CD4+ T-cell engagement by both wild-type and variant HCV peptides modulates the conversion of viral clearing helper T cells to Tregs

PubMed Central

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

Inhibition of IKKβ in enterocytes exacerbates sepsis-induced intestinal injury and worsens mortality.

PubMed

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.

The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females

PubMed Central

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

The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females.

PubMed

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.

Reduced 4-Aminobiphenyl-Induced Liver Tumorigenicity but not DNA Damage in Arylamine N-Acetyltransferase Null Mice

PubMed Central

Sugamori, Kim S.; Brenneman, Debbie; Sanchez, Otto; Doll, Mark A.; Hein, David W.; Pierce, William M.; Grant, Denis M.

2012-01-01

The aromatic amine 4-aminobiphenyl (ABP) is a liver procarcinogen in mice, requiring enzymatic bioactivation to exert its tumorigenic effect. To assess the role of arylamine N-acetyltransferase (NAT)-dependent acetylation capacity in the risk for ABP-induced liver tumors, we compared 1-year liver tumor incidence following the postnatal exposure of wild-type and NAT-deficient Nat1/2(−/−) mice to ABP. At an ABP exposure of 1200 nmoles, male Nat1/2(−/−) mice had a liver tumor incidence of 36% compared to 69% in wild-type males, and at 600 nmoles there was a complete absence of tumors compared to 60% in wild-type mice. Only one female wild-type mouse had a tumor using this exposure protocol. However, levels of N-deoxyguanosin-8-yl-ABP-DNA adducts did not correlate with either the strain or sex differences in tumor incidence. These results suggest that female sex and NAT deficiency reduce risk for ABP-induced liver tumors, but by mechanisms unrelated to differences in DNA-damaging events. PMID:22193722

Comprehensive and differential long-term characterization of the alpha-galactosidase A deficient mouse model of Fabry disease focusing on the sensory system and pain development

PubMed Central

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

Changes in fatty acid content and composition between wild type and CsHMA3 overexpressing Camelina sativa under heavy-metal stress.

PubMed

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.

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

The MAP kinase JNK2 mediates cigarette smoke-induced arterial thrombosis.

PubMed

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.

Alternative Oxidase in Resistance to Biotic Stresses: Nicotiana attenuata AOX Contributes to Resistance to a Pathogen and a Piercing-Sucking Insect But Not Manduca sexta Larvae1[W][OA

PubMed Central

Zhang, Lu; Oh, Youngjoo; Li, Hongyu; Baldwin, Ian T.; Galis, Ivan

2012-01-01

The role of the alternative respiratory pathway in the protection of plants against biotic stress was examined in transgenic tobacco (Nicotiana attenuata) plants (irAOX) silenced in the expression of ALTERNATIVE OXIDASE (AOX) gene. Wild-type and irAOX plants were independently challenged with (1) chewing herbivores (Manduca sexta), (2) piercing-sucking insects (Empoasca spp.), and (3) bacterial pathogens (Pseudomonas syringae pv tomato DC3000), showing that all these treatments can strongly elicit accumulation of AOX gene transcripts in wild-type plants. When N. attenuata chemical defenses and resistance were examined, irAOX plants showed wild-type levels of defense-related phytohormones, secondary metabolites, and resistance to M. sexta. In contrast, piercing-sucking leafhoppers (Empoasca spp.) caused more leaf damage and induced significantly higher salicylic acid levels in irAOX compared with wild-type plants in the field and/or glasshouse. Subsequently, irAOX plants accumulated lower levels of defense metabolites, 17-hydroxygeranyllinalool diterpene glycosides, caffeoylputrescine, and nicotine compared with wild-type plants under prolonged attack of Empoasca spp. in the glasshouse. Finally, an accelerated cell death phenotype was observed in irAOX plants infected with P. syringae, which correlated with higher levels of salicylic acid and hydrogen peroxide levels in pathogen-infected irAOX compared with wild-type leaves. Overall, the AOX-associated changes in phytohormone and/or redox levels appear to support the resistance of N. attenuata plants against cell piercing-sucking insects and modulate the progression of cell death in pathogen-infected tissues but are not effective against rapidly feeding specialist herbivore M. sexta. PMID:22961128

Contribution of Secretory Antibodies to Intestinal Mucosal Immunity against Helicobacter pylori

PubMed Central

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

FIBRILLIN4 Is Required for Plastoglobule Development and Stress Resistance in Apple and Arabidopsis1[W][OA

PubMed Central

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

Pharmacologic Treatment Assigned for Niemann Pick Type C1 Disease Partly Changes Behavioral Traits in Wild-Type Mice.

PubMed

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.

Cross-neutralization between three mumps viruses & mapping of haemagglutinin-neuraminidase (HN) epitopes.

PubMed

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.

Hyperactive mutant of a wheat plasma membrane Na+/H+ antiporter improves the growth and salt tolerance of transgenic tobacco.

PubMed

Zhou, Yang; Lai, Zesen; Yin, Xiaochang; Yu, Shan; Xu, Yuanyuan; Wang, Xiaoxiao; Cong, Xinli; Luo, Yuehua; Xu, Haixia; Jiang, Xingyu

2016-12-01

Wheat SOS1 (TaSOS1) activity could be relieved upon deletion of the C-terminal 168 residues (the auto-inhibitory domain). This truncated form of wheat SOS1 (TaSOS1-974) was shown to increase compensation (compared to wild-type TaSOS1) for the salt sensitivity of a yeast mutant strain, AXT3K, via increased Na + transportation out of cells during salinity stress. Expression of the plasma membrane proteins TaSOS1-974 or TaSOS1 improved the growth of transgenic tobacco plants compared with wild-type plants under normal conditions. However, plants expressing TaSOS1-974 grew better than TaSOS1-transformed plants. Upon salinity stress, Na + efflux and K + influx rates in the roots of transgenic plants expressing TaSOS1-974 or TaSOS1 were greater than those of wild-type plants. Furthermore, compared to TaSOS1-transgenic plants, TaSOS1-974-expressing roots showed faster Na + efflux and K + influx, resulting in less Na + and more K + accumulation in TaSOS1-974-transgenic plants compared to TaSOS1-transgenic and wild-type plants. TaSOS1-974-expressing plants had the lowest MDA content and electrolyte leakage among all tested plants, indicating that TaSOS1-974 might protect the plasma membrane against oxidative damage generated by salt stress. Overall, TaSOS1-974 conferred higher salt tolerance in transgenic plants compared to TaSOS1. Consistent with this result, transgenic plants expressing TaSOS1-974 showed a better growth performance than TaSOS1-expressing and wild-type plants under saline conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Deficiency of eNOS exacerbates early-stage NAFLD pathogenesis by changing the fat distribution.

PubMed

Nozaki, Yuichi; Fujita, Koji; Wada, Koichiro; Yoneda, Masato; Shinohara, Yoshiyasu; Imajo, Kento; Ogawa, Yuji; Kessoku, Takaomi; Nakamuta, Makoto; Saito, Satoru; Masaki, Naohiko; Nagashima, Yoji; Terauchi, Yasuo; Nakajima, Atsushi

2015-12-17

Although many factors and molecules that are closely associated with non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) have been reported, the role of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) in the pathogenesis of NAFLD/NASH remains unclear. We therefore investigated the role of eNOS-derived NO in NAFLD pathogenesis using systemic eNOS-knockout mice fed a high-fat diet. eNOS-knockout and wild-type mice were fed a basal diet or a high-fat diet for 12 weeks. Lipid accumulation and inflammation were evaluated in the liver, and various factors that are closely associated with NAFLD/NASH and hepatic tissue blood flow were analyzed. Lipid accumulation and inflammation were more extensive in the liver and lipid accumulation was less extensive in the visceral fat tissue in eNOS-knockout mice, compared with wild-type mice, after 12 weeks of being fed a high-fat diet. While systemic insulin resistance was comparable between the eNOS-knockout and wild-type mice fed a high-fat diet, hepatic tissue blood flow was significantly suppressed in the eNOS-knockout mice, compared with the wild-type mice, in mice fed a high-fat diet. The microsomal triglyceride transfer protein activity was down-regulated in eNOS-knockout mice, compared with wild-type mice, in mice fed a high-fat diet. A deficiency of eNOS-derived NO may exacerbate the early-stage of NASH pathogenesis by changing the fat distribution in a mouse model via the regulation of hepatic tissue blood flow.

Adenosine A2B Receptor Deficiency Promotes Host Defenses against Gram-Negative Bacterial Pneumonia

PubMed Central

Barletta, Kathryn E.; Cagnina, R. Elaine; Burdick, Marie D.; Linden, Joel

2012-01-01

Rationale: Activation of the adenosine A2B receptor (A2BR) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. Objectives: To test the hypothesis that absence of adenosine A2B receptor signaling promotes host defense against bacterial pneumonia. Methods: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A2BR. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. Measurements and Main Results: A2BR–/– mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow–derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A2BR–/– and wild-type mice, but A2BR–/– neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A2BR–/– mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. Conclusions: These data suggest that the absence of A2BR signaling enhances antimicrobial activity in gram-negative bacterial pneumonia. PMID:22997203

Repair of dentin defects from DSPP knockout mice by PILP mineralization

PubMed Central

Nurrohman, H.; Saeki, K.; Carneiro, K.; Chien, Y.C.; Djomehri, S.; Ho, S.P.; Qin, C.; Marshall, S.J.; Gower, L.B.; Marshall, G.W.; Habelitz, S.

2016-01-01

Dentinogenesis imperfecta type II (DGI-II) lacks intrafibrillar mineral with severe compromise of dentin mechanical properties. A Dspp knockout (Dspp−/−) mouse, with a phenotype similar to that of human DGI-II, was used to determine if poly-L-aspartic acid [poly(ASP)] in the “polymer-induced liquid-precursor” (PILP) system can restore its mechanical properties. Dentin from six-week old Dspp−/− and wild-type mice was treated with CaP solution containing poly(ASP) for up to 14 days. Elastic modulus and hardness before and after treatment were correlated with mineralization from Micro x-ray computed tomography (Micro-XCT). Transmission electron microscopy (TEM)/Selected area electron diffraction (SAED) were used to compare matrix mineralization and crystallography. Mechanical properties of the Dspp−/− dentin were significantly less than wild-type dentin and recovered significantly (P < 0.05) after PILP-treatment, reaching values comparable to wild-type dentin. Micro-XCT showed mineral recovery similar to wild-type dentin after PILP-treatment. TEM/SAED showed repair of patchy mineralization and complete mineralization of defective dentin. This approach may lead to new strategies for hard tissue repair. PMID:27239097

DNA Repair Modulates The Vulnerability of The Developing Brain to Alkylating Agents

PubMed Central

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

CytR Homolog of Pectobacterium carotovorum subsp. carotovorum Controls Air-Liquid Biofilm Formation by Regulating Multiple Genes Involved in Cellulose Production, c-di-GMP Signaling, Motility, and Type III Secretion System in Response to Nutritional and Environmental Signals.

PubMed

Haque, M M; Oliver, M M H; Nahar, Kamrun; Alam, Mohammad Z; Hirata, Hisae; Tsuyumu, Shinji

2017-01-01

Pectobacterium carotovorum subsp. carotovorum [Pcc (formerly Erwinia carotovora subsp. carotovora )] PC1 causes soft-rot disease in a wide variety of plant species by secreting multiple pathogenicity-related traits. In this study, regulatory mechanism of a ir- l iquid (AL) biofilm formation was studied using a cytR homolog gene deletion mutant (Δ cytR ) of Pcc PC1. Compared to the wild type (Pcc PC1), the Δ cytR mutant produced fragile and significantly ( P < 0.001) lower amounts of AL biofilm on s alt- o ptimized b roth plus 2% g lycerol (SOBG), yeast peptone dextrose adenine, and also on King's B at 27°C after 72 h incubation in static condition. The wild type also produced significantly higher quantities of AL biofilm on SOBGMg - (magnesium deprived) containing Cupper (Cu 2+ ), Zinc (Zn 2+ ), Manganese (Mn 2+ ), Magnesium (Mg 2+ ), and Calcium (Ca 2+ ) compared to the Δ cytR mutant. Moreover, the wild type was produced higher amounts of biofilms compared to the mutant while responding to pH and osmotic stresses. The Δ fliC (encoding flagellin), flhD ::Tn5 (encoding a master regulator) and Δ motA (a membrane protein essential for flagellar rotation) mutants produced a lighter and more fragile AL biofilm on SOBG compared to their wild counterpart. All these mutants resulted in having weak bonds with the cellulose specific dye (Calcofluor) producing lower quantities of cellulose compared to the wild type. Gene expression analysis using mRNA collected from the AL biofilms showed that Δ cytR mutant significantly ( P < 0.001) reduced the expressions of multiple genes responsible for cellulose production ( bcsA, bcsE , and adrA ), motility ( flhD, fliA, fliC , and motA ) and type III secretion system ( hrpX, hrpL, hrpA , and hrpN ) compared to the wild type. The CytR homolog was therefore, argued to be able to regulate the AL biofilm formation by controlling cellulose production, motility and T3SS in Pcc PC1. In addition, all the mutants exhibited poorer attachment to radish sprouts and AL biofilm cells of the wild type was resistant than stationary-phase and planktonic cells to acidity and oxidative stress compared to the same cells of the Δ cytR mutant. The results of this study therefore suggest that CytR homolog is a major determinant of Pcc PC1's virulence, attachment and its survival mechanism.

CytR Homolog of Pectobacterium carotovorum subsp. carotovorum Controls Air-Liquid Biofilm Formation by Regulating Multiple Genes Involved in Cellulose Production, c-di-GMP Signaling, Motility, and Type III Secretion System in Response to Nutritional and Environmental Signals

PubMed Central

Haque, M. M.; Oliver, M. M. H.; Nahar, Kamrun; Alam, Mohammad Z.; Hirata, Hisae; Tsuyumu, Shinji

2017-01-01

Pectobacterium carotovorum subsp. carotovorum [Pcc (formerly Erwinia carotovora subsp. carotovora)] PC1 causes soft-rot disease in a wide variety of plant species by secreting multiple pathogenicity-related traits. In this study, regulatory mechanism of air-liquid (AL) biofilm formation was studied using a cytR homolog gene deletion mutant (ΔcytR) of Pcc PC1. Compared to the wild type (Pcc PC1), the ΔcytR mutant produced fragile and significantly (P < 0.001) lower amounts of AL biofilm on salt-optimized broth plus 2% glycerol (SOBG), yeast peptone dextrose adenine, and also on King’s B at 27°C after 72 h incubation in static condition. The wild type also produced significantly higher quantities of AL biofilm on SOBGMg– (magnesium deprived) containing Cupper (Cu2+), Zinc (Zn2+), Manganese (Mn2+), Magnesium (Mg2+), and Calcium (Ca2+) compared to the ΔcytR mutant. Moreover, the wild type was produced higher amounts of biofilms compared to the mutant while responding to pH and osmotic stresses. The ΔfliC (encoding flagellin), flhD::Tn5 (encoding a master regulator) and ΔmotA (a membrane protein essential for flagellar rotation) mutants produced a lighter and more fragile AL biofilm on SOBG compared to their wild counterpart. All these mutants resulted in having weak bonds with the cellulose specific dye (Calcofluor) producing lower quantities of cellulose compared to the wild type. Gene expression analysis using mRNA collected from the AL biofilms showed that ΔcytR mutant significantly (P < 0.001) reduced the expressions of multiple genes responsible for cellulose production (bcsA, bcsE, and adrA), motility (flhD, fliA, fliC, and motA) and type III secretion system (hrpX, hrpL, hrpA, and hrpN) compared to the wild type. The CytR homolog was therefore, argued to be able to regulate the AL biofilm formation by controlling cellulose production, motility and T3SS in Pcc PC1. In addition, all the mutants exhibited poorer attachment to radish sprouts and AL biofilm cells of the wild type was resistant than stationary-phase and planktonic cells to acidity and oxidative stress compared to the same cells of the ΔcytR mutant. The results of this study therefore suggest that CytR homolog is a major determinant of Pcc PC1’s virulence, attachment and its survival mechanism. PMID:28620360

Phase II Study of the Dual EGFR/HER3 Inhibitor Duligotuzumab (MEHD7945A) versus Cetuximab in Combination with FOLFIRI in Second-Line RAS Wild-Type Metastatic Colorectal Cancer.

PubMed

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.

Mannose-specific interaction of Lactobacillus plantarum with porcine jejunal epithelium.

PubMed

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.

Effects of Elevated Pax6 Expression and Genetic Background on Mouse Eye Development

PubMed Central

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

A genetically engineered human Kunitz protease inhibitor with increased kallikrein inhibition in an ovine model of cardiopulmonary bypass.

PubMed

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.

Effect of Trehalose and Trehalose Transport on the Tolerance of Clostridium perfringens to Environmental Stress in a Wild Type Strain and Its Fluoroquinolone-Resistant Mutant

PubMed Central

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

Toll-Like Receptor 3 Is Critical for Coxsackievirus B4-Induced Type 1 Diabetes in Female NOD Mice

PubMed Central

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

Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii.

PubMed

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.

Post-Transcriptional Dysregulation by miRNAs Is Implicated in the Pathogenesis of Gastrointestinal Stromal Tumor [GIST

PubMed Central

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

Post-transcriptional dysregulation by miRNAs is implicated in the pathogenesis of gastrointestinal stromal tumor [GIST].

PubMed

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.

Cross-neutralization between three mumps viruses & mapping of haemagglutinin-neuraminidase (HN) epitopes

PubMed Central

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

Sex- and age-related differences in the chronic pressure-natriuresis relationship: role of the angiotensin type 2 receptor.

PubMed

Mirabito, Katrina M; Hilliard, Lucinda M; Kett, Michelle M; Brown, Russell D; Booth, Sean C; Widdop, Robert E; Moritz, Karen M; Evans, Roger G; Denton, Kate M

2014-10-15

Sex hormones regulate the renin-angiotensin system. For example, estrogen enhances expression of the angiotensin type 2 receptor. We hypothesized that activation of the angiotensin type 2 receptor shifts the chronic pressure-natriuresis relationship leftward in females compared with males and that this effect is lost with age. Mean arterial pressure was measured by radiotelemetry in adult (4 mo old) and aged (14 mo old) wild-type and angiotensin type 2 receptor knockout male and female mice. Chronic pressure-natriuresis curves were constructed while mice were maintained on a normal-salt (0.26%) diet and following 6 days of high salt (5.0%) diet. Mean arterial pressure was lower in adult wild-type females than males (88 ± 1 and 97 ± 1 mmHg, respectively), a difference that was maintained with age, but was absent in adult knockout mice. In wild-type females, the chronic pressure-natriuresis relationship was shifted leftward compared with knockout females, an effect that was lost with age. In males, the chronic pressure-natriuresis relationship was not influenced by angiotensin type 2 receptor deficiency. Compared with age-matched females, the chronic pressure-natriuresis relationships in male mice were shifted rightward. Renal expression of the angiotensin type 2 receptor was fourfold greater in adult wild-type females than males. With age, the angiotensin type 2 receptor-to-angiotensin type 1 receptor balance was reduced in females. Conversely, in males, angiotensin receptor expression did not vary significantly with age. In conclusion, the angiotensin type 2 receptor modulates the chronic pressure-natriuresis relationship in an age- and sex-dependent manner. Copyright © 2014 the American Physiological Society.

Alterations of neutral glycolipids in cells infected with syncytium-producing mutants of herpes simplex virus type 1

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

Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

PubMed Central

Zieg, J; Maples, V F; Kushner, S R

1978-01-01

Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products. PMID:350859

Importance of cell damage causing growth delay for high pressure inactivation of Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Nanba, Masaru; Nomura, Kazuki; Nasuhara, Yusuke; Hayashi, Manabu; Kido, Miyuki; Hayashi, Mayumi; Iguchi, Akinori; Shigematsu, Toru; Hirayama, Masao; Ueno, Shigeaki; Fujii, Tomoyuki

2013-06-01

A high pressure (HP) tolerant (barotolerant) mutant a2568D8 and a variably barotolerant mutant a1210H12 were generated from Saccharomyces cerevisiae using ultra-violet mutagenesis. The two mutants, a barosensitive mutant a924E1 and the wild-type strain, were pressurized (225 MPa), and pressure inactivation behavior was analyzed. In the wild-type strain, a proportion of the growth-delayed cells were detected after exposure to HP. In a924E1, the proportion of growth-delayed cells significantly decreased compared with the wild-type. In a2568D8, the proportion of growth-delayed cells increased and the proportion of inactivated cells decreased compared with the wild-type. In a1210H12, the growth-delayed cells could not be detected within 120 s of exposure to HP. The proportion of growth-delayed cells, which incurred the damage, would affect the survival ratio by HP. These results suggested that cellular changes in barotolerance caused by mutations are remarkably affected by the ability to recover from cellular damage, which results in a growth delay.

Structural, molecular motions, and free-energy landscape of Leishmania sterol-14α-demethylase wild type and drug resistant mutant: a comparative molecular dynamics study.

PubMed

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.

MLH1-deficient Colorectal Carcinoma With Wild-type BRAF and MLH1 Promoter Hypermethylation Harbor KRAS Mutations and Arise From Conventional Adenomas.

PubMed

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.

Self-Assembling Protein Materials for Metal Nanoparticle Templation

DTIC Science & Technology

2015-05-01

conductivity of the wild type biofilm compared to a control of buffer and PilA-deficient mutant strain (c).146 .............................. 113...micrograph of biofilm and pili nanofilaments grown on the electrode surface (b) and the conductivity of the wild type biofilm compared to a control of buffer ...to obtain conductive amyloid fibers deposited on Si3N4 viewed by AFM (b) and TEM (c), where diameters of coated fibers were enlarged from 50 nm to 100

Requirement for erythroblast-macrophage protein (Emp) in definitive erythropoiesis.

PubMed

Soni, Shivani; Bala, Shashi; Hanspal, Manjit

2008-01-01

Emp, erythroblast-macrophage protein was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation. More recent studies have shown that targeted disruption of Emp leads to abnormal erythropoiesis in the fetal liver, and fetal demise. To further address the activity of Emp in the hematopoietic lineage in adult bone marrow, we conducted fetal liver HSC reconstitution assay. Emp null fetal liver cells were transplanted into lethally irradiated wild-type sibling mice, and assessed the erythropoietic activity. We found that Emp null cells rescued lethally irradiated mice with efficiency comparable to that of wild-type cells. However, the recipients of Emp null cells showed abnormal erythropoiesis as indicated by the presence of persistent anemia, extensive extramedullary erythropoiesis, and increased apoptosis of erythroid precursors. Extramedullary erythropoiesis suggests perturbed interactions between the Emp-deficient hematopoietic cells and the wild-type niche. Furthermore, in spleen colony-forming unit assays, proliferation rates of the Emp null cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Emp null livers. Morphologic examination showed that Emp null CFU-E-derived erythroblasts were immature compared to those derived from wild-type CFU-Es, suggesting that loss of Emp function in erythroid cells results in impaired proliferation and terminal differentiation. These results demonstrate that Emp plays a cell intrinsic role in the erythroid lineage.

Hepcidin regulation in wild-type and Hfe knockout mice in response to alcohol consumption: evidence for an alcohol-induced hypoxic response.

PubMed

Heritage, Mandy L; Murphy, Therese L; Bridle, Kim R; Anderson, Gregory J; Crawford, Darrell H G; Fletcher, Linda M

2009-08-01

Expression of Hamp1, the gene encoding the iron regulatory peptide hepcidin, is inappropriately low in HFE-associated hereditary hemochromatosis and Hfe knockout mice (Hfe(-/-)). Since chronic alcohol consumption is also associated with disturbances in iron metabolism, we investigated the effects of alcohol consumption on hepcidin mRNA expression in Hfe(-/-) mice. Hfe(-/-) and C57BL/6 (wild-type) mice were pair-fed either an alcohol liquid diet or control diet for up to 8 weeks. The mRNA levels of hepcidin and ferroportin were measured at the mRNA level by RT-PCR and protein expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was measured by western blot. Hamp1 mRNA expression was significantly decreased and duodenal ferroportin expression was increased in alcohol-fed wild-type mice at 8 weeks. Time course experiments showed that the decrease in hepcidin mRNA was not immediate, but was significant by 4 weeks. Consistent with the genetic defect, Hamp1 mRNA was decreased and duodenal ferroportin mRNA expression was increased in Hfe(-/-) mice fed on the control diet compared with wild-type animals and alcohol further exacerbated these effects. HIF-1alpha protein levels were elevated in alcohol-fed wild-type animals compared with controls. Alcohol may decrease Hamp1 gene expression independently of the HFE pathway possibly via alcohol-induced hypoxia.

Loss of T cells influences sex differences in behavior and brain structure.

PubMed

Rilett, Kelly C; Friedel, Miriam; Ellegood, Jacob; MacKenzie, Robyn N; Lerch, Jason P; Foster, Jane A

2015-05-01

Clinical and animal studies demonstrate that immune-brain communication influences behavior and brain function. Mice lacking T cell receptor β and δ chains were tested in the elevated plus maze, open field, and light-dark test and showed reduced anxiety-like behavior compared to wild type. Interestingly sex differences were observed in the behavioural phenotype of TCRβ-/-δ- mice. Specifically, female TCRβ-/-δ- mice spent more time in the light chamber compared to wild type females, whereas male TCRβ-/-δ- spent more time in the center of the open field compared to wild type males. In addition, TCRβ-/-δ- mice did not show sex differences in activity-related behaviors observed in WT mice. Ex vivo brain imaging (7 Tesla MRI) revealed volume changes in hippocampus, hypothalamus, amygdala, periaqueductal gray, and dorsal raphe and other brain regions between wild type and T cell receptor knockout mice. There was also a loss of sexual dimorphism in brain volume in the bed nucleus of the stria terminalis, normally the most sexually dimorphic region in the brain, in immune compromised mice. These data demonstrate the presence of T cells is important in the development of sex differences in CNS circuitry and behavior. Copyright © 2015 Elsevier Inc. All rights reserved.

Increased susceptibility to fatigue of slow- and fast-twitch muscles from mice lacking the MG29 gene.

PubMed

Nagaraj, R Y; Nosek, C M; Brotto, M A; Nishi, M; Takeshima, H; Nosek, T M; Ma, J

2000-11-09

Mitsugumin 29 (MG29), a major protein component of the triad junction in skeletal muscle, has been identified to play roles in the formation of precise junctional membrane structures important for efficient signal conversion in excitation-contraction (E-C) coupling. We carried out several experiments to not only study the role of MG29 in normal muscle contraction but also to determine its role in muscle fatigue. We compared the in vitro contractile properties of three muscles types, extensor digitorum longus (EDL) (fast-twitch muscle), soleus (SOL) (slow-twitch muscle), and diaphragm (DPH) (mixed-fiber muscle), isolated from mice lacking the MG29 gene and wild-type mice prior to and after fatigue. Our results indicate that the mutant EDL and SOL muscles, but not DPH, are more susceptible to fatigue than the wild-type muscles. The mutant muscles not only fatigued to a greater extent but also recovered significantly less than the wild-type muscles. Following fatigue, the mutant EDL and SOL muscles produced lower twitch forces than the wild-type muscles; in addition, fatiguing produced a downward shift in the force-frequency relationship in the mutant mice compared with the wild-type controls. Our results indicate that fatiguing affects the E-C components of the mutant EDL and SOL muscles, and the effect of fatigue in these mutant muscles could be primarily due to an alteration in the intracellular Ca homeostasis.

Exacerbation of Diabetic Renal Alterations in Mice Lacking Vasohibin-1

PubMed Central

Hinamoto, Norikazu; Maeshima, Yohei; Yamasaki, Hiroko; Nasu, Tatsuyo; Saito, Daisuke; Watatani, Hiroyuki; Ujike, Haruyo; Tanabe, Katsuyuki; Masuda, Kana; Arata, Yuka; Sugiyama, Hitoshi; Sato, Yasufumi; Makino, Hirofumi

2014-01-01

Vasohibin-1 (VASH1) is a unique endogenous inhibitor of angiogenesis that is induced in endothelial cells by pro-angiogenic factors. We previously reported renoprotective effect of adenoviral delivery of VASH1 in diabetic nephropathy model, and herein investigated the potential protective role of endogenous VASH1 by using VASH1-deficient mice. Streptozotocin-induced type 1 diabetic VASH1 heterozygous knockout mice (VASH1+/−) or wild-type diabetic mice were sacrificed 16 weeks after inducing diabetes. In the diabetic VASH1+/− mice, albuminuria were significantly exacerbated compared with the diabetic wild-type littermates, in association with the dysregulated distribution of glomerular slit diaphragm related proteins, nephrin and ZO-1, glomerular basement membrane thickning and reduction of slit diaphragm density. Glomerular monocyte/macrophage infiltration and glomerular nuclear translocation of phosphorylated NF-κB p65 were significantly exacerbated in the diabetic VASH1+/− mice compared with the diabetic wild-type littermates, accompanied by the augmentation of VEGF-A, M1 macrophage-derived MCP-1 and phosphorylation of IκBα, and the decrease of angiopoietin-1/2 ratio and M2 macrophage-derived Arginase-1. The glomerular CD31+ endothelial area was also increased in the diabetic VASH1+/− mice compared with the diabetic-wild type littermates. Furthermore, the renal and glomerular hypertrophy, glomerular accumulation of mesangial matrix and type IV collagen and activation of renal TGF-β1/Smad3 signaling, a key mediator of renal fibrosis, were exacerbated in the diabetic VASH1+/− mice compared with the diabetic wild-type littermates. In conditionally immortalized mouse podocytes cultured under high glucose condition, transfection of VASH1 small interfering RNA (siRNA) resulted in the reduction of nephrin, angiopoietin-1 and ZO-1, and the augmentation of VEGF-A compared with control siRNA. These results suggest that endogenous VASH1 may regulate the development of diabetic renal alterations, partly via direct effects on podocytes, and thus, a strategy to recover VASH1 might potentially lead to the development of a novel therapeutic approach for diabetic nephropathy. PMID:25255225

Inhibition of the promotion of hepatocarcinogenesis by 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) by the deletion of the p50 subunit of NF-{kappa}B in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glauert, Howard P.; Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40506; Tharappel, Job C.

Polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental chemicals that bioaccumulate and have hepatic tumor promoting activity in rodents. The present study examined the effect of deleting the p50 subunit of NF-{kappa}B on the hepatic tumor promoting activity of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) in mice. Both wild-type and p50-/- male mice were injected i.p. with diethylnitrosamine (DEN, 90 mg/kg) and then subsequently injected biweekly with 20 i.p. injections of PCB-153 (300 {mu}mol/kg/injection). p50 deletion decreased the tumor incidence in both PCB- and vehicle-treated mice, whereas PCB-153 slightly (P = 0.09) increased the tumor incidence in wild-type and p50-/- mice. PCB-153 increased themore » total tumor volume in both wild-type and p50-/- mice, but the total tumor volume was not affected by p50 deletion in either PCB- or vehicle-treated mice. The volume of tumors that were positive for glutamine synthetase (GS), which is indicative of mutations in the beta-catenin gene, was increased in both wild-type and p50-/- mice administered PCB-153 compared to vehicle controls, and inhibited in p50-/- mice compared to wild-type mice (in both PCB- and vehicle-treated mice). The volume of tumors that were negative for GS was increased in p50-/- mice compared to wild-type mice but was not affected by PCB-153. PCB-153 increased cell proliferation in normal hepatocytes in wild-type but not p50-/- mice; this increase was inhibited in p50-/- mice. In hepatic tumors, the rate of cell proliferation was much higher than in normal hepatocytes, but was not affected by PCB treatment or p50 deletion. The rate of apoptosis, as measured by the TUNEL assay, was not affected by PCB-153 or p50 deletion in normal hepatocytes. In hepatic tumors, the rate of apoptosis was lower than in normal hepatocytes; PCB-153 slightly (P = 0.10) increased apoptosis in p50-/- but not wild-type mice; p50 deletion had no effect. Taken together, these data indicate that the absence of the NF-{kappa}B p50 subunit inhibits the promoting activity of PCB-153 and alters the proliferative and apoptotic changes in mouse liver in the response to PCBs.« less

Influence of developmental stage and genotype on liver mRNA levels among wild, domesticated, and hybrid rainbow trout (Oncorhynchus mykiss).

PubMed

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.

The cell adhesion molecule L1 regulates the expression of choline acetyltransferase and the development of septal cholinergic neurons

PubMed Central

Cui, Xuezhi; Weng, Ying-Qi; Frappé, Isabelle; Burgess, Alison; Girão da Cruz, M Teresa; Schachner, Melitta; Aubert, Isabelle

2011-01-01

Mutations in the L1 gene cause severe brain malformations and mental retardation. We investigated the potential roles of L1 in the regulation of choline acetyltransferase (ChAT) and in the development of septal cholinergic neurons, which are known to project to the hippocampus and play key roles in cognitive functions. Using stereological approaches, we detected significantly fewer ChAT-positive cholinergic neurons in the medial septum and vertical limb of the diagonal band of Broca (MS/VDB) of 2-week-old L1-deficient mice compared to wild-type littermates (1644 ± 137 vs. 2051 ± 165, P = 0.038). ChAT protein levels in the septum were 53% lower in 2-week-old L1-deficient mice compared to wild-type littermates. ChAT activity in the septum was significantly reduced in L1-deficient mice compared to wild-type littermates at 1 (34%) and 2 (40%) weeks of age. In vitro, increasing doses of L1-Fc induced ChAT activity in septal neurons with a significant linear trend (*P = 0.0065). At 4 weeks of age in the septum and at all time points investigated in the caudate-putamen (CPu), the number of ChAT-positive neurons and the levels of ChAT activity were not statistically different between L1-deficient mice and wild-type littermates. The total number of cells positive for the neuronal nuclear antigen (NeuN) in the MS/VDB and CPu was not statistically different in L1-deficient mice compared to wild-type littermates, and comparable expression of the cell cycle marker Ki67 was observed. Our results indicate that L1 is required for the timely maturation of septal cholinergic neurons and that L1 promotes the expression and activity of ChAT in septal neurons. PMID:22399087

Protein S is protective in pulmonary fibrosis.

PubMed

Urawa, M; Kobayashi, T; D'Alessandro-Gabazza, C N; Fujimoto, H; Toda, M; Roeen, Z; Hinneh, J A; Yasuma, T; Takei, Y; Taguchi, O; Gabazza, E C

2016-08-01

Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar epithelial cells in vitro. Conclusions These observations suggest clinical relevance and a protective role of protein S in pulmonary fibrosis. © 2016 International Society on Thrombosis and Haemostasis.

Nuclear factor erythroid 2-related factor 2 deletion impairs glucose tolerance and exacerbates hyperglycemia in type 1 diabetic mice.

PubMed

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.

A truncated F-box protein confers the dwarfism in cucumber

USDA-ARS?s Scientific Manuscript database

Dwarfism is an important plant architecture trait for cucumber breeding. In the present study, we identified a dwarf mutant 406M in cucumber which showed a shorter internode length as compared with its wild type. In a BC1F2 population from the cross of 406M with its wild type parental line 406, the ...

Salivary Gland Hypofunction in tyrosylprotein sulfotransferase-2 Knockout Mice Is Due to Primary Hypothyroidism

PubMed Central

Westmuckett, Andrew D.; Siefert, Joseph C.; Tesiram, Yasvir A.; Pinson, David M.; Moore, Kevin L.

2013-01-01

Background Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2). We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI) to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice. Methodology/Principal Findings Quantitative MRI imaging documented that salivary glands in Tpst2-/- females were ≈ 30% smaller than wild type or Tpst1-/- mice and that the granular convoluted tubules in Tpst2-/- submandibular glands were less prominent and were almost completely devoid of exocrine secretory granules compared to glands from wild type or Tpst1-/- mice. In addition, pilocarpine–induced salivary flow and salivary α-amylase activity in Tpst2-/- mice of both sexes was substantially lower than in wild type and Tpst1-/- mice. Anti-sulfotyrosine Western blots of salivary gland extracts and saliva showed no differences between wild type, Tpst1-/-, and Tpst2-/- mice, suggesting that the salivary gland hypofunction is due to factor(s) extrinsic to the salivary glands. Finally, we found that all indicators of hypothyroidism (serum T4, body weight) and salivary gland hypofunction (salivary flow, salivary α-amylase activity, histological changes) were restored to normal or near normal by thyroid hormone supplementation. Conclusions/Significance Our findings conclusively demonstrate that low body weight and salivary gland hypofunction in Tpst2-/- mice is due solely to primary hypothyroidism. PMID:23951251

Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine

PubMed Central

Barreno, Ramon X.; Richards, Jeremy B.; Schneider, Daniel J.; Cromar, Kevin R.; Nadas, Arthur J.; Hernandez, Christopher B.; Hallberg, Lance M.; Price, Roger E.; Hashmi, Syed S.; Blackburn, Michael R.; Haque, Ikram U.

2013-01-01

Inhalation of ozone (O3), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O3 (2 parts/million). In wild-type mice, O3 exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O3-exposed wild-type mice. O3 exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O3 exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-β-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O3 exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O3 and functionally contributes to the development of O3-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine. PMID:23666750

Profiling of anthocyanins in transgenic purple-fleshed sweet potatoes by HPLC-MS/MS.

PubMed

Ge, Jingqiu; Hu, Yijie; Wang, Hongxia; Huang, Yuanshe; Zhang, Peng; Liao, Zhihua; Chen, Min

2017-11-01

Anthocyanins in purple-fleshed sweet potato (PSP) are beneficial to human health. The leaf color (Lc) gene is a transcription factor involved in regulating anthocyanin biosynthesis. The anthocyanin profiles of wild-type PSP of Ayamurasaki and its three Lc-transgenic lines were investigated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). In vitro antioxidant activities of wild-type and Lc-transgenic lines, including reducing power activity, DPPH radical scavenging activity, hydroxyl radical scavenging activity, linoleic acid autoxidation inhibition activity, ABTS free radical scavenging activity and oxygen radical absorbance capacity activity, were measured. The results showed that the total anthocyanin contents increased 1.5-1.9 times in three transgenic lines compared with that in wild-type PSP. Seventeen anthocyanins were found in wild-type PSP, while 19 in Lc-transgenic lines including cyanidin-based, peonidin-based and pelargonidin-based anthocyanins. Three pelargonidin-based anthocyanins were detected in three Lc-transgenic lines. Among them, the relative contents of cyanidin-based and pelargonidin-based anthocyanins increased 1.9-2.0 and 3.4-4.5 times respectively, while peonidin-based anthocyanins decreased 1.8-1.9 times in Lc-transgenic lines, compared with wild-type PSP. PSP from wild-type Ayamurasaki and three Lc-transgenic lines exhibited potent antioxidant activities, whereas there was no distinct difference among them. The transgene Lc significantly increased the content of total anthocyanins and remarkably changed the anthocyanin profiles in Ayamurasaki. Such novel and high content of anthocyanins obtained in the Lc-transgenic lines with potent antioxidant activities may provide unique functional products with potential helpful for human health. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Matrilysin (Matrix Metalloproteinase-7) Regulates Anti-Inflammatory and Antifibrotic Pulmonary Dendritic Cells That Express CD103 (αEβ7-Integrin)

PubMed Central

Manicone, Anne M.; Huizar, Isham; McGuire, John K.

2009-01-01

The E-cadherin receptor CD103 (αEβ7-integrin) is expressed on specific populations of pulmonary dendritic cells (DC) and T cells. However, CD103 function in the lung is not well understood. Matrilysin (MMP-7) expression is increased in lung injury and cleaves E-cadherin from injured lung epithelium. Thus, to assess matrilysin effects on CD103-E-cadherin interactions in lung injury, wild-type, CD103−/−, and Mmp7−/− mice, in which E-cadherin isn’t cleaved in the lung, were treated with bleomycin or bleomycin with nFMLP to reverse the defect in acute neutrophil influx seen in Mmp7−/− mice. Pulmonary CD103+ DC were significantly increased in injured wild-type compared with Mmp7−/− mice, and CD103+ leukocytes showed significantly enhanced interaction with E-cadherin on injured wild-type epithelium than with Mmp7−/− epithelium in vitro and in vivo. Bleomycin-treated CD103−/− mice had persistent neutrophilic inflammation, increased fibrosis, and increased mortality compared with wild-type mice, a phenotype that was partially recapitulated in bleomycin/nFMLP-treated Mmp7−/− mice. Soluble E-cadherin increased IL-12 and IL-10 and reduced IL-6 mRNA expression in wild-type bone marrow-derived DC but not in CD103−/− bone marrow-derived DC. Similar mRNA patterns were seen in lungs of bleomycin-injured wild-type, but not CD103−/− or Mmp7−/−, mice. In conclusion, matrilysin regulates pulmonary localization of DC that express CD103, and E-cadherin cleavage may activate CD103+ DC to limit inflammation and inhibit fibrosis. PMID:19893044

Retinal ganglion cell responses to voltage and current stimulation in wild-type and rd1 mouse retinas

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.

Gibberellins regulate the stem elongation rate without affecting the mature plant height of a quick development mutant of winter wheat (Triticum aestivum L.).

PubMed

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.

Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

PubMed Central

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

Activation Of Wild-Type Hras Suppresses The Earliest Stages Of Pancreatic Cancer.

PubMed

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.

Impaired removal of Vβ8(+) lymphocytes aggravates colitis in mice deficient for B cell lymphoma-2-interacting mediator of cell death (Bim).

PubMed

Leucht, K; Caj, M; Fried, M; Rogler, G; Hausmann, M

2013-09-01

We investigated the role of B cell lymphoma (BCL)-2-interacting mediator of cell death (Bim) for lymphocyte homeostasis in intestinal mucosa. Lymphocytes lacking Bim are refractory to apoptosis. Chronic colitis was induced in Bim-deficient mice (Bim(-/-) ) with dextran sulphate sodium (DSS). Weight loss and colonoscopic score were increased significantly in Bim(-/-) mice compared to wild-type mice. As Bim is induced for the killing of autoreactive cells we determined the role of Bim in the regulation of lymphocyte survival at mucosal sites. Upon chronic dextran sulphate sodium (DSS)-induced colitis, Bim(-/-) animals exhibited an increased infiltrate of lymphocytes into the mucosa compared to wild-type mice. The number of autoreactive T cell receptor (TCR) Vβ8(+) lymphocytes was significantly higher in Bim(-/-) mice compared to wild-type controls. Impaired removal of autoreactive lymphocytes in Bim(-/-) mice upon chronic DSS-induced colitis may therefore contribute to aggravated mucosal inflammation. © 2013 British Society for Immunology.

Isolation and Characterization of Mutants of Common Ice Plant Deficient in Crassulacean Acid Metabolism1[W][OA

PubMed Central

Cushman, John C.; Agarie, Sakae; Albion, Rebecca L.; Elliot, Stewart M.; Taybi, Tahar; Borland, Anne M.

2008-01-01

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM. PMID:18326789

BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

PubMed

Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

2010-12-01

During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

[Upregulation of P2X3 receptors in dorsal root ganglion of TRPV1 knockout female mice].

PubMed

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.

DNA-dependent protein kinase is a molecular target for the development of noncytotoxic radiation-sensitizing drugs.

PubMed

Shinohara, Eric T; Geng, Ling; Tan, Jiahui; Chen, Heidi; Shir, Yu; Edwards, Eric; Halbrook, James; Kesicki, Edward A; Kashishian, Adam; Hallahan, Dennis E

2005-06-15

DNA-dependent protein kinase (DNA-PK)-defective severe combined immunodeficient (SCID) mice have a greater sensitivity to ionizing radiation compared with wild-type mice due to deficient repair of DNA double-strand break. SCID cells were therefore studied to determine whether radiosensitization by the specific inhibitor of DNA-PK, IC87361, is eliminated in the absence of functional DNA-PK. IC87361 enhanced radiation sensitivity in wild-type C57BL6 endothelial cells but not in SCID cells. The tumor vascular window model was used to assess IC87361-induced radiosensitization of SCID and wild-type tumor microvasculature. Vascular density was 5% in irradiated SCID host compared with 50% in C57BL6 mice (P < 0.05). IC87361 induced radiosensitization of tumor microvasculature in wild-type mice that resembled the radiosensitive phenotype of tumor vessels in SCID mice. Radiosensitization by IC87361 was eliminated in SCID tumor vasculature, which lack functional DNA-PK. Irradiated LLC and B16F0 tumors implanted into SCID mice showed greater tumor growth delay compared with tumors implanted into either wild-type C57BL6 or nude mice. Furthermore, LLC tumors treated with radiation and IC87361 showed tumor growth delay that was significantly greater than tumors treated with radiation alone (P < 0.01 for 3 Gy alone versus 3 Gy + IC87361). DNA-PK inhibitors induced no cytotoxicity and no toxicity in mouse normal tissues. Mouse models deficient in enzyme activity are useful to assess the specificity of novel kinase inhibitors. DNA-PK is an important target for the development of novel radiation-sensitizing drugs that have little intrinsic cytotoxicity.

Noxious heat threshold temperature and pronociceptive effects of allyl isothiocyanate (mustard oil) in TRPV1 or TRPA1 gene-deleted mice.

PubMed

Tékus, Valéria; Horváth, Ádám; Hajna, Zsófia; Borbély, Éva; Bölcskei, Kata; Boros, Melinda; Pintér, Erika; Helyes, Zsuzsanna; Pethő, Gábor; Szolcsányi, János

2016-06-01

To investigate the roles of TRPV1 and TRPA1 channels in baseline and allyl isothiocyanate (AITC)-evoked nociceptive responses by comparing wild-type and gene-deficient mice. In contrast to conventional methods of thermonociception measuring reflex latencies, we used our novel methods to determine the noxious heat threshold. It was revealed that the heat threshold of the tail measured by an increasing-temperature water bath is significantly higher in TRPV1(-/-), but not TRPA1(-/-), mice compared to respective wild-types. There was no difference between the noxious heat thresholds of the hind paw as measured by an increasing-temperature hot plate in TRPV1(-/-), TRPA1(-/-) and the corresponding wild-type mice. The withdrawal latency of the tail from 0°C water was prolonged in TRPA1(-/-), but not TRPV1(-/-), mice compared to respective wild-types. In wild-type animals, dipping the tail or paw into 1% AITC induced an 8-14°C drop of the noxious heat threshold (heat allodynia) of both the tail and paw, and 40-50% drop of the mechanonociceptive threshold (mechanical allodynia) of the paw measured by dynamic plantar esthesiometry. These AITC-evoked responses were diminished in TRPV1(-/-), but not TRPA1(-/-), mice. Tail withdrawal latency to 1% AITC was significantly prolonged in both gene-deleted strains. Different heat sensors determine the noxious heat threshold in distinct areas: a pivotal role for TRPV1 on the tail is contrasted with no involvement of either TRPV1 or TRPA1 on the hind paw. Noxious heat threshold measurement appears appropriate for preclinical screening of TRP channel ligands as novel analgesics. Copyright © 2016 Elsevier Inc. All rights reserved.

Endogenous PTH deficiency impairs fracture healing and impedes the fracture-healing efficacy of exogenous PTH(1-34).

PubMed

Ren, Yongxin; Liu, Bo; Feng, Yuxu; Shu, Lei; Cao, Xiaojian; Karaplis, Andrew; Goltzman, David; Miao, Dengshun

2011-01-01

Although the capacity of exogenous PTH1-34 to enhance the rate of bone repair is well established in animal models, our understanding of the mechanism(s) whereby PTH induces an anabolic response during skeletal repair remains limited. Furthermore it is unknown whether endogenous PTH is required for fracture healing and how the absence of endogenous PTH would influence the fracture-healing capacity of exogenous PTH. Closed mid-diaphyseal femur fractures were created and stabilized with an intramedullary pin in 8-week-old wild-type and Pth null (Pth(-/-)) mice. Mice received daily injections of vehicle or of PTH1-34 (80 µg/kg) for 1-4 weeks post-fracture, and callus tissue properties were analyzed at 1, 2 and 4 weeks post-fracture. Cartilaginous callus areas were reduced at 1 week post-fracture, but were increased at 2 weeks post-fracture in vehicle-treated and PTH-treated Pth(-/-) mice compared to vehicle-treated and PTH-treated wild-type mice respectively. The mineralized callus areas, bony callus areas, osteoblast number and activity, osteoclast number and surface in callus tissues were all reduced in vehicle-treated and PTH-treated Pth(-/-) mice compared to vehicle-treated and PTH-treated wild-type mice, but were increased in PTH-treated wild-type and Pth(-/-) mice compared to vehicle-treated wild-type and Pth(-/-) mice. Absence of endogenous PTH1-84 impedes bone fracture healing. Exogenous PTH1-34 can act in the absence of endogenous PTH but callus formation, including accelerated endochondral bone formation and callus remodeling as well as mechanical strength of the bone are greater when endogenous PTH is present. Results of this study suggest a complementary role for endogenous PTH1-84 and exogenous PTH1-34 in accelerating fracture healing.

Impaired ventilatory acclimatization to hypoxia in mice lacking the immediate early gene fos B.

PubMed

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.

Photo- and heterotrophic nitrogenase activity by the cyano-bacterium Nostoc in symbiosis with the bryophyte Anthoceros

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinberg, N.A.; Meeks, J.C.

1987-04-01

In symbiosis with Anthoceros, Nostoc is thought to do little or no photosynthesis. However, light-dependent /sup 14/CO/sub 2/ fixation by symbiotic Nostoc, freshly isolated from pure cultures of the reconstituted Anthoceros-Nostoc association, was 16% of that by free-living Nostoc. A DCMU-resistant mutant of Nostoc was isolated that fixed CO/sub 2/ at rates comparable to wild-type in both symbiotic and free-living growth states. To determine if symbiotic Nostoc can use its photosynthate directly to fix nitrogen, acetylene reduction by Anthoceros associations reconstituted with wild-type Nostoc was compared to associations with the DCMU-resistant mutant. In wild-type Anthoceros-Nostoc acetylene reduction was inhibited 97%more » by 5 ..mu..M DCMU, while inhibition of the DCMU-resistant Nostoc association was only 63%. Additions of glucose, fructose, maltose or sucrose to wild-type associations completely restored DCMU-inhibited acetylene reduction in the light. Acetylene reduction in the dark was stimulated by glucose, attaining 84% of the uninhibited light-dependent value. The authors conclude that symbiotic Nostoc maintains a pool of photosynthate which supports nitrogenase activity. The pool can also be supplemented from plant sources.« less

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

PubMed

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)

Direct comparison of progenitor cells derived from adipose, muscle, and bone marrow from wild-type or craniosynostotic rabbits

PubMed Central

GM, Cooper; EL, Lensie; JJ, Cray; MR, Bykowski; GE, DeCesare; MA, Smalley; MP, Mooney; PG, Campbell; JE, Losee

2010-01-01

Background Reports have identified cells capable of osteogenic differentiation in bone marrow, muscle, and adipose tissues, but there are few direct comparisons of these different cell-types. Also, few have investigated the potential connection between a tissue-specific pathology and cells derived from seemingly unrelated tissues. Here, we compare cells isolated from wild-type rabbits or rabbits with nonsyndromic craniosynostosis, defined as the premature fusion of one or more of the cranial sutures. Methods Cells were derived from bone marrow, adipose, and muscle of 10 day-old wild-type rabbits (WT; n=17) or from age-matched rabbits with familial nonsyndromic craniosynostosis (CS; n=18). Cells were stimulated with bone morphogenetic protein 4 (BMP4) and alkaline phosphatase expression and cell proliferation were assessed. Results In WT rabbits, cells derived from muscle had more alkaline phosphatase activity than cells derived from either adipose or bone marrow. The cells derived from CS rabbit bone marrow and muscle were significantly more osteogenic than WT. Adipose-derived cells demonstrated no significant differences. While muscle-derived cells were most osteogenic in WT rabbits, bone marrow-derived cells were most osteogenic in CS rabbits. Conclusions Results suggest that cells from different tissues have different potentials for differentiation. Furthermore, cells derived from rabbits with craniosynostosis were different from wild-type derived cells. Interestingly, cells derived from the craniosynostotic rabbits were not uniformly more responsive compared with wild-type cells, suggesting that specific tissue-derived cells may react differently in individuals with craniosynostosis. PMID:20871482

Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations.

PubMed

Song, Wei; Guo, Jun-Tao

2015-01-01

Transcription factors regulate gene expression through binding to specific DNA sequences. How transcription factors achieve high binding specificity is still not well understood. In this paper, we investigated the role of protein flexibility in protein-DNA-binding specificity by comparative molecular dynamics (MD) simulations. Protein flexibility has been considered as a key factor in molecular recognition, which is intrinsically a dynamic process involving fine structural fitting between binding components. In this study, we performed comparative MD simulations on wild-type and F10V mutant P22 Arc repressor in both free and complex conformations. The F10V mutant has lower DNA-binding specificity though both the bound and unbound main-chain structures between the wild-type and F10V mutant Arc are highly similar. We found that the DNA-binding motif of wild-type Arc is structurally more flexible than the F10V mutant in the unbound state, especially for the six DNA base-contacting residues in each dimer. We demonstrated that the flexible side chains of wild-type Arc lead to a higher DNA-binding specificity through forming more hydrogen bonds with DNA bases upon binding. Our simulations also showed a possible conformational selection mechanism for Arc-DNA binding. These results indicate the important roles of protein flexibility and dynamic properties in protein-DNA-binding specificity.

Mutants of Arabidopsis thaliana with decreased amplitude in their phototropic response

NASA Technical Reports Server (NTRS)

Khurana, J. P.; Ren, Z.; Steinitz, B.; Parks, B.; Best, T. R.; Poff, K. L.

1989-01-01

Two mutants of Arabidopsis thaliana have been identified with decreased phototropism to 450-nanometer light. Fluence-response relationships for these strains (ZR8 and ZR19) to single and multiple flashes of light show thresholds, curve shapes, and fluence for maximum curvature in first positive' phototropism which are the same as those of the wild type. Similarly, there is no alteration from the wild type in the kinetics of curvature or in the optimum dark period separating sequential flashes in a multiple flash regimen. In addition, in both strains, gravitropism is decreased compared to the wild type by an amount which is comparable to the decrease in phototropism. Based on reciprocal backcrosses, it appears that the alteration is due to a recessive nuclear mutation. It is suggested that ZR8 and ZR19 represent alterations in some step analogous to an amplifier, downstream of the photoreceptor pigment, and common to both phototropism and gravitropism.

Fermentation of Acid-pretreated Corn Stover to Ethanol Without Detoxification Using Pichia stipitis

NASA Astrophysics Data System (ADS)

Agbogbo, Frank K.; Haagensen, Frank D.; Milam, David; Wenger, Kevin S.

In this work, the effect of adaptation on P. stipitis fermentation using acidpretreated corn stover hydrolyzates without detoxification was examined. Two different types of adaptation were employed, liquid hydrolyzate and solid state agar adaptation. Fermentation of 12.5% total solids undetoxified acid-pretreated corn stover was performed in shake flasks at different rotation speeds. At low rotation speed (100 rpm), both liquid hydrolyzate and solid agar adaptation highly improved the sugar consumption rate as well as ethanol production rate compared to the wild-type strains. The fermentation rate was higher for solid agar-adapted strains compared to liquid hydrolyzate-adapted strains. At a higher rotation speed (150 rpm), there was a faster sugar consumption and ethanol production for both the liquid-adapted and the wild-type strains. However, improvements in the fermentation rate between the liquid-adapted and wild strains were less pronounced at the high rotation speed.

Balance of Go1α and Go2α expression regulates motor function via the striatal dopaminergic system.

PubMed

Baron, J; Bilbao, A; Hörtnagl, H; Birnbaumer, L; Leixner, S; Spanagel, R; Ahnert-Hilger, G; Brunk, I

2018-05-10

The heterotrimeric G-protein Go with its splice variants, Go1α and Go2α, seems to be involved in the regulation of motor function but isoform specific effects are still unclear. We found that Go1α-/- knockouts performed worse on the rota-rod than Go2α-/- and wild type (WT) mice. In Go1+2α-/- mice motor function was partially recovered. Furthermore, Go1+2α-/- mice showed an increased spontaneous motor activity. Compared to wild types or Go2α-/- mice, Go1+2α-/- mice developed increased behavioural sensitization following repetitive cocaine treatment, but failed to develop conditioned place preference. Analysis of dopamine concentration and expression of D1 and D2 receptors unravelled splice-variant specific imbalances in the striatal dopaminergic system: In Go1α-/- mice dopamine concentration and vesicular monoamine uptake were increased compared to wild types. The expression of the D2 receptor was higher in Go1α-/- compared to wild type littermates, but unchanged in Go2α-/- mice. Deletion of both Go1α and Go2α re-established both dopamine and D2 receptor levels comparable to those in the wild type. Cocaine treatment had no effect on the ratio of D1 receptor to D2 receptor in Go1+2α-/- mutants, but decreased this ratio in Go2α-/- mice. Finally, we observed that the deletion of Go1α led to a threefold higher striatal expression of Go2α. Taken together our data suggest that a balance in the expression of Go1α and Go2α sustains normal motor function. Deletion of either splice variant results in divergent behavioural and molecular alterations in the striatal dopaminergic system. Deletion of both splice variants partially restores the behavioural and molecular changes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Microarray and differential display identify genes involved in jasmonate-dependent anther development.

PubMed

Mandaokar, Ajin; Kumar, V Dinesh; Amway, Matt; Browse, John

2003-07-01

Jasmonate (JA) is a signaling compound essential for anther development and pollen fertility in Arabidopsis. Mutations that block the pathway of JA synthesis result into male sterility. To understand the processes of anther and pollen maturation, we used microarray and differential display approaches to compare gene expression pattern in anthers of wild-type Arabidopsis and the male-sterile mutant, opr3. Microarray experiment revealed 25 genes that were up-regulated more than 1.8-fold in wild-type anthers as compared to mutant anthers. Experiments based on differential display identified 13 additional genes up-regulated in wild-type anthers compared to opr3 for a total of 38 differentially expressed genes. Searches of the Arabidopsis and non-redundant databases disclosed known or likely functions for 28 of the 38 genes identified, while 10 genes encode proteins of unknown function. Northern blot analysis of eight representative clones as probes confirmed low expression in opr3 anthers compared with wild-type anthers. JA responsiveness of these same genes was also investigated by northern blot analysis of anther RNA isolated from wild-type and opr3 plants, In these experiments, four genes were induced in opr3 anthers within 0.5-1 h of JA treatment while the remaining genes were up-regulated only 1-8 h after JA application. None of these genes was induced by JA in anthers of the coil mutant that is deficient in JA responsiveness. The four early-induced genes in opr3 encode lipoxygenase, a putative bHLH transcription factor, epithiospecifier protein and an unknown protein. We propose that these and other early components may be involved in JA signaling and in the initiation of developmental processes. The four late genes encode an extensin-like protein, a peptide transporter and two unknown proteins, which may represent components required later in anther and pollen maturation. Transcript profiling has provided a successful approach to identify genes involved in anther and pollen maturation in Arabidopsis.

Polymerase ε (POLE) ultra-mutation in uterine tumors correlates with T lymphocyte infiltration and increased resistance to platinum-based chemotherapy in vitro

PubMed Central

Bellone, Stefania; Eliana, Bignotti; Lonardi, Silvia; Ferrari, Francesca; Centritto, Floriana; Masserdotti, Alice; Pettinella, Francesca; Black, Jonathan; Menderes, Gulden; Altwerger, Gary; Hui, Pei; Lopez, Salvatore; de Haydu, Christopher; Bonazzoli, Elena; Predolini, Federica; Zammataro, Luca; Cocco, Emiliano; Ferrari, Federico; Ravaggi, Antonella; Romani, Chiara; Facchettie, Fabio; Sartori, Enrico; Odicino, Franco E.; Silasi, Dan-Arin; Litkouhi, Babak; Ratner, Elena; Azodi, Masoud; Schwartz, Peter E.; Santin, Alessandro D.

