-deficient (nar) insertion mutants and wild-type strains of Escherichia coli K-12. The bacterial molybdenum cofactor was assayed by its ability to restore activity to the cofactor-deficient nitrate reductase found in the nit-1 strain of Neurospora crassa. In the wild-type E. coli strains, molybdenum cofactor was synthesized constitutively and found in both cytoplasmic and membrane fractions. Cofactor was found in two forms: the demolybdo form required additional molybdate in the assay mix for detection, whereas the molybdenum-containing form was active without additional molybdate. The chlA and chlE mutants had no detectable cofactor. The chlB and the narG, narI, narK, and narL (previously designated chlC) strains had cofactor levels similar to those of the wild-type strains, except the chlB strains had two to threefold more membrane-bound cofactor. Cofactor levels in the chlD and chlG strains were sensitive to molybdate. When grown in 1 microM molybdate, the chlD strains had only 15 to 20% of the wild-type levels of the demolybdo and molybdenum-containing forms of the cofactor. In contrast, the chlG strains had near wild-type levels of demolybdo cofactor when grown in 1 microM molybdate, but none of the molybdenum-containing form of the cofactor. Near wild-type levels of both forms of the cofactor were restored to the chlD and chlG strains by growth in 1 mM molybdate. PMID:6307982
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Do Cultivated Varieties of Native Plants Have the Ability to Outperform Their Wild Relatives?
Schröder, Roland; Prasse, Rüdiger
2013-01-01
Vast amounts of cultivars of native plants are annually introduced into the semi-natural range of their wild relatives for re-vegetation and restoration. As cultivars are often selected towards enhanced biomass production and might transfer these traits into wild relatives by hybridization, it is suggested that cultivars and the wild × cultivar hybrids are competitively superior to their wild relatives. The release of such varieties may therefore result in unintended changes in native vegetation. In this study we examined for two species frequently used in re-vegetation (Plantago lanceolata and Lotus corniculatus) whether cultivars and artificially generated intra-specific wild × cultivar hybrids may produce a higher vegetative and generative biomass than their wilds. For that purpose a competition experiment was conducted for two growing seasons in a common garden. Every plant type was growing (a.) alone, (b.) in pairwise combination with a similar plant type and (c.) in pairwise interaction with a different plant type. When competing with wilds cultivars of both species showed larger biomass production than their wilds in the first year only and hybrids showed larger biomass production than their wild relatives in both study years. As biomass production is an important factor determining fitness and competitive ability, we conclude that cultivars and hybrids are competitively superior their wild relatives. However, cultivars of both species experienced large fitness reductions (nearly complete mortality in L. corniculatus) due to local climatic conditions. We conclude that cultivars are good competitors only as long as they are not subjected to stressful environmental factors. As hybrids seemed to inherit both the ability to cope with the local climatic conditions from their wild parents as well as the enhanced competitive strength from their cultivars, we regard them as strong competitors and assume that they are able to outperform their wilds at least over the short-term. PMID:23951081
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Süssenbacher, Iris; Kreutz, Christoph R; Christ, Bastien; Hörtensteiner, Stefan; Kräutler, Bernhard
2015-08-10
1-Formyl-19-oxobilin-type tetrapyrroles are characteristic, abundant products of chlorophyll breakdown in senescent leaves. However, in some leaves, 1,19-dioxobilin-type chlorophyll catabolites (DCCs) lacking the formyl group accumulate instead. A P450 enzyme was identified in in vitro studies that removed the formyl group of a primary fluorescent chlorophyll catabolite (pFCC) and generated fluorescent DCCs. These DCCs are precursors of isomeric nonfluorescent DCCs (NDCCs). Here, we report a structural investigation of the NDCCs in senescent leaves of wild-type Arabidopsis thaliana. Four new NDCCs were characterized, two of which carried a stereoselectively added hydroxymethyl group. Such formal DCC hydroxymethylations were previously found in DCCs in leaves of a mutant of A. thaliana. They are now indicated to be a feature of chlorophyll breakdown in A. thaliana, associated with the specific in vivo deformylation of pFCC en route to NDCCs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Ge, Yu-Zheng; Xu, Lu-Wei; Zhou, Chang-Cheng; Lu, Tian-Ze; Yao, Wen-Tao; Wu, Ran; Zhao, You-Cai; Xu, Xiao; Hu, Zhi-Kai; Wang, Min; Yang, Xiao-Bing; Zhou, Liu-Hua; Zhong, Bing; Xu, Zheng; Li, Wen-Cheng; Zhu, Jia-Geng; Jia, Rui-Peng
2017-01-01
Background: Clear cell renal cell carcinoma (ccRCC) is the most prevalent histologic subtype of kidney cancers in adults, which could be divided into two distinct subgroups according to the BRCA1 associated protein-1 (BAP1) mutation status. In the current study, we comprehensively analyzed the genome-wide microRNA (miRNA) expression profiles in ccRCC, with the aim to identify the differentially expressed miRNAs between BAP1 mutant and wild-type tumors, and generate a BAP1 mutation-specific miRNA signature for ccRCC patients with wild-type BAP1. Methods: The BAP1 mutation status and miRNA profiles in BAP1 mutant and wild-type tumors were analyzed. Subsequently, the association of the differentially expressed miRNAs with patient survival was examined, and a BAP1 mutation-specific miRNA signature was generated and examined with Kaplan-Meier survival, univariate and multivariate Cox regression analyses. Finally, the bioinformatics methods were adopted for the target prediction of selected miRNAs and functional annotation analyses. Results: A total of 350 treatment-naïve primary ccRCC patients were selected from The Cancer Genome Atlas project, among which 35 (10.0%) subjects carried mutant BAP1 and had a shorter overall survival (OS) time. Furthermore, 33 miRNAs were found to be differentially expressed between BAP1 mutant and wild-type tumors, among which 11 (miR-149, miR-29b-2, miR-182, miR-183, miR-21, miR-365-2, miR-671, miR-365-1, miR-10b, miR-139, and miR-181a-2) were significantly associated with OS in ccRCC patients with wild-type BAP1. Finally, a BAP1 mutation-specific miRNA signature consisting of 11 miRNAs was generated and validated as an independent prognostic parameter. Conclusions: In summary, our study identified a total of 33 miRNAs differentially expressed between BAP1 mutant and wild-type tumors, and generated a BAP1 mutation-specific miRNA signature including eleven miRNAs, which could serve as a novel prognostic biomarker for ccRCC patients with wild-type BAP1. PMID:28900502
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deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R
2003-02-01
Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function. Copyright 2003 Elsevier Science (USA)
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Keller, Judith I; Shriver, W Gregory
2014-01-01
Campylobacter jejuni is responsible for the majority of bacterial foodborne gastroenteritis in the US, usually due to the consumption of undercooked poultry. Research on which avian species transmit the bacterium is limited, especially in the US. We sampled wild birds in three families-Anatidae, Scolopacidae, and Laridae-in eastern North America to determine the prevalence and specific strains of Campylobacter. The overall prevalence of Campylobacter spp. was 9.2% for all wild birds sampled (n = 781). Campylobacter jejuni was the most prevalent species (8.1%), while Campylobacter coli and Campylobacter lari prevalence estimates were low (1.4% and 0.3%, respectively). We used multilocus sequence typing PCR specific to C. jejuni to characterize clonal complexes and sequence types isolated from wild bird samples and detected 13 novel sequence types, along with a clonal complex previously only associated with human disease (ST-658). Wild birds share an increasing amount of habitat with humans as more landscapes become fragmented and developed for human needs. Wild birds are and will remain an important aspect of public health due to their ability to carry and disperse emerging zoonotic pathogens or their arthropod vectors. As basic information such as prevalence is limited or lacking from a majority of wild birds in the US, this study provides further insight into Campylobacter epidemiology, host preference, and strain characterization of C. jejuni.
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Aleksunes, Lauren M; Reisman, Scott A; Yeager, Ronnie L; Goedken, Michael J; Klaassen, Curtis D
2010-04-01
The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces a battery of cytoprotective genes after oxidative stress. Nrf2 aids in liver regeneration by altering insulin signaling; however, whether Nrf2 participates in hepatic glucose homeostasis is unknown. Compared with wild-type mice, mice lacking Nrf2 (Nrf2-null) have lower basal serum insulin and prolonged hyperglycemia in response to an intraperitoneal glucose challenge. In the present study, blood glucose, serum insulin, urine flow rate, and hepatic expression of glucose-related genes were quantified in male diabetic wild-type and Nrf2-null mice. Type 1 diabetes was induced with a single intraperitoneal dose (200 mg/kg) of streptozotocin (STZ). Histopathology and serum insulin levels confirmed depleted pancreatic beta-cells in STZ-treated mice of both genotypes. Five days after STZ, Nrf2-null mice had higher blood glucose levels than wild-type mice. Nine days after STZ, polyuria occurred in both genotypes with more urine output from Nrf2-null mice (11-fold) than wild-type mice (7-fold). Moreover, STZ-treated Nrf2-null mice had higher levels of serum beta-hydroxybutyrate, triglycerides, and fatty acids 10 days after STZ compared with wild-type mice. STZ reduced hepatic glycogen in both genotypes, with less observed in Nrf2-null mice. Increased urine output and blood glucose in STZ-treated Nrf2-null mice corresponded with enhanced gluconeogenesis (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase)- and reduced glycolysis (pyruvate kinase)-related mRNA expression in their livers. Furthermore, the Nrf2 activator oltipraz lowered blood glucose in wild-type but not Nrf2-null mice administered STZ. Collectively, these data indicate that the absence of Nrf2 worsens hyperglycemia in type I diabetic mice and Nrf2 may represent a therapeutic target for reducing circulating glucose levels.