2016-01-01

Objective Up to 12 % of all endometrial-carcinomas (EC) harbor DNA-polymerase-ε-(POLE) mutations. It is currently unknown whether the favorable prognosis of POLE-mutated EC is derived from their low metastatic capability, extraordinary number of somatic mutations thus imparting immunogenicity, or a high sensitivity to chemotherapy. Methods Polymerase-chain-reaction-amplification and Sanger-sequencing were used to test for POLE exonuclease-domain-mutations (exons 9–14) 131 EC. Infiltration of CD4+ and CD8+ T-lymphocytes (TIL) and PD-1-expression in POLE-mutated vs POLE wild-type EC was studied by immunohistochemistry (IHC) and the correlations between survival and molecular features were investigated. Finally, primary POLE-mutated and POLE-wild-type EC cell lines were established and compared in-vitro for their sensitivity to chemotherapy. Results Eleven POLE-mutated EC (8.5%) were identified. POLE-mutated tumors were associated with improved progression-free-survival (P<0.05) and displayed increased numbers of CD4+ (44.5 vs 21.8; P = .001) and CD8+ (32.8 vs 13.5; P < .001) TILs when compared to wild-type POLE EC. PD-1 receptor was overexpressed in TILs from POLE-mutated vs wild-type-tumors (81% vs 28%; P < .001). Primary POLE tumor cell lines were significantly more resistant to platinum-chemotherapy in-vitro when compared to POLE-wild-type tumors (P < 0.004). Conclusions POLE ultra-mutated EC are heavily infiltrated with CD4+/CD8+ TIL, overexpress PD-1 immune-check-point (i.e., features consistent with chronic antigen-exposure), and have a better prognosis when compared to other molecular subtypes of EC patients. POLE-mutated tumor-cell lines are resistant to platinum-chemotherapy in-vitro suggesting that the better prognosis of POLE-patients is not secondary to a higher sensitivity to chemotherapy but likely linked to enhanced immunogenicity. PMID:27894751

The receptor for advanced glycation end products impairs collateral formation in both diabetic and non-diabetic mice.

PubMed

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.

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

DTIC Science & Technology

2011-07-01

type and mutant plants via chromatin immunoprecipitation (ChIP). Additionally, differences in centromere structure between wild-type and VIM1 RING...contexts. The proposed work is ongoing, and so far the major accomplishments include creation of relevant plant lines and development of in vitro assays...a comparative proteomics approach in wild-type plants and RING domain mutants (Months 1 - 18) This work is in early stages, with the main

Comparison of fermentative capacities of industrial baking and wild-type yeasts of the species Saccharomyces cerevisiae in different sugar media.

PubMed

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.

Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST)

PubMed Central

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

Analysis by rotavirus gene 6 reverse transcriptase-polymerase chain reaction assay of rotavirus-positive gastroenteritis cases observed during the vaccination phase of the Rotavirus Efficacy and Safety Trial (REST).

PubMed

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.

S-Nitrosation and regulation of inducible nitric oxide synthase.

PubMed

Mitchell, Douglas A; Erwin, Phillip A; Michel, Thomas; Marletta, Michael A

2005-03-29

The inducible isoform of nitric oxide synthase (iNOS) and three zinc tetrathiolate mutants (C104A, C109A, and C104A/C109A) were expressed in Escherichia coli and purified. The mutants were found by ICP-AES and the zinc-specific PAR colorimetric assay to be zinc free, whereas the wild-type iNOS zinc content was 0.38 +/- 0.01 mol of Zn/mol of iNOS dimer. The cysteine mutants (C104A and C109A) had an activity within error of wild-type iNOS (2.24 +/- 0.12 micromol of NO min(-1) mg(-1)), but the double cysteine mutant had a modestly decreased activity (1.75 +/- 0.14 micromol of NO min(-1) mg(-1)). To determine if NO could stimulate release of zinc and dimer dissociation, wild-type protein was allowed to react with an NO donor, DEA/NO, followed by buffer exchange. ICP-AES of samples treated with 10 microM DEA/NO showed a decrease in zinc content (0.23 +/- 0.01 to 0.09 +/- 0.01 mol of Zn/mol of iNOS dimer) with no loss of heme iron. Gel filtration of wild-type iNOS treated similarly resulted in approximately 20% more monomeric iNOS compared to a DEA-treated sample. Only wild-type iNOS had decreased activity (42 +/- 2%) after reaction with 50 microM DEA/NO compared to a control sample. Using the biotin switch method under the same conditions, only wild-type iNOS had increased levels of S-biotinylation. S-Biotinylation was mapped to C104 and C109 on wild-type iNOS using LysC digestion and MALDI-TOF/TOF MS. Immunoprecipitation of iNOS from the mouse macrophage cell line, RAW-264.7, and the biotin switch method were used to confirm endogenous S-nitrosation of iNOS. The data show that S-nitrosation of the zinc tetrathiolate cysteine results in zinc release from the dimer interface and formation of inactive monomers, suggesting that this mode of inhibition might occur in vivo.

Peptidomics of Cpefat/fat mouse brain regions: Implications for neuropeptide processing

PubMed Central

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

Long-chain fatty acid uptake by skeletal muscle is impaired in homozygous, but not heterozygous, heart-type-FABP null mice.

PubMed

Luiken, J J F P; Koonen, D P Y; Coumans, W A; Pelsers, M M A L; Binas, B; Bonen, A; Glatz, J F C

2003-04-01

Previous studies with cardiac myocytes from homozygous heart-type fatty acid (FA)-binding protein (H-FABP) -/- mice have indicated that this intracellular receptor protein for long-chain FA is involved in the cellular uptake of these substrates. Based on the knowledge that muscle FA uptake is a process highly sensitive to regulation by hormonal and mechanical stimuli, we studied whether H-FABP would play a role in this regulation. A suitable model system to answer this question is provided by H-FABP +/- mice, because in hindlimb muscles the content of H-FABP was measured to be 34% compared to wild-type mice. In these H-FABP +/- skeletal muscles, just as in H-FABP -/- muscles, contents of FA transporters, i.e., 43-kDa FABPpm and 88-kDa FAT/CD36, were similar compared to wild-type muscles, excluding possible compensatory mechanisms at the sarcolemmal level. Palmitate uptake rates were measured in giant vesicles prepared from hindlimb muscles of H-FABP -/-, H-FABP +/-, and H-FABP +/+ mice. For comparison, giant vesicles were isolated from liver, the tissue of which expresses a distinct type of FABP (i.e., L-FABP). Whereas in H-FABP -/- skeletal muscle FA uptake was reduced by 42-45%, FA uptake by H-FABP +/- skeletal muscle was not different from that in wild-type mice. In contrast, in liver from H-FABP -/- and from H-FABP +/- mice, FA uptake was not altered compared to wild-type animals, indicating that changes in FA uptake are restricted to H-FABP expressing tissues. It is concluded that H-FABP plays an important, yet merely permissive, role in FA uptake into muscle tissues.

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol.

PubMed

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.

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil.

PubMed

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.

Ecological Value of Native Plant Cultivars Versus Wild-Type Native Plants for Promoting Hemipteran Diversity in Suburban Areas.

PubMed

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.

A Universal Role for Inositol 1,4,5-Trisphosphate-Mediated Signaling in Plant Gravitropism1[W

PubMed Central

Perera, Imara Y.; Hung, Chiu-Yueh; Brady, Shari; Muday, Gloria K.; Boss, Wendy F.

2006-01-01

Inositol 1,4,5-trisphosphate (InsP3) has been implicated in the early signaling events of plants linking gravity sensing to the initiation of the gravitropic response. However, at present, the contribution of the phosphoinositide signaling pathway in plant gravitropism is not well understood. To delineate the role of InsP3 in plant gravitropism, we generated Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes InsP3. The transgenic plants show no significant differences in growth and life cycle compared to wild-type plants, although basal InsP3 levels are reduced by greater than 90% compared to wild-type plants. With gravistimulation, InsP3 levels in inflorescence stems of transgenic plants show no detectable change, whereas in wild-type plant inflorescences, InsP3 levels increase approximately 3-fold within the first 5 to 15 min of gravistimulation, preceding visible bending. Furthermore, gravitropic bending of the roots, hypocotyls, and inflorescence stems of the InsP 5-ptase transgenic plants is reduced by approximately 30% compared with the wild type. Additionally, the cold memory response of the transgenic plants is attenuated, indicating that InsP3 contributes to gravisignaling in the cold. The transgenic roots were shown to have altered calcium sensitivity in controlling gravitropic response, a reduction in basipetal indole-3-acetic acid transport, and a delay in the asymmetric auxin-induced β-glucuronidase expression with gravistimulation as compared to the controls. The compromised gravitropic response in all the major axes of growth in the transgenic Arabidopsis plants reveals a universal role for InsP3 in the gravity signal transduction cascade of plants. PMID:16384898

High-Fat-Diet Intake Enhances Cerebral Amyloid Angiopathy and Cognitive Impairment in a Mouse Model of Alzheimer's Disease, Independently of Metabolic Disorders.

PubMed

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.

Economic Analysis of Panitumumab Compared With Cetuximab in Patients With Wild-type KRAS Metastatic Colorectal Cancer That Progressed After Standard Chemotherapy.

PubMed

Graham, Christopher N; Maglinte, Gregory A; Schwartzberg, Lee S; Price, Timothy J; Knox, Hediyyih N; Hechmati, Guy; Hjelmgren, Jonas; Barber, Beth; Fakih, Marwan G

2016-06-01

In this analysis, we compared costs and explored the cost-effectiveness of subsequent-line treatment with cetuximab or panitumumab in patients with wild-type KRAS (exon 2) metastatic colorectal cancer (mCRC) after previous chemotherapy treatment failure. Data were used from ASPECCT (A Study of Panitumumab Efficacy and Safety Compared to Cetuximab in Patients With KRAS Wild-Type Metastatic Colorectal Cancer), a Phase III, head-to-head randomized noninferiority study comparing the efficacy and safety of panitumumab and cetuximab in this population. A decision-analytic model was developed to perform a cost-minimization analysis and a semi-Markov model was created to evaluate the cost-effectiveness of panitumumab monotherapy versus cetuximab monotherapy in chemotherapy-resistant wild-type KRAS (exon 2) mCRC. The cost-minimization model assumed equivalent efficacy (progression-free survival) based on data from ASPECCT. The cost-effectiveness analysis was conducted with the full information (uncertainty) from ASPECCT. Both analyses were conducted from a US third-party payer perspective and calculated average anti-epidermal growth factor receptor doses from ASPECCT. Costs associated with drug acquisition, treatment administration (every 2 weeks for panitumumab, weekly for cetuximab), and incidence of infusion reactions were estimated in both models. The cost-effectiveness model also included physician visits, disease progression monitoring, best supportive care, and end-of-life costs and utility weights estimated from EuroQol 5-Dimension questionnaire responses from ASPECCT. The cost-minimization model results demonstrated lower projected costs for patients who received panitumumab versus cetuximab, with a projected cost savings of $9468 (16.5%) per panitumumab-treated patient. In the cost-effectiveness model, the incremental cost per quality-adjusted life-year gained revealed panitumumab to be less costly, with marginally better outcomes than cetuximab. These economic analyses comparing panitumumab and cetuximab in chemorefractory wild-type KRAS (exon 2) mCRC suggest benefits in favor of panitumumab. ClinicalTrials.gov identifier: NCT01001377. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

PubMed Central

Rockenstein, Edward; Nuber, Silke; Overk, Cassia R.; Ubhi, Kiren; Mante, Michael; Patrick, Christina; Adame, Anthony; Trejo-Morales, Margarita; Gerez, Juan; Picotti, Paola; Jensen, Poul H.; Campioni, Silvia; Riek, Roland; Winkler, Jürgen; Gage, Fred H.; Winner, Beate

2014-01-01

In Parkinson’s disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinson’s disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction. PMID:24662516

Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo.

PubMed

Rockenstein, Edward; Nuber, Silke; Overk, Cassia R; Ubhi, Kiren; Mante, Michael; Patrick, Christina; Adame, Anthony; Trejo-Morales, Margarita; Gerez, Juan; Picotti, Paola; Jensen, Poul H; Campioni, Silvia; Riek, Roland; Winkler, Jürgen; Gage, Fred H; Winner, Beate; Masliah, Eliezer

2014-05-01

In Parkinson's disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinson's disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction.

Perception of sweet taste is important for voluntary alcohol consumption in mice.

PubMed

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.

Dose-response assessment for influenza A virus based on data sets of infection with its live attenuated reassortants.

PubMed

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.

Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

PubMed

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.

Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections▿

PubMed Central

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

PERCEPTION OF SWEET TASTE IS IMPORTANT FOR VOLUNTARY ALCOHOL CONSUMPTION IN MICE

PubMed Central

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

Sebacina vermifera Promotes the Growth and Fitness of Nicotiana attenuata by Inhibiting Ethylene Signaling1[W

PubMed Central

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

Characterization of an ntrX mutant of Neisseria gonorrhoeae reveals a response regulator that controls expression of respiratory enzymes in oxidase-positive proteobacteria.

PubMed

Atack, John M; Srikhanta, Yogitha N; Djoko, Karrera Y; Welch, Jessica P; Hasri, Norain H M; Steichen, Christopher T; Vanden Hoven, Rachel N; Grimmond, Sean M; Othman, Dk Seti Maimonah Pg; Kappler, Ulrike; Apicella, Michael A; Jennings, Michael P; Edwards, Jennifer L; McEwan, Alastair G

2013-06-01

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

Deletion of the Dynein Heavy-Chain Gene DYN1 Leads to Aberrant Nuclear Positioning and Defective Hyphal Development in Candida albicans

PubMed Central

Martin, R.; Walther, A.; Wendland, J.

2004-01-01

Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1. PMID:15590831

Clinical efficacy of icotinib in lung cancer patients with different EGFR mutation status: a meta-analysis

PubMed Central

Xu, Ping; Xiang, Da-Xiong; Yang, Rui; Wei, Wei; Qu, Qiang

2017-01-01

Icotinib is a novel and the third listed epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), which exerts a good anti-tumor efficacy on non-small cell lung cancer (NSCLC). The efficacy of EGFR-TKIs has been shown to be associated with the EGFR mutation status, especially exon 19 deletion (19Del) and exon 21 L858R mutation. Therefore, a meta-analysis was performed to assess the efficacy of icotinib in NSCLC patients harboring EGFR mutations (19Del or L858R) and wild type (19Del and L858R loci wild type). A total of 24 studies were included for comparing the objective response rate (ORR) in the EGFR wild type and mutant patients treated with icotinib. The ORRs of EGFR mutant patients (19Del or L858R) are better than those of EGFR wild type patients (OR = 7.03(5.09–9.71), P < 0.00001). The pooling ORs from 21 studies on the disease control rate (DCR) in EGFR mutant patients are better than those of EGFR wild type patients (OR = 10.54(5.72–19.43), P < 0.00001). Moreover, the ORRs of EGFR 19Del patients are better than those of EGFR L858R patients after pooling ORs of 12 studies (OR = 2.04(1.12–3.73), P = 0.019). However, there was no significant difference on DCRs of EGFR 19Del patients and those of EGFR L858R patients (OR = 2.01(0.94–4.32), P = 0.072). Our findings indicated that compared with EGFR wild type patients, EGFR mutant patients have better ORRs and DCRs after icotinib treatment; EGFR 19Del patients treated with icotinib have better ORRs than EGFR L858R patients. EGFR mutation status is a useful biomarker for the evaluation of icotinib efficacy in NSCLC patients. PMID:28430623

Clinical efficacy of icotinib in lung cancer patients with different EGFR mutation status: a meta-analysis.

PubMed

Qu, Jian; Wang, Ya-Nan; Xu, Ping; Xiang, Da-Xiong; Yang, Rui; Wei, Wei; Qu, Qiang

2017-05-16

Icotinib is a novel and the third listed epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), which exerts a good anti-tumor efficacy on non-small cell lung cancer (NSCLC). The efficacy of EGFR-TKIs has been shown to be associated with the EGFR mutation status, especially exon 19 deletion (19Del) and exon 21 L858R mutation. Therefore, a meta-analysis was performed to assess the efficacy of icotinib in NSCLC patients harboring EGFR mutations (19Del or L858R) and wild type (19Del and L858R loci wild type). A total of 24 studies were included for comparing the objective response rate (ORR) in the EGFR wild type and mutant patients treated with icotinib. The ORRs of EGFR mutant patients (19Del or L858R) are better than those of EGFR wild type patients (OR = 7.03(5.09-9.71), P < 0.00001). The pooling ORs from 21 studies on the disease control rate (DCR) in EGFR mutant patients are better than those of EGFR wild type patients (OR = 10.54(5.72-19.43), P < 0.00001). Moreover, the ORRs of EGFR 19Del patients are better than those of EGFR L858R patients after pooling ORs of 12 studies (OR = 2.04(1.12-3.73), P = 0.019). However, there was no significant difference on DCRs of EGFR 19Del patients and those of EGFR L858R patients (OR = 2.01(0.94-4.32), P = 0.072). Our findings indicated that compared with EGFR wild type patients, EGFR mutant patients have better ORRs and DCRs after icotinib treatment; EGFR 19Del patients treated with icotinib have better ORRs than EGFR L858R patients. EGFR mutation status is a useful biomarker for the evaluation of icotinib efficacy in NSCLC patients.

A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana.

PubMed Central

Howden, R; Andersen, C R; Goldsbrough, P B; Cobbett, C S

1995-01-01

The roots of the cadmium-sensitive mutant of Arabidopsis thaliana, cad1-1, become brown in the presence of cadmium. A new cadmium-sensitive mutant affected at a second locus, cad2, has been identified using this phenotype. Genetic analysis has grown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Assays of cadmium accumulation by intact plants indicated that the mutant is deficient in its ability to sequester cadmium. Undifferentiated callus tissue was also cadmium sensitive, suggesting that the mutant phenotype is expressed at the cellular level. The level of cadmium-binding complexes formed in vivo was decreased compared with the wild type and accumulation of phytochelatins was about 10% of that in the wild type. The level of glutathione, the substrate for phytochelatin biosynthesis, in tissues of the mutant was decreased to about 15 to 30% of that in the wild type. Thus, the deficiency in phytochelatin biosynthesis can be explained by a deficiency in glutathione. PMID:7770518

Root graviresponsiveness and cellular differentiation in wild-type and a starchless mutant of Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Moore, R.

1989-01-01

Primary roots of a starchless mutant of Arabidopsis thaliana L. are strongly graviresponsive despite lacking amyloplasts in their columella cells. The ultrastructures of calyptrogen and peripheral cells in wild-type as compared to mutant seedlings are not significantly different. The largest difference in cellular differentiation in caps of mutant and wild-type roots is the relative volume of plastids in columella cells. Plastids occupy 12.3% of the volume of columella cells in wild-type seedlings, but only 3.69% of columella cells in mutant seedlings. These results indicate that: (1) amyloplasts and starch are not necessary for root graviresponsiveness; (2) the increase in relative volume of plastids that usually accompanies differentiation of columella cells is not necessary for root graviresponsiveness; and (3) the absence of starch and amyloplasts does not affect the structure of calyptrogen (i.e. meristematic) and secretory (i.e. peripheral) cells in root caps. These results are discussed relative to proposed models for root gravitropism.

Functional verification of a porcine myostatin propeptide mutant.

PubMed

Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

2015-10-01

Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells.

Human thrombomodulin knock-in mice reveal differential effects of human thrombomodulin on thrombosis and atherosclerosis.

PubMed

Raife, Thomas J; Dwyre, Denis M; Stevens, Jeff W; Erger, Rochelle A; Leo, Lorie; Wilson, Katina M; Fernández, Jose A; Wilder, Jennifer; Kim, Hyung-Suk; Griffin, John H; Maeda, Nobuyo; Lentz, Steven R

2011-11-01

We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo. Knock-in mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knock-in mice exhibited decreased protein C activation in the aorta (P<0.01) and lung (P<0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knock-in mice compared with wild-type mice (P<0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knock-in mice (12±3 minutes) than in wild-type mice (31±6 minutes; P<0.05). No differences in serum cytokine levels were detected between knock-in and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knock-in mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis. Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This humanized animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.

Knockout mutations of insulin-like peptide genes enhance sexual receptivity in Drosophila virgin females.

PubMed

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.

Differential proteomic and behavioral effects of long-term voluntary exercise in wild-type and APP-overexpressing transgenics.

PubMed

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.

Cdx mutant axial progenitor cells are rescued by grafting to a wild type environment.

PubMed

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.

Biliary excretion of technetium-99m-sestamibi in wild-type dogs and in dogs with intrinsic (ABCB1-1Delta mutation) and extrinsic (ketoconazole treated) P-glycoprotein deficiency.

PubMed

Coelho, J C; Tucker, R; Mattoon, J; Roberts, G; Waiting, D K; Mealey, K L

2009-10-01

P-glycoprotein (P-gp), the product of ABCB1 gene, is thought to play a role in the biliary excretion of a variety of drugs, but specific studies in dogs have not been performed. Because a number of endogenous (ABCB1 polymorphisms) and exogenous (pharmacological P-gp inhibition) factors can interfere with normal P-gp function, a better understanding of P-gp's role in biliary drug excretion is crucial in preventing adverse drug reactions and drug-drug interactions in dogs. The objectives of this study were to compare biliary excretion of technetium-99m-sestamibi ((99m)Tc-MIBI), a radio-labelled P-gp substrate, in wild-type dogs (ABCB1 wild/wild), and dogs with intrinsic and extrinsic deficiencies in P-gp function. Dogs with intrinsic P-gp deficiency included ABCB1 mut/mut dogs, and dogs with presumed intermediate P-gp phenotype (ABCB1 mut/wild). Dogs with extrinsic P-gp deficiency were considered to be ABCB1 wild/wild dogs treated with the P-gp inhibitor ketoconazole (5 mg/kg PO q12h x 9 doses). Results from this study indicate that ABCB1 mut/mut dogs have significantly decreased biliary excretion of (99m)Tc-MIBI compared with ABCB1 wild/wild dogs. Treatment with ketoconazole significantly decreased biliary excretion of (99m)Tc-MIBI in ABCB1 wild/wild dogs. P-gp appears to play an important role in the biliary excretion of (99m)Tc-MIBI in dogs. It is likely that concurrent administration of a P-gp inhibitor such as ketoconazole will decrease P-gp-mediated biliary excretion of other substrate drugs as well.

Impairment of Vision in a Mouse Model of Usher Syndrome Type III.

PubMed

Tian, Guilian; Lee, Richard; Ropelewski, Philip; Imanishi, Yoshikazu

2016-03-01

The purpose of this study was to obtain an Usher syndrome type III mouse model with retinal phenotype. Speed congenic method was used to obtain Clrn1 exon 1 knockout (Clrn1-/-) and Clrn1N48K knockin (Clrn1N48K/N48K) mice under A/J background. To study the retinal functions of these mice, we measured scotopic and photopic ERG responses. To observe if there are any structural abnormalities, we conducted light and transmission electron microscopy of fixed retinal specimens. In 3-month-old Clrn1-/- mice, scotopic b-wave amplitude was reduced by more than 25% at the light intensities from -2.2 to 0.38 log cd·s/m2, but scotopic a-wave amplitudes were comparable to those of age-matched wild type mice at all the light intensities tested. In 9-month-old Clrn1-/- mice, scotopic b-wave amplitudes were further reduced by more than 35%, and scotopic a-wave amplitude also showed a small decline as compared with wild type mice. Photopic ERG responses were comparable between Clrn1-/- and wild type mice. Those electrophysiological defects were not associated with a loss of rods. In Clrn1N48K/N48K mice, both a- and b-wave amplitudes were not discernable from those of wild type mice aged up to 10 months. Mutations that are Clrn1-/- biallelic cause visual defects when placed under A/J background. The absence of apparent rod degeneration suggests that the observed phenotype is due to functional defects, and not due to loss of rods. Biallelic Clrn1N48K/N48K mutations did not cause discernible visual defects, suggesting that Clrn1- allele is more severely dysfunctional than ClrnN48K allele.

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

PubMed Central

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants. PMID:27602024

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

PubMed

Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

2016-01-01

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants.

HLA-B27 and Human β2-Microglobulin Affect the Gut Microbiota of Transgenic Rats

PubMed Central

Lin, Phoebe; Bach, Mary; Asquith, Mark; Lee, Aaron Y.; Akileswaran, Lakshmi; Stauffer, Patrick; Davin, Sean; Pan, Yuzhen; Cambronne, Eric D.; Dorris, Martha; Debelius, Justine W.; Lauber, Christian L.; Ackermann, Gail; Baeza, Yoshiki V.; Gill, Tejpal; Knight, Rob; Colbert, Robert A.; Taurog, Joel D.; Van Gelder, Russell N.; Rosenbaum, James T.

2014-01-01

The HLA-B27 gene is a major risk factor for clinical diseases including ankylosing spondylitis, acute anterior uveitis, reactive arthritis, and psoriatic arthritis, but its mechanism of risk enhancement is not completely understood. The gut microbiome has recently been shown to influence several HLA-linked diseases. However, the role of HLA-B27 in shaping the gut microbiome has not been previously investigated. In this study, we characterize the differences in the gut microbiota mediated by the presence of the HLA-B27 gene. We identified differences in the cecal microbiota of Lewis rats transgenic for HLA-B27 and human β2-microglobulin (hβ2m), compared with wild-type Lewis rats, using biome representational in situ karyotyping (BRISK) and 16S rRNA gene sequencing. 16S sequencing revealed significant differences between transgenic animals and wild type animals by principal coordinates analysis. Further analysis of the data set revealed an increase in Prevotella spp. and a decrease in Rikenellaceae relative abundance in the transgenic animals compared to the wild type animals. By BRISK analysis, species-specific differences included an increase in Bacteroides vulgatus abundance in HLA-B27/hβ2m and hβ2m compared to wild type rats. The finding that HLA-B27 is associated with altered cecal microbiota has not been shown before and can potentially provide a better understanding of the clinical diseases associated with this gene. PMID:25140823

HLA-B27 and human β2-microglobulin affect the gut microbiota of transgenic rats.

PubMed

Lin, Phoebe; Bach, Mary; Asquith, Mark; Lee, Aaron Y; Akileswaran, Lakshmi; Stauffer, Patrick; Davin, Sean; Pan, Yuzhen; Cambronne, Eric D; Dorris, Martha; Debelius, Justine W; Lauber, Christian L; Ackermann, Gail; Baeza, Yoshiki V; Gill, Tejpal; Knight, Rob; Colbert, Robert A; Taurog, Joel D; Van Gelder, Russell N; Rosenbaum, James T

2014-01-01

The HLA-B27 gene is a major risk factor for clinical diseases including ankylosing spondylitis, acute anterior uveitis, reactive arthritis, and psoriatic arthritis, but its mechanism of risk enhancement is not completely understood. The gut microbiome has recently been shown to influence several HLA-linked diseases. However, the role of HLA-B27 in shaping the gut microbiome has not been previously investigated. In this study, we characterize the differences in the gut microbiota mediated by the presence of the HLA-B27 gene. We identified differences in the cecal microbiota of Lewis rats transgenic for HLA-B27 and human β2-microglobulin (hβ2m), compared with wild-type Lewis rats, using biome representational in situ karyotyping (BRISK) and 16S rRNA gene sequencing. 16S sequencing revealed significant differences between transgenic animals and wild type animals by principal coordinates analysis. Further analysis of the data set revealed an increase in Prevotella spp. and a decrease in Rikenellaceae relative abundance in the transgenic animals compared to the wild type animals. By BRISK analysis, species-specific differences included an increase in Bacteroides vulgatus abundance in HLA-B27/hβ2m and hβ2m compared to wild type rats. The finding that HLA-B27 is associated with altered cecal microbiota has not been shown before and can potentially provide a better understanding of the clinical diseases associated with this gene.

Reduced emotional and corticosterone responses to stress in μ-opioid receptor knockout mice

PubMed Central

Ide, Soichiro; Sora, Ichiro; Ikeda, Kazutaka; Minami, Masabumi; Uhl, George R.; Ishihara, Kumatoshi

2014-01-01

The detailed mechanisms of emotional modulation in the nervous system by opioids remain to be elucidated, although the opioid system is well known to play important roles in the mechanisms of analgesia and drug dependence. In the present study, we conducted behavioral tests of anxiety and depression and measured corticosterone concentrations in both male and female μ-opioid receptor knockout (MOP-KO) mice to reveal the involvement of μ-opioid receptors in stress-induced emotional responses. MOP-KO mice entered more and spent more time in the open arms of the elevated plus maze compared with wild-type mice. MOP-KO mice also displayed significantly decreased immobility in a 15 min tail-suspension test compared with wild-type mice. Similarly, MOP-KO mice exhibited significantly decreased immobility on days 2, 3, and 4 in a 6 min forced swim test conducted for 5 consecutive days. The increase in plasma corticosterone concentration induced by tail-suspension, repeated forced swim, or restraint stress was reduced in MOP-KO mice compared with wild-type mice. Corticosterone levels were not different between wild-type and MOP-KO mice before stress exposure. In contrast, although female mice tended to exhibit fewer anxiety-like responses in the tail-suspension test in both genotypes, no significant gender differences were observed in stress-induced emotional responses. These results suggest that MOPs play an important facilitatory role in emotional responses to stress, including anxiety- and depression-like behavior and corticosterone levels. PMID:19596019

Detection of MEF-1 laboratory reference strain of poliovirus type 2 in children with poliomyelitis in India in 2002 & 2003.

PubMed

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.

Resistance to collagen-induced arthritis in SHPS-1 mutant mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okuzawa, Chie; Kaneko, Yoriaki; Murata, Yoji

SHPS-1 is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Here we show that mice expressing a mutant form of SHPS-1 fail to develop type-II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans. Histological examinations of the arthritic paws from immunized wild-type mice revealed that cartilage was destroyed in association with marked mononuclear cell infiltration, while only mild cell infiltration was observed in immunized SHPS-1 mutant mice. Consistently, the serum levels of both IgG and IgG2a specific to CII andmore » of IL-1{beta} in immunized SHPS-1 mutant mice were markedly reduced compared with those apparent for wild-type mice. The CII-induced proliferation of, and production of cytokines by, T cells from immunized SHPS-1 mutant mice were reduced compared to wild-type cells. These results suggest that SHPS-1 is essential for development of CIA.« less

Contrasting colonization and plant growth promoting capacity between wild type and gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar

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

Differences in Fusarium Species in brown midrib Sorghum and in Air Populations in Production Fields.

PubMed

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.

Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization.

PubMed

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.

1,3-Propanediol dehydrogenases in Lactobacillus reuteri: impact on central metabolism and 3-hydroxypropionaldehyde production.

PubMed

Stevens, Marc J A; Vollenweider, Sabine; Meile, Leo; Lacroix, Christophe

2011-08-03

Lactobacillus reuteri metabolizes glycerol to 3-hydroxypropionaldehyde (3-HPA) and further to 1,3-propanediol (1,3-PDO), the latter step catalysed by a propanediol dehydrogenase (PDH). The last step in this pathway regenerates NAD+ and enables therefore the energetically more favourable production of acetate over ethanol during growth on glucose. A search throughout the genome of L. reuteri DSM 20016 revealed two putative PDHs encoded by ORFs lr_0030 and lr_1734. ORF lr_1734 is situated in the pdu operon encoding the glycerol conversion machinery and therefore likely involved in 1,3-PDO formation. ORF lr_0030 has not been associated with PDH-activity so far. To elucidate the role of these two PDHs, gene deletion mutant strains were constructed. Growth behaviour on glucose was comparable between the wild type and both mutant strains. However, on glucose + glycerol, the exponential growth rate of Δlr_0030 was lower compared to the wild type and the lr_1734 mutant. Furthermore, glycerol addition resulted in decreased ethanol production in the wild type and Δlr_1734, but not in Δlr_0030. PDH activity measurements using 3-HPA as a substrate revealed lower activity of Δlr_0030 extracts from exponential growing cells compared to wild type and Δlr_1734 extracts.During biotechnological 3-HPA production using non-growing cells, the ratio 3-HPA to 1,3-PDO was approximately 7 in the wild type and Δlr_0030, whereas this ratio was 12.5 in the mutant Δlr_1734. The enzyme encoded by lr_0030 plays a pivotal role in 3-HPA conversion in exponential growing L. reuteri cells. The enzyme encoded by lr_1734 is active during 3-HPA production by non-growing cells and this enzyme is a useful target to enhance 3-HPA production and minimize formation of the by-product 1,3-PDO.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Cebula, Patricia

2005-11-03

The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Deltatap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.

The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora

PubMed Central

Nowrousian, Minou; Cebula, Patricia

2005-01-01

Background The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Results Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Δtap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. Conclusion The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora. PMID:16266439

Modulation of interaction of mutant TP53 and wild type BRCA1 by alkaloids: a computational approach towards targeting protein-protein interaction as a futuristic therapeutic intervention strategy for breast cancer impediment.

PubMed

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.

Mutants of Arabidopsis thaliana with decreased amplitude in their phototropic response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khurana, J.P.; Ren, Zhangling; Steinitz, B.

1989-10-01

Two mutants of Arabidopsis thaliana have been identified with decreased phototropism to 450-nanometer light. Fluence-response relationships for these strains (ZR8 and ZR19) to single and multiple flashes of light show thresholds, curve shapes, and fluence for maximum curvature in first positive phototropism which are the same as those of the wild type. Similarly, there is no alteration from the wild type in the kinetics of curvature or in the optimum dark period separating sequential flashes in a multiple flash regimen. In addition, in both strains, gravitropism is decreased compared to the wild type by an amount which is comparable tomore » the decrease in phototropism. Based on reciprocal backcrosses, it appears that the alteration is due to a recessive nuclear mutation. It is suggested that ZR8 and ZR19 represent alterations in some step analogous to an amplifier, downstream of the photoreceptor pigment, and common to both phototropism and gravitropism.« less

Transglutaminase-2 differently regulates cartilage destruction and osteophyte formation in a surgical model of osteoarthritis.

PubMed

Orlandi, A; Oliva, F; Taurisano, G; Candi, E; Di Lascio, A; Melino, G; Spagnoli, L G; Tarantino, U

2009-04-01

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodeling. Transglutaminases catalyze a calcium-dependent transamidation reaction that produces covalent cross-linking of available substrate glutamine residues and modifies the extracellular matrix. Increased transglutaminases-mediated activity is reported in osteoarthritis, but the relative contribution of transglutaminases-2 (TG2) is uncertain. We describe TG2 expression in human femoral osteoarthritis and in wild-type and homozygous TG2 knockout mice after surgically-induced knee joint instability. Increased TG2 levels were observed in human and wild-type murine osteoarthritic cartilage compared to the respective controls. Histomorphometrical but not X-ray investigation documented in osteoarthritic TG2 knockout mice reduced cartilage destruction and an increased osteophyte formation compared to wild-type mice. These differences were associated with increased TGFbeta-1 expression. In addition to confirming its important role in osteoarthritis development, our results demonstrated that TG2 expression differently influences cartilage destruction and bone remodeling, suggesting new targeted TG2-related therapeutic strategies.

Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum.

PubMed

Okuwa, T; Katayama, T; Takano, A; Kodaira, K; Yasukawa, H

2001-12-01

Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.

Females with a mutation in a nuclear-encoded mitochondrial protein pay a higher cost of survival than do males in Drosophila.

PubMed

Melvin, Richard G; Ballard, J William O

2011-07-01

Males and females age at different rates in a variety of species, but the mechanisms underlying the difference is not understood. In this study, we investigated sex-specific costs of a naturally occurring mildly deleterious deletion (DTrp85, DVal86) in cytochrome c oxidase subunit 7A (cox7A) in Drosophila simulans. We observed that females and males homozygous for the mutation had 30% and 26% reduced Cox activity, respectively, compared with wild type. Furthermore, 4-day-old females had 34%-42% greater physical activity than males. Greater physical activity in mutant females was correlated with a 19% lower 50% survival compared with wild-type females. Mutant and wild-type males had equal survival. These data suggest that females paid a higher cost of the mutation than did males. The data demonstrate linking population genetics and structural modeling to experimental manipulations that lead to functional predictions of mitochondrial bioenergetics and organism aging.

IgA is Important for Clearance and Critical for Protection from Rotavirus Infection

PubMed Central

Blutt, Sarah E; Miller, Amber D.; Salmon, Sharon L.; Metzger, Dennis W.; Conner, Margaret E

2012-01-01

Based on a lack of severe phenotype in human IgA deficiency syndromes, the role of IgA in controlling respiratory and gastrointestinal (GI) infections has not been clearly defined. C57BL/6 and BALB/c mice lacking IgA (IgA−/−) were developed and used to address this question. When exposed to a common GI virus, rotavirus, IgA−/− mice exhibited a substantial and significant delay in clearance of the initial infection compared to wild type mice. IgA−/− mice excreted rotavirus in stool up to three weeks after the initial exposure compared to ten days observed in wild type mice. Importantly, IgA−/− mice failed to develop protective immunity against multiple repeat exposures to the virus. All IgA−/− mice excreted virus in the stool upon re-exposure to rotavirus while wild type mice were completely protected against re-infection. These findings clearly indicate a critical role for IgA in the establishment of immunity against a GI viral pathogen. PMID:22739233

Arabidopsis myrosinases link the glucosinolate-myrosinase system and the cuticle

PubMed Central

Ahuja, Ishita; de Vos, Ric C. H.; Rohloff, Jens; Stoopen, Geert M.; Halle, Kari K.; Ahmad, Samina Jam Nazeer; Hoang, Linh; Hall, Robert D.; Bones, Atle M.

2016-01-01

Both physical barriers and reactive phytochemicals represent two important components of a plant’s defence system against environmental stress. However, these two defence systems have generally been studied independently. Here, we have taken an exclusive opportunity to investigate the connection between a chemical-based plant defence system, represented by the glucosinolate-myrosinase system, and a physical barrier, represented by the cuticle, using Arabidopsis myrosinase (thioglucosidase; TGG) mutants. The tgg1, single and tgg1 tgg2 double mutants showed morphological changes compared to wild-type plants visible as changes in pavement cells, stomatal cells and the ultrastructure of the cuticle. Extensive metabolite analyses of leaves from tgg mutants and wild-type Arabidopsis plants showed altered levels of cuticular fatty acids, fatty acid phytyl esters, glucosinolates, and indole compounds in tgg single and double mutants as compared to wild-type plants. These results point to a close and novel association between chemical defence systems and physical defence barriers. PMID:27976683

The disruption of two salt bridges of the cold-active xylanase XynGR40 results in an increase in activity, but a decrease in thermostability.

PubMed

Wang, Guozeng; Wu, Jingjing; Lin, Juan; Ye, Xiuyun; Yao, Bin

2016-12-02

Cold-active xylanases are of great interest due to their large potential for application in the food industry. In this study, salt bridges of the eight glycoside hydrolase (GH) family 10 cold-active xylanases reported to date were predicted and the salt bridges specific to the cold-active xylanase XynGR40 were identified. Seven mutants were constructed to disrupt salt bridges specific to XynGR40. The results suggested that five mutants lost their xylanase activity, while the other two mutants, D30N and D83N, displayed different properties when compared with the wild-type XynGR40. First, both mutations showed an obvious decrease in thermostability, with the T 1/2 of D30N and D83N at 50 °C being about one half and one sixth of the wild-type, respectively. Second, both D30N and D83N had a higher specific activity than the wild-type, with activities about 13 and 163% higher, respectively. Third, both D30N and D83N had high k cat and K m values, which resulted in a higher catalytic efficiency of the mutant D83N, but a lower catalytic efficiency of the mutant D30N compared to the wild-type. Our results suggested that salt bridges play important roles in both the activity and thermostability of the cold-active xylanase XynGR40. The mutant D83N had a higher k cat and higher relative activity at low temperatures than the wild-type, and is a good candidate for application in the food industry. Copyright © 2016 Elsevier Inc. All rights reserved.

Stability of Iowa mutant and wild type Aβ-peptide aggregates

NASA Astrophysics Data System (ADS)

Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.; Hansmann, Ulrich H. E.

2014-11-01

Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

Mycoviruses as Triggers and Targets of RNA Silencing in White Mold Fungus Sclerotinia sclerotiorum.

PubMed

Mochama, Pauline; Jadhav, Prajakta; Neupane, Achal; Lee Marzano, Shin-Yi

2018-04-22

This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, dicer genes ( dcl-1, dcl-2 , and both dcl-1 / dcl-2 ) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum ; however, the double dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, Δdcl-1 and Δdcl-2 . Complementation of a single dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that S. sclerotiorum has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus.

Changes in blood carnitine and acylcarnitine profiles of very long-chain acyl-CoA dehydrogenase-deficient mice subjected to stress.

PubMed

Spiekerkoetter, U; Tokunaga, C; Wendel, U; Mayatepek, E; Exil, V; Duran, M; Wijburg, F A; Wanders, R J A; Strauss, A W

2004-03-01

In humans with deficiency of the very long-chain acyl-CoA dehydrogenase (VLCAD), C14-C18 acylcarnitines accumulate. In this paper we have used the VLCAD knockout mouse as a model to study changes in blood carnitine and acylcarnitine profiles under stress. VLCAD knockout mice exhibit stress-induced hypoglycaemia and skeletal myopathy; symptoms resembling human VLCADD. To study the extent of biochemical derangement in response to different stressors, we determined blood carnitine and acylcarnitine profiles after exercise on a treadmill, fasting, or exposure to cold. Even in a nonstressed, well-fed state, knockout mice presented twofold higher C14-C18 acylcarnitines and a lower free carnitine of 72% as compared to wild-type littermates. After 1 h of intense exercise, the C14-C18 acylcarnitines in blood significantly increased, but free carnitine remained unchanged. After 8 h of fasting at 4 degrees C, the long-chain acylcarnitines were elevated 5-fold in knockout mice in comparison with concentrations in unstressed wild-type mice (P < 0.05), and four out of 12 knockout mice died. Free carnitine decreased to 44% as compared with unstressed wild-type mice. An increase in C14-C18 acylcarnitines and a decrease of free carnitine were also observed in fasted heterozygous and wild-type mice. Long-chain acylcarnitines in blood increase in knockout mice in response to different stressors and concentrations correlate with the clinical condition. A decrease in blood free carnitine in response to severe stress is observed in knockout mice but also in wild-type littermates. Monitoring blood acylcarnitine profiles in response to different stressors may allow systematic analysis of therapeutic interventions in VLCAD knockout mice.

Erlotinib for Patients with EGFR Wild-Type Metastatic NSCLC: a Retrospective Biomarkers Analysis.

PubMed

Inno, Alessandro; Di Noia, Vincenzo; Martini, Maurizio; D'Argento, Ettore; Di Salvatore, Mariantonietta; Arena, Vincenzo; Schinzari, Giovanni; Orlandi, Armando; Larocca, Luigi Maria; Cassano, Alessandra; Barone, Carlo

2018-03-20

Erlotinib is approved for the treatment of patients with EGFR mutation positive, metastatic NSCLC. It is also approved as second/third line therapy for EGFR mutation negative patients, but in this setting the benefit of erlotinib is modest and there is no validated biomarker for selecting EGFR wild-type patients who may benefit the most from the treatment. We retrospectively assessed EGFR and K-RAS mutational status, and EGFR, c-MET and IGF1-R expression in tumor samples of 72 patients with metastatic NSCLC treated with erlotinib after at least one prior line of chemotherapy, from 2008 to 2012. We analyzed the association between biomarkers and outcome (RR, PFS, and OS). EGFR mutated patients achieved a better RR (56% vs 8%, p = .002), PFS (10 vs 3 months, HR 0.53, p = 0.48) and OS (20 vs 6 months, HR 0.55, p = .07), compared to EGFR wild-type patients. Among 63 EGFR wild-type patients, those with EGFR high-expression had a better outcome in terms of RR (40% vs 2%, p = .002), PFS (7.5 vs 2 months, HR 0.45, p = .007) and OS (30 vs 5 months, HR 0.34, p < .001) compared to patients with EGFR intermediate or low/negative-expression. IGF1-R expression, c-MET expression and K-RAS mutational status did not significantly affect the outcome; however, no patients with K-RAS mutation or c-MET high-expression achieved an objective response. In patients with metastatic, chemo-refractory EGFR wild-type NSCLC, EGFR high-expression may represent a positive predictor of activity for erlotinib, whereas K-RAS mutation and c-MET high-expression may predict lack of activity. These findings deserve further prospective evaluation.

Modeling and Re-Engineering of Azotobacter vinelandii Alginate Lyase to Enhance Its Catalytic Efficiency for Accelerating Biofilm Degradation.

PubMed

Jang, Chul Ho; Piao, Yu Lan; Huang, Xiaoqin; Yoon, Eun Jeong; Park, So Hee; Lee, Kyoung; Zhan, Chang-Guo; Cho, Hoon

2016-01-01

Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL) might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A) were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate) compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT) and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect.

Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder

PubMed Central

Pak, K. J.; Ostrom, R. S.; Matsui, M.

2010-01-01

We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg−1) 2–24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC50 value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 µM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M2 function is enhanced following streptozotocin treatment. PMID:20349044

Impaired M3 and enhanced M2 muscarinic receptor contractile function in a streptozotocin model of mouse diabetic urinary bladder.

PubMed

Pak, K J; Ostrom, R S; Matsui, M; Ehlert, F J

2010-05-01

We investigated the contractile roles of M2 and M3 muscarinic receptors in urinary bladder from streptozotocin-treated mice. Wild-type and M2 muscarinic receptor knockout (M2 KO) mice were given a single injection of vehicle or streptozotocin (125 mg kg(-1)) 2-24 weeks prior to bladder assays. The effect of forskolin on contractions elicited to the muscarinic agonist, oxotremorine-M, was measured in isolated urinary bladder (intact or denuded of urothelium). Denuded urinary bladder from vehicle-treated wild-type and M2 KO mice exhibited similar contractile responses to oxotremorine-M, when contraction was normalized relative to that elicited by KCl (50 mM). Eight to 9 weeks after streptozotocin treatment, the EC(50) value of oxotremorine-M increased 3.1-fold in urinary bladder from the M2 KO mouse (N = 5) compared to wild type (N = 6; P < 0.001). Analogous changes were observed in intact bladder. In denuded urinary bladder from vehicle-treated mice, forskolin (5 microM) caused a much greater inhibition of contraction in M2 KO bladder compared to wild type. Following streptozotocin treatment, this forskolin effect increased 1.6-fold (P = 0.032). At the 20- to 24-week time point, the forskolin effect increased 1.7-fold for denuded as well as intact bladders (P = 0.036, 0.01, respectively). Although streptozotocin treatment inhibits M3 receptor-mediated contraction in denuded urinary bladder, muscarinic contractile function is maintained in wild-type bladder by enhanced M2 contractile function. M2 receptor activation opposes forskolin-induced relaxation of the urinary bladder, and this M(2) function is enhanced following streptozotocin treatment.

Cystathionine-gamma-lyase deficient mice are protected against the development of multiorgan failure and exhibit reduced inflammatory response during burn.

PubMed

Ahmad, Akbar; Druzhyna, Nadiya; Szabo, Csaba

2017-08-01

Considering the role of H 2 S in critical illness, the aim of this study was to compare the outcome of burn in wild-type mice and in mice deficient in CSE, one of the principal mammalian H 2 S-generating enzymes. Animals were subjected to scald burn. Outcome variables included indices of organ injury, clinical chemistry parameters and plasma levels of inflammatory mediators. Plasma levels of H 2 S significantly increased in response to burn in wild-type mice, but remained unchanged in CSE -/- mice. Expression of the three H 2 S-producing enzymes (CSE, CBS and 3-MST) in the lung and liver, and the capacity of tissue homogenates to produce H 2 S, however, was not affected by burn. In CSE deficient mice there was a significant amelioration of burn-induced accumulation of myeloperoxidase levels in heart, lung, liver and kidney and significantly lower degree of malon dialdehyde accumulation in the heart, lung and kidney than in wild-type mice. CSE deficient mice, compared to wild-type mice, showed a significant attenuation of the burn-induced elevation in circulating alkaline aminotransferase and blood urea nitrogen and creatinine levels, indicative of protective effects of CSE deficiency against burn-induced hepatic, and renal functional impairment. Multiple burn-induced inflammatory mediators (TNF-α, IL-1β, IL-4, IL-6, IL-10 and IL-12) were significantly lower in the plasma of CSE -/- animals after burn than in the plasma of wild-type controls subjected to burns. In conclusion, CSE deficiency improves organ function and attenuates the inflammatory response in a murine model of burn. Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.

Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts

PubMed Central

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

RAS testing of liquid biopsy correlates with the outcome of metastatic colorectal cancer patients treated with first-line FOLFIRI plus cetuximab in the CAPRI-GOIM trial.

PubMed

Normanno, N; Esposito Abate, R; Lambiase, M; Forgione, L; Cardone, C; Iannaccone, A; Sacco, A; Rachiglio, A M; Martinelli, E; Rizzi, D; Pisconti, S; Biglietto, M; Bordonaro, R; Troiani, T; Latiano, T P; Giuliani, F; Leo, S; Rinaldi, A; Maiello, E; Ciardiello, F

2018-01-01

Liquid biopsy is an alternative to tissue for RAS testing in metastatic colorectal carcinoma (mCRC) patients. Little information is available on the predictive role of liquid biopsy RAS testing in patients treated with first-line anti-EGFR monoclonal antibody-based therapy. In the CAPRI-GOIM trial, 340 KRAS exon-2 wild-type mCRC patients received first-line cetuximab plus FOLFIRI. Tumor samples were retrospectively assessed by next generation sequencing (NGS). Baseline plasma samples were analyzed for KRAS and NRAS mutations using beads, emulsion, amplification, and magnetics digital PCR (BEAMing). Discordant cases were solved by droplet digital PCR (ddPCR) or deep-sequencing. A subgroup of 92 patients with available both NGS data on tumor samples and baseline plasma samples were included in this study. Both NGS analysis of tumor tissue and plasma testing with BEAMing identified RAS mutations in 33/92 patients (35.9%). However, 10 cases were RAS tissue mutant and plasma wild-type, and additional 10 cases were tissue wild-type and plasma mutant, resulting in a concordance rate of 78.3%. Analysis of plasma samples with ddPCR detected RAS mutations in 2/10 tissue mutant, plasma wild-type patients. In contrast, in all tissue wild-type and plasma mutant cases, ddPCR or deep-sequencing analysis of tumor tissue confirmed the presence of RAS mutations at allelic frequencies ranging between 0.15% and 1.15%. The median progression-free survival of RAS mutant and wild-type patients according to tissue (7.9 versus 12.6 months; P = 0.004) and liquid biopsy testing (7.8 versus 13.8 moths; P < 0.001) were comparable. Similar findings were observed for the median overall survival of RAS mutant and wild-type patients based on tissue (22.1 versus 35.8 months; P = 0.016) and plasma (19.9 versus 35.8 months; P = 0.013) analysis. This study indicates that RAS testing of liquid biopsy results in a similar outcome when compared with tissue testing in mCRC patients receiving first-line anti-EGFR monoclonal antibodies. © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Altered Expression of OsNLA1 Modulates Pi Accumulation in Rice (Oryza sativa L.) Plants

PubMed Central

Zhong, Sihui; Mahmood, Kashif; Bi, Yong-Mei; Rothstein, Steven J.; Ranathunge, Kosala

2017-01-01

Current agricultural practices rely on heavy use of fertilizers for increased crop productivity. However, the problems associated with heavy fertilizer use, such as high cost and environmental pollution, require the development of crop species with increased nutrient use efficiency. In this study, by using transgenic approaches, we have revealed the critical role of OsNLA1 in phosphate (Pi) accumulation of rice plants. When grown under sufficient Pi and nitrate levels, OsNLA1 knockdown (Osnla1-1, Osnla1-2, and Osnla1-3) lines accumulated higher Pi content in their shoot tissues compared to wild-type, whereas, over-expression lines (OsNLA1-OE1, OsNLA1-OE2, and OsNLA1-OE3) accumulated the least levels of Pi. However, under high Pi levels, knockdown lines accumulated much higher Pi content compared to wild-type and exhibited Pi toxicity symptoms in the leaves. In contrast, the over-expression lines had 50–60% of the Pi content of wild-type and did not show such symptoms. When grown under limiting nitrate levels, OsNLA1 transgenic lines also displayed a similar pattern in Pi accumulation and Pi toxicity symptoms compared to wild-type suggesting an existence of cross-talk between nitrogen (N) and phosphorous (P), which is regulated by OsNLA1. The greater Pi accumulation in knockdown lines was a result of enhanced Pi uptake/permeability of roots compared to the wild-type. The cross-talk between N and P was found to be nitrate specific since the knockdown lines failed to over-accumulate Pi under low (sub-optimal) ammonium level. Moreover, OsNLA1 was also found to interact with OsPHO2, a known regulator of Pi homeostasis, in a Yeast Two-Hybrid (Y2H) assay. Taken together, these results show that OsNLA1 is involved in Pi homeostasis regulating Pi uptake and accumulation in rice plants and may provide an opportunity to enhance P use efficiency by manipulating nitrate supply in the soil. PMID:28626465

Assessment of the Toxicity of CuO Nanoparticles by Using Saccharomyces cerevisiae Mutants with Multiple Genes Deleted

PubMed Central

Bao, Shaopan; Lu, Qicong; Dai, Heping; Zhang, Chao

2015-01-01

To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P < 0.05). When the toxicity of the CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles. PMID:26386067

Helicobacter pylori HP1512 Is a Nickel-Responsive NikR-Regulated Outer Membrane Protein▿

PubMed Central

Davis, Gregg S.; Flannery, Erika L.; Mobley, Harry L. T.

2006-01-01

Helicobacter pylori is dependent upon the production of the highly abundant and active metalloenzyme urease for colonization of the human stomach. Thus, H. pylori has an absolute requirement for the transition metal nickel, a required cofactor for urease. To investigate the contribution of genes that are factors in this process, microarray analysis comparing the transcriptome of wild-type H. pylori 26695 cultured in brucella broth containing fetal calf serum (BBF) alone or supplemented with 100 μM NiCl2 suggested that HP1512 is repressed in the presence of 100 μM supplemental nickel. When measured by comparative real-time quantitative PCR (qPCR), HP1512 transcription was reduced 43-fold relative to the value for the wild type when cultured in BBF supplemented with 10 μM NiCl2. When grown in unsupplemented BBF, urease activity of an HP1512::cat mutant was significantly reduced compared to the wild type, 4.9 ± 0.5 μmol/min/mg of protein (n = 7) and 17.1 ± 4.9 μmol/min/mg of protein (n = 13), respectively (P < 0.0001). In silico analysis of the HP1511-HP1512 (HP1511-1512) intergenic region identified a putative NikR operator upstream of HP1512. Gel shift analysis with purified recombinant NikR verified nickel-dependent binding of H. pylori NikR to the HP1511-1512 intergenic region. Furthermore, comparative real-time qPCR of four nickel-related genes suggests that mutation of HP1512 results in reduced intracellular nickel concentration relative to wild-type H. pylori 26695. Taken together, these data suggest that HP1512 encodes a NikR-nickel-regulated outer membrane protein. PMID:17030579

Comparative study on fermentation performance in the genome shuffled Candida versatilis and wild-type salt tolerant yeast strain.

PubMed

Qi, Wei; Guo, Hong-Lian; Wang, Chun-Ling; Hou, Li-Hua; Cao, Xiao-Hong; Liu, Jin-Fu; Lu, Fu-Ping

2017-01-01

The fermentation performance of a genome-shuffled strain of Candida versatilis S3-5, isolated for improved tolerance to salt, and wild-type (WT) strain were analysed. The fermentation parameters, such as growth, reducing sugar, ethanol, organic acids and volatile compounds, were detected during soy sauce fermentation process. The results showed that ethanol produced by the genome shuffled strain S3-5 was increasing at a faster rate and to a greater extent than WT. At the end of the fermentation, malic acid, citric acid and succinic acid formed in tricarboxylic acid cycle after S3-5 treatment elevated by 39.20%, 6.85% and 17.09% compared to WT, respectively. Moreover, flavour compounds such as phenethyl acetate, ethyl vanillate, ethyl acetate, isoamyl acetate, ethyl myristate, ethyl pentadecanoate, ethyl palmitate and phenylacetaldehyde produced by S3-5 were 2.26, 2.12, 2.87, 34.41, 6.32, 13.64, 2.23 and 78.85 times as compared to WT. S3-5 exhibited enhanced metabolic ability as compared to the wild-type strain, improved conversion of sugars to ethanol, metabolism of organic acid and formation of volatile compounds, especially esters, Moreover, S3-5 might be an ester-flavour type salt-tolerant yeast. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Maize pollen coat xylanase facilitates pollen tube penetration into silk during sexual reproduction.

PubMed

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.

Research on the ultrafast fluorescence property of thylakoid membranes of the wild-type and mutant rice

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.

The effect of IDH1 mutation on the structural connectome in malignant astrocytoma.

PubMed

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.

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

Maternal eNOS deficiency determines a fatty liver phenotype of the offspring in a sex dependent manner

PubMed Central

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

Stimulus control by 5methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice

PubMed Central

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

Hyperresponsive febrile reactions to interleukin (IL) 1α and IL-1β, and altered brain cytokine mRNA and serum cytokine levels, in IL-1β-deficient mice

PubMed Central

Alheim, Katarina; Chai, Zhen; Fantuzzi, Giamila; Hasanvan, Homa; Malinowsky, David; Di Santo, Elena; Ghezzi, Pietro; Dinarello, Charles A.; Bartfai, Tamas

1997-01-01

IL-1β is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1α and IL-1β, and compared these with response to LPS (i.p.) in wild-type and IL-1β-deficient mice. The IL-1β deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1β, IL-1α, or LPS induced hyperresponsive fevers in the IL-1β-deficient mice. We also observed phenotypic differences between wild-type and IL-1β-deficient mice in hypothalamic basal mRNA levels for IL-1α and IL-6, but not for IL-1β-converting enzyme or IL-1 receptor type I or type II. The IL-1α mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1β-deficient mice as compared with wild-type mice. The IL-1β-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type α levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1β plays an important but not obligatory role in fever induction by LPS or IL-1α, as well as in the induction of serum tumor necrosis factor type α and corticosterone responses either by LPS or by IL-1α or IL-1β. PMID:9122256

The SLICK hair locus derived from Senepol cattle confers thermotolerance to intensively managed lactating Holstein cows.

PubMed

Dikmen, S; Khan, F A; Huson, H J; Sonstegard, T S; Moss, J I; Dahl, G E; Hansen, P J

2014-09-01

The SLICK haplotype (http://omia.angis.org.au/OMIA001372/9913/) in cattle confers animals with a short and sleek hair coat. Originally identified in Senepol cattle, the gene has been introduced into Holsteins. The objectives of the current study were to determine (1) whether lactating Holsteins with the slick hair phenotype have superior ability for thermoregulation compared with wild-type cows or relatives not inheriting the SLICK haplotype, and (2) whether seasonal depression in milk yield would be reduced in SLICK cows. In experiment 1, diurnal variation in vaginal temperature in the summer was monitored for cows housed in a freestall barn with fans and sprinklers. Vaginal temperatures were lower in slick-haired cows than in relatives and wild-type cows. In experiment 2, acute responses to heat stress were monitored after cows were moved to a dry lot in which the only heat abatement was shade cloth. The increases in rectal temperature and respiration rate caused by heat stress during the day were lower for slick cows than for relatives or wild-type cows. Moreover, sweating rate was higher for slick cows than for cows of the other 2 types. In experiment 3, effects of season of calving (summer vs. winter) on milk yield and composition were determined. Compared with milk yield of cows calving in winter, milk yield during the first 90 d in milk was lower for cows calving in the summer. However, this reduction was less pronounced for slick cows than for wild-type cows. In conclusion, Holsteins with slick hair have superior thermoregulatory ability compared with non-slick animals and experience a less drastic depression in milk yield during the summer. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis

PubMed Central

Xia, Yunfeng; Yan, Jingyin; Jin, Xiaogao; Entman, Mark L.; Wang, Yanlin

2014-01-01

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. Since chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly fewer bone marrow-derived fibroblasts accumulated in the kidney of CXCR6 knockout mice in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type mice. CXCR6 deficiency inhibited total collagen deposition and suppressed expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, wild type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Transplant of wild type bone marrow into CXCR6−/− recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may play important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis. PMID:24646857

Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection

PubMed Central

Nguyen, Bao-Ngoc H.; Azimzadeh, Agnes M.; Schroeder, Carsten; Buddensick, Thomas; Zhang, Tianshu; Laaris, Amal; Cochrane, Megan; Schuurman, Henk-Jan; Sachs, David H.; Allan, James S.; Pierson, Richard N.

2012-01-01

Background Galactosyl transferase gene knock-out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection. Methods We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild-type and human decay-accelerating factor-transgenic (hDAF+/+) pig lungs. Results GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild-type lungs (P = 0.001) and 45 ± 60 min for hDAF+/+ lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild-type or hDAF+/+ lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild-type or hDAF+/+ lungs. However, GalTKO lungs adsorbed cytotoxic anti-non-Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. Conclusions In summary, GalTKO lungs are substantially protected from injury but, in addition to anti-non-Gal antibody and complement, platelet adhesion and non-physiologic intravascular coagulation contribute to Gal-independent lung injury mechanisms. PMID:21496117

Two Novel Functions of Hyaluronidase from Streptococcus agalactiae Are Enhanced Intracellular Survival and Inhibition of Proinflammatory Cytokine Expression

PubMed Central

Wang, Zhaofei; Guo, Changming; Xu, Yannan; Liu, Guangjin; Lu, Chengping

2014-01-01

Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl+ isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl+ strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity. PMID:24711564

Two novel functions of hyaluronidase from Streptococcus agalactiae are enhanced intracellular survival and inhibition of proinflammatory cytokine expression.