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Complex history of admixture during citrus domestication revealed by genome analysis
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wu, G. Albert; Prochnik, Simon; Jenkins, Jerry
Although Citrus is the most globally significant tree fruit, its domestication history is poorly understood. Cultivated citrus types are believed to comprise selections from and/or hybrids of several wild progenitor species, but the identities of these progenitors, and their contribution to modern cultivars, remain controversial. Here we report the genomes of a collection of mandarins, pummelos, and oranges, including a high quality reference sequence from a haploid Clementine mandarin. By comparative genome analysis we show that these cultivated types can be derived from two progenitor species. Cultivated pummelos represent selections from a single progenitor species C. maxima. Unexpectedly, however, wemore » find that cultivated mandarins are introgressions of C. maxima into a distinct second population that we identify with the ancestral wild mandarin species C. reticulata. Sweet and sour oranges are found to be interspecific hybrids. Sweet orange, the most widely cultivated citrus, arose as the offspring of previously admixed individuals. In contrast, sour (or Seville) orange is an F1 hybrid of pure C. maxima and C. reticulata parents, implying that wild mandarins were part of the early breeding germplasm. Surprisingly, we also find that a wild Chinese mandarin from Mangshan, China shows substantial sequence divergence from C. reticulata and appears to represent a distinct taxon. Understanding the relationships and phylogeny of cultivated citrus through genome analysis will clarify taxonomic relationships and enable previously inconceivable opportunities for sequence-directed genetic improvement. Citrus are widely consumed worldwide as juice or fresh fruit, providing important sources of vitamin C and other health-promoting compounds. Global production in 2012 exceeded 86 million metric tons, with an estimated value of US$9 billion (http://www.fas.usda.gov/psdonline/circulars/citrus.pdf). The very narrow genetic diversity of cultivated citrus makes it highly vulnerable to disease outbreaks, including citrus greening disease (also known as Huanglongbing) that is rapidly spreading throughout the world's major citrus producing regions1. Understanding the population genomics and domestication of citrus will enable strategies for improvements to citrus including resistance to greening and other diseases. The domestication and distribution of edible citrus types began several thousand years ago in Southeast Asia and spread globally following ancient land and sea routes. The lineages that gave rise to most modern cultivated varieties, however, are lost in undocumented antiquity, and their identities remain controversial2, 3. Several features of Citrus biology and cultivation make deciphering these origins difficult. Cultivated varieties are typically propagated clonally by grafting and through asexual seed production (apomixis via nucellar polyembryony) to maintain desirable combinations of traits (Fig. 1). Thus many important cultivar groups have characteristic basic genotypes that presumably arose through interspecific hybridization and/or successive introgressive hybridizations of wild ancestral species. These domestication events predated the global expansion of citrus cultivation by hundreds or perhaps thousands of years, with no record of the domestication process. Diversity within such groups arises through accumulated somatic mutations, generally without sexual recombination, either as limb sports on trees or variants among apomictic seedling progeny. Two wild species are believed to have contributed to domesticated pummelos, mandarins and oranges. Based on morphology and genetic markers, pummelos have generally been identified with the wild species C. maxima (Burm.) Merrill that is indigenous to Southeast Asia. Although mandarins are similarly widely identified with the species C. reticulata Blanco 4-6, wild populations of C. reticulata have not been definitively described. Various authors have taken dif« less
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Condit, Richard C.; Williamson, Anna-Lise; Sheets, Rebecca; Seligman, Stephen J.; Monath, Thomas P.; Excler, Jean-Louis; Gurwith, Marc; Bok, Karin; Robertson, James S.; Kim, Denny; Hendry, Michael; Singh, Vidisha; Mac, Lisa M.; Chen, Robert T.
2016-01-01
In 2003 and 2013, the World Health Organization convened informal consultations on characterization and quality aspects of vaccines based on live virus vectors. In the resulting reports, one of several issues raised for future study was the potential for recombination of virus-vectored vaccines with wild type pathogenic virus strains. This paper presents an assessment of this issue formulated by the Brighton Collaboration. To provide an appropriate context for understanding the potential for recombination of virus-vectored vaccines, we review briefly the current status of virus vectored vaccines, mechanisms of recombination between viruses, experience with recombination involving live attenuated vaccines in the field, and concerns raised previously in the literature regarding recombination of virus-vectored vaccines with wild type virus strains. We then present a discussion of the major variables that could influence recombination between a virus-vectored vaccine and circulating wild type virus and the consequences of such recombination, including intrinsic recombination properties of the parent virus used as a vector; sequence relatedness of vector and wild virus; virus host range, pathogenesis and transmission; replication competency of vector in target host; mechanism of vector attenuation; additional factors potentially affecting virulence; and circulation of multiple recombinant vectors in the same target population. Finally, we present some guiding principles for vector design and testing intended to anticipate and mitigate the potential for and consequences of recombination of virus-vectored vaccines with wild type pathogenic virus strains. PMID:27346303
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Vijayakumar, Saravanan; Das, Pradeep
2018-04-18
Sterol-14α-demethylase (CYP51) is an ergosterol pathway enzyme crucial for the survival of infectious Leishmania parasite. Recent high-throughput metabolomics and whole genome sequencing study revealed amphotericin B resistance in Leishmania is indeed due to mutation in CYP51. The residue of mutation (asparagine 176) is conserved across the kinetoplastidae and not in yeast or humans, portraying its functional significance. In order to understand the possible cause for the resistance, knowledge of structural changes due to mutation is of high importance. To shed light on the structural changes of wild and mutant CYP51, we conducted comparative molecular dynamics simulation study. The active site, substrate biding cavity, substrate channel entrance (SCE), and cavity involving the mutated site were studied based on basic parameters and large concerted molecular motions derived from essential dynamics analyses of 100 ns simulation. Results indicated that mutant CYP51 is stable and less compact than the wild type. Correspondingly, the solvent accessible surface area (SASA) of the mutant was found to be increased, especially in active site and cavities not involving the mutation site. Free-energy landscape analysis disclosed mutant to have a rich conformational diversity than wild type, with various free-energy conformations of mutant having SASA greater than wild type with SCE open. More residues were found to interact with the mutant CYP51 upon docking of substrate to both the wild and mutant CYP51. These results indicate that, relative to wild type, the N176I mutation of CYP51 in Leishmania mexicana could possibly favor increased substrate binding efficiency.
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Origins of domestication and polyploidy in oca (Oxalis tuberosa : Oxalidaceae): nrDNA ITS data.
Emshwiller, E; Doyle, J
1998-07-01
As part of a study aimed at elucidating the origins of the octoploid tuber crop "oca," Oxalis tuberosa, DNA sequences of the internal trancribed spacer of nuclear ribosomal DNA (nrDNA ITS) were determined for oca and several wild Oxalis species, mostly from Bolivia. Phylogenetic analysis of these data supports a group of these species as being close relatives of oca, in agreement with morphology and cytology, but at odds with traditional infrageneric taxonomy. Variation in ITS sequences within this group is quite low (0-7 substitutions in the entire ITS region), contrasting with the highly divergent (unalignable in some cases) sequences within the genus overall. Some groups of morphologically differentiated species were found to have identical sequences, notably a group that includes oca, wild populations of Oxalis that bear small tubers, and several other clearly distinct species. The presence of a second, minor sequence type in at least some oca accessions suggests a possible contribution from a second genome donor, also from within this same species group. ITS data lack sufficient variation to elucidate the origins of oca precisely, but have identified a pool of candidate species and so can be used as a tool to screen yet unsampled species for possible progenitors.
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Ding, Jianqiang; Yannam, Govardhana R.; Roy-Chowdhury, Namita; Hidvegi, Tunda; Basma, Hesham; Rennard, Stephen I.; Wong, Ronald J.; Avsar, Yesim; Guha, Chandan; Perlmutter, David H.; Fox, Ira J.; Roy-Chowdhury, Jayanta
2011-01-01
α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z–expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%–98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z–expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals. PMID:21505264
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DNA Repair Modulates The Vulnerability of The Developing Brain to Alkylating Agents
Kisby, G.E.; Olivas, A.; Park, T.; Churchwell, M.; Doerge, D.; Samson, L. D.; Gerson, S.L.; Turker, M.S.