PubMed

Wang, Zhaofei; Guo, Changming; Xu, Yannan; Liu, Guangjin; Lu, Chengping; Liu, Yongjie

2014-06-01

Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl(+) isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl(+) strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.

Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy

PubMed Central

Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

2014-01-01

The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knockin mice expressing non-phosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knockin mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knockin mice compared to their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knockin mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knockin and wild type mice, indicating that mTORC1 was still activated in the knockin mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knockin mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth. PMID:25229342

Resistin deficiency in mice has no effect on pulmonary responses induced by acute ozone exposure

PubMed Central

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

Matrix metalloproteinase (MMP)-9 in cancer-associated fibroblasts (CAFs) is suppressed by omega-3 polyunsaturated fatty acids in vitro and in vivo.

PubMed

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.

Changes in oxidative stress in transgenic RNAi ACO1 tomato fruit during ripening

NASA Astrophysics Data System (ADS)

Eglous, Najat Mohamed; Ali, Zainon Mohd; Hassan, Maizom; Zainal, Zamri

2013-11-01

Tomato (Solanum Lycopersicum L.) is the second most cultivated vegetable in the world and widely used as a system for studying the role of ethylene during fruit ripening. Our objective was to study the oxidative stress and antioxidative metabolism during ripening of non transgenic tomato and transgenic line-21 tomato which reduced ethylene. The line-21 of transgenic tomato plants (RNAi ACO1) had lower ethylene production and longer shelf-life more than 32 days as compared to the wild-type fruits which have very short shelf-life. In this study, tomato fruit were divided into five different stages (MG: mature green 5%, B: breaker 25%, T: turning 50%, O: orange75%, RR: red ripe100%). The activity of lipoxygenase (LOX) and lipid peroxidation (MDA) were measured to assess changes in oxidative stress. The LOX activity and MDA content decreased significantly obtaining 2.6-fold and 1.2-fold, respectively, as compared to the wild type fruit. However, superoxide dismutase (SOD) and catalase (CAT) activities were increased to 1.9 and 1.2 folds from the mature green to the fully ripe stage in transgenic tomatoes. Furthermore, the wild type tomato increases 1.3 in SOD and 1.6 in CAT activities. The overall results indicate that the wild type tomato fruit showed a faster rate of ripening, parallel to decline in the rate of enzymatic antioxidative systems as compared to the transgenic line-21 tomato fruit. In addition, the results show that the antioxidant capacity is improved during the ripening process and is accompanied by an increase in the oxidative stress.

Curcumin Affects Phase II Disposition of Resveratrol Through Inhibiting Efflux Transporters MRP2 and BCRP

PubMed Central

Ge, Shufan; Yin, Taijun; Xu, Beibei; Gao, Song; Hu, Ming

2015-01-01

Purpose To evaluate the impact of curcumin on the disposition of resveratrol phase II metabolites in vivo, and explain the observations by performing in vitro studies in transporter-overexpressed cells. Methods Pharmacokinetic studies of resveratrol with and without the co-administration of curcumin were performed in both FVB wild-type and Bcrp1 (−/−) mice. Human UGT1A9-overexpressing HeLa cells and human MRP2-overexpressing MDCK II-UGT1A1 cells were used as in vitro tools to further determine the impact of curcumin as a transporter inhibitor on resveratrol metabolites. Results We observed higher exposure of resveratrol conjugates in Bcrp1 (−/−) mice compared to wild-type mice. In wild-type mice, curcumin increased the AUC of resveratrol glucuronide by 4-fold compared to the mice treated without curcumin. The plasma levels of resveratrol and its sulfate conjugate also increased moderately. In Bcrp1 (−/−) mice, there was a further increase (6-fold increase) in AUC of resveratrol glucuronide observed when curcumin was co-administered compared to AUC values obtained in wild-type mice without curcumin treatment. In the presence of 50nM curcumin, the clearance of resveratrol-3-O-glucuronide and resveratrol-3-O-sulfate reduced in both MRP2-overexpressing MDCKII-UGT1A1 cells and Human UGT1A9-overexpressing HeLa cells. Conclusions These results suggest that curcumin alters the phase II distribution of resveratrol through inhibiting efflux transporters including MRP2 and BCRP. PMID:26502886

Chir99021 and Valproic acid reduce the proliferative advantage of Apc mutant cells.

PubMed

Langlands, Alistair J; Carroll, Thomas D; Chen, Yu; Näthke, Inke

2018-02-15

More than 90% of colorectal cancers carry mutations in Apc that drive tumourigenesis. A 'just-right' signalling model proposes that Apc mutations stimulate optimal, but not excessive Wnt signalling, resulting in a growth advantage of Apc mutant over wild-type cells. Reversal of this growth advantage constitutes a potential therapeutic approach. We utilised intestinal organoids to compare the growth of Apc mutant and wild-type cells. Organoids derived from Apc Min/+ mice recapitulate stages of intestinal polyposis in culture. They eventually form spherical cysts that reflect the competitive growth advantage of cells that have undergone loss of heterozygosity (LOH). We discovered that this emergence of cysts was inhibited by Chiron99021 and Valproic acid, which potentiates Wnt signalling. Chiron99021 and Valproic acid restrict the growth advantage of Apc mutant cells while stimulating that of wild-type cells, suggesting that excessive Wnt signalling reduces the relative fitness of Apc mutant cells. As a proof of concept, we demonstrated that Chiron99021-treated Apc mutant organoids were rendered susceptible to TSA-induced apoptosis, while wild-type cells were protected.

Enhancement of lipid production in Scenedesmus sp. by UV mutagenesis and hydrogen peroxide treatment.

PubMed

Sivaramakrishnan, Ramachandran; Incharoensakdi, Aran

2017-07-01

The high potential UV mutagenized Scenedesmus sp. was obtained in which the cells had a higher biomass and lipid content than the wild type with an increase from 1.9 to 2.4g/L and from 40 to 55% of dry cell weight respectively after 12days. Oxidative stress imposed by H 2 O 2 treatment decreased the biomass of both the wild type and the mutant. The H 2 O 2 treated mutant when grown in BG11 medium showed an increase in biomass which was in contrast to a decreased biomass observed in the H 2 O 2 treated wild type. A 3-fold increase in lipid yield of 1.63g/L was obtained in the oxidative stress-induced mutant compared to the wild type. Overall results indicate that prior treatment of UV-mutagenized Scenedesmus with oxidative stress can increase the total lipid production which, due to its derived methyl ester having acceptable biodiesel properties, can be potentially utilized for biodiesel production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development

NASA Technical Reports Server (NTRS)

Simske, Steven J.; Bateman, Ted A.; Smith, Erin E.; Ferguson, Virginia L.; Chapes, Stephen K.

2002-01-01

We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls.

Exploratory biomarker analysis for treatment response in KRAS wild type metastatic colorectal cancer patients who received cetuximab plus irinotecan.

PubMed

Kim, Seung Tae; Ahn, Tae Jin; Lee, Eunjin; Do, In-Gu; Lee, Su Jin; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Kim, Suk Hyeong; Lee, Jeeyun; Kim, Hee Cheol

2015-10-20

More than half of the patients selected based on KRAS mutation status fail to respond to the treatment with cetuximab in metastatic colorectal cancer (mCRC). We designed a study to identify additional biomarkers that could act as indicators for cetuximab treatment in mCRC. We investigated 58 tumor samples from wild type KRAS CRC patients treated with cetuximab plus irinotecan (CI). We conducted the genotyping for mutations in either BRAF or PIK3CA and profiled comprehensively the expression of 522 kinase genes. BRAF mutation was detected in 5.1 % (3/58) of patients. All 50 patients showed wild type PIK3CA. Gene expression patterns that categorized patients with or without the disease control to CI were compared by supervised classification analysis. PSKH1, TLK2 and PHKG2 were overexpressed significantly in patients with the disease control to IC. The higher expression value of PSKH1 (r = 0.462, p < 0.001) and TLK2 (r = 0.361, p = 0.005) had the significant correlation to prolonged PFS. The result of this work demonstrated that expression nature of kinase genes such as PSKH1, TLK2 and PHKG2 may be informative to predict the efficacy of CI in wild type KRAS CRC. Mutations in either BRAF or PIK3CA were rare subsets in wild type KRAS CRC.

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit.

PubMed

Liu, Cheng-Qian; Hu, Kang-Di; Li, Ting-Ting; Yang, Ying; Yang, Feng; Li, Yan-Hong; Liu, He-Ping; Chen, Xiao-Yan; Zhang, Hua

2017-01-01

Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade plant cell wall. To evaluate whether the deletion of an exo-polygalacturonase gene pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.15 (wild type) via homologous recombination. The ΔpgxB mutant showed similar growth behavior compared with the wild type. Pectin medium induced significant higher expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. However, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused by ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, gene expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results prove that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple fruit, suggesting that pgxB plays an important role in the infection process on the apple fruit.

Arthritis is developed in Borrelia-primed and -infected mice deficient of interleukin-17.

PubMed

Kuo, Joseph; Warner, Thomas F; Munson, Erik L; Nardelli, Dean T; Schell, Ronald F

2016-10-01

Interleukin-17 (IL-17) has been shown to participate in the development of Lyme arthritis in experimental mice. For example, neutralization of IL-17 with antibodies inhibits induction of arthritis in Borrelia-primed and -infected C57BL/6 wild-type mice. We hypothesized that mice lacking IL-17 would fail to develop Borrelia-induced arthritis. IL-17-deficient and wild-type C57BL/6 mice were primed with heat-inactivated Borrelia and then infected with viable spirochetes 3 weeks later. No swelling or major histopathological changes of the hind paws were detected in IL-17-deficient or wild-type mice that were primed with Borrelia or infected with viable spirochetes. By contrast, IL-17-deficient and wild-type mice that were primed and subsequently infected with heterologous Borrelia developed severe swelling and histopathological changes of the hind paws. In addition, Borrelia-primed and -infected IL-17-deficient mice exhibited elevated gamma-interferon (IFN-γ) levels in sera and increased frequencies of IFN-γ-expressing lymphocytes in popliteal lymph nodes compared to Borrelia-primed and -infected wild-type mice. These results demonstrate that IL-17 is not required for development of severe pathology in response to infection with Borrelia burgdorferi, but may contribute to disease through an interaction with IFN-γ. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Role of the glyoxylate pathway in acetic acid production by Acetobacter aceti.

PubMed

Sakurai, Kenta; Yamazaki, Shoko; Ishii, Masaharu; Igarashi, Yasuo; Arai, Hiroyuki

2013-01-01

Wild-type Acetobacter aceti NBRC 14818 possesses genes encoding isocitrate lyase (aceA) and malate synthase (glcB), which constitute the glyoxylate pathway. In contrast, several acetic acid bacteria that are utilized for vinegar production lack these genes. Here, an aceA-glcB knockout mutant of NBRC 14818 was constructed and used for investigating the role of the glyoxylate pathway in acetate productivity. In medium containing ethanol as a carbon source, the mutant grew normally during ethanol oxidation to acetate, but exhibited slower growth than that of the wild-type strain as the accumulated acetate was oxidized. The mutant grew similarly to that of the wild-type strain in medium containing glucose as a carbon source, indicating that the glyoxylate pathway was not necessary for glucose utilization. However, in medium containing both ethanol and glucose, the mutant exhibited significantly poorer growth and lower glucose consumption compared to the wild-type strain. Notably, the mutant oxidized ethanol nearly stoichiometrically to acetate, which was retained in the medium for a longer period of time than the acetate produced by wild-type strain. The features of the aceA-glcB knockout mutant revealed here indicate that the lack of the glyoxylate pathway is advantageous for industrial vinegar production by A. aceti. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines.

PubMed

Tanami, Hideaki; Imoto, Issei; Hirasawa, Akira; Yuki, Yasuhiro; Sonoda, Itaru; Inoue, Jun; Yasui, Kohichiro; Misawa-Furihata, Akiko; Kawakami, Yutaka; Inazawa, Johji

2004-11-18

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.

The neuropathology observed in wild-type mice inoculated with human poliovirus mirrors human paralytic poliomyelitis.

PubMed

Ford, Dayton J; Ropka, Stacie L; Collins, George H; Jubelt, Burk

2002-09-01

Human paralytic poliomyelitis results from the destruction of spinal cord anterior horn motor neurons by human poliovirus (PV). CNS disease pathology similar to human poliomyelitis has been observed in experimentally infected chimpanzees, monkeys and wild-type mice. In this study we present a detailed examination of the clinical and histopathological features in the wild-type mouse after intracranial (i.c.) and novel intramuscular (i.m.) injection of poliovirus. Either route of poliovirus administration results in a clinical disease characterized predominately by flaccid paralysis. The observed histopathological features are compared with the histopathology reported for human paralytic poliomyelitis, experimentally infected chimpanzees, monkeys and transgenic mice expressing the human poliovirus receptor (hPVR). The observation of flaccid paralysis and anterior horn motor neuron destruction mirrors what is observed in human paralytic poliomyelitis. Our results suggest that the neuropathology observed in the wild-type mouse model is similar to what has been observed in both the human disease and in other experimental animal models, with the possible exception of the transgenic mouse model. The observed neuropathology of the wild-type mouse model more closely reflects what has been observed in human poliomyelitis, as well as in experimentally infected chimpanzees and monkeys, than does the hPVR transgenic mouse model. The previously reported poliovirus-induced white matter demyelinating disease was not observed.

Suicide Inhibitors of Reverse Transcriptase in the Therapy of AIDS and Other Retroviruses

DTIC Science & Technology

1990-07-01

I and 10 nanonolar) and compared to the E . Coli recombinant HIV-RT (Kindly donated by Dr. Steven Hughes Fort Detrick M.D.) and the wild type HIV-RT...Both the wild type and E . Coli HIV-RT’s were resistant to PFA showing essentially no inhibition at the lOnM level. Previous studies have shown that...10 nanomolar PFA. j, Sentivitv of Recombinant HIV-Reverse Transcriotase to Foscarnet. RECOMBINANT HIV RT ( E . COLI) + FOSCARNET 350001R 300000 PFA .001

Three Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutants with Distinct and Asymmetric Effects on Virulence in Mice Compared with Rabbits

PubMed Central

Perng, Guey-Chuen; Esmaili, Daniel; Slanina, Susan M.; Yukht, Ada; Ghiasi, Homayon; Osorio, Nelson; Mott, Kevin R.; Maguen, Barak; Jin, Ling; Nesburn, Anthony B.; Wechsler, Steven L.

2001-01-01

Herpes simplex virus type 1 latency-associated transcript (LAT)-null mutants have decreased reactivation but normal virulence in rabbits and mice. We report here on dLAT1.5, a mutant with LAT nucleotides 76 to 1667 deleted. Following ocular infection of rabbits, dLAT1.5 reactivated at a lower rate than its wild-type parent McKrae (6.1 versus 11.8%; P = 0.0025 [chi-square test]). Reactivation was restored in the marker-rescued virus dLAT1.5R (12.6%; P = 0.53 versus wild type), confirming the importance of the deleted region in spontaneous reactivation. Compared with wild-type or marker-rescued virus, dLAT1.5 had similar or slightly reduced virulence in rabbits (based on survival following ocular infection). In contrast, in mice, dLAT1.5 had increased virulence (P < 0.0001). Thus, deletion of LAT nucleotides 76 to 1667 increased viral virulence in mice but not in rabbits. In contrast, we also report here that LAT2.9A, a LAT mutant that we previously reported to have increased virulence in rabbits (G. C. Perng, S. M. Slanina, A. Yuhkt, B. S. Drolet, W. J. Keleher, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 73:920–929, 1999), had decreased virulence in mice (P = 0.03). In addition, we also found that dLAT371, a LAT mutant that we previously reported to have wild-type virulence in rabbits (G. C. Perng, S. M. Slanina, H. Ghiasi, A. B. Nesburn, and S. L. Wechsler, J. Virol. 70:2014–2018, 1996), had decreased virulence in mice (P < 0.05). Thus, these three mutants, each of which encodes a different LAT RNA, have different virulence phenotypes. dLAT1.5 had wild-type virulence in rabbits but increased virulence in mice. In contrast, LAT2.9A had increased virulence in rabbits but decreased virulence in mice, and dLAT371 had wild-type virulence in rabbits but decreased virulence in mice. Taken together, these results suggest that (i) the 5′ end of LAT and/or a gene that overlaps part of this region is involved in viral virulence, (ii) this virulence appears to have species-specific effects, and (iii) regulation of this virulence may be complex. PMID:11533165

Fecundity of transgenic wild-crop hybrids of Cucurbita pepo (Cucurbitaceae): implications for crop-to-wild gene flow.

PubMed

Spencer, L J; Snow, A A

2001-06-01

Hybridization between crops and their weedy or wild relatives is an area of concern because the widespread use of genetically engineered crops may allow novel, beneficial transgenes to enter nearby populations. We compared fitness components of wild Cucurbita pepo from Arkansas, USA, with wild-crop hybrids derived from yellow squash (a cultivar of C. pepo with transgenic resistance to two viruses). Wild and hybrid progeny were grown in agricultural fields in Arkansas (1996-98) and Ohio (1996) in six similar experiments. Cross types (wild and hybrid) did not differ significantly in seedling survival, which exceeded 85% in all cases. In Ohio, where more detailed observations were made, hybrid plants produced 41% as many male flowers, 21% as many female flowers, and 28% as many seeds as wild plants. At all sites, flowering periods of the two cross types overlapped extensively. Putative virus symptoms were more common in wild plants than in hybrids. Lifetime fecundity varied considerably among sites and years. The average fecundity of hybrids ranged from 453 to 4497 seeds per plant and represented 15% - 53% of the numbers of seeds produced by wild plants in the same experiments. These results suggest that the F1 generation does not represent a strong barrier to the introgression of neutral or beneficial crop genes into free-living populations of C. pepo.

Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

PubMed Central

Oliveira, Sofia A.; Park, Se-Ho; Lee, Peter; Bendelac, Albert; Shenk, Thomas E.

2002-01-01

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient γc/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance. PMID:11752177

Silencing NaTPI Expression Increases Nectar Germin, Nectarins, and Hydrogen Peroxide Levels and Inhibits Nectar Removal from Plants in Nature1[W][OA

PubMed Central

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

Critical role of toll-like receptor 9 in morphine and Mycobacterium tuberculosis-Induced apoptosis in mice.

PubMed

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.

Cellular and molecular mechanisms of autosomal dominant form of progressive hearing loss, DFNA2.

PubMed

Kim, Hyo Jeong; Lv, Ping; Sihn, Choong-Ryoul; Yamoah, Ebenezer N

2011-01-14

Despite advances in identifying deafness genes, determination of the underlying cellular and functional mechanisms for auditory diseases remains a challenge. Mutations of the human K(+) channel hKv7.4 lead to post-lingual progressive hearing loss (DFNA2), which affects world-wide population with diverse racial backgrounds. Here, we have generated the spectrum of point mutations in the hKv7.4 that have been identified as diseased mutants. We report that expression of five point mutations in the pore region, namely L274H, W276S, L281S, G285C, and G296S, as well as the C-terminal mutant G321S in the heterologous expression system, yielded non-functional channels because of endoplasmic reticulum retention of the mutant channels. We mimicked the dominant diseased conditions by co-expressing the wild-type and mutant channels. As compared with expression of wild-type channel alone, the blend of wild-type and mutant channel subunits resulted in reduced currents. Moreover, the combinatorial ratios of wild type:mutant and the ensuing current magnitude could not be explained by the predictions of a tetrameric channel and a dominant negative effect of the mutant subunits. The results can be explained by the dependence of cell surface expression of the mutant on the wild-type subunit. Surprisingly, a transmembrane mutation F182L, which has been identified in a pre-lingual progressive hearing loss patient in Taiwan, yielded cell surface expression and functional features that were similar to that of the wild type, suggesting that this mutation may represent redundant polymorphism. Collectively, these findings provide traces of the cellular mechanisms for DFNA2.

NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.

PubMed Central

De Lorimier, R.; Hellinga, H. W.; Spicer, L. D.

1996-01-01

Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two- and three-dimensional NMR. Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain. The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout. PMID:8976564

Stimulus control by 5-methoxy-N,N-dimethyltryptamine in wild-type and CYP2D6-humanized mice.

PubMed

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.

Dexamethasone-Mediated Changes in Adipose Triacylglycerol Metabolism Are Exaggerated, Not Diminished, in the Absence of a Functional GR Dimerization Domain

PubMed Central

Roohk, Donald J.; Mascharak, Smita; Khambatta, Cyrus; Leung, Ho; Hellerstein, Marc

2013-01-01

The glucocorticoid (GC) receptor (GR) has multiple effector mechanisms, including dimerization-mediated transactivation of target genes via DNA binding and transcriptional repression mediated by protein-protein interactions. Much attention has been focused on developing selective GR modulators that would dissociate adverse effects from therapeutic anti-inflammatory effects. The GRdim/dim mouse has a mutation in the dimerization domain of GR and has been shown to have attenuated transactivation with intact repression. To understand the role of GR dimerization-dependent targets in multiple tissues, we measured metabolic fluxes through several disease-relevant GC target pathways using heavy water labeling and mass spectrometry in wild-type and GRdim/dim mice administered the potent GC dexamethasone (DEX). Absolute triglyceride synthesis was increased in both wild-type and GRdim/dim mice by DEX in the inguinal and epididymal fat depots. GRdim/dim mice showed an exaggerated response to DEX in both depots. De novo lipogenesis was also greatly increased in both depots in response to DEX in GRdim/dim, but not wild-type mice. In contrast, the inhibitory effect of DEX on bone and skin collagen synthesis rates was greater in wild-type compared with GRdim/dim mice. Wild-type mice were more sensitive to DEX-dependent decreases in insulin sensitivity than GRdim/dim mice. Wild-type and GRdim/dim mice were equally sensitive to DEX-dependent decreases in muscle protein synthesis. Chronic elevation of GCs in GRdim/dim mice results in severe runting and lethality. In conclusion, some metabolic effects of GC treatment are exaggerated in adipose tissue of GRdim/dim mice, suggesting that selective GR modulators based on dissociating GR transactivation from repression should be evaluated carefully. PMID:23493372

Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq

PubMed Central

Loeffler-Wirth, Henry; Hopp, Lydia; Schadendorf, Dirk; Schartl, Manfred; Anderegg, Ulf; Camp, Gray; Treutlein, Barbara; Binder, Hans; Kunz, Manfred

2017-01-01

Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy. PMID:27903987

Plant defence responses in oilseed rape MINELESS plants after attack by the cabbage moth Mamestra brassicae

PubMed Central

Ahuja, Ishita; van Dam, Nicole Marie; Winge, Per; Trælnes, Marianne; Heydarova, Aysel; Rohloff, Jens; Langaas, Mette; Bones, Atle Magnar

2015-01-01

The Brassicaceae family is characterized by a unique defence mechanism known as the ‘glucosinolate–myrosinase’ system. When insect herbivores attack plant tissues, glucosinolates are hydrolysed by the enzyme myrosinase (EC 3.2.1.147) into a variety of degradation products, which can deter further herbivory. This process has been described as ‘the mustard oil bomb’. Additionally, insect damage induces the production of glucosinolates, myrosinase, and other defences. Brassica napus seeds have been genetically modified to remove myrosinase-containing myrosin cells. These plants are termed MINELESS because they lack myrosin cells, the so-called toxic mustard oil mines. Here, we examined the interaction between B. napus wild-type and MINELESS plants and the larvae of the cabbage moth Mamestra brassicae. No-choice feeding experiments showed that M. brassicae larvae gained less weight and showed stunted growth when feeding on MINELESS plants compared to feeding on wild-type plants. M. brassicae feeding didn’t affect myrosinase activity in MINELESS plants, but did reduce it in wild-type seedlings. M. brassicae feeding increased the levels of indol-3-yl-methyl, 1-methoxy-indol-3-yl-methyl, and total glucosinolates in both wild-type and MINELESS seedlings. M. brassicae feeding affected the levels of glucosinolate hydrolysis products in both wild-type and MINELESS plants. Transcriptional analysis showed that 494 and 159 genes were differentially regulated after M. brassicae feeding on wild-type and MINELESS seedlings, respectively. Taken together, the outcomes are very interesting in terms of analysing the role of myrosin cells and the glucosinolate–myrosinase defence system in response to a generalist cabbage moth, suggesting that similar studies with other generalist or specialist insect herbivores, including above- and below-ground herbivores, would be useful. PMID:25563968

Interactions between cycloguanil derivatives and wild type and resistance-associated mutant Plasmodium falciparum dihydrofolate reductases

NASA Astrophysics Data System (ADS)

Maitarad, Phornphimon; Kamchonwongpaisan, Sumalee; Vanichtanankul, Jarunee; Vilaivan, Tirayut; Yuthavong, Yongyuth; Hannongbua, Supa

2009-04-01

Comparative molecular field analysis (CoMFA) and quantum chemical calculations were performed on cycloguanil (Cyc) derivatives of the wild type and the quadruple mutant (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) of Plasmodium falciparum dihydrofolate reductase ( PfDHFR). The represented CoMFA models of wild type ( r_{{cv}}2 = 0.727 and r 2 = 0.985) and mutant type ( r_{{cv}}2 = 0.786 and r 2 = 0.979) can describe the differences of the Cyc structural requirements for the two types of PfDHFR enzymes and can be useful to guide the design of new inhibitors. Moreover, the obtained particular interaction energies between the Cyc and the surrounding residues in the binding pocket indicated that Asn108 of mutant enzyme was the cause of Cyc resistance by producing steric clash with p-Cl of Cyc. Consequently, comparing the energy contributions with the potent flexible WR99210 inhibitor, it was found that the key mutant residue, Asn108, demonstrates attractive interaction with this inhibitor and some residues, Leu46, Ile112, Pro113, Phe116, and Leu119, seem to perform as second binding site with WR99210. Therefore, quantum chemical calculations can be useful for investigating residue interactions to clarify the cause of drug resistance.

Mixed Lineage Kinase 3 deficiency delays viral clearance in the lung and is associated with diminished influenza-induced cytopathic effect in infected cells

PubMed Central

Desmet, Emily A.; Hollenbaugh, Joseph A.; Sime, Patricia J.; Wright, Terry W.; Topham, David J.; Sant, Andrea J.; Takimoto, Toru; Dewhurst, Stephen; Maggirwar, Sanjay B.

2010-01-01

Influenza virus leads to acute respiratory disease resulting in seasonal epidemics and periodic pandemics. Little is known about the signaling events that regulate host defense to influenza. One particular pathway, the c-Jun amino-terminal kinase (JNK) cascade is activated following influenza infection and blocking JNK leads to enhanced viral replication. We hypothesize that Mixed Lineage Kinase 3 (MLK3), an upstream regulator of JNK, is involved in the host response to influenza. To test this, wild-type and MLK3−/− mice were infected with pathogenic strain of influenza A virus, A/PR/8/34 (PR8). Although, cellular and humoral immune responses were similar between wild-type and MLK3−/− hosts, the viral load in the lungs was comparatively higher in MLK3−/− mice at day 8 post infection. Consistent with this, MLK3−/− murine lung fibrobalsts had prolonged survival and increased virion production following infection compared to wild-type. These findings support a role for MLK3 in viral production during influenza infection. PMID:20185156

Non-small cell lung cancer patients with EML4-ALK fusion gene are insensitive to cytotoxic chemotherapy.

PubMed

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.

EML1 (CNG-Modulin) Controls Light Sensitivity in Darkness and under Continuous Illumination in Zebrafish Retinal Cone Photoreceptors

PubMed Central

Mehta, Milap; Tserentsoodol, Nomingerel; Postlethwait, John H.; Rebrik, Tatiana I.

2013-01-01

The ligand sensitivity of cGMP-gated (CNG) ion channels in cone photoreceptors is modulated by CNG-modulin, a Ca2+-binding protein. We investigated the functional role of CNG-modulin in phototransduction in vivo in morpholino-mediated gene knockdown zebrafish. Through comparative genomic analysis, we identified the orthologue gene of CNG-modulin in zebrafish, eml1, an ancient gene present in the genome of all vertebrates sequenced to date. We compare the photoresponses of wild-type cones with those of cones that do not express the EML1 protein. In the absence of EML1, dark-adapted cones are ∼5.3-fold more light sensitive than wild-type cones. Previous qualitative studies in several nonmammalian species have shown that immediately after the onset of continuous illumination, cones are less light sensitive than in darkness, but sensitivity then recovers over the following 15–20 s. We characterize light sensitivity recovery in continuously illuminated wild-type zebrafish cones and demonstrate that sensitivity recovery does not occur in the absence of EML1. PMID:24198367

EML1 (CNG-modulin) controls light sensitivity in darkness and under continuous illumination in zebrafish retinal cone photoreceptors.

PubMed

Korenbrot, Juan I; Mehta, Milap; Tserentsoodol, Nomingerel; Postlethwait, John H; Rebrik, Tatiana I

2013-11-06

The ligand sensitivity of cGMP-gated (CNG) ion channels in cone photoreceptors is modulated by CNG-modulin, a Ca(2+)-binding protein. We investigated the functional role of CNG-modulin in phototransduction in vivo in morpholino-mediated gene knockdown zebrafish. Through comparative genomic analysis, we identified the orthologue gene of CNG-modulin in zebrafish, eml1, an ancient gene present in the genome of all vertebrates sequenced to date. We compare the photoresponses of wild-type cones with those of cones that do not express the EML1 protein. In the absence of EML1, dark-adapted cones are ∼5.3-fold more light sensitive than wild-type cones. Previous qualitative studies in several nonmammalian species have shown that immediately after the onset of continuous illumination, cones are less light sensitive than in darkness, but sensitivity then recovers over the following 15-20 s. We characterize light sensitivity recovery in continuously illuminated wild-type zebrafish cones and demonstrate that sensitivity recovery does not occur in the absence of EML1.

Hydrogen Cyanide Produced by Pseudomonas chlororaphis O6 Exhibits Nematicidal Activity against Meloidogyne hapla

PubMed Central

Kang, Beom Ryong; Anderson, Anne J.; Kim, Young Cheol

2018-01-01

Root-knot nematodes (Meloidogyne spp.) are parasites that attack many field crops and orchard trees, and affect both the quantity and quality of the products. A root-colonizing bacterium, Pseudomonas chlororaphis O6, possesses beneficial traits including strong nematicidal activity. To determine the molecular mechanisms involved in the nematicidal activity of P. chlororaphis O6, we constructed two mutants; one lacking hydrogen cyanide production, and a second lacking an insecticidal toxin, FitD. Root drenching with wild-type P. chlororaphis O6 cells caused juvenile mortality in vitro and in planta. Efficacy was not altered in the fitD mutant compared to the wild-type but was reduced in both bioassays for the mutant lacking hydrogen cyanide production. The reduced number of galls on tomato plants caused by the wild-type strain was comparable to that of a standard chemical nematicide. These findings suggest that hydrogen cyanide-producing root colonizers, such as P. chlororaphis O6, could be formulated as “green” nematicides that are compatible with many crops and offer agricultural sustainability. PMID:29422786

Substance P and central respiratory activity: a comparative in vitro study in NK1 receptor knockout and wild-type mice.

PubMed

Ptak, K; Hunt, S P; Monteau, R

2000-07-01

Neurokinin-1 receptors (NK1) are present within the respiratory medullary network and in the phrenic nucleus, which controls the diaphragm. We compared the efficacy of substance P (SP) at inducing changes in respiratory frequency or the amplitude of the respiratory motor output between NK1 knockout (NK1-/-) and wild-type mice, using the in vitro brainstem-spinal cord preparation. The in vitro respiratory frequency, as well as the variability of the rhythm and the amplitude of the motor output were similar in both lines. In wild-type mice, application of exogenous SP induced either an increase in respiratory frequency (superfusion of the medulla) or an increase of the inspiratory motor output, as defined by the integral of C4 cervical ventral root activity (superfusion of the spinal cord). These two effects were not apparent in NK1-/- mice. In conclusion, NK1 receptors mediate the respiratory responses to SP but the lack of NK1 receptors in newborn NK1-/- mice does not change the respiratory activity.

Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

PubMed

Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

2014-07-01

A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

Overexpression of inducible 70-kDa heat shock protein in mouse improves structural and functional recovery of skeletal muscles from atrophy.

PubMed

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.

Targeted overexpression of endothelial nitric oxide synthase in endothelial cells improves cerebrovascular reactivity in Ins2Akita-type-1 diabetic mice.

PubMed

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.

The effects of genetic manipulation, dieldrin treatment and irradiation on the mating competitiveness of male Anopheles arabiensis in field cages.

PubMed

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.

Mating competitiveness of sterile genetic sexing strain males (GAMA) under laboratory and semi-field conditions: Steps towards the use of the Sterile Insect Technique to control the major malaria vector Anopheles arabiensis in South Africa.

PubMed

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.

Inactivation of ferric uptake regulator (Fur) attenuates Helicobacter pylori J99 motility by disturbing the flagellar motor switch and autoinducer-2 production.

PubMed

Lee, Ai-Yun; Kao, Cheng-Yen; Wang, Yao-Kuan; Lin, Ssu-Yuan; Lai, Tze-Ying; Sheu, Bor-Shyang; Lo, Chien-Jung; Wu, Jiunn-Jong

2017-08-01

Flagellar motility of Helicobacter pylori has been shown to be important for the bacteria to establish initial colonization. The ferric uptake regulator (Fur) is a global regulator that has been identified in H. pylori which is involved in the processes of iron uptake and establishing colonization. However, the role of Fur in H. pylori motility is still unclear. Motility of the wild-type, fur mutant, and fur revertant J99 were determined by a soft-agar motility assay and direct video observation. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. Single bacterial motility and flagellar switching were observed by phase-contrast microscopy. Autoinducer-2 (AI-2) production in bacterial culture supernatant was analyzed by a bioluminescence assay. The fur mutant showed impaired motility in the soft-agar assay compared with the wild-type J99 and fur revertant. The numbers and lengths of flagellar filaments on the fur mutant cells were similar to those of the wild-type and revertant cells. Phenotypic characterization showed similar swimming speed but reduction in switching rate in the fur mutant. The AI-2 production of the fur mutant was dramatically reduced compared with wild-type J99 in log-phase culture medium. These results indicate that Fur positively modulates H. pylori J99 motility through interfering with bacterial flagellar switching. © 2017 John Wiley & Sons Ltd.

Single-Cell Microfluidics to Study the Effects of Genome Deletion on Bacterial Growth Behavior.

PubMed

Yuan, Xiaofei; Couto, Jillian M; Glidle, Andrew; Song, Yanqing; Sloan, William; Yin, Huabing

2017-12-15

By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and nongrowing cells) of the clean genome population was also lower. Interestingly, after the random mutations induced by a glucose starvation treatment, for the clean genome population mutants that had survived the competition of chemostat culture, each parameter markedly improved (i.e., the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased). However, this effect was not seen in the wild type strain; the wild type mutants cultured in a chemostat retained a high diversity of growth phenotypes. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population.

Functional mechanism of C-terminal tail in the enzymatic role of porcine testicular carbonyl reductase: a combined experiment and molecular dynamics simulation study of the C-terminal tail in the enzymatic role of PTCR.

PubMed

Son, Minky; Bang, Woo Young; Park, Chanin; Lee, Yuno; Kwon, Seul Gi; Kim, Sam Woong; Kim, Chul Wook; Lee, Keun Woo

2014-01-01

Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR) superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C-terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV). Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT) were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily.

Detrimental Effects of Centrally Administered Angiotensin II are Enhanced in a Mouse Model of Alzheimer Disease Independently of Blood Pressure.

PubMed

Takane, Koki; Hasegawa, Yu; Lin, Bowen; Koibuchi, Nobutaka; Cao, Cheng; Yokoo, Takashi; Kim-Mitsuyama, Shokei

2017-04-20

The significance of brain angiotensin II in Alzheimer disease (AD) is unclear. To examine the role of brain angiotensin II in AD, intracerebroventricular angiotensin II infusion was performed on 5XFAD mice, a mouse model of AD, and wild-type mice, and the detrimental effects of brain angiotensin II was compared between the 2 strains of mice. Intracerebroventricular angiotensin II infusion significantly impaired cognitive function in 5XFAD mice but not in wild-type mice. This vulnerability of 5XFAD mice to brain angiotensin II was associated with enhancement of hippocampal inflammation and oxidative stress and with increased cerebrovascular amyloid β deposition. We also compared the effect of brain angiotensin II on the heart and skeletal muscle between the 2 strains because AD is associated with heart failure and sarcopenia. We found that cardiac compensatory response of 5XFAD mice to brain angiotensin II-induced hypertension was less than that of wild-type mice. Brain angiotensin II caused skeletal muscle atrophy and injury in 5XFAD mice more than in wild-type mice. Brain angiotensin II seems to be involved in cognitive impairment and brain injury in AD, which is associated with oxidative stress, inflammation, and cerebral amyloid angiopathy. Further, brain angiotensin II may participate in cardiac disease and sarcopenia observed in AD. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

The Klebsiella pneumoniae O Antigen Contributes to Bacteremia and Lethality during Murine Pneumonia

PubMed Central

Shankar-Sinha, Sunita; Valencia, Gabriel A.; Janes, Brian K.; Rosenberg, Jessica K.; Whitfield, Chris; Bender, Robert A.; Standiford, Ted J.; Younger, John G.

2004-01-01

Bacterial surface carbohydrates are important pathogenic factors in gram-negative pneumonia infections. Among these factors, O antigen has been reported to protect pathogens against complement-mediated killing. To examine further the role of O antigen, we insertionally inactivated the gene encoding a galactosyltransferase necessary for serotype O1 O-antigen synthesis (wbbO) from Klebsiella pneumoniae 43816. Analysis of the mutant lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the absence of O antigen. In vitro, there were no detectable differences between wild-type K. pneumoniae and the O-antigen-deficient mutant in regard to avid binding by murine complement C3 or resistance to serum- or whole-blood-mediated killing. Nevertheless, the 72-h 50% lethal dose of the wild-type strain was 30-fold greater than that of the mutant (2 × 103 versus 6 × 104 CFU) after intratracheal injection in ICR strain mice. Despite being less lethal, the mutant organism exhibited comparable intrapulmonary proliferation at 24 h compared to the level of the wild type. Whole-lung chemokine expression (CCL3 and CXCL2) and bronchoalveolar inflammatory cell content were also similar between the two infections. However, whereas the wild-type organism produced bacteremia within 24 h of infection in every instance, bacteremia was not seen in mutant-infected mice. These results suggest that during murine pneumonia caused by K. pneumoniae, O antigen contributes to lethality by increasing the propensity for bacteremia and not by significantly changing the early course of intrapulmonary infection. PMID:14977947

Functional Mechanism of C-Terminal Tail in the Enzymatic Role of Porcine Testicular Carbonyl Reductase: A Combined Experiment and Molecular Dynamics Simulation Study of the C-Terminal Tail in the Enzymatic Role of PTCR

PubMed Central

Park, Chanin; Lee, Yuno; Kwon, Seul Gi; Kim, Sam Woong; Kim, Chul Wook; Lee, Keun Woo

2014-01-01

Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR) superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C- terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV). Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT) were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily. PMID:24646606

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hitomi, Toshiaki; Habu, Toshiyuki; Kobayashi, Hatasu

Highlights: •Overexpression of RNF213 R4810K inhibited cell proliferation. •Overexpression of RNF213 R4810K had the time of mitosis 4-fold and mitotic failure. •R4810K formed a complex with MAD2 more readily than wild-type. •iPSECs from the MMD patients had elevated mitotic failure compared from the control. •RNF213 R4810K induced mitotic abnormality and increased risk of aneuploidy. -- Abstract: Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cellmore » cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n = 3, 61.0 ± 8.2%) compared with wild-type subjects (n = 6, 13.1 ± 7.7%; p < 0.01). Aneuploidy was observed more frequently in fibroblasts (p < 0.01) and induced pluripotent stem cells (iPSCs) (p < 0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p < 0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p < 0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.« less

Adaptive evolution of Desulfovibrio alaskensis G20 for developing resistance to perchlorate

NASA Astrophysics Data System (ADS)

Mehta-Kolte, M. G.; Youngblut, M.; Redford, S.; Gregoire, P.; Carlson, H. K.; Coates, J. D.

2015-12-01

Due to its toxic, explosive, and corrosive nature, inadvertent biological H2S production by sulfate reducing microorganisms (SRM) poses significant health and industrial operational risks. Anthropogenic sources are dominated by the oil industry where H2S in reservoir gases and fluids has an associated annual cost estimated at $90 billion globally. Our previous studies have identified perchlorate (ClO4-) as a selective and potent inhibitor of SRM in pure culture and complex microbial ecosystems. However, constant addition of inhibitors like perchlorate to natural ecosystems may result in a new adaptive selective pressure on SRM populations. With this in mind we investigated the ability of Desulfovibrio alaskensis G20, a model oil reservoir SRM, to adapt to perchlorate and develop a resistance. Serial transfers of three parallel cultures with increasing concentrations of perchlorate up to 100 mM were generated and compared to wild-type strains that were transferred for same number of generations in absence of perchlorate. Genome sequencing revealed that all three adapted strains had single non-synonymous single-nucleotide polymorphisms in the same gene, Dde_2265, the sulfate adenylytransferase (ATP sulfurylase (ATPS)) (EC 2.7.7.4). ATPS catalyzes the first committed step in sulfate reduction and is essential in all SRM. IC50s against growth for these evolved strains demonstrated a three-fold increased resistance to perchlorate compared to wild-type controls. These evolved strains also had 5x higher transcriptional abundance of Dde_2265 compared to the wild-type strain. Biochemical characterization of the purified ATPS enzyme from both wild-type and the evolved strain showed that the mutant ATPS from the evolved strain was resistant to perchlorate inhibition of ATP turnover with a KI for perchlorate that was 3x greater relative to the wild-type ATPS. These results demonstrate that a single-base pair mutation in ATPS can have a significant impact on developing resistance to perchlorate and suggest that adaptive evolution is a valuable tool to understand potential responses of microorganism to any environmental perturbations imposed during oil production.

Toll-like receptor 4 regulates lipopolysaccharide-induced inflammation and lactation insufficiency in a mouse model of mastitis.

PubMed

Glynn, Danielle J; Hutchinson, Mark R; Ingman, Wendy V

2014-05-01

Lactation mastitis is a debilitating inflammatory breast disease in postpartum women. Disease severity is associated with markers of inflammation rather than bacterial load, suggesting that immune-signaling pathways activated in the host are important in the disease pathology. The role of the innate pattern recognition receptor toll-like receptor 4 (TLR4) in progression and resolution of mastitislike disease was investigated in a mouse model. Lipopolysaccharide in Matrigel (10 μg/10 μl) was administered into the teat canal of lactating Tlr4 null mutant and wild-type mice to induce a localized area of inflammation. Mastitis induction resulted in a marked influx of RB6-positive neutrophils and F4/80-positive macrophages, which was higher in Tlr4(-/-) mice compared to wild-type mice. Tlr4 null mutation resulted in an altered immune-signaling fingerprint following induction of mastitis, with attenuated serum cytokines, including CXCL1, CCL2, interleukin 1 beta, and tumor necrosis factor alpha compared to wild-type mice. In both genotypes, the localized area of inflammation had resolved after 7 days, and milk protein was evident. However, the mammary glands of wild-type mice exhibited reduced capacity for milk production, with decreased percent area populated with glandular epithelium and decreased abundance of nuclear phosphorylated signal transducer and activator of transcription 5 compared to Tlr4 null mice. This study demonstrates that inflammatory pathways activated in the host are critically important in mastitis disease progression and suggests that lactation insufficiency associated with mastitis may be a consequence of TLR4-mediated inflammation, rather than the bacterial infection itself.

The role of strigolactones in photomorphogenesis of pea is limited to adventitious rooting.

PubMed

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.

A CAM- and starch-deficient mutant of the facultative CAM species Mesembryanthemum crystallinum reconciles sink demands by repartitioning carbon during acclimation to salinity

PubMed Central

Haider, Muhammad Sajjad; Barnes, Jeremy D.; Cushman, John C.; Borland, Anne M.

2012-01-01

In the halophytic species Mesembryanthemum crystallinum, the induction of crassulacean acid metabolism (CAM) by salinity requires a substantial investment of resources in storage carbohydrates to provide substrate for nocturnal CO2 uptake. Acclimation to salinity also requires the synthesis and accumulation of cyclitols as compatible solutes, maintenance of root respiration, and nitrate assimilation. This study assessed the hierarchy and coordination of sinks for carbohydrate in leaves and roots during acclimation to salinity in M. crystallinum. By comparing wild type and a CAM-/starch-deficient mutant of this species, it was sought to determine if other metabolic sinks could compensate for a curtailment in CAM and enable acclimation to salinity. Under salinity, CAM deficiency reduced 24 h photosynthetic carbon gain by >50%. Cyclitols were accumulated to comparable levels in leaves and roots of both the wild type and mutant, but represented only 5% of 24 h carbon balance. Dark respiration of leaves and roots was a stronger sink for carbohydrate in the mutant compared with the wild type and implied higher maintenance costs for the metabolic processes underpinning acclimation to salinity when CAM was curtailed. CAM required the nocturnal mobilization of >70% of primary carbohydrate in the wild type and >85% of carbohydrate in the mutant. The substantial allocation of carbohydrate to CAM limited the export of sugars to roots, and the root:shoot ratio declined under salinity. The data suggest a key role for the vacuole in regulating the supply and demand for carbohydrate over the day/night cycle in the starch-/CAM-deficient mutant. PMID:22219316

Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

NASA Technical Reports Server (NTRS)

Ishikawa, H.; Evans, M. L.

1997-01-01

In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.

Manipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin

PubMed Central

Lawrence, Sara L.; Feil, Susanne C.; Holien, Jessica K.; Kuiper, Michael J.; Doughty, Larissa; Dolezal, Olan; Mulhern, Terrence D.; Tweten, Rodney K.; Parker, Michael W.

2012-01-01

The cholesterol-dependent cytolysins (CDCs) attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modeling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases. PMID:23181061

Norrin mediates angiogenic properties via the induction of insulin-like growth factor-1.

PubMed

Zeilbeck, Ludwig F; Müller, Birgit B; Leopold, Stephanie A; Senturk, Berna; Langmann, Thomas; Tamm, Ernst R; Ohlmann, Andreas

2016-04-01

Norrin is an angiogenic signaling molecule that activates canonical Wnt/β-catenin signaling, and is involved in capillary formation in retina and brain. Moreover, Norrin induces vascular repair following an oxygen-induced retinopathy (OIR), the model of retinopathy of prematurity in mice. Since insulin-like growth factor (IGF)-1 is a very potent angiogenic molecule, we investigated if IGF-1 is a downstream mediator of Norrin's angiogenic properties. In retinae of transgenic mice with an ocular overexpression of Norrin (βB1-Norrin), we found at postnatal day (P)11 a significant increase of IGF-1 mRNA compared to wild-type littermates. In addition, after treatment of cultured Müller cells or dermal microvascular endothelial cells with Norrin we observed an increase of IGF-1 and its mRNA, an effect that could be blocked with DKK-1, an inhibitor of Wnt/β-catenin signaling. When OIR was induced, the expression of IGF-1 was significantly suppressed in both transgenic βB1-Norrin mice and wild-type littermates when compared to wild-type animals that were housed in room air. Furthermore, at P13, one day after the mice had returned to normoxic conditions, IGF-1 levels were significantly higher in transgenic mice compared to wild-type littermates. Finally, after intravitreal injections of inhibitory α-IGF-1 antibodies at P12 or at P12 and P14, the Norrin-mediated vascular repair was significantly attenuated. We conclude that Norrin induces the expression of IGF-1 via an activation of the Wnt/β-catenin signaling pathway, an effect that significantly contributes to the protective effects of Norrin against an OIR. Copyright © 2015 Elsevier Ltd. All rights reserved.

Theiler's virus-infected L-selectin-deficient mice have decreased infiltration of CD8(+) T lymphocytes in central nervous system but clear the virus.

PubMed

Zhang, X; Brewer, L; Walcheck, B; Johnson, A; Pease, L R; Njenga, M K

2001-06-01

Mice with targeted deletion of L-selectin gene (L-sel(-/-)) were used to investigate the role of adhesion molecule in immunologic responses following virus infection in the central nervous system (CNS). L-Sel(-/-) mice from a resistant H-2(b) genetic background and parental wild-type H-2(b) (C57BL/6) mice were infected with Theiler's murine encephalomyelitis virus (TMEV) intracerebrally and the kinetics of virus replication and infiltration of immune cells in the CNS determined. The levels of infectious TMEV, as measured by plaque assay at 3, 7, 14, and 28 days after infection were between 4 and 6 log(10) PFU of virus per gram of CNS tissues at days 3 and 7 post-infection, and then decreased to undetectable levels by day 14 after infection in both strains of mice. The L-sel(-/-) mice had decreased numbers of CD8(+) T lymphocytes (17.72%+/-2.4) infiltrating into the CNS at 7 days post-infection when compared to wild-type mice (31.02%+/-7.5). In addition, the L-sel(-/-) mice had significantly lower levels of TMEV-specific serum IgG resulting in lower virus neutralizing activity of the serum when compared to wild-type mice. However, the L-sel(-/-) mice had 2.5-fold increase in B lymphocytes in the CNS (8.29%+/-1.1) when compared to wild-type mice (3.2%+/-0.4). Taken together, these data indicate that L-selectin plays a role in recruitment of B and CD8(+) T lymphocytes into the CNS following virus infection, which, however, did not affect the ability of the mice to clear TMEV infection.

Methionine flux to transsulfuration is enhanced in the long living Ames dwarf mouse

PubMed Central

Uthus, Eric O.; Brown-Borg, Holly M.

2007-01-01

Long-lived Ames dwarf mice lack growth hormone, prolactin, and thyroid stimulating hormone. Additionally the dwarf mice have enzyme activities and levels that combat oxidative stress more efficiently than those of normal mice. We have shown that methionine metabolism in Ames mice is markedly different than in their wild type littermates. In our previous work we hypothesized that the flux of methionine to the transsulfuration pathway is enhanced in the dwarf mice. The current study was designed to determine whether the flux of methionine to the transsulfuration pathway is increased. We did this by injecting either l-[methyl-3H]-methionine or l-[35S]-methionine into dwarf or normal mice and then determined retained label (in form of S-adenosylmethionine) 45 min later. The amount of retained hepatic 3H and 35S label was significantly reduced in the dwarf mice; at 45 min the specific radioactivity of SAM (pCi/nmol SAM) was 56% lower (p < 0.05) for 3H-label and 64% lower (p < 0.005) for 35S-label in dwarf than wild type mice. Retention of 35S was significantly lower in the brain (37%, p < 0.04) and kidney (47%, p < 0.02) of the dwarf compared to wild type mice; there was no statistical difference in retained 3H-label in either brain or kidney. This suggests that both the methyl-moiety and the carbon chain of methionine are lost much faster in the dwarf compared to the wild type mouse, implying that both transmethylation in the liver and transsulfuration in the liver, brain, and kidney are increased in the dwarf mice. As further support, we determined by real-time RT PCR the expression of methionine metabolism genes in livers of mice. Compared to wild type, the Ames dwarf had increased expression of methionine adenosyltransferase 1a (2.3-fold, p = 0.013), glycine N-methyltransferase (3.8-fold, p = 0.023), betaine homocysteine methyltransferase (5.5-fold, p = 0.0006), S-adenosylhomocysteine hydrolase (3.8-fold, p = 0.0005), and cystathionase (2.6-fold; tended to be increased, p = 0.055). Methionine synthase expression was significantly decreased in dwarf compared to wild type (0.48-fold, p = 0.023). These results confirm that the flux of methionine to transsulfuration is enhanced in the Ames dwarf. This, along with data from previous studies support the hypothesis that altered methionine metabolism plays a significant role in the oxidative defense of the dwarf mouse and that the mechanism for the enhanced oxidative defense may be through altered GSH metabolism as a result of the distinctive methionine metabolism. PMID:16519922

Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

PubMed

Noguchi, Fumihito; Nakajima, Takeshi; Inui, Shigeki; Reddy, Janardan K; Itami, Satoshi

2014-01-01

MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair follicle stem cells to epidermal regeneration after wounding in 6-month-old Med1(epi-/-) mice. This study sheds light on the novel function of MED1 in keratinocytes and suggests a possible new therapeutic approach for skin wound healing and aging.

First report of wild boar susceptibility to Porcine circovirus type 3: High prevalence in the Colli Euganei Regional Park (Italy) in the absence of clinical signs.

PubMed

Franzo, Giovanni; Tucciarone, Claudia Maria; Drigo, Michele; Cecchinato, Mattia; Martini, Marco; Mondin, Alessandra; Menandro, Maria Luisa

2018-05-18

The genus Circovirus includes one of the most relevant infectious agents affecting domestic pigs, Porcine circovirus type 2 (PCV-2). The wild boar susceptibility to this pathogen has also been demonstrated although the actual epidemiological role of wild populations is still debated. In recent times, a new circovirus, Porcine circovirus type 3 (PCV-3), has been discovered and reported in the presence of several clinical conditions. However, no information is currently available about PCV-3 circulation and prevalence in wild boar. To fill this gap, 187 wild boar serum samples were collected in the Colli Euganei Regional Park (Northern Italy) and screened for PCV-3, demonstrating a high viral prevalence (approximately 30%). No gender differences were demonstrated while a lower infection prevalence was observed in animals younger than 12 months compared to older ones, differently from what described in commercial pigs. Almost all sampled animals were in good health conditions and no association was proven between PCV-3 status and clinical syndromes in wild animals. The genetic characterization of selected strains enlightened a relevant variability and the absence of closely related strains originating from domestic pigs. Therefore, the observed scenario is suggestive of multiple introductions from other wild or domestic swine populations followed by prolonged circulation and independent evolution. Worldwide, this study reports for the first time the high susceptibility of the wild boar to PCV-3 infection. The high prevalence and the absence of association with clinical signs support the marginal role of this virus in the wild boar population ecology. However, its epidemiological role as reservoir endangering commercial swine cannot be excluded and will require further investigations. © 2018 Blackwell Verlag GmbH.

Plaque and growth characteristics of different polioviruses isolated from acute flaccid paralysis in Northern Nigeria.

PubMed

Sule, W F; Oyedele, O I; Osei-Kwasi, M; Odoom, J K; Adu, F D

2008-03-01

To determine some virulent trait-related properties of poliovirus isolates from children with acute flaccid paralysis following vaccination with oral polio vaccine (OPV). Six polioviruses earlier characterised into wild, vaccine-derived and OPV-like were studied using the plaque morphology and growth kinetics at supra-optimal temperature. Department of Virology, University of Ibadan, Nigeria. Polio isolates from six children who developed acute flaccid paralysis following vaccinations with various doses of OPV were used. All the children were located in the Northern part of the country where poliovirus is still circulating. The two vaccine-derived polioviruses acquired wild type characteristics. All the six poliovirus isolates developed different forms of plaques ranging from tiny, small and large. The plaque formed could however not be used to identify the different isolates. Growth of the different isolates at supra-optimal temperature showed that the three wild polioviruses grew to a higher titre when compared with the Sabin 2 control. The two vaccine derived isolates behaved like the wild poliovirus while the OPV-like virus acquired an intermediate characteristics between wild and sabin. The wild polioviruses represented in this study are among the last vestiges of the circulating polioviruses found in the world. It is possible that the observed biological properties of wild types 1 and 3 described in the study are typical of the West African polioviruses. These properties will provide useful previews to the final identification of some important clinical isolates especially type 1 which may grow rapidly in cell culture.

Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase.

PubMed

Miyazawa, Ken; Yoshimi, Akira; Zhang, Silai; Sano, Motoaki; Nakayama, Mayumi; Gomi, Katsuya; Abe, Keietsu

2016-09-01

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.

Cloning and expression of cyclodextrin glycosyltransferase gene from Paenibacillus sp. T16 isolated from hot spring soil in northern Thailand.

PubMed

Charoensakdi, Ratiya; Murakami, Shuichiro; Aoki, Kenji; Rimphanitchayakit, Vichien; Limpaseni, Tipaporn

2007-05-31

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.

Distinct Motion of GFP-Tagged Histone Expressing Cells Under AC Electrokinetics in Electrode-Multilayered Microfluidic Device.

PubMed

Yao, Jiafeng; Sugawara, Michiko; Obara, Hiromichi; Mizutani, Takeomi; Takei, Masahiro

2017-12-01

The distinct motion of GFP-tagged histone expressing cells (Histone-GFP type cells) has been investigated under ac electrokinetics in an electrode-multilayered microfluidic device as compared with Wild type cells and GFP type cells in terms of different intracellular components. The Histone-GFP type cells were modified by the transfection of green fluorescent protein-fused histone from the human lung fibroblast cell line. The velocity of the Histone-GFP type cells obtained by particle tracking velocimetry technique is faster than Wild type cells by 24.9% and GFP type cells by 57.1%. This phenomenon is caused by the more amount of proteins in the intracellular of single Histone-GFP type cell than that of the Wild type and GFP type cells. The more amount of proteins in the Histone-GFP type cells corresponds to a lower electric permittivity ϵ c of the cells, which generates a lower dielectrophoretic force exerting on the cells. The velocity of Histone-GFP type cells is well agreed with Eulerian-Lagrangian two-phase flow simulation by 4.2% mean error, which proves that the fluid motion driven by thermal buoyancy and electrothermal force dominates the direction of cells motion, while the distinct motion of Histone-GFP type cells is caused by dielectrophoretic force. The fluid motion does not generate a distinct drag motion for Histone-GFP type cells because the Histone-GFP type cells have the same size to the Wild type and GFP type cells. These results clarified the mechanism of cells motion in terms of intracellular components, which helps to improve the cell manipulation efficiency with electrokinetics.

Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance.

PubMed

Nishimoto, Sachiko; Fukuda, Daiju; Higashikuni, Yasutomi; Tanaka, Kimie; Hirata, Yoichiro; Murata, Chie; Kim-Kaneyama, Joo-Ri; Sato, Fukiko; Bando, Masahiro; Yagi, Shusuke; Soeki, Takeshi; Hayashi, Tetsuya; Imoto, Issei; Sakaue, Hiroshi; Shimabukuro, Michio; Sata, Masataka

2016-03-01

Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9 (-/-) ) macrophages. Fat-fed Tlr9 (-/-) mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9 (-/-) mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography-determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance.

wPip Wolbachia contribution to Aedes albopictus SIT performance: Advantages under intensive rearing.

PubMed

Puggioli, Arianna; Calvitti, Maurizio; Moretti, Riccardo; Bellini, Romeo

2016-12-01

As a part of a project aiming at the suppression of the mosquito vector Aedes albopictus, a specific Ae. albopictus line producing sterile males, ARwP, was tested for its suitability to intense rearing conditions compatible with mass production and field release. This line was developed by the Italian National Agency for New Technologies, Energy and Sustainable Economic Development thanks to the artificial infection with a heterologous Wolbachia strain, resulting in a bidirectional incompatibility pattern with wild-type Ae. albopictus. ARwP was reared under Standard Operating Procedures at the Centro Agricoltura Ambiente and compared with a wild-type strain in terms of time of pupation onset, production of male pupae in the following 24h and mechanical sexing efficacy. Mating competitiveness of ARwP males was also evaluated in comparison with irradiated wild-type males in large field enclosures. ARwP males demonstrated a significantly shorter time of pupation onset, a higher rate of production of male pupae in the following 24h and a lower percentage of residual contaminant females when applying mechanical sexing procedures. In addition, ARwP males were more efficient than wild-types in competing for wild-type females in large enclosures, thus inducing a level of sterility significantly higher than that expected for an equal mating competitiveness. These results encourage the use of this Ae. albopictus strain as suppression tool against Ae. albopictus based on considerations thoroughly discussed in the manuscript. Copyright © 2016 Elsevier B.V. All rights reserved.

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit

PubMed Central

Yang, Ying; Yang, Feng; Li, Yan-Hong; Liu, He-Ping; Chen, Xiao-Yan

2017-01-01

Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade plant cell wall. To evaluate whether the deletion of an exo-polygalacturonase gene pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.15 (wild type) via homologous recombination. The ΔpgxB mutant showed similar growth behavior compared with the wild type. Pectin medium induced significant higher expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. However, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused by ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, gene expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results prove that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple fruit, suggesting that pgxB plays an important role in the infection process on the apple fruit. PMID:28257463

Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum.

PubMed

Otero, Joel H; Lizák, Beata; Feige, Matthias J; Hendershot, Linda M

2014-10-03

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential proteomic responses of selectively bred and wild-type Sydney rock oyster populations exposed to elevated CO2.