2009-01-01
Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag-/-) or O6-methylguanine methyltransferase (Mgmt-/-), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt-/- neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag-/- neurons were for the most part significantly less sensitive than wild type or Mgmt-/- neurons to MAM and HN2. Aag-/- neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt-/- mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM treated Aag-/- or MGMT overexpressing (MgmtTg+) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in MgmtTg+ mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant. PMID:19162564
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How MMPs Impact Bone Responses to Metastatic Prostate Cancer
2009-02-01
STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Using an animal model of prostate tumor...prostate tumor progression in the bone since no difference in osteolytic or osteoblastic responses between wild type and MMP-9 deficient animals were...described as the ‘vicious cycle’ (4). Using an animal model of 4 tumor progression in the bone, we have previously identified a group of enzymes known as
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How MMPs Impact Bone Responses to Metastatic Prostate Cancer
2010-02-01
13. SUPPLEMENTARY NOTES 14. ABSTRACT Using an animal model of prostate tumor progression in the bone we have previously shown that MMPs...in osteolytic or osteoblastic responses between wild type and MMP-9 deficient animals were detected by Faxitron, CT, SPECT and histomorphometry...as the ‘vicious cycle’ (4). Using an animal model of 2 tumor progression in the bone, we have previously identified a group of enzymes known as
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Genetics of Eosinophilic Esophagitis
2012-03-01
cells, left panel, and differential cell counts , right panel) in bronchoalveolar lavage fluid (BALF) in IL- 21R-/- mice compared to wild-type (WT). A...positive skin tests. A third group (30%) had multiple sensitivities to foods and pollens (GM total IgE 285 IU/ ml). Tests for IgE to carbohydrate antigens...milk sensitized, and those with multiple pollen allergies. The frequent occurrence of multiple associated sensitivities to grains, legumes, molds, and
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Krishna, Gopala; Ganiger, Shivaputhrappa; Kannan, Kamala; Gopalakrishnan, Gopa; Goel, Saryu
2015-12-01
The purpose of this study was to better understand the biological effects of increased prolactin levels induced in mice by dopamine D2 receptor antagonist molindone treatment. Toxicokinetics, prolactin levels, and reproductive tissue histology were evaluated in Tg rasH2 wild-type mice treated orally with molindone at 0, 5, 15, and 50mg/kg/day for 6 months, followed by a 2-month posttreatment recovery period. A greater than dose-proportional increase in molindone exposure ([AUC]0‒24) was observed on Day 180 for both sexes. Statistically significant (P<0.01) increases in prolactin levels were observed in most treatment groups compared with controls at 0.5h postdose on Days 1 and 180. Prolactin levels returned to baseline levels during the recovery period. Microscopic changes attributable to hyperprolactinemia, including corpora lutea enlargement and interstitial cell atrophy in the ovaries, and atrophy of the uterus and vagina were observed on Day 180. These changes were reversed during the recovery period in the 5- and 15-mg/kg/day treatment groups. Mice receiving molindone at 50mg/kg/day also showed signs of reversal on histologic examination. Copyright © 2015 Elsevier Inc. All rights reserved.
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A Genetic Analysis of the Suppressor 2 of Zeste Complex of Drosophila Melanogaster
Wu, C. T.; Howe, M.
1995-01-01
The zeste(1) (z(1)) mutation of Drosophila melanogaster produces a mutant yellow eye color instead of the wild-type red. Genetic and molecular data suggest that z(1) achieves this change by altering expression of the wild-type white gene in a manner that exhibits transvection effects. There exist suppressor and enhancer mutations that modify the z(1) eye color, and this paper summarizes our studies of those belonging to the Suppressor 2 of zeste complex [Su(z)2-C]. The Su(z)2-C consists of at least three subregions called Psc (Posterior sex combs), Su(z)2 and Su(z)2D (Distal). The products of these subregions are proposed to act at the level of chromatin. Complementation analyses predict that the products are functionally similar and interacting. The alleles of Psc define two overlapping phenotypic classes, the hopeful and hapless. The distinctions between these two classes and the intragenic complementation seen among some of the Psc alleles are consistent with a multidomain structure for the product of Psc. Psc is a member of the homeotic Polycomb group of genes. A general discussion of the Polycomb and trithorax group of genes, position-effect variegation, transvection, chromosome pairing and chromatin structure is presented. PMID:7635282
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Faridounnia, Maryam; Wienk, Hans; Kovačič, Lidija; Folkers, Gert E; Jaspers, Nicolaas G J; Kaptein, Robert; Hoeijmakers, Jan H J; Boelens, Rolf
2015-08-14
The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Klomtong, P; Chaweewan, K; Phasuk, Y; Duangjinda, M
2015-10-19
Mutations in melanocortin 1 receptor (MC1R) gene and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) gene have been shown to affect coat color patterns in pigs. Additional functional marker genes, such as insulin like growth factor-2 (IGF2) and orphan nuclear receptor, germ cell nuclear factor (NR6A1), have been described for variations in factors such as fat deposition, litter size, and vertebra number in pigs. In this study, we investigated 129 pigs representing 4 breeds: Thai indigenous, classified into black (similar to Raad or Ka done pig) and black and white (similar to the Hailum and Kwai pig) coat color types; wild boar; Duroc; and Chinese Meishan. Mutations of MC1R, KIT, IGF2, and NR6A1 were detected using polymerase chain reaction-restriction fragment length polymorphism. The genotypes variation in MC1R and KIT genes could be used to differentiate four groups of coat color: solid black, black and white, red, and wild type. For IGF2, the GG genotype was present in wild boar only; for NR6A1 the TT genotype was found only in Duroc pigs. We identified novel 14-bp deletions in KIT that were associated with black and white coat color in Thai indigenous pigs. Insights into variations in genes presented in this study will be useful in future developmental breeding programs for the Thai native pig.
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Barratt, Daniel T; Coller, Janet K; Hallinan, Richard; Byrne, Andrew; White, Jason M; Foster, David JR; Somogyi, Andrew A
2012-01-01
Background: Genetic variability in ABCB1, encoding the P-glycoprotein efflux transporter, has been linked to altered methadone maintenance treatment dose requirements. However, subsequent studies have indicated that additional environmental or genetic factors may confound ABCB1 pharmacogenetics in different methadone maintenance treatment settings. There is evidence that genetic variability in OPRM1, encoding the mu opioid receptor, and ABCB1 may interact to affect morphine response in opposite ways. This study aimed to examine whether a similar gene-gene interaction occurs for methadone in methadone maintenance treatment. Methods: Opioid-dependent subjects (n = 119) maintained on methadone (15–300 mg/day) were genotyped for five single nucleotide polymorphisms of ABCB1 (61A > G; 1199G > A; 1236C > T; 2677G > T; 3435C > T), as well as for the OPRM1 118A > G single nucleotide polymorphism. Subjects’ methadone doses and trough plasma (R)-methadone concentrations (Ctrough) were compared between ABCB1 haplotypes (with and without controlling for OPRM1 genotype), and between OPRM1 genotypes (with and without controlling for ABCB1 haplotype). Results: Among wild-type OPRM1 subjects, an ABCB1 variant haplotype group (subjects with a wild-type and 61A:1199G:1236C:2677T:3435T haplotype combination, or homozygous for the 61A:1199G:1236C:2677T:3435T haplotype) had significantly lower doses (median ± standard deviation 35 ± 5 versus 180 ± 65 mg/day, P < 0.01) and Ctrough (78 ± 22 versus 177 ± 97 ng/mL, P < 0.05) than ABCB1 wild-type subjects. Among subjects with the most common ABCB1 haplotype combination (wild-type with 61A:1199G:1236T:2677T:3435T), the OPRM1 118 A/G genotype was associated with a significantly higher Ctrough than 118 A/A (250 ± 126 versus 108 ± 36 ng/mL, P = 0.016). No ABCB1 haplotype group or OPRM1 genotype was associated with dose or Ctrough without taking into account confounding genetic variability at the other locus. Therefore, two interacting pharmacogenetic determinants of methadone maintenance treatment response were identified, ie, ABCB1, where variants are associated with lower methadone requirements, and OPRM1, where the variant is associated with higher methadone requirements. Conclusion: These opposing pharmacogenetic effects therefore need to be considered in combination when assessing methadone maintenance treatment pharmacogenetics. PMID:23226062
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Bai, Hanwen
2011-01-01
The majority of understanding of root gravity responses comes from the study of primary roots, even though lateral roots make a far greater contribution to root system architecture. The focus of this report is the analysis of gravitropic responses in lateral roots of wild-type background and pgm-1 mutants. Despite the significant reduction in gravitropic response of primary roots of pgm-1 mutants, the lateral roots of this mutant demonstrate wild-type rates of gravitropism, suggesting a significant difference in gravity signal transduction between primary and lateral roots. PMID:21921698
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Bai, Hanwen; Wolverton, Chris
2011-10-01
The majority of understanding of root gravity responses comes from the study of primary roots, even though lateral roots make a far greater contribution to root system architecture. The focus of this report is the analysis of gravitropic responses in lateral roots of wild-type background and pgm-1 mutants. Despite the significant reduction in gravitropic response of primary roots of pgm-1 mutants, the lateral roots of this mutant demonstrate wild-type rates of gravitropism, suggesting a significant difference in gravity signal transduction between primary and lateral roots.
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Genetic conversion of a fungal plant pathogen to a non-pathogenic, endophytic mutualist
Freeman, Stanley; Rodriguez, Rusty J.
1993-01-01
The filamentous fungal ascomycete Colletotrichum magna causes anthracnose in cucurbit plants. Isolation of a nonpathogenic mutant of this species (path-1) resulted in maintained wild-type levels of in vitro sporulation, spore adhesion, appressorial formation, and infection. Path-1 grew throughout host tissues as an endophyte and retained the wild-type host range, which indicates that the genetics involved in pathogenicity and host specificity are distinct. Prior infection with path-1 protected plants from disease caused by Colletotrichum and Fusarium.Genetic analysis of a cross between path-1 and wild-type strains indicated mutation of a single locus.
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1996-08-01
J-4030 TITLE: The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle PRINCIPAL...The In Vivo DNA Binding Properties of 5. FUNDING NUMBERS Wild-Type and Mutant p53 Proteins in Mammary Cell Lines DAMD17-94-J-4030 During the Course of...ABSTRACT (Maximum 200 Using a pair of murine cell lines, one lacking p53 and a derivative cell line containing temperature sensitive p53 val 135
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Weyens N.; van der Lelie D.; Boulet, J.