PubMed

Thompson, E L; O'Connor, W; Parker, L; Ross, P; Raftos, D A

2015-03-01

Previous work suggests that larvae from Sydney rock oysters that have been selectively bred for fast growth and disease resistance are more resilient to the impacts of ocean acidification than nonselected, wild-type oysters. In this study, we used proteomics to investigate the molecular differences between oyster populations in adult Sydney rock oysters and to identify whether these form the basis for observations seen in larvae. Adult oysters from a selective breeding line (B2) and nonselected wild types (WT) were exposed for 4 weeks to elevated pCO2 (856 μatm) before their proteomes were compared to those of oysters held under ambient conditions (375 μatm pCO2 ). Exposure to elevated pCO2 resulted in substantial changes in the proteomes of oysters from both the selectively bred and wild-type populations. When biological functions were assigned, these differential proteins fell into five broad, potentially interrelated categories of subcellular functions, in both oyster populations. These functional categories were energy production, cellular stress responses, the cytoskeleton, protein synthesis and cell signalling. In the wild-type population, proteins were predominantly upregulated. However, unexpectedly, these cellular systems were downregulated in the selectively bred oyster population, indicating cellular dysfunction. We argue that this reflects a trade-off, whereby an adaptive capacity for enhanced mitochondrial energy production in the selectively bred population may help to protect larvae from the effects of elevated CO2 , whilst being deleterious to adult oysters. © 2015 John Wiley & Sons Ltd.

The Arabidopsis aba4-1 mutant reveals a specific function for neoxanthin in protection against photooxidative stress.

PubMed

Dall'Osto, Luca; Cazzaniga, Stefano; North, Helen; Marion-Poll, Annie; Bassi, Roberto

2007-03-01

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions.

The Arabidopsis aba4-1 Mutant Reveals a Specific Function for Neoxanthin in Protection against Photooxidative Stress[W

PubMed Central

Dall'Osto, Luca; Cazzaniga, Stefano; North, Helen; Marion-Poll, Annie; Bassi, Roberto

2007-01-01

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions. PMID:17351115

Dissection of Structural and Functional Requirements That Underlie the Interaction of ERdj3 Protein with Substrates in the Endoplasmic Reticulum*

PubMed Central

Otero, Joel H.; Lizák, Beata; Feige, Matthias J.; Hendershot, Linda M.

2014-01-01

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates. PMID:25143379

A recurrent KCNQ2 pore mutation causing early onset epileptic encephalopathy has a moderate effect on M current but alters subcellular localization of Kv7 channels.

PubMed

Abidi, Affef; Devaux, Jérôme J; Molinari, Florence; Alcaraz, Gisèle; Michon, François-Xavier; Sutera-Sardo, Julie; Becq, Hélène; Lacoste, Caroline; Altuzarra, Cécilia; Afenjar, Alexandra; Mignot, Cyril; Doummar, Diane; Isidor, Bertrand; Guyen, Sylvie N; Colin, Estelle; De La Vaissière, Sabine; Haye, Damien; Trauffler, Adeline; Badens, Catherine; Prieur, Fabienne; Lesca, Gaetan; Villard, Laurent; Milh, Mathieu; Aniksztejn, Laurent

2015-08-01

Mutations in the KCNQ2 gene encoding the voltage-dependent potassium M channel Kv7.2 subunit cause either benign epilepsy or early onset epileptic encephalopathy (EOEE). It has been proposed that the disease severity rests on the inhibitory impact of mutations on M current density. Here, we have analyzed the phenotype of 7 patients carrying the p.A294V mutation located on the S6 segment of the Kv7.2 pore domain (Kv7.2(A294V)). We investigated the functional and subcellular consequences of this mutation and compared it to another mutation (Kv7.2(A294G)) associated with a benign epilepsy and affecting the same residue. We report that all the patients carrying the p.A294V mutation presented the clinical and EEG characteristics of EOEE. In CHO cells, the total expression of Kv7.2(A294V) alone, assessed by western blotting, was only 20% compared to wild-type. No measurable current was recorded in CHO cells expressing Kv7.2(A294V) channel alone. Although the total Kv7.2(A294V) expression was rescued to wild-type levels in cells co-expressing the Kv7.3 subunit, the global current density was still reduced by 83% compared to wild-type heteromeric channel. In a configuration mimicking the patients' heterozygous genotype i.e., Kv7.2(A294V)/Kv7.2/Kv7.3, the global current density was reduced by 30%. In contrast to Kv7.2(A294V), the current density of homomeric Kv7.2(A294G) was not significantly changed compared to wild-type Kv7.2. However, the current density of Kv7.2(A294G)/Kv7.2/Kv7.3 and Kv7.2(A294G)/Kv7.3 channels were reduced by 30% and 50% respectively, compared to wild-type Kv7.2/Kv7.3. In neurons, the p.A294V mutation induced a mislocalization of heteromeric mutant channels to the somato-dendritic compartment, while the p.A294G mutation did not affect the localization of the heteromeric channels to the axon initial segment. We conclude that this position is a hotspot of mutation that can give rise to a severe or a benign epilepsy. The p.A294V mutation does not exert a dominant-negative effect on wild-type subunits but alters the preferential axonal targeting of heteromeric Kv7 channels. Our data suggest that the disease severity is not necessarily a consequence of a strong inhibition of M current and that additional mechanisms such as abnormal subcellular distribution of Kv7 channels could be determinant. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of various physiological traits in flies (Drosophila melanogaster) of wild origin, infected or uninfected by the hereditary Rhabdovirus sigma.

PubMed

Fleuriet, A

1981-01-01

Flies infected or uninfected by the hereditary Rhabdovirus sigma have been collected in the natural French populations of Drosophila melanogaster. The have been compared for various physiological traits: male and female fertility, female longevity, sexual selection and egg viability. The only significant difference was the lower viability of eggs laid by infected females. For all the other traits, infected and uninfected flies were quite comparable. The viral types found in flies of wild origin, thus appear to be almost harmless for their carriers. This result can be connected with previous findings which gave evidence for the relative infrequency of infectious particles in stabilized flies of wild origin.

Attenuated Stress Response to Acute Restraint and Forced Swimming Stress in Arginine Vasopressin 1b Receptor Subtype (Avpr1b) Receptor Knockout Mice and Wild-Type Mice Treated with a Novel Avpr1b Receptor Antagonist

PubMed Central

Roper, J A; Craighead, M; O’Carroll, A-M; Lolait, S J

2010-01-01

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. PMID:20846299

Attenuated stress response to acute restraint and forced swimming stress in arginine vasopressin 1b receptor subtype (Avpr1b) receptor knockout mice and wild-type mice treated with a novel Avpr1b receptor antagonist.

PubMed

Roper, J A; Craighead, M; O'Carroll, A-M; Lolait, S J

2010-11-01

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. © 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.

A quorum sensing-defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems.

PubMed

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.

Mumps-specific cross-neutralization by MMR vaccine-induced antibodies predicts protection against mumps virus infection.

PubMed

Gouma, Sigrid; Ten Hulscher, Hinke I; Schurink-van 't Klooster, Tessa M; de Melker, Hester E; Boland, Greet J; Kaaijk, Patricia; van Els, Cécile A C M; Koopmans, Marion P G; van Binnendijk, Rob S

2016-07-29

Similar to other recent mumps genotype G outbreaks worldwide, most mumps patients during the recent mumps genotype G outbreaks in the Netherlands had received 2 doses of measles, mumps and rubella (MMR) vaccine during childhood. Here, we investigate the capacity of vaccine-induced antibodies to neutralize wild type mumps virus strains, including mumps virus genotype G. In this study, we tested 105 pre-outbreak serum samples from students who had received 2 MMR vaccine doses and who had no mumps virus infection (n=76), symptomatic mumps virus infection (n=10) or asymptomatic mumps virus infection (n=19) during the mumps outbreaks. In all samples, mumps-specific IgG concentrations were measured by multiplex immunoassay and neutralization titers were measured against the Jeryl Lynn vaccine strain and against wild type genotype G and genotype D mumps virus strains. The correlation between mumps-specific IgG concentrations and neutralization titers against Jeryl Lynn was poor, which suggests that IgG concentrations do not adequately represent immunological protection against mumps virus infection by antibody neutralization. Pre-outbreak neutralization titers in infected persons were significantly lower against genotype G than against the vaccine strain. Furthermore, antibody neutralization of wild type mumps virus genotype G and genotype D was significantly reduced in pre-outbreak samples from infected persons as compared with non-infected persons. No statistically significant difference was found for the vaccine strain. The sensitivity/specificity ratio was largest for neutralization of the genotype G strain as compared with the genotype D strain and the vaccine strain. The reduced neutralization of wild type mumps virus strains in MMR vaccinated persons prior to infection indicates that pre-outbreak mumps virus neutralization is partly strain-specific and that neutralization differs between infected and non-infected persons. Therefore, we recommend the use of wild type mumps virus neutralization assays as preferred tool for surveillance of protection against mumps virus infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

PubMed

Stephen, Abish S; Millhouse, Emma; Sherry, Leighann; Aduse-Opoku, Joseph; Culshaw, Shauna; Ramage, Gordon; Bradshaw, David J; Burnett, Gary R; Allaker, Robert P

2016-01-01

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.

Regulation of cytokine polarization and T cell recruitment to inflamed paws in mouse collagen-induced arthritis by the chemokine receptor CXCR6.

PubMed

Slauenwhite, Drew; Gebremeskel, Simon; Doucette, Carolyn D; Hoskin, David W; Johnston, Brent

2014-11-01

The chemokine receptor CXCR6 is highly expressed on lymphocytes isolated from the synovium of patients with rheumatoid arthritis, psoriatic arthritis, or juvenile idiopathic arthritis, suggesting that CXCR6 regulates immune cell activation or infiltration into arthritic joints. This study was undertaken to examine the role of CXCR6 in T cell activation and arthritis development. A collagen-induced arthritis model was used to examine arthritis development in wild-type and CXCR6(-/-) mice. CXCR6 expression, lymphocyte accumulation, and intracellular cytokine production were examined by flow cytometry. Collagen-specific antibodies were measured in the serum. Collagen-specific recall responses were examined in vitro via proliferation and cytokine release assays. T cell homing to inflamed joints was examined using competitive adoptive transfer of dye-labeled lymphocytes from wild-type and CXCR6(-/-) mice. The numbers of CXCR6+ T cells were increased in the paws and draining lymph nodes of arthritic mice. The incidence of arthritis, disease severity, extent of T cell accumulation, and levels of collagen-specific IgG2a antibodies were significantly reduced in CXCR6(-/-) mice compared to wild-type mice. T cells from wild-type mice exhibited Th1 (interferon-γ [IFNγ]) polarization in the inguinal lymph nodes following immunization. At disease peak, this shifted to a Th17 (interleukin-17A [IL-17A]) response in the popliteal lymph nodes. T cells in CXCR6(-/-) mice exhibited impaired cytokine polarization, resulting in a decreased frequency and number of IL-17A- and IFNγ-producing cells. Recruitment of activated CXCR6(-/-) mouse T cells to the inflamed paws was impaired compared to recruitment of wild-type mouse T cells. These experiments demonstrate that CXCR6 plays important roles in the pathogenesis of arthritis through its effects on both T cell cytokine polarization and homing of T cells to inflamed joints. Copyright © 2014 by the American College of Rheumatology.

Disturbed hepatic carbohydrate management during high metabolic demand in medium-chain acyl-CoA dehydrogenase (MCAD)-deficient mice.

PubMed

Herrema, Hilde; Derks, Terry G J; van Dijk, Theo H; Bloks, Vincent W; Gerding, Albert; Havinga, Rick; Tietge, Uwe J F; Müller, Michael; Smit, G Peter A; Kuipers, Folkert; Reijngoud, Dirk-Jan

2008-06-01

Medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) catalyzes crucial steps in mitochondrial fatty acid oxidation, a process that is of key relevance for maintenance of energy homeostasis, especially during high metabolic demand. To gain insight into the metabolic consequences of MCAD deficiency under these conditions, we compared hepatic carbohydrate metabolism in vivo in wild-type and MCAD(-/-) mice during fasting and during a lipopolysaccharide (LPS)-induced acute phase response (APR). MCAD(-/-) mice did not become more hypoglycemic on fasting or during the APR than wild-type mice did. Nevertheless, microarray analyses revealed increased hepatic peroxisome proliferator-activated receptor gamma coactivator-1alpha (Pgc-1alpha) and decreased peroxisome proliferator-activated receptor alpha (Ppar alpha) and pyruvate dehydrogenase kinase 4 (Pdk4) expression in MCAD(-/-) mice in both conditions, suggesting altered control of hepatic glucose metabolism. Quantitative flux measurements revealed that the de novo synthesis of glucose-6-phosphate (G6P) was not affected on fasting in MCAD(-/-) mice. During the APR, however, this flux was significantly decreased (-20%) in MCAD(-/-) mice compared with wild-type mice. Remarkably, newly formed G6P was preferentially directed toward glycogen in MCAD(-/-) mice under both conditions. Together with diminished de novo synthesis of G6P, this led to a decreased hepatic glucose output during the APR in MCAD(-/-) mice; de novo synthesis of G6P and hepatic glucose output were maintained in wild-type mice under both conditions. APR-associated hypoglycemia, which was observed in wild-type mice as well as MCAD(-/-) mice, was mainly due to enhanced peripheral glucose uptake. Our data demonstrate that MCAD deficiency in mice leads to specific changes in hepatic carbohydrate management on exposure to metabolic stress. This deficiency, however, does not lead to reduced de novo synthesis of G6P during fasting alone, which may be due to the existence of compensatory mechanisms or limited rate control of MCAD in murine mitochondrial fatty acid oxidation.

Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study.

PubMed

Secundo, Francesco; Russo, Consiglia; Giordano, Antonietta; Carrea, Giacomo; Rossi, Mosè; Raia, Carlo A

2005-08-23

A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the K(SV) increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K(M) thermodependence measured with cyclohexanol and NAD(+) at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and coenzyme binding.

Comparative characteristics of the VP7 and VP4 antigenic epitopes of the rotaviruses circulating in Russia (Nizhny Novgorod) and the Rotarix and RotaTeq vaccines.

PubMed

Morozova, O V; Sashina, T A; Fomina, S G; Novikova, N A

2015-07-01

Two live, attenuated rotavirus A (RVA) vaccines, Rotarix and RotaTeq, have been successfully introduced into national immunization programs worldwide. The parent strains of both vaccines were obtained more than 30 years ago. Nonetheless, only very limited data are available on the molecular similarity of the vaccine strains and their genetic relationships to the wild-type strains circulating within the territory of Russian Federation. In this study, we have determined the nucleotide sequences of the genes encoding the viral proteins VP7 and VP4 (the globular domain VP8*) of vaccine strains and natural isolates of rotaviruses in Nizhny Novgorod, Russia. The VP7 and VP4 proteins contain antigenic sites that are the main targets of neutralizing antibodies. Phylogenetic analysis based on VP4 and VP7 showed that the majority of the natural RVA isolates from Nizhny Novgorod and the vaccine strains belong to different clusters. Four amino acids within the VP7 antigenic sites were common in both the wild-type and vaccine strains. The largest number of amino acid differences was found between the vaccine strain Rotarix and the Nizhny Novgorod G2 strains (19 residues out of 29). From 3 to 5 amino acid differences per strain were identified in the antigenic sites of VP4 (domain VP8*) between wild-type strains and the vaccine RotaTeq, and 6-8 substitutions were found when they were compared with the vaccine strain Rotarix. For the first time, immunodominant T-cell epitopes of VP7 were analyzed, and differences in the sequences between the vaccine and the wild-type strains were found. The accumulation of amino acid substitutions in the VP7 and VP4 antigenic sites may potentially reduce the immune protection of vaccinated children from wild-type strains of rotavirus.

Genetic ablation or pharmacological blockade of dipeptidyl peptidase IV does not impact T cell-dependent immune responses

PubMed Central

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

CD248/endosialin critically regulates hepatic stellate cell proliferation during chronic liver injury via a PDGF-regulated mechanism.

PubMed

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/

Bone material elasticity in a murine model of osteogenesis imperfecta.

PubMed

Mehta, S S; Antich, P P; Landis, W J

1999-01-01

To investigate the source of bone brittleness in the disease osteogenesis imperfecta (OI), biomechanical properties have been measured in the femurs from a homozygous (oim/oim) mutant mouse model of OI, its heterozygous littermates, and wild-type animals. The novel technique of ultrasound critical-angle reflectometry (UCR) was used to determine bone material elasticity matrix from measurements of the pressure and shear wave velocity at different orientations about selected points of the bone specimens. This nondestructive method is the only available means for obtaining measurements of this nature from a single surface. The ultrasound pressure wave velocity showed an increased isotropy in the homozygous compared to the wild-type specimens. This was reflected in a significant decrease in the principal elastic modulus measured along the length of the oim/oim bones (E33) while the modulus along the width (E11) did not change significantly, compared to wild-type specimens. The Poisson's ratio, v12, also had a significantly increased value in oim/oim bones. Measurements of these parameters in heterozygous animals generally fell between those from homozygous and control mice. The differences in the elasticity components in oim/oim bones indicate an altered stress distribution and a modified elastic response to loads, compared to normal bone.

Increased Melanoma Growth and Metastasis Spreading in Mice Overexpressing Placenta Growth Factor

PubMed Central

Marcellini, Marcella; De Luca, Naomi; Riccioni, Teresa; Ciucci, Alessandro; Orecchia, Angela; Lacal, Pedro Miguel; Ruffini, Federica; Pesce, Maurizio; Cianfarani, Francesca; Zambruno, Giovanna; Orlandi, Augusto; Failla, Cristina Maria

2006-01-01

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family, plays an important role in adult pathological angiogenesis. To further investigate PlGF functions in tumor growth and metastasis formation, we used transgenic mice overexpressing PlGF in the skin under the control of the keratin 14 promoter. These animals showed a hypervascularized phenotype of the skin and increased levels of circulating PlGF with respect to their wild-type littermates. Transgenic mice and controls were inoculated intradermally with B16-BL6 melanoma cells. The tumor growth rate was fivefold increased in transgenic animals compared to wild-type mice, in the presence of a similar percentage of tumor necrotic tissue. Tumor vessel area was increased in transgenic mice as compared to controls. Augmented mobilization of endothelial and hematopoietic stem cells from the bone marrow was observed in transgenic animals, possibly contributing to tumor vascularization. The number and size of pulmonary metastases were significantly higher in transgenic mice compared to wild-type littermates. Finally, PlGF promoted tumor cell invasion of the extracellular matrix and increased the activity of selected matrix metalloproteinases. These findings indicate that PlGF, in addition to enhancing tumor angiogenesis and favoring tumor growth, may directly influence melanoma dissemination. PMID:16877362

Cloning of a MADS box gene (GhMADS3) from cotton and analysis of its homeotic role in transgenic tobacco.

PubMed

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.

Comparative 13C Metabolic Flux Analysis of Pyruvate Dehydrogenase Complex-Deficient, l-Valine-Producing Corynebacterium glutamicum▿†

PubMed Central

Bartek, Tobias; Blombach, Bastian; Lang, Siegmund; Eikmanns, Bernhard J.; Wiechert, Wolfgang; Oldiges, Marco; Nöh, Katharina; Noack, Stephan

2011-01-01

l-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by 13C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an l-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for l-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP. PMID:21784914

Tumour mutation status and melanoma recurrence following a negative sentinel lymph node biopsy.

PubMed

Adler, Nikki R; Wolfe, Rory; McArthur, Grant A; Kelly, John W; Haydon, Andrew; McLean, Catriona A; Mar, Victoria J

2018-05-14

A proportion of patients develop recurrence following a tumour-negative sentinel lymph node biopsy (SLNB). This study aimed to explore whether melanoma patients with BRAF or NRAS mutant tumours have an increased risk of developing disease recurrence following a negative SLNB compared to patients with wild-type tumours. Prospective cohort study of melanoma patients at three tertiary referral centres in Melbourne, who underwent SLNB. Clinical, pathological and molecular characteristics and recurrence data were prospectively recorded. Multivariate Cox proportional hazards regression models estimated the adjusted hazard ratio (aHR) and corresponding 95% confidence interval (CI) for the association between mutation status and development of recurrence following a negative-SLNB. Overall, 344/477 (72.1%) patients had a negative SLNB. Of these, 54 (15.7%) developed subsequent recurrence. The risk of disease recurrence following a negative SLNB was increased for patients with either a BRAF or NRAS mutant tumour compared to wild-type tumours (aHR 1.92, 95% CI: 1.02-3.60, p = 0.04). Melanoma patients with BRAF or NRAS mutant tumours had an increased risk compared to patients with BRAF/NRAS wild-type tumours of developing disease recurrence following a tumour-negative SLNB. The findings also confirm the importance of continued surveillance to monitor for disease recurrence among SLNB-negative patients.

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

PubMed Central

Moseley, B. E. B.

1969-01-01

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision. PMID:5773016

The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism

PubMed Central

Labrador, Juan Pablo; Azcoitia, Valeria; Tuckermann, Jan; Lin, Calvin; Olaso, Elvira; Mañes, Santos; Brückner, Katja; Goergen, Jean-Louis; Lemke, Greg; Yancopoulos, George; Angel, Peter; Martínez-A, Carlos; Klein, Rüdiger

2001-01-01

The discoidin domain receptor 2 (DDR2) is a member of a subfamily of receptor tyrosine kinases whose ligands are fibrillar collagens, and is widely expressed in postnatal tissues. We have generated DDR2-deficient mice to establish the in vivo functions of this receptor, which have remained obscure. These mice exhibit dwarfism and shortening of long bones. This phenotype appears to be caused by reduced chondrocyte proliferation, rather than aberrant differentiation or function. In a skin wound healing model, DDR2–/– mice exhibit a reduced proliferative response compared with wild-type littermates. In vitro, fibroblasts derived from DDR2–/– mutants proliferate more slowly than wild-type fibroblasts, a defect that is rescued by introduction of wild-type but not kinase-dead DDR2 receptor. Together our results suggest that DDR2 acts as an extracellular matrix sensor to modulate cell proliferation. PMID:11375938

Postprandial fatty acid uptake and adipocyte remodeling in angiotensin type 2 receptor-deficient mice fed a high-fat/high-fructose diet

PubMed Central

Noll, Christophe; Labbé, Sébastien M.; Pinard, Sandra; Shum, Michael; Bilodeau, Lyne; Chouinard, Lucie; Phoenix, Serge; Lecomte, Roger; Carpentier, André C.; Gallo-Payet, Nicole

2016-01-01

ABSTRACT The role of the angiotensin type-2 receptor in adipose physiology remains controversial. The aim of the present study was to demonstrate whether genetic angiotensin type-2 receptor-deficiency prevents or worsens metabolic and adipose tissue morphometric changes observed following a 6-week high-fat/high-fructose diet with injection of a small dose of streptozotocin. We compared tissue uptake of nonesterified fatty acid and dietary fatty acid in wild-type and angiotensin type-2 receptor-deficient mice by using the radiotracer 14(R,S)-[18F]-fluoro-6-thia-heptadecanoic acid in mice fed a standard or high-fat diet. Postprandial fatty acid uptake in the heart, liver, skeletal muscle, kidney and adipose tissue was increased in wild-type mice after a high-fat diet and in angiotensin type-2 receptor-deficient mice on both standard and high-fat diets. Compared to the wild-type mice, angiotensin type-2 receptor-deficient mice had a lower body weight, an increase in fasting blood glucose and a decrease in plasma insulin and leptin levels. Mice fed a high-fat diet exhibited increased adipocyte size that was prevented by angiotensin type-2 receptor-deficiency. Angiotensin type-2 receptor-deficiency abolished the early hypertrophic adipocyte remodeling induced by a high-fat diet. The small size of adipocytes in the angiotensin type-2 receptor-deficient mice reflects their inability to store lipids and explains the increase in fatty acid uptake in non-adipose tissues. In conclusion, a genetic deletion of the angiotensin type-2 receptor is associated with metabolic dysfunction of white adipose depots, and indicates that adipocyte remodeling occurs before the onset of insulin resistance in the high-fat fed mouse model. PMID:27144096

Sex-Specific Regulation of Depression, Anxiety-Like Behaviors and Alcohol Drinking in Mice Lacking ENT1

PubMed Central

Ruby, Christina L.; Walker, Denise L.; An, Joyce; Kim, Jason; Choi, Doo-Sup

2012-01-01

Objectives Adenosine signaling has been implicated in the pathophysiology of several psychiatric disorders including alcoholism, depression, and anxiety. Adenosine levels are controlled in part by transport across the cell membrane by equilibrative nucleoside transporters (ENTs). Recent evidence showed that a polymorphism in the gene encoding ENT1 is associated with comorbid depression and alcoholism in women. We have previously shown that deletion of ENT1 reduces ethanol intoxication and elevates alcohol intake in mice. Interestingly, ENT1 null mice display decreased anxiety-like behavior compared to wild-type littermates. However, our behavioral studies were performed only in male mice. Here, we extend our research to include female mice, and test the effect of ENT1 knockout on other behavioral correlates of alcohol drinking, including depressive and compulsive behavior, in mice. Methods To assess depression-like behavior, we used a forced swim test modified for mice. We examined anxiety-like behavior and locomotor activity in open field chambers, and perseverant behavior using the marble-burying test. Finally, we investigated alcohol consumption and preference in female mice using a two-bottle choice paradigm. Results ENT1 null mice of both sexes showed reduced immobility time in the forced swim test and increased time in the center of the open field compared to wild-type littermates. ENT1 null mice of both sexes showed similar locomotor activity levels and habituation to the open field chambers. Female ENT1 null mice displayed increased marble-burying compared to female wild-types, but no genotype difference was evident in males. Female ENT1 null mice showed increased ethanol consumption and preference compared to female wild-types. Conclusions Our findings suggest that ENT1 contributes to several important behaviors involved in psychiatric disorders. Inhibition of ENT1 may be beneficial in treating depression and anxiety, while enhancement of ENT1 function may reduce compulsive behavior and drinking, particularly in females. PMID:23101030

Blinded histopathological characterisation of POLE exonuclease domain-mutant endometrial cancers: sheep in wolf's clothing.

PubMed

Van Gool, Inge C; Ubachs, Jef E H; Stelloo, Ellen; de Kroon, Cor D; Goeman, Jelle J; Smit, Vincent T H B M; Creutzberg, Carien L; Bosse, Tjalling

2018-01-01

POLE exonuclease domain mutations identify a subset of endometrial cancer (EC) patients with an excellent prognosis. The use of this biomarker has been suggested to refine adjuvant treatment decisions, but the necessary sequencing is not widely performed and is relatively expensive. Therefore, we aimed to identify histopathological and immunohistochemical characteristics to aid in the detection of POLE-mutant ECs. Fifty-one POLE-mutant endometrioid, 67 POLE-wild-type endometrioid and 15 POLE-wild-type serous ECs were included (total N = 133). An expert gynaecopathologist, blinded to molecular features, evaluated each case (two or more slides) for 16 morphological characteristics. Immunohistochemistry was performed for p53, p16, MLH1, MSH2, MSH6, and PMS2. POLE-mutant ECs were characterised by a prominent immune infiltrate: 80% showed peritumoral lymphocytes and 59% showed tumour-infiltrating lymphocytes, as compared with 43% and 28% of POLE-wild-type endometrioid ECs, and 27% and 13% of their serous counterparts (P < 0.01, all comparisons). Of POLE-mutant ECs, 33% contained tumour giant cells; this proportion was significantly higher than that in POLE-wild-type endometrioid ECs (10%; P = 0.003), but not significantly different from that in serous ECs (53%). Serous-like features were as often (focally) present in POLE-mutant as in POLE-wild-type endometrioid ECs (6-24%, depending on the feature). The majority of POLE-mutant ECs showed wild-type p53 (86%), negative/focal p16 (82%) and normal mismatch repair protein expression (90%). A simple combination of morphological and immunohistochemical characteristics (tumour type, grade, peritumoral lymphocytes, MLH1, and p53 expression) can assist in prescreening for POLE exonuclease domain mutations in EC, increasing the probability of a mutation being detected from 7% to 33%. This facilitates the use of this important prognostic biomarker in routine pathology. © 2017 John Wiley & Sons Ltd.

Characteristics of colonic migrating motor complexes in neuronal NOS (nNOS) knockout mice.

PubMed

Spencer, Nick J

2013-01-01

It is well established that the intrinsic pacemaker mechanism that generates cyclical colonic migrating motor complexes (CMMCs) does not require endogenous nitric oxide (NO). However, pharmacological blockade of endogenous NO production potently increases the frequency of CMMCs, suggesting that endogenous NO acts normally to inhibit the CMMC pacemaker mechanism. In this study, we investigated whether mice with a life long genetic deletion of the neuronal nitric oxide synthase (nNOS) gene would show similar CMMC characteristics as wild type mice that have endogenous NO production acutely inhibited. Intracellular electrophysiological and mechanical recordings were made from circular muscle cells of isolated whole mouse colon in wild type and nNOS knockout (KO) mice at 35°C. In wild type mice, the NOS inhibitor, L-NA (100 μM) caused a significant increase in CMMC frequency and a significant depolarization of the CM layer. However, unexpectedly, the frequency of CMMCs in nNOS KO mice was not significantly different from control mice. Also, the resting membrane potential of CM cells in nNOS KO mice was not depolarized compared to controls; and the amplitude of the slow depolarization phase underlying MCs was of similar amplitude between KO and wild type offspring. These findings show that in nNOS KO mice, the major characteristics of CMMCs and their electrical correlates are, at least in adult mice, indistinguishable from wild type control offspring. One possibility why the major characteristics of CMMCs were no different between both types of mice is that nNOS KO mice may compensate for their life long deletion of the nNOS gene, and their permanent loss of neuronal NO production. In this regard, we suggest caution should be exercised when assuming that data obtained from adult nNOS KO mice can be directly extrapolated to wild type mice, that have been acutely exposed to an inhibitor of NOS.

Collagen VI Null Mice as a Model for Early Onset Muscle Decline in Aging.