2011-06-09
This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promotemore » growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619::gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.« less
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Differences in Fusarium Species in brown midrib Sorghum and in Air Populations in Production Fields.
Funnell-Harris, Deanna L; Scully, Erin D; Sattler, Scott E; French, Roy C; O'Neill, Patrick M; Pedersen, Jeffrey F
2017-11-01
Several Fusarium spp. cause sorghum (Sorghum bicolor) grain mold, resulting in deterioration and mycotoxin production in the field and during storage. Fungal isolates from the air (2005 to 2006) and from leaves and grain from wild-type and brown midrib (bmr)-6 and bmr12 plants (2002 to 2003) were collected from two locations. Compared with the wild type, bmr plants have reduced lignin content, altered cell wall composition, and different levels of phenolic intermediates. Multilocus maximum-likelihood analysis identified two Fusarium thapsinum operational taxonomic units (OTU). One was identified at greater frequency in grain and leaves of bmr and wild-type plants but was infrequently detected in air. Nine F. graminearum OTU were identified: one was detected at low levels in grain and leaves while the rest were only detected in air. Wright's F statistic (F ST ) indicated that Fusarium air populations differentiated between locations during crop anthesis but did not differ during vegetative growth, grain development, and maturity. F ST also indicated that Fusarium populations from wild-type grain were differentiated from those in bmr6 or bmr12 grain at one location but, at the second location, populations from wild-type and bmr6 grain were more similar. Thus, impairing monolignol biosynthesis substantially effected Fusarium populations but environment had a strong influence.
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Hill, Andrew G; Findlay, Michael P; Burge, Matthew E; Jackson, Christopher; Alfonso, Pilar Garcia; Samuel, Leslie; Ganju, Vinod; Karthaus, Meinolf; Amatu, Alessio; Jeffery, Mark; Bartolomeo, Maria Di; Bridgewater, John; Coveler, Andrew L; Hidalgo, Manuel; Kapp, Amy V; Sufan, Roxana I; McCall, Bruce B; Hanley, William D; Penuel, Elicia M; Pirzkall, Andrea; Tabernero, Josep
2018-05-15
Purpose: Duligotuzumab is a dual-action antibody directed against EGFR and HER3. Experimental Design: Metastatic colorectal cancer (mCRC) patients with KRAS ex2 wild-type received duligotuzumab or cetuximab and FOLFIRI until progression or intolerable toxicity. Mandatory tumor samples underwent mutation and biomarker analysis. Efficacy analysis was conducted in patients with RAS exon 2/3 wild-type tumors. Results: Of 134 randomly assigned patients, 98 had RAS ex2/3 wild-type. Duligotuzumab provided no progression-free survival (PFS) or overall survival (OS) benefit compared with cetuximab, although there was a trend for a lower objective response rate (ORR) in the duligotuzumab arm. No relationship was seen between PFS or ORR and ERBB3, NRG1, or AREG expression. There were fewer skin rash events for duligotuzumab but more diarrhea. Although the incidence of grade ≥3 AEs was similar, the frequency of serious AEs was higher for duligotuzumab. Conclusions: Duligotuzumab plus FOLFIRI did not appear to improve the outcomes in patients with RAS exon 2/3 wild-type mCRC compared with cetuximab + FOLFIRI. Clin Cancer Res; 24(10); 2276-84. ©2018 AACR . ©2018 American Association for Cancer Research.
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Mannose-specific interaction of Lactobacillus plantarum with porcine jejunal epithelium.
Gross, Gabriele; van der Meulen, Jan; Snel, Johannes; van der Meer, Roelof; Kleerebezem, Michiel; Niewold, Theo A; Hulst, Marcel M; Smits, Mari A
2008-11-01
Host-microorganism interactions in the intestinal tract are complex, and little is known about specific nonpathogenic microbial factors triggering host responses in the gut. In this study, mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig Small Intestinal Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were compared with those of two corresponding mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of the wild-type strain associated with the intestinal surface could be observed after 8 h of perfusion when a mixture of wild-type and msa-mutant strain had been applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended to induce a decrease in jejunal net fluid absorption compared with control conditions. Furthermore, after 8 h of perfusion expression of the host gene encoding pancreatitis-associated protein, a protein with proposed bactericidal properties, was found to be upregulated by the wild-type strain only. These observations suggest a role of Msa in the induction of host responses in the pig intestine.
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Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization.
Balsanelli, Eduardo; Serrato, Rodrigo V; de Baura, Valter A; Sassaki, Guilherme; Yates, Marshall G; Rigo, Liu Un; Pedrosa, Fábio O; de Souza, Emanuel M; Monteiro, Rose A
2010-08-01
In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.
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Donnison, Iain S; Gay, Alan P; Thomas, Howard; Edwards, Keith J; Edwards, David; James, Caron L; Thomas, Ann M; Ougham, Helen J
2007-01-01
A maize (Zea mays) senescence-associated legumain gene, See2beta, was characterized at the physiological and molecular levels to determine its role in senescence and resource allocation. A reverse-genetics screen of a maize Mutator (Mu) population identified a Mu insertion in See2beta. Maize plants homozygous for the insertion were produced. These See2 mutant and sibling wild-type plants were grown under high or low quantities of nitrogen (N). The early development of both genotypes was similar; however, tassel tip and collar emergence occurred earlier in the mutant. Senescence of the mutant leaves followed a similar pattern to that of wild-type leaves, but at later sampling points mutant plants contained more chlorophyll than wild-type plants and showed a small extension in photosynthetic activity. Total plant weight was higher in the wild-type than in the mutant, and there was a genotype x N interaction. Mutant plants under low N maintained cob weight, in contrast to wild-type plants under the same treatment. It is concluded, on the basis of transposon mutagenesis, that See2beta has an important role in N-use and resource allocation under N-limited conditions, and a minor but significant function in the later stages of senescence.
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Root Formation in Ethylene-Insensitive Plants1
Clark, David G.; Gubrium, Erika K.; Barrett, James E.; Nell, Terril A.; Klee, Harry J.
1999-01-01
Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia × hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more belowground root mass but fewer aboveground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated taproots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli. PMID:10482660
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ruhlig, M.A.; Person, S.
1977-11-01
The isolation of syncytium-producing mutants of herpes simplex virus type 1 (KOS strain), which cause extensive cell fusion during otherwise normal infections, has been reported previously (S. Person, R.W. Knowles, G.S. Read, S.C. Warner, and V.C. Bond, J. Virol. 17:183-190, 1976). Seven of these mutants, plus two syncytial strains obtained elsewhere were used to compare the incorporation of labeled galactose into neutral glycolipids of mock-infected, wild-type-infected, and syncytially infected human embryonic lung cells. Five predominant cellular glycolipid species were observed, denoted GL-1 through GL-5 in order of increasing oligosaccharide chain length; for example, GL-1 and GL-2 correspond to glycolipids thatmore » contain mono- and disaccharide units, respectively. Wild-type virus infection caused an increase in galactose incorporation into GL-1 and GL-2 relative to GL-3 through GL-5. For a single labeling interval from 4 to 10 h after adsorption, syncytial infections generally resulted in a relatively greater incorporation into more complex glycolipids than did wild-type infections. One mutant, syn 20, was compared with wild-type virus throughout infection by using a series of shorter labeling pulses and appeared to delay by at least 2 h the alterations observed during wild-type infections. These alterations are apparently due to defects in synthesis, since prelabeled cellular glycolipids were not differentially degraded during mock or virus infection.« less
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Hughes, L A; Wigley, P; Bennett, M; Chantrey, J; Williams, N
2010-10-01
Recent studies have suggested that Salmonella Typhimurium strains associated with mortality in UK garden birds are significantly different from strains that cause disease in humans and livestock and that wild bird strains may be host adapted. However, without further genomic characterization of these strains, it is not possible to determine whether they are host adapted. The aim of this study was to characterize a representative sample of Salm. Typhimurium strains detected in wild garden birds using multi-locus sequence typing (MLST)to investigate evolutionary relationships between them. Multi-locus sequence typing was performed on nine Salm. Typhimurium strains isolated from wild garden birds. Two sequence types were identified, the most common of which was ST568. Examination of the public Salmonella enterica MLST database revealed that only three other ST568 isolates had been cultured from a human in Scotland. Two further isolates of Salm. Typhimurium were determined to be ST19. Results of MLST analysis suggest that there is a predominant strain of Salm. Typhimurium circulating among garden bird populations in the United Kingdom, which is rarely detected in other species, supporting the hypothesis that this strain is host adapted. Host-pathogen evolution is often assumed to lead to pathogens becoming less virulent to avoid the death of their host; however, infection with ST568 led to high mortality rates among the wild birds examined, which were all found dead at wild bird-feeding stations. We hypothesize that by attracting unnaturally high densities of birds, wild bird-feeding stations may facilitate the transmission of ST568 between wild birds, therefore reducing the evolutionary cost of this pathogen killing its host, resulting in a host-adapted strain with increased virulence.
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Abu-Amero, Khaled K; Al-Boudari, Olayan M; Mohamed, Gamal H; Dzimiri, Nduna
2006-01-01
Background The role of the Beta2-adrenoceptor (beta2-AR) Gln27Glu polymorphism in the manifestation of cardiovascular diseases is still unclear. Methods In the present study, we evaluated the potential relevance of the c.79 C>G (p.Gln27Glu) polymorphism of this receptor gene for coronary artery disease (CAD) and its associated risk factors in Saudi Arabs. Genotyping was performed by PCR using the confronting two-pair primer (PCR-CTPP) method. Results In the general population group (BD) (n = 895), 68.5% were homozygous wild-type C/C, 28.3% were heterozygous C/G and 3.2% were homozygous mutant G/G. Among the CAD patients (n = 773), 50.6% were homozygous wild-type C/C, 43.6% were heterozygous C/G and 5.8% were homozygous mutant G/G, while in the angiographed control group (CON) (n = 528), 71.8% were C/C, 24.4% C/G and 3.8% G/G genotypes. These results indicate that both the C/G (p = < .001) and G/G (p = .005) genotypes are significantly associated with CAD, when compared to the CON group. In addition, C/G (p = < .001) and G/G (p = < .001) were significantly associated with CAD, when compared to the BD group. Furthermore, stepwise logistic regression showed that the genotype [C/G (p < .001) and G/G (p < .001)] increase the risk of CAD. Conclusion These results shows that the Gln27Glu genotypes (homo- or heterozygous) of the beta2-AR may be independent predictors of severe CAD. PMID:16573811
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Xie, Yongjing; Min, Soyoung; Harte, Níal P; Kirk, Hannah; O'Brien, John E; Voorheis, H Paul; Svanborg, Catharina; Hun Mok, K
2013-01-01
Human α-lactalbumin made lethal to tumor cells (HAMLET) and its analogs are partially unfolded protein-oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge-specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively-charged lysine residues to negatively-charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild-type α-lactalbumin-oleic acid complex. With the addition of OA, the wild-type and guanidinated α-lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α-lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively-charged basic groups on α-lactalbumin and the negatively-charged carboxylate groups on OA molecules play an essential role in the binding of OA to α-lactalbumin and that these interactions appear to be as important as hydrophobic interactions. Copyright © 2012 Wiley Periodicals, Inc.
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Bigley, Andrew N; Xu, Chengfu; Henderson, Terry J; Harvey, Steven P; Raushel, Frank M
2013-07-17
The V-type nerve agents (VX and VR) are among the most toxic substances known. The high toxicity and environmental persistence of VX make the development of novel decontamination methods particularly important. The enzyme phosphotriesterase (PTE) is capable of hydrolyzing VX but with an enzymatic efficiency more than 5 orders of magnitude lower than with its best substrate, paraoxon. PTE has previously proven amenable to directed evolution for the improvement of catalytic activity against selected compounds through the manipulation of active-site residues. Here, a series of sequential two-site mutational libraries encompassing 12 active-site residues of PTE was created. The libraries were screened for catalytic activity against a new VX analogue, DEVX, which contains the same thiolate leaving group of VX coupled to a diethoxyphosphate core rather than the ethoxymethylphosphonate core of VX. The evolved catalytic activity with DEVX was enhanced 26-fold relative to wild-type PTE. Further improvements were facilitated by targeted error-prone PCR mutagenesis of loop-7, and additional PTE variants were identified with up to a 78-fold increase in the rate of DEVX hydrolysis. The best mutant hydrolyzed the racemic nerve agent VX with a value of kcat/Km = 7 × 10(4) M(-1) s(-1), a 230-fold improvement relative to wild-type PTE. The highest turnover number achieved by the mutants created for this investigation was 137 s(-1), an enhancement of 152-fold relative to wild-type PTE. The stereoselectivity for the hydrolysis of the two enantiomers of VX was relatively low. These engineered mutants of PTE are the best catalysts ever reported for the hydrolysis of nerve agent VX.
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A Novel SLC27A4 Splice Acceptor Site Mutation in Great Danes with Ichthyosis.
Metzger, Julia; Wöhlke, Anne; Mischke, Reinhard; Hoffmann, Annalena; Hewicker-Trautwein, Marion; Küch, Eva-Maria; Naim, Hassan Y; Distl, Ottmar
2015-01-01
Ichthyoses are a group of various different types of hereditary disorders affecting skin cornification. They are characterized by hyperkeratoses of different severity levels and are associated with a dry and scaling skin. Genome-wide association analysis of nine affected and 13 unaffected Great Danes revealed a genome-wide significant peak on chromosome 9 at 57-58 Mb in the region of SLC27A4. Sequence analysis of genomic DNA of SLC27A4 revealed the non-synonymous SNV SLC27A4:g.8684G>A in perfect association with ichthyosis-affection in Great Danes. The mutant transcript of SLC27A4 showed an in-frame loss of 54 base pairs in exon 8 probably induced by a new splice acceptor site motif created by the mutated A- allele of the SNV. Genotyping 413 controls from 35 different breeds of dogs and seven wolves revealed that this mutation could not be found in other populations except in Great Danes. Affected dogs revealed high amounts of mutant transcript but only low levels of the wild type transcript. Targeted analyses of SLC27A4 protein from skin tissues of three affected and two unaffected Great Danes indicated a markedly reduced or not detectable wild type and truncated protein levels in affected dogs but a high expression of wild type SLC27A4 protein in unaffected controls. Our data provide evidence of a new splice acceptor site creating SNV that results in a reduction or loss of intact SLC27A4 protein and probably explains the severe skin phenotype in Great Danes. Genetic testing will allow selective breeding to prevent ichthyosis-affected puppies in the future.
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Vaid, Mudit; Singh, Tripti; Prasad, Ram; Elmets, Craig A; Xu, Hui; Katiyar, Santosh K
2013-03-01
Ultraviolet (UV) radiation-induced immunosuppression has been implicated in skin carcinogenesis. Grape seed proanthocyanidins (GSPs) have anti-skin carcinogenic effects in mice and GSPs-fed mice exhibit a reduction in UV-induced suppression of allergic contact hypersensitivity (CHS), a prototypic T-cell-mediated response. Here, we report that dietary GSPs did not inhibit UVB-induced suppression of CHS in xeroderma pigmentosum complementation group A (XPA)-deficient mice, which lack nucleotide excision repair mechanisms. GSPs enhanced repair of UVB-induced DNA damage (cyclobutane pyrimidine dimers) in wild-type, but not XPA-deficient, dendritic cells (DC). Co-culture of CD4(+) T cells with DCs from UVB-irradiated wild-type mice resulted in suppression of T-cell proliferation and secretion of T-helper (TH) 1-type cytokines that was ameliorated when the DCs were obtained from GSP-fed mice, whereas DCs obtained from GSP-fed XPA-KO mice failed to restore T-cell proliferation. In adoptive transfer experiments, donor DCs were positively selected from the draining lymph nodes of UVB-exposed donor mice that were sensitized to 2,4,-dinitrofluorobenzene were transferred into naïve recipient mice and the CHS response assessed. Naïve recipients that received DCs from UVB-exposed wild-type donors that had been fed GSPs exhibited a full CHS response, whereas no significant CHS was observed in mice that received DCs from XPA-KO mice fed GSPs. These results suggest that GSPs prevent UVB-induced immunosuppression through DNA repair-dependent functional activation of dendritic cells in mice. Cancer Prev Res; 6(3); 242-52. ©2013 AACR. ©2013 AACR.
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Bell, P J; Higgins, V J; Attfield, P V
2001-04-01
To compare the fermentative capacity of wild and domesticated isolates of the genus Saccharomyces. The fermentative capacity of yeasts from a variety of wild and domesticated sources was tested in synthetic dough media that mimic major bread dough types. Domesticated yeast strains were found to have better maltose-utilizing capacity than wild yeast strains. The capacity to ferment sugars under high osmotic stress was randomly distributed amongst wild and baking strains of Saccharomyces. The domestication of bakers' yeast has enhanced the ability of yeasts to ferment maltose, without a similar impact on the fermentative capacity under high osmotic conditions. This study, combined with molecular studies of both wild and domesticated yeast, showed that domestication of bakers' yeast has resulted in improved maltose utilization, apparently via the duplication and mutation of the MAL genes.
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Murine study of portal hypertension associated endothelin-1 hypo-response.
Theodorakis, Nicholas; Maluccio, Mary; Skill, Nicholas
2015-04-28
To investigate endothelin-1 hypo-responsive associated with portal hypertension in order to improve patient treatment outcomes. Wild type, eNOS(-/-) and iNOS(-/-) mice received partial portal vein ligation surgery to induce portal hypertension or sham surgery. Development of portal hypertension was determined by measuring the splenic pulp pressure, abdominal aortic flow and portal systemic shunting. To measure splenic pulp pressure, a microtip pressure transducer was inserted into the spleen pulp. Abdominal aortic flow was measured by placing an ultrasonic Doppler flow probe around the abdominal aorta between the diaphragm and celiac artery. Portal systemic shunting was calculated by injection of fluorescent microspheres in to the splenic vein and determining the percentage accumulation of spheres in liver and pulmonary beds. Endothelin-1 hypo-response was evaluated by measuring the change in abdominal aortic flow in response to endothelin-1 intravenous administration. In addition, thoracic aorta endothelin-1 contraction was measured in 5 mm isolated thoracic aorta rings ex-vivo using an ADI small vessel myograph. In wild type and iNOS(-/-) mice splenic pulp pressure increased from 7.5 ± 1.1 mmHg and 7.2 ± 1 mmHg to 25.4 ± 3.1 mmHg and 22 ± 4 mmHg respectively. In eNOS(-/-) mice splenic pulp pressure was increased after 1 d (P = NS), after which it decreased and by 7 d was not significantly elevated when compared to 7 d sham operated controls (6.9 ± 0.6 mmHg and 7.3 ± 0.8 mmHg respectively, P = 0.3). Abdominal aortic flow was increased by 80% and 73% in 7 d portal vein ligated wild type and iNOS when compared to shams, whereas there was no significant difference in 7 d portal vein ligated eNOS(-/-) mice when compared to shams. Endothelin-1 induced a rapid reduction in abdominal aortic blood flow in wild type, eNOS(-/-) and iNOS(-/-) sham mice (50% ± 8%, 73% ± 9% and 47% ± 9% respectively). Following portal vein ligation endothelin-1 reduction in blood flow was significantly diminished in each mouse group. Abdominal aortic flow was reduced by 19% ± 9%, 32% ± 10% and 9% ± 9% in wild type, eNOS(-/-) and iNOS(-/-) mice respectively. Aberrant endothelin-1 response in murine portal hypertension is NOS isoform independent. Moreover, portal hypertension in the portal vein ligation model is independent of ET-1 function.