PubMed

Capitanio, Daniele; Moriggi, Manuela; De Palma, Sara; Bizzotto, Dario; Molon, Sibilla; Torretta, Enrica; Fania, Chiara; Bonaldo, Paolo; Gelfi, Cecilia; Braghetta, Paola

2017-01-01

Collagen VI is an extracellular matrix (ECM) protein playing a key role in skeletal muscles and whose deficiency leads to connective tissue diseases in humans and in animal models. However, most studies have been focused on skeletal muscle features. We performed an extensive proteomic profiling in two skeletal muscles (diaphragm and gastrocnemius) of wild-type and collagen VI null ( Col6a1 -/- ) mice at different ages, from 6- (adult) to 12- (aged) month-old to 24 (old) month-old. While in wild-type animals the number of proteins and the level of modification occurring during aging were comparable in the two analyzed muscles, Col6a1 -/- mice displayed a number of muscle-type specific variations. In particular, gastrocnemius displayed a limited number of dysregulated proteins in adult mice, while in aged muscles the modifications were more pronounced in terms of number and level. In diaphragm, the differences displayed by 6-month-old Col6a1 -/- mice were more pronounced compared to wild-type mice and persisted at 12 months of age. In adult Col6a1 -/- mice, the major variations were found in the enzymes belonging to the glycolytic pathway and the tricarboxylic acid (TCA) cycle, as well as in autophagy-related proteins. When compared to wild-type animals Col6a1 -/- mice displayed a general metabolic rewiring which was particularly prominent the diaphragm at 6 months of age. Comparison of the proteomic features and the molecular analysis of metabolic and autophagic pathways in adult and aged Col6a1 -/- diaphragm indicated that the effects of aging, culminating in lipotoxicity and autophagic impairment, were already present at 6 months of age. Conversely, the effects of aging in Col6a1 -/- gastrocnemius were similar but delayed becoming apparent at 12 months of age. A similar metabolic rewiring and autophagic impairment was found in the diaphragm of 24-month-old wild-type mice, confirming that fatty acid synthase (FASN) increment and decreased microtubule-associated proteins 1A/1B light chain 3B (LC3B) lipidation are hallmarks of the aging process. Altogether these data indicate that the diaphragm of Col6a1 -/- animal model can be considered as a model of early skeletal muscle aging.

Collagen VI Null Mice as a Model for Early Onset Muscle Decline in Aging

PubMed Central

Capitanio, Daniele; Moriggi, Manuela; De Palma, Sara; Bizzotto, Dario; Molon, Sibilla; Torretta, Enrica; Fania, Chiara; Bonaldo, Paolo; Gelfi, Cecilia; Braghetta, Paola

2017-01-01

Collagen VI is an extracellular matrix (ECM) protein playing a key role in skeletal muscles and whose deficiency leads to connective tissue diseases in humans and in animal models. However, most studies have been focused on skeletal muscle features. We performed an extensive proteomic profiling in two skeletal muscles (diaphragm and gastrocnemius) of wild-type and collagen VI null (Col6a1−/−) mice at different ages, from 6- (adult) to 12- (aged) month-old to 24 (old) month-old. While in wild-type animals the number of proteins and the level of modification occurring during aging were comparable in the two analyzed muscles, Col6a1−/− mice displayed a number of muscle-type specific variations. In particular, gastrocnemius displayed a limited number of dysregulated proteins in adult mice, while in aged muscles the modifications were more pronounced in terms of number and level. In diaphragm, the differences displayed by 6-month-old Col6a1−/− mice were more pronounced compared to wild-type mice and persisted at 12 months of age. In adult Col6a1−/− mice, the major variations were found in the enzymes belonging to the glycolytic pathway and the tricarboxylic acid (TCA) cycle, as well as in autophagy-related proteins. When compared to wild-type animals Col6a1−/− mice displayed a general metabolic rewiring which was particularly prominent the diaphragm at 6 months of age. Comparison of the proteomic features and the molecular analysis of metabolic and autophagic pathways in adult and aged Col6a1−/− diaphragm indicated that the effects of aging, culminating in lipotoxicity and autophagic impairment, were already present at 6 months of age. Conversely, the effects of aging in Col6a1−/− gastrocnemius were similar but delayed becoming apparent at 12 months of age. A similar metabolic rewiring and autophagic impairment was found in the diaphragm of 24-month-old wild-type mice, confirming that fatty acid synthase (FASN) increment and decreased microtubule-associated proteins 1A/1B light chain 3B (LC3B) lipidation are hallmarks of the aging process. Altogether these data indicate that the diaphragm of Col6a1−/− animal model can be considered as a model of early skeletal muscle aging. PMID:29114203

Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

PubMed

Liu, Da-Fei; Liu, Chun-Guo; Tian, Jin; Jiang, Yi-Tong; Zhang, Xiao-Zhan; Chai, Hong-Liang; Yang, Tian-Kuo; Yin, Xiu-Chen; Zhang, Hong-Ying; Liu, Ming; Hua, Yu-Ping; Qu, Lian-Dong

2015-06-01

Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine. Copyright © 2015 Elsevier B.V. All rights reserved.

Covalent attachment of Arc repressor subunits by a peptide linker enhances affinity for operator DNA.

PubMed

Robinson, C R; Sauer, R T

1996-01-09

By designing a recombinant gene containing tandem copies of the arc coding sequence with intervening DNA encoding the linker sequence GGGSGGGTGGGSGGG, the two subunits of the P22 Are repressor dimer have been covalently linked to form a single-chain protein called Arc-L1-Arc. The 15-residue linker joins the C-terminus of one monomer to the N-terminus of the second, a distance of approximately 45 A in the Arc-operator cocrystal structure. Arc-L1-Arc is expressed at high levels in Escherichia coli, with no evidence of degradation or proteolytic clipping of the linker, and is more active than wild-type Arc in repression assays. The purified Arc-L1-Arc protein has the molecular weight expected for the designed protein and unfolds cooperatively, reversibly, and with no concentration dependence in thermal-denaturation studies. Arc-L1-Arc protects operator DNA in a manner indistinguishable from that of wild-type Arc in DNase I and copper-phenanthroline footprinting studies, but the covalent attachment of the two monomers results in enhanced affinity for operator DNA. Arc-L1-Arc binds operator DNA half-maximally at a concentration of 1.7 pM, compared with the wild-type value of 185 pM, and also binds DNA fragments containing the left or right operator half-sites more tightly than wild type. Because wild-type Arc is monomeric at sub-nanomolar concentrations and must dimerize before binding to the operator, it was anticipated that Arc-L1-Arc would exhibit a lower half-maximal binding concentration. However, even when the change from a monomeric to a dimeric species is taken into account, the affinity of Arc-L1-Arc for operator and half-operator DNA is greater than the wild-type affinity. This tighter binding appears to result from slower dissociation, as Arc-L1-Arc DNA complexes with full or half-site operators dissociate at rates 5-10 times slower than the corresponding Arc--DNA complexes. Hence, the activity of the designed Arc-L1-Arc protein is substantially increased relative to wild-type Arc in a variety of assays.

HFE Gene Mutations and Iron Status in 100 Healthy Polish Children.

PubMed

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.

Structure-Based Engineering of Methionine Residues in the Catalytic Cores of Alkaline Amylase from Alkalimonas amylolytica for Improved Oxidative Stability

PubMed Central

Yang, Haiquan; Wang, Mingxing; Li, Jianghua; Wang, Nam Sun; Du, Guocheng

2012-01-01

This work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H2O2 at 35°C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, the kcat values of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 × 104 to 5.6 × 104 min−1. The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries. PMID:22865059

Mdr1a plays a crucial role in regulating the analgesic effect and toxicity of aconitine by altering its pharmacokinetic characteristics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Lijun; Wu, Jinjun; Zhao, Min

Aconitine (AC) is the primary bioactive/toxic alkaloid in plants of the Aconitum species. Our previous study demonstrated that Mdr1 was involved in efflux of AC. However, the mechanism by which Mdr1 regulates the efficacy/toxicity of AC in vivo remains unclear. The present study aimed to determine the effects of Mdr1a on the efficacy/toxicity and pharmacokinetics of AC in wild-type and Mdr1a{sup −/−} FVB mice. After oral administration of AC, significantly higher analgesic effect was observed in Mdr1a{sup −/−} mice (49% to 105%) compared to wild-type mice (P < 0.05). The levels of s100-β protein and creatine kinase, which indicate cerebralmore » and myocardial damage, respectively, were also significantly increased (P < 0.05) in Mdr1a{sup −/−} mice. Histopathological examination revealed that the Mdr1a{sup −/−} mice suffered from evident cerebral and myocardial damages, but the wild-type mice did not. These findings suggested that Mdr1a deficiency significantly promoted the analgesic effect of AC and exacerbated its toxicity. Pharmacokinetic experiments showed that T{sub 1/2} of AC in the Mdr1a{sup −/−} mice was significantly higher (from 87% to 300%) than that in wild-type mice (P < 0.05). The distribution of AC in the brain of Mdr1a{sup −/−} mice was 2- to 32-fold higher than that in the brains of wild-type mice (P < 0.05). Toxic reactions were more severe in Mdr1a{sup −/−} mice compared to wild-type mice. In conclusion, Mdr1a deficiency significantly enhanced the analgesic effect of AC and exacerbated its toxicity by upregulating its distribution to the brain and decreasing its plasma elimination rate. Thus, Mdr1a dysfunction may cause severe AC poisoning. - Highlights: • The efficacy and toxicity of aconitine were significantly enhanced in Mdr1a{sup −/−} mice. • The distribution of aconitine to the brain was remarkably increased in Mdr1a{sup −/−} mice. • The elimination rate of aconitine was significantly decreased in Mdr1a{sup −/−} mice.« less

Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

PubMed

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.

Engineering of Helicobacter pylori L-Asparaginase: Characterization of Two Functionally Distinct Groups of Mutants

PubMed Central

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

Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells.

PubMed

Ge, Y; Li, Z H; Marshall, M S; Broxmeyer, H E; Lu, L

1998-06-01

To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34 cells which are highly enriched for hematopoietic stem/progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34 cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

Comparative Response of the Hepatic Transcriptomes of Domesticated and Wild Turkey to Aflatoxin B₁.

PubMed

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.

Comparative transcriptomic analysis of key genes involved in flavonoid biosynthetic pathway and identification of a flavonol synthase from Artemisia annua L.

PubMed

Liu, S; Liu, L; Tang, Y; Xiong, S; Long, J; Liu, Z; Tian, N

2017-07-01

The regulatory mechanism of flavonoids, which synergise anti-malarial and anti-cancer compounds in Artemisia annua, is still unclear. In this study, an anthocyanidin-accumulating mutant callus was induced from A. annua and comparative transcriptomic analysis of wild-type and mutant calli performed, based on the next-generation Illumina/Solexa sequencing platform and de novo assembly. A total of 82,393 unigenes were obtained and 34,764 unigenes were annotated in the public database. Among these, 87 unigenes were assigned to 14 structural genes involved in the flavonoid biosynthetic pathway and 37 unigenes were assigned to 17 structural genes related to metabolism of flavonoids. More than 30 unigenes were assigned to regulatory genes, including R2R3-MYB, bHLH and WD40, which might regulate flavonoid biosynthesis. A further 29 unigenes encoding flavonoid biosynthetic enzymes or transcription factors were up-regulated in the mutant, while 19 unigenes were down-regulated, compared with the wild type. Expression levels of nine genes involved in the flavonoid pathway were compared using semi-quantitative RT-PCR, and results were consistent with comparative transcriptomic analysis. Finally, a putative flavonol synthase gene (AaFLS1) was identified from enzyme assay in vitro and in vivo through heterogeneous expression, and confirmed comparative transcriptomic analysis of wild-type and mutant callus. The present work has provided important target genes for the regulation of flavonoid biosynthesis in A. annua. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

Regulation of drought tolerance by the F-box protein MAX2 in Arabidopsis.

PubMed

Bu, Qingyun; Lv, Tianxiao; Shen, Hui; Luong, Phi; Wang, Jimmy; Wang, Zhenyu; Huang, Zhigang; Xiao, Langtao; Engineer, Cawas; Kim, Tae Houn; Schroeder, Julian I; Huq, Enamul

2014-01-01

MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions.

The secreted esterase of Propionibacterium freudenreichii has a major role in cheese lipolysis.

PubMed

Abeijón Mukdsi, María Claudia; Falentin, Hélène; Maillard, Marie-Bernadette; Chuat, Victoria; Medina, Roxana Beatriz; Parayre, Sandrine; Thierry, Anne

2014-01-01

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1(T) for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis.

The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

PubMed Central

Abeijón Mukdsi, María Claudia; Falentin, Hélène; Maillard, Marie-Bernadette; Chuat, Victoria; Medina, Roxana Beatriz; Parayre, Sandrine

2014-01-01

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis. PMID:24242250

Identification and comparative profiling of miRNAs in an early flowering mutant of trifoliate orange and its wild type by genome-wide deep sequencing.

PubMed

Sun, Lei-Ming; Ai, Xiao-Yan; Li, Wen-Yang; Guo, Wen-Wu; Deng, Xiu-Xin; Hu, Chun-Gen; Zhang, Jin-Zhi

2012-01-01

MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes.

Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity

PubMed Central

Manley, Sharon; Ni, Hong-Min; Williams, Jessica A.; Kong, Bo; DiTacchio, Luciano; Guo, Grace; Ding, Wen-Xing

2014-01-01

Alcoholic liver disease encompasses a wide spectrum of pathogenesis including steatosis, fibrosis, cirrhosis, and alcoholic steatohepatitis. Autophagy is a lysosomal degradation process that degrades cellular proteins and damaged/excess organelles, and serves as a protective mechanism in response to various stresses. Acute alcohol treatment induces autophagy via FoxO3a-mediated autophagy gene expression and protects against alcohol-induced steatosis and liver injury in mice. Farnesoid X Receptor (FXR) is a nuclear receptor that regulates cellular bile acid homeostasis. In the present study, wild type and FXR knockout (KO) mice were treated with acute ethanol for 16 h. We found that ethanol treated-FXR KO mice had exacerbated hepatotoxicity and steatosis compared to wild type mice. Furthermore, we found that ethanol treatment had decreased expression of various essential autophagy genes and several other FoxO3 target genes in FXR KO mice compared with wild type mice. Mechanistically, we did not find a direct interaction between FXR and FoxO3. Ethanol-treated FXR KO mice had increased Akt activation, increased phosphorylation of FoxO3 resulting in decreased FoxO3a nuclear retention and DNA binding. Furthermore, ethanol treatment induced hepatic mitochondrial spheroid formation in FXR KO mice but not in wild type mice, which may serve as a compensatory alternative pathway to remove ethanol-induced damaged mitochondria in FXR KO mice. These results suggest that lack of FXR impaired FoxO3a-mediated autophagy and in turn exacerbated alcohol-induced liver injury. PMID:25460735

Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK.

PubMed

Chae, Yun Jeong; Zhang, Jianan; Au, Paul; Sabbadini, Marta; Xie, Guo-Xi; Yost, C Spencer

2010-12-01

We investigated the role of tandem pore potassium ion channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice. Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wild type, knockout, or heterozygote) was determined using polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared with wild-type littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane, and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype. With the exception of decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test), TRESK knockout mice had healthy development and behavior. TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared with wild-type littermates. Sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice after initial anesthesia testing was nearly threefold higher than that of wild-type littermates. TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogeneous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the theory that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.

Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samplesmore » is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.« less

Cardiac remodeling in response to chronic iron deficiency: role of the erythropoietin receptor.

PubMed

Naito, Yoshiro; Sawada, Hisashi; Oboshi, Makiko; Iwasaku, Toshihiro; Okuhara, Yoshitaka; Morisawa, Daisuke; Eguchi, Akiyo; Hirotani, Shinichi; Mano, Toshiaki; Tsujino, Takeshi; Masuyama, Tohru

2015-06-01

Anemia is a common comorbidity of patients with heart failure, and iron deficiency is known as one of the causes of anemia in heart failure. Recent studies have shown that iron deficiency alone, without overt anemia, is associated with poor outcomes in patients with heart failure. Thus, to minimize the mortality in patients with heart failure, it is important to understand the link between iron deficiency and cardiac function. Chronic untreated iron deficiency results in cardiac remodeling, and we have previously reported that erythropoietin (Epo) and cardiac Epo receptor (EpoR) signaling may be associated with its remodeling. However, the link between EpoR signaling and its remodeling remains to be elucidated. Herein, we investigated the role of EpoR signaling on cardiac remodeling in response to chronic iron deficiency. Wild-type mice and transgene-rescued EpoR-null mutant mice, which express EpoR only in the hematopoietic lineage (EpoR-restricted mice), were fed with either a normal or an iron-restricted diet, and the molecular mechanisms were investigated. Dietary iron restriction gradually induced anemia, Epo secretion, and cardiac hypertrophy in wild-type mice. In contrast, EpoR-restricted mice fed with an iron-restricted diet exhibited anemia, left ventricular dilatation, and cardiac dysfunction compared with wild-type mice. Interestingly, altered cardiac mitochondrial biogenesis was observed in EpoR-restricted mice following iron deficiency. Moreover, cardiac p53 expression was increased in EpoR-restricted mice compared with wild-type mice following iron deficiency. These data indicate that EpoR signaling is associated with cardiac remodeling following chronic iron deficiency.

Mycobacterium tuberculosis TlyA Protein Negatively Regulates T Helper (Th) 1 and Th17 Differentiation and Promotes Tuberculosis Pathogenesis*

PubMed Central

Rahman, Md. Aejazur; Sobia, Parveen; Dwivedi, Ved Prakash; Bhawsar, Aakansha; Singh, Dhiraj Kumar; Sharma, Pawan; Moodley, Prashini; Van Kaer, Luc; Bishai, William R; Das, Gobardhan

2015-01-01

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient pathogen and a major cause of death worldwide. Although various virulence factors of M. tuberculosis have been identified, its pathogenesis remains incompletely understood. TlyA is a virulence factor in several bacterial infections and is evolutionarily conserved in many Gram-positive bacteria, but its function in M. tuberculosis pathogenesis has not been elucidated. Here, we report that TlyA significantly contributes to the pathogenesis of M. tuberculosis. We show that a TlyA mutant M. tuberculosis strain induces increased IL-12 and reduced IL-1β and IL-10 cytokine responses, which sharply contrasts with the immune responses induced by wild type M. tuberculosis. Furthermore, compared with wild type M. tuberculosis, TlyA-deficient M. tuberculosis bacteria are more susceptible to autophagy in macrophages. Consequently, animals infected with the TlyA mutant M. tuberculosis organisms exhibited increased host-protective immune responses, reduced bacillary load, and increased survival compared with animals infected with wild type M. tuberculosis. Thus, M. tuberculosis employs TlyA as a host evasion factor, thereby contributing to its virulence. PMID:25847237

Overexpression of TGF-alpha increases lung tissue hysteresivity in transgenic mice.

PubMed

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.

Metabonomic Profiling of TASTPM Transgenic Alzheimer's Disease Mouse Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Zeping; Browne, Edward R.; Liu, Tao

2012-12-07

Identification of molecular mechanisms underlying early stage Alzheimer’s disease (AD) is important for the development of new therapies against and diagnosis of AD. In this study, non-targeted metabotyping of TASTPM transgenic AD mice was performed. The metabolic profiles of both brain and plasma of TASTPM mice were characterized using gas chromatography-mass spectrometry and compared to those of wild type C57BL/6J mice. TASTPM mice were metabolically distinct compared to wild type mice (Q28 Y = 0.587 and 0.766 for PLS-DA models derived from brain and plasma, respectively). A number of metabolites were found to be perturbed in TASTPM mice in bothmore » brain (D11 fructose, L-valine, L-serine, L-threonine, zymosterol) and plasma (D-glucose, D12 galactose, linoleic acid, arachidonic acid, palmitic acid and D-gluconic acid). In addition, enzyme immunoassay confirmed that selected endogenous steroids were significantly perturbed in brain (androstenedione and 17-OH-progesterone) and plasma (cortisol and testosterone) of TASTPM mice. Ingenuity pathway analysis revealed that perturbations related to amino acid metabolism (brain), steroid biosynthesis (brain), linoleic acid metabolism (plasma) and energy metabolism (plasma) accounted for the differentiation of TASTPM and wild-type« less

Cancer astrocytes have a more conserved molecular status in long recurrence free survival (RFS) IDH1 wild-type glioblastoma patients: new emerging cancer players

PubMed Central

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

Expression of Haemophilus ducreyi Collagen Binding Outer Membrane Protein NcaA Is Required for Virulence in Swine and Human Challenge Models of Chancroid

PubMed Central

Fulcher, Robert A.; Cole, Leah E.; Janowicz, Diane M.; Toffer, Kristen L.; Fortney, Kate R.; Katz, Barry P.; Orndorff, Paul E.; Spinola, Stanley M.; Kawula, Thomas H.

2006-01-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection. PMID:16622201

Defective Generation of a Humoral Immune Response Is Associated with a Reduced Incidence and Severity of Collagen-Induced Arthritis in Microsomal Prostaglandin E Synthase-1 Null Mice1

PubMed Central

Kojima, Fumiaki; Kapoor, Mohit; Yang, Lihua; Fleishaker, Erica L.; Ward, Martin R.; Monrad, Seetha U.; Kottangada, Ponnappa C.; Pace, Charles Q.; Clark, James A.; Woodward, Jerold G.; Crofford, Leslie J.

2008-01-01

Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase and specifically catalyzes the conversion of PGH2 to PGE2. The present study demonstrates the effect of genetic deletion of mPGES-1 on the developing immunologic responses and its impact on the clinical model of bovine collagen-induced arthritis. mPGES-1 null and heterozygous mice exhibited decreased incidence and severity of arthritis compared with wild-type mice in a gene dose-dependent manner. Histopathological examination revealed significant reduction in lining hyperplasia and tissue destruction in mPGES-1 null mice compared with their wild-type littermates. mPGES-1 deficient mice also exhibited attenuation of mechanical nociception in a gene dose-dependent manner. In addition, mPGES-1 null and heterozygous mice showed a marked reduction of serum IgG against type II collagen (CII), including subclasses IgG1, IgG2a, IgG2b, IgG2c, and IgG3, compared with wild-type mice, which correlated with the reduction in observed inflammatory features. These results demonstrate for the first time that deficiency of mPGES-1 inhibits the development of collagen-induced arthritis, at least in part, by blocking the development of a humoral immune response against type II collagen. Pharmacologic inhibition of mPGES-1 may therefore impact both the inflammation and the autoimmunity associated with human diseases such as rheumatoid arthritis. PMID:18523303

Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

PubMed

Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

2006-05-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

The causes and prognoses of different types of fractures in wild koalas submitted to wildlife hospitals.

PubMed

Henning, Joerg; Hannon, Christabel; McKinnon, Allan; Larkin, Rebecca; Allavena, Rachel

2015-12-01

Fractures are a major problem in wild koalas of great veterinary and conservation importance as their occurrence in different locations of the body might result in varying healing success. The aim of this study was to determine the fracture types (defined by location of the fracture) occurring in wild koalas, temporal patterns, possible causes and risk factors of fracture types, and the prognosis for successfully releasing kolas with healed fracture types into the wild. Data from a total of 2031 wild koalas submitted to wildlife hospitals in South-East Queensland, Australia, over a period of 13 years were analysed. Approximately 56.7% of koalas experienced head fractures, 13.4% had torso fractures, 14.9% had limb fractures and 15% had combination fractures. A total of 84.1% of fractures were caused by vehicle collisions, 9.1% by dog attacks, 3.3% by falls from trees, 1.3% by train collisions, 0.2% by livestock trampling and 1.8% due to unknown causes. Multinominal logistic regression was used to identify risk factors (cause of fracture, age category, sex, year, three-year admission period and season of fracture event) by fracture type. The type of fracture was associated with both the cause of the fracture and the season when it occurred: for example torso fractures (compared to combination fractures) were associated with dog attacks (OR=10.98; 95% CI6.03, 20.01) and falls from trees (OR=4.79; 95% CI2.26, 10.19) relative to vehicle collisions. More submissions of koalas with head fractures due to vehicle collisions occurred in spring compared to autumn and winter, coinciding with the breeding season of koalas and increased animal movement. Prognosis for koalas with fractures was poor, with approximately 63.8% of koalas admitted dead on arrival, 34.2% euthanised, and only 2.0% of koalas able to be released. Given this data, further research into mitigation strategies to decrease the risk of fractures and to increase the observed low recovery rate should be considered. Copyright © 2015 Elsevier B.V. All rights reserved.

Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

NASA Technical Reports Server (NTRS)

Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

2002-01-01

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

Up-Regulation of Phosphoinositide Metabolism in Tobacco Cells Constitutively Expressing the Human Type I Inositol Polyphosphate 5-Phosphatase1

PubMed Central

Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.

2002-01-01

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP3) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP3. The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP3 compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP3 in both wild-type and transgenic cells. However, even with stimulation, InsP3 levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP3 signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP2), the lipid precursor of InsP3, was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP2 metabolism showed that the activity of the PtdInsP2-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of 32P into PtdInsP2 in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP2 synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP2 synthesis as a regulatory step in this system. PMID:12177493

The wild type as concept and in experimental practice: A history of its role in classical genetics and evolutionary theory.

PubMed

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.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE PAGES

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.; ...

2016-03-03

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

MicroRNA-146a Deficiency Protects against Listeria monocytogenes Infection by Modulating the Gut Microbiota.

PubMed

Du, Chong-Tao; Gao, Wei; Ma, Ke; Yu, Shui-Xing; Li, Na; Yan, Shi-Qing; Zhou, Feng-Hua; Liu, Zhen-Zhen; Chen, Wei; Lei, Lian-Cheng; Yang, Yong-Jun; Han, Wen-Yu

2018-03-26

The gut microbiota and microRNAs play important roles in the defense against infection. However, the role of miR-146a in L. monocytogenes infection and gut microbiota remains unclear. We tried to determine whether miR-146a controlled L. monocytogenes infection by regulating the gut microbiota. Wild-type and miR-146a-deficient mice or macrophages were used to characterize the impact of miR-146a on animal survival, cell death, bacterial clearance, and gut microbiota following L. monocytogenes challenge. We found that L. monocytogenes infection induced miR-146a expression both in vitro and in vivo. When compared to wild-type mice, miR-146a-deficient mice were more resistant to L. monocytogenes infection. MiR-146a deficiency in macrophages resulted in reduced invasion and intracellular survival of L. monocytogenes . High-throughput sequencing of 16S rRNA revealed that the gut microbiota composition differed between miR-146a-deficient and wild-type mice. Relative to wild-type mice, miR-146a-deficient mice had decreased levels of the Proteobacteria phylum, Prevotellaceae family, and Parasutterella genus, and significantly increased short-chain fatty acid producing bacteria, including the genera Alistipes , Blautia , Coprococcus_1, and Ruminococcus_1 . Wild-type mice co-housed with miR-146a-deficient mice had increased resistance to L. monocytogenes , indicating that miR-146a deficiency guides the gut microbiota to alleviate infection. Together, these results suggest that miR-146a deficiency protects against L. monocytogenes infection by regulating the gut microbiota.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Wild-type and 'a' epitope variants in chronic hepatitis B virus carriers positive for hepatitis B surface antigen and antibody.

PubMed

Mesenas, Steven J; Chow, Wan C; Zhao, Yi; Lim, Gek K; Oon, Chong J; Ng, Han S

2002-02-01

This study aims to examine the genomic variants of the 'a' epitope in chronic hepatitis B virus (HBV) carriers positive for both hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs). Eighteen HBV carriers were studied. Hepatitis B virus (HBV) DNA was extracted and the 'a' epitope region was amplified and sequenced. Eighteen Chinese asymptomatic HBV carriers were studied. There were 13 patients who were positive for both HBsAg and anti-HBs. Of these, one patient had only wild-type HBV, three had a viral mixture, and five had only 'a' epitope variant HBV. Of the three patients with a viral mixture, all had variants in the less conserved region (123-137). Of the five patients with pure HBsAg mutants, three had variants in the less conserved region while two had variants in the highly conserved region. In this study with a limited number of patients, the serum alanine aminotransferase (ALT) levels were higher in patients with wild-type HBV, compared with those with either 'a' epitope variants or a viral mixture consisting of wild type and variants. Eight of the nine (89%) patients positive for both HBsAg and anti-HBs harbored an 'a' epitope variant. The lower ALT levels seen in patients who had either pure 'a' epitope variant or a mixture of wild type and mutants suggest that a closer monitoring of these 'a' epitope variants should be required, as patients carrying these infectious viral strains may remain asymptomatic.

GABAA Receptor Regulation of Voluntary Ethanol Drinking Requires PKCε

PubMed Central

Besheer, Joyce; Lepoutre, Veronique; Mole, Beth; Hodge, Clyde W.

2010-01-01

Protein kinase C (PKC) regulates a variety of neural functions, including ion channel activity, neurotransmitter release, receptor desensitization and differentiation. We have shown previously that mice lacking the ε-isoform of PKC (PKCε) self-administer 75% less ethanol and exhibit supersensitivity to acute ethanol and allosteric positive modulators of GABAA receptors when compared with wild-type controls. The purpose of the present study was to examine involvement of PKCε in GABAA receptor regulation of voluntary ethanol drinking. To address this question, PKCε null-mutant and wild-type control mice were allowed to drink ethanol (10% v/v) vs. water on a two-bottle continuous access protocol. The effects of diazepam (nonselective GABAA BZ positive modulator), zolpidem (GABAA α1 agonist), L-655,708 (BZ-sensitive GABAA α5 inverse agonist), and flumazenil (BZ antagonist) were then tested on ethanol drinking. Ethanol intake (grams/kg/day) by wild-type mice decreased significantly after diazepam or zolpidem but increased after L-655,708 administration. Flumazenil antagonized diazepam-induced reductions in ethanol drinking in wild-type mice. However, ethanol intake by PKCε null mice was not altered by any of the GABAergic compounds even though effects were seen on water drinking in these mice. Increased acute sensitivity to ethanol and diazepam, which was previously reported, was confirmed in PKCε null mice. Thus, results of the present study show that PKCε null mice do not respond to doses of GABAA BZ receptor ligands that regulate ethanol drinking by wild-type control mice. This suggests that PKCε may be required for GABAA receptor regulation of chronic ethanol drinking. PMID:16881070

Peptide Transporter 1 is Responsible for Intestinal Uptake of the Dipeptide Glycylsarcosine: Studies in Everted Jejunal Rings from Wild-type and Pept1 Null Mice

PubMed Central

Ma, Katherine; Hu, Yongjun; Smith, David E.

2010-01-01

The purpose of this study was to determine the relative importance of PEPT1 in the uptake of peptides/mimetics from mouse small intestine using glycylsarcosine (GlySar). After isolating jejunal tissue from wild-type and Pept1 null mice, 2-cm intestinal segments were everted and mounted on glass rods for tissue uptake studies. [14C]GlySar (4 μM) was studied as a function of time, temperature