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Cabezón, O; Colom-Cadena, A; Muñoz-González, S; Pérez-Simó, M; Bohórquez, J A; Rosell, R; Marco, I; Domingo, M; Lavín, S; Ganges, L
2017-04-01
In this study, fifteen wild boar piglets were intranasally inoculated <10 h after birth with the moderately virulent classical swine fever virus (CSFV) strain Catalonia 01. At 5 days post-inoculation, seven other animals within 48 h of birth were put in contact with them. Viral replication and innate and specific immune responses were evaluated. Of the inoculated animals, 46.67% remained post-natally persistently infected and were apparently healthy with neither humoral nor cellular immunological responses specific to CSFV and with high viral loads in their blood, organs and body secretions. Moreover, the present data extend the time period to 48 h after birth when a moderately virulent CSFV strain could lead to post-natal persistent infection given the generation of persistently infected wild boars in the contact group (33.33%). The innate immune response to the virus, as measured by type I IFN-α in serum, was mostly not impaired in the persistently infected wild boars. Interestingly, a decrease and lack of IFN-γ-producing cells against CSFV and PHA was observed. In endemic countries where wild swine species are increasing and low and moderate virulence CSFV strains are prevalent, the possible generation of this form of disease cannot be ruled out. © 2015 Blackwell Verlag GmbH.
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Vidal, Natalia P; Manzanos, María J; Goicoechea, Encarnación; Guillén, María D
2016-03-15
Farmed and wild European sea bass (Dicentrarchus labrax) could be distinguished by its volatile metabolites, an issue not addressed until now. The aim of this work was to study these metabolites by solid-phase microextraction followed by gas chromatography/mass spectrometry (SPME-GC/MS). Both farmed and wild sea bass have a great number of volatile metabolites, most of them being in low concentrations. These include alcohols, aldehydes, ketones, alkylfurans, acids, aliphatic and aromatic hydrocarbons, terpenes, sulfur and nitrogen derivatives, 2,6-di-tert-butyl-4-methylphenol and one derived compound, as well as 2,4,7,9-tetramethyl-5-decyne-4,7-diol, this latter compound presumably resulting from environmental contamination. Important differences have been detected between both types of sea bass, and also among individuals inside each group. Farmed specimens are richer in volatile metabolites than the wild counterparts; however, these latter, in general, contain a high number and abundance of metabolites resulting from microbial and enzymatic non-oxidative activity than the former. Clear differences in the volatile metabolites of wild and farmed sea bass have been found. A great deal of valuable information on sea bass volatile metabolites has been obtained, which can be useful in understanding certain aspects of the quality and safety of raw and processed sea bass. © 2015 Society of Chemical Industry.
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Yamamoto, Itaru; Nosho, Katsuhiko; Kanno, Shinichi; Igarashi, Hisayoshi; Kurihara, Hiroyoshi; Ishigami, Keisuke; Ishiguro, Kazuya; Mitsuhashi, Kei; Maruyama, Reo; Koide, Hideyuki; Okuda, Hiroyuki; Hasegawa, Tadashi; Sukawa, Yasutaka; Okita, Kenji; Takemasa, Ichiro; Yamamoto, Hiroyuki; Shinomura, Yasuhisa; Nakase, Hiroshi
2017-03-14
The polycomb group protein enhancer of zeste homolog 2 (EZH2) is a methyltransferase that suppresses microRNA-31 (miR-31) in various human malignancies including colorectal cancer. We recently suggested that miR-31 regulates the signaling pathway downstream of epidermal growth factor receptor (EGFR) in colorectal cancer. Therefore, we conducted this study for assessing the relationship between EZH2 expression and clinical outcomes in patients with colorectal cancer treated with anti-EGFR therapeutics. We immunohistochemically evaluated EZH2 expression and assessed miR-31 and gene mutations [KRAS (codon 61/146), NRAS (codon 12/13/61), and BRAF (codon 600)] in 109 patients with colorectal cancer harboring KRAS (codon 12/13) wild-type. We also evaluated the progression-free survival (PFS) and overall survival (OS). In the result, low EZH2 expression was significantly associated with shorter PFS (log-rank test: P = 0.023) and OS (P = 0.036) in patients with colorectal cancer. In the low-miR-31-expression group and the KRAS (codon 61/146), NRAS, and BRAF wild-type groups, a significantly shorter PFS (P = 0.022, P = 0.039, P = 0.021, and P = 0.036, respectively) was observed in the EZH2 low-expression groups than in the high-expression groups. In the multivariate analysis, low EZH2 expression was associated with a shorter PFS (P = 0.046), independent of the mutational status and miR-31. In conclusion, EZH2 expression was associated with survival in patients with colorectal cancer who were treated with anti-EGFR therapeutics. Moreover, low EZH2 expression was independently associated with shorter PFS in patients with cancer, suggesting that EZH2 expression is a useful additional prognostic biomarker for anti-EGFR therapy.
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The effect of polymorphic metabolism enzymes on serum phenytoin level.
Ozkaynakci, Aydan; Gulcebi, Medine Idrizoglu; Ergeç, Deniz; Ulucan, Korkut; Uzan, Mustafa; Ozkara, Cigdem; Guney, Ilter; Onat, Filiz Yilmaz
2015-03-01
Phenytoin has a widespread use in epilepsy treatment and is mainly metabolized by hepatic cytochrome P450 enzymes (CYP). We have investigated CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3 allelic variants in a Turkish population of patients on phenytoin therapy. Patients on phenytoin therapy (n = 102) for the prevention of epileptic seizures were included. Polymorphic alleles were analyzed by restriction fragment length polymorphism method. Serum concentrations of phenytoin were measured by fluorescence polarization immune assay method. The most frequent genotype was detected for CYP2C9 wild-type alleles (78.43 %), whereas CYP2C19*2/*2 (5.88 %) was the least frequent genotype group. According to the classification made with both enzyme polymorphisms, CYP2C9*1/*1-CYP2C19*1/*1 (G1: 41.17 %) genotype group was the most frequent whereas CYP2C9*1/*2-CYP2C19*1/*3 (G7: 0.98 %) was the least frequent one. The highest mean phenytoin level (27.95 ± 1.85 µg/ml) was detected in the G8 genotype group (CYP2C9*1/*3-CYP2C19*2/*3) and the G1 genotype group showed the lowest mean phenytoin level (7.43 ± 0.73 µg/ml). The mean serum concentration of phenytoin of the polymorphic patients with epilepsy was higher than that for the wild-type alleles both in the monotherapy and polytherapy patients. These results show the importance of the genetic polymorphism analysis of the main metabolizing enzyme groups of phenytoin for the dose adjustment.
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Jiang, Lin; Ma, Jing; Zhang, Yi; Zhou, Chun-Ni; Zhang, Lei; Chao, Feng-Lei; Chen, Lin-Mu; Jiang, Rong; Wu, Hong; Tang, Yong
2018-08-01
To investigate the effect of running exercise on the number of the neurons in the hippocampus of young APP/PS1 mice, twenty 6-month-old male APP/ PS1 transgenic mice were randomly divided into the APP/PS1 control (AD control) group and the APP/PS1 running (AD running) group (10 mice per group), and ten wild-type mice of the littermate were regarded as the wild-type (WT) group. The AD running mice ran on motorized treadmill machiene for 4 months, while the WT mice and AD control mice were housed in standard condition without running. Then, Morris water maze tests (MWM) were used to assess the special learning and memory abilities of mice in three groups. The stereological methods were used to quantitatively evaluate the volume of the hippocampus, CA1/2, CA3 and the dentate gyrus (DG) and count the number of the neurons in CA1/2, CA3 and DG. We found that 4-month running effectively shortened the escape latency of young APP/PS1 control mice in MWM. More importantly, 4-month running effectively increased the volumes of the hippocampus, CA1/2, CA3 and DG and increased the number of neurons in CA1/2, CA3 and DG in young APP/PS1 mice. The present results suggested that 4-month running has significant beneficial effects on the spatial learning and memory capacities of young APP/PS1 mice and could delay the progress of atrophy of hippocampus and the neuron death in CA1/2, CA3 and DG in young APP/PS1 mice. Copyright © 2018 Elsevier B.V. All rights reserved.
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Kawahara, Kazuki; Oki, Hiroya; Fukakusa, Shunsuke; Maruno, Takahiro; Kobayashi, Yuji; Motooka, Daisuke; Taniguchi, Tooru; Honda, Takeshi; Iida, Tetsuya; Nakamura, Shota; Ohkubo, Tadayasu
2015-06-01
Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.
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Kenessey, István; Kói, Krisztina; Horváth, Orsolya; Cserepes, Mihály; Molnár, Dávid; Izsák, Vera; Dobos, Judit; Hegedűs, Balázs
2016-01-01
Background In non-small cell lung cancer (NSCLC) KRAS-mutant status is a negative prognostic and predictive factor. Nitrogen-containing bisphosphonates inhibit prenylation of small G-proteins (e.g. Ras, Rac, Rho) and thus may affect proliferation and migration. In our preclinical work, we investigated the effect of an aminobisphosphonate compound (zoledronic acid) on mutant and wild type KRAS-expressing human NSCLC cell lines. Results We confirmed that zoledronic acid was unable to inhibit the prenylation of mutant K-Ras unlike in the case of wild type K-Ras. In case of in vitro proliferation, the KRAS-mutant human NSCLC cell lines showed resistance to zoledronic acid wild-type KRAS-cells proved to be sensitive. Combinatory application of zoledronic acid enhanced the cytostatic effect of cisplatin. Zoledronic acid did not induce significant apoptosis. In xenograft model, zoledronic acid significantly reduced the weight of wild type KRAS-EGFR-expressing xenograft tumor by decreasing the proliferative capacity. Futhermore, zoledronic acid induced VEGF expression and improved in vivo tumor vascularization. Materials and methods Membrane association of K-Ras was examined by Western-blot. In vitro cell viability, apoptotic cell death and migration were measured in NSCLC lines with different molecular background. The in vivo effect of zoledronic acid was investigated in a SCID mouse subcutaneous xenograft model. Conclusions The in vitro and in vivo inhibitory effect of zoledronic acid was based on the blockade of cell cycle in wild type KRAS-expressing human NSCLC cells. The zoledronic acid induced vascularization supported in vivo cytostatic effect. Our preclinical investigation suggests that patients with wild type KRAS-expressing NSCLC could potentially benefit from aminobisphosphonate therapy. PMID:27780929
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Shoaib, M; Gommans, J; Morley, A; Stolerman, I P; Grailhe, R; Changeux, J-P
2002-03-01
The subtypes of nicotinic receptors at which the behavioural effects of nicotine originate are not fully understood. These experiments use mice lacking the beta2 subunit of nicotinic receptors to investigate its role in nicotine discrimination and conditioned taste aversion (CTA). Wild-type and mutant mice were trained either in a two-lever nicotine discrimination procedure using a tandem schedule of food reinforcement, or in a counterbalanced two-flavour CTA procedure. Rates of lever-pressing of wild-type and mutant mice did not differ. Wild-type mice acquired discrimination of nicotine (0.4 or 0.8 mg/kg) rapidly and exhibited steep dose-response curves. Mutant mice failed to acquire these nicotine discriminations and exhibited flat dose-response curves. Both wild-type and mutant mice acquired discrimination of nicotine (1.6 mg/kg) although discrimination performance was weak in the mutants. Nicotine initially reduced response rates in wild-type and mutant mice, and tolerance developed to this effect in each genotype. Both genotypes acquired discrimination of morphine (3 mg/kg) with similar degrees of accuracy, and dose-response curves for morphine discrimination in the two genotypes were indistinguishable. Nicotine produced dose-related CTA in both genotypes, but the magnitude of the effect was less in the mutants than in the wild-type controls. It is concluded that nicotinic receptors containing the beta2 subunit play a major role in the discriminative stimulus and taste aversion effects of nicotine that may reflect psychological aspects of tobacco dependence. Such receptors appear to have a less crucial role in the response-rate, reducing effects of nicotine and in nicotine tolerance.
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CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor*
Matsushita, Yuki; Sakamoto, Kei; Tamamura, Yoshihiro; Shibata, Yasuaki; Minamizato, Tokutaro; Kihara, Tasuku; Ito, Masako; Katsube, Ken-ichi; Hiraoka, Shuichi; Koseki, Haruhiko; Harada, Kiyoshi; Yamaguchi, Akira
2013-01-01
CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy. PMID:23653360
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Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase
Keereetaweep, Jantana; Liu, Hui; Zhai, Zhiyang; ...
2018-04-06
The first committed step in fatty acid synthesis is mediated by Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback-regulated by short-term (reversible) and longer-term (irreversible) inhibition upon oversupply of fatty acids (FA) provided by Tween80 (predominantly containing oleic acid; 18:1). Biotin-Attachment-Domain-Containing (BADC) proteins are inactive analogs of biotin carboxyl transfer protein (BCCP) that lack biotin and their incorporation into ACCase downregulates it by displacing active (biotin-containing) BCCP subunits. Individual T-DNA insertion lines of BADC1, BADC2, and BADC3 were used to generate badc1badc2 and badc1badc3. The badc1badc3 mutant and wild-type exhibited normal growthmore » and development, however ACCase activity was 26% higher in badc1badc3 relative to wild-type and its seeds contained 30.1 %DW more FA and 32.6 %DW more TAG than wild-type. Cell suspension cultures were generated from leaves of badc1badc3 and wild-type plants to test whether BADC contributes to the irreversible phase of ACCase inhibition resulting from culture in medium containing 10mM Tween80. While the reversible phase of ACCase inhibition after two days of Tween80 feeding was equivalent for badc1badc3 and wild-type, the irreversible phase of inhibition following four days of Tween80 feeding was reduced by 50% in badc1badc3 relative to wild-type. In this work we present evidence for two important homeostatic roles for BADC proteins in downregulating ACCase activity: during normal growth and development, and by contributing to its long-term irreversible feedback inhibition resulting from oversupply of fatty acids.« less
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Abe, Hiroshi; Tateishi, Ken; Seo, Shigemi; Kugimiya, Soichi; Hirai, Masami Yokota; Sawada, Yuji; Murata, Yoshiyuki; Yara, Kaori; Shimoda, Takeshi; Kobayashi, Masatomo
2013-11-01
Here, we analyzed the interaction between Arabidopsis (Arabidopsis thaliana) and the American serpentine leafminer (Liriomyza trifolii), an important and intractable herbivore of many cultivated plants. We examined the role of the immunity-related plant hormone jasmonate (JA) in the plant response and resistance to leafminer feeding to determine whether JA affects host suitability for leafminers. The expression of marker genes for the JA-dependent plant defense was induced by leafminer feeding on Arabidopsis wild-type plants. Analyses of JA-insensitive coi1-1 mutants suggested the importance of JA in the plant response to leafminer feeding. The JA content of wild-type plants significantly increased after leafminer feeding. Moreover, coi1-1 mutants showed lower feeding resistance against leafminer attack than did wild-type plants. The number of feeding scars caused by inoculated adult leafminers in JA-insensitive coi1-1 mutants was higher than that in wild-type plants. In addition, adults of the following generation appeared only from coi1-1 mutants and not from wild-type plants, suggesting that the loss of the JA-dependent plant defense converted nonhost plants to accessible host plants. Interestingly, the glucosinolate-myrosinase defense system may play at most a minor role in this conversion, indicating that this major antiherbivore defense of Brassica species plants probably does not have a major function in plant resistance to leafminer. Application of JA to wild-type plants before leafminer feeding enhanced feeding resistance in Chinese cabbage (Brassica rapa), tomato (Solanum lycopersicum), and garland chrysanthemum (Chrysanthemum coronarium). Our results indicate that JA plays an important role in the plant response and resistance to leafminers and, in so doing, affects host plant suitability for leafminers.
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White, Stephanie; Marquez de Prado, Blanca; Russo, Andrew F; Hammond, Donna L
2014-01-01
This study examined whether mice with a deficiency of neurofibromin, a Ras GTPase activating protein, exhibit a nociceptive phenotype and probed a possible contribution by calcitonin gene-related peptide. In the absence of inflammation, Nf1+/- mice (B6.129S6 Nf1
/J) and wild type littermates responded comparably to heat or mechanical stimuli, except for a subtle enhanced mechanical sensitivity in female Nf1+/- mice. Nociceptive phenotype was also examined after inflammation induced by capsaicin and formalin, which release endogenous calcitonin gene-related peptide. Intraplantar injection of capsaicin evoked comparable heat hyperalgesia and mechanical hypersensitivity in Nf1+/- and wild type mice of both genders. Formalin injection caused a similar duration of licking in male Nf1+/- and wild type mice. Female Nf1+/- mice licked less than wild type mice, but displayed other nociceptive behaviors. In contrast, intraplantar injection of CGRP caused greater heat hyperalgesia in Nf1+/- mice of both genders compared to wild type mice. Male Nf1+/- mice also exhibited greater mechanical hypersensitivity; however, female Nf1+/- mice exhibited less mechanical hypersensitivity than their wild type littermates. Transcripts for calcitonin gene-related peptide were similar in the dorsal root ganglia of both genotypes and genders. Transcripts for receptor activity-modifying protein-1, which is rate-limiting for the calcitonin gene-related peptide receptor, in the spinal cord were comparable for both genotypes and genders. The increased responsiveness to intraplantar calcitonin gene-related peptide suggests that the peripheral actions of calcitonin gene-related peptide are enhanced as a result of the neurofibromin deficit. The analgesic efficacy of calcitonin gene-related peptide receptor antagonists may therefore merit investigation in neurofibromatosis patients. -
Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes.
Johansen, L; Bryn, K; Stormer, F C
1975-01-01
Aerobacter (Enterobacter) aerogenes wild type and three mutants deficient in the formation of acetoin and 2,3-butanediol were grown in a glucose minimal medium. Culture densities, pH, and diacetyl, acetoin, and 2,3-butanediol levels were recorded. The pH in wild-type cultures dropped from 7.0 to 5.8, remained constant while acetoin and 2,3-butanediol were formed, and increased to pH 6.5 after exhaustion of the carbon source. More 2,3-butanediol than acetoin was formed initially, but after glucose exhaustion reoxidation to acetoin occurred. The three mutants differed from the wild type in yielding acid cultures (pH below 4.5). The wild type and one of the mutants were grown exponentially under aerobic and anaerobic conditions with the pH fixed at 7.0, 5.8, and 5.0, respectively. Growth rates decreased with decreasing pH values. Aerobically, this effect was weak, and the two strains were affected to the same degree. Under anaerobic conditions, the growth rates were markedly inhibited at a low pH, and the mutant was slightly more affected than the wild type. Levels of alcohol dehydrogenase were low under all conditions, indicating that the enzyme plays no role during exponential growth. The levels of diacetyl (acetoin) reductase, lactate dehydrogenase, and phosphotransacetylase were independent of the pH during aerobic growth of the two strains. Under anaerobic conditions, the formation of diacetyl (acetoin) reductase was pH dependent, with much higher levels of the enzyme at pH 5.0 than at pH 7.0. Lactate dehydrogenase and phosphotransacetylase revealed the same pattern of pH-dependent formation in the mutant, but not in the wild type. PMID:239921
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Bezzi, Siham; Kessler, Danny; Diezel, Celia; Muck, Alexander; Anssour, Samir; Baldwin, Ian T.
2010-01-01
Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellant, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H2O2) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H2O2 compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H2O2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H2O2 levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H2O2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators. PMID:20190094
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Chen, Lin; Shi, Wanliang; Li, Hui; Sun, Xiuli; Fan, Xionglin; Lesage, Gene; Li, Hui; Li, Yi; Zhang, Yi; Zhang, Xiumei; Zhang, Ying; Yin, Deling
2010-02-19
Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive. We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-alpha, IL-1beta, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3beta in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3beta in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration. These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.
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Mutation and repair in an ultraviolet-sensitive Chinese hamster ovary cell line
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wood, R.D.
1981-11-01
An ultraviolet (UV) light-sensitive mutant of Chinese hamster ovary cells (CHO) has been isolated and characterized with respect to a number of post-irradiation responses. The UV-sensitive mutant, termed 43-3B, has the same growth rate and chromosome number as the wild-type CHO-9. 43-3B is hypersensitive to the lethal effects of UV light (D/sub 0/ of 0.3 J/m/sup 2/ as compared to 3.2 J/m/sup 2/ for the wild-type). A marked UV-hypermutability is observed in 43-3B as compared to the wild-type, as measured with markers for induced resistance to 6-thioguanine, ouabain, and diphtheria toxin. A factor of 38 to 65 more mutations aremore » induced per unit fluence in 43-3B than in CHO-9. The UV-sensitive mutant is also sensitive to killing by simulated solar light, although the D/sub 0/ ratio is not as great as for germicidal UV. 43-3B exhibits only about 17% of the wild-type level of UV-stimulated DNA repair synthesis, as measured by autoradiography of G/sub 1/ phase cells. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild-type CHO-9, but little recovery was seen in 43-3B. The present investigation demonstrates that a sensitive/wild-type pair of CHO cell lines can be used in comparative studies to determine the involvement of repair in a wide range of post-irradiation phenomena.« less
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The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.
Breitenstein, Alexander; Stämpfli, Simon F; Reiner, Martin F; Shi, Yi; Keller, Stephan; Akhmedov, Alexander; Schaub Clerigué, Ariane; Spescha, Remo D; Beer, Hans-Jürg; Lüscher, Thomas F; Tanner, Felix C; Camici, Giovanni G
2017-01-05
Despite public awareness of its deleterious effects, smoking remains a major cause of death. Indeed, it is a risk factor for atherothrombotic complications and in line with this, the introduction of smoking ban in public areas reduced smoking-associated cardiovascular complications. Nonetheless, smoking remains a major concern, and molecular mechanisms by which it causes cardiovascular disease are not known. Peripheral blood monocytes from healthy smokers displayed increased JNK2 and tissue factor (TF) gene expression compared to non-smokers (n=15, p<0.05). Similarly, human aortic endothelial cells exposed to cigarette smoke total particulate matter (CS-TPM) revealed increased TF expression mediated by JNK2 (n=4; p<0.05). Wild-type and JNK2 -/- mice were exposed to cigarette smoke for two weeks after which arterial thrombosis was investigated. Wild-type mice exposed to smoke displayed reduced time to thrombotic arterial occlusion (n=8; p<0.05) and increased tissue factor activity (n=7; p<0.05) as compared to wild-type controls (n=6), while JNK2 -/- mice exposed to smoke maintained an unaltered thrombotic potential (n=8; p=NS) and tissue factor activity (n=8) comparable to that of JNK2 -/- and wild-type controls (n=6; p=NS). Smoking caused an increased production of reactive oxygen species (ROS) in wild-type but not in JNK2 -/- mice (n=7; p<0.05 for wild-type mice and n=5-6; p=NS for JNK2 -/- mice). In conclusion, the MAP kinase JNK2 mediates cigarette smoke-induced TF activation, arterial thrombosis and ROS production. These results underscore a major role of JNK2 in smoke-mediated thrombus formation and may offer an attractive target to prevent smoke-related thrombosis in those subjects which do not manage quitting.
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Biotin Attachment Domain-Containing Proteins Irreversibly Inhibit Acetyl CoA Carboxylase
DOE Office of Scientific and Technical Information (OSTI.GOV)
Keereetaweep, Jantana; Liu, Hui; Zhai, Zhiyang
The first committed step in fatty acid synthesis is mediated by Acetyl-CoA carboxylase (ACCase), a biotin-dependent enzyme that carboxylates acetyl-CoA to produce malonyl-CoA. ACCase can be feedback-regulated by short-term (reversible) and longer-term (irreversible) inhibition upon oversupply of fatty acids (FA) provided by Tween80 (predominantly containing oleic acid; 18:1). Biotin-Attachment-Domain-Containing (BADC) proteins are inactive analogs of biotin carboxyl transfer protein (BCCP) that lack biotin and their incorporation into ACCase downregulates it by displacing active (biotin-containing) BCCP subunits. Individual T-DNA insertion lines of BADC1, BADC2, and BADC3 were used to generate badc1badc2 and badc1badc3. The badc1badc3 mutant and wild-type exhibited normal growthmore » and development, however ACCase activity was 26% higher in badc1badc3 relative to wild-type and its seeds contained 30.1 %DW more FA and 32.6 %DW more TAG than wild-type. Cell suspension cultures were generated from leaves of badc1badc3 and wild-type plants to test whether BADC contributes to the irreversible phase of ACCase inhibition resulting from culture in medium containing 10mM Tween80. While the reversible phase of ACCase inhibition after two days of Tween80 feeding was equivalent for badc1badc3 and wild-type, the irreversible phase of inhibition following four days of Tween80 feeding was reduced by 50% in badc1badc3 relative to wild-type. In this work we present evidence for two important homeostatic roles for BADC proteins in downregulating ACCase activity: during normal growth and development, and by contributing to its long-term irreversible feedback inhibition resulting from oversupply of fatty acids.« less
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Abe, Hiroshi; Tateishi, Ken; Seo, Shigemi; Kugimiya, Soichi; Hirai, Masami Yokota; Sawada, Yuji; Murata, Yoshiyuki; Yara, Kaori; Shimoda, Takeshi; Kobayashi, Masatomo
2013-01-01
Here, we analyzed the interaction between Arabidopsis (Arabidopsis thaliana) and the American serpentine leafminer (Liriomyza trifolii), an important and intractable herbivore of many cultivated plants. We examined the role of the immunity-related plant hormone jasmonate (JA) in the plant response and resistance to leafminer feeding to determine whether JA affects host suitability for leafminers. The expression of marker genes for the JA-dependent plant defense was induced by leafminer feeding on Arabidopsis wild-type plants. Analyses of JA-insensitive coi1-1 mutants suggested the importance of JA in the plant response to leafminer feeding. The JA content of wild-type plants significantly increased after leafminer feeding. Moreover, coi1-1 mutants showed lower feeding resistance against leafminer attack than did wild-type plants. The number of feeding scars caused by inoculated adult leafminers in JA-insensitive coi1-1 mutants was higher than that in wild-type plants. In addition, adults of the following generation appeared only from coi1-1 mutants and not from wild-type plants, suggesting that the loss of the JA-dependent plant defense converted nonhost plants to accessible host plants. Interestingly, the glucosinolate-myrosinase defense system may play at most a minor role in this conversion, indicating that this major antiherbivore defense of Brassica species plants probably does not have a major function in plant resistance to leafminer. Application of JA to wild-type plants before leafminer feeding enhanced feeding resistance in Chinese cabbage (Brassica rapa), tomato (Solanum lycopersicum), and garland chrysanthemum (Chrysanthemum coronarium). Our results indicate that JA plays an important role in the plant response and resistance to leafminers and, in so doing, affects host plant suitability for leafminers. PMID:24022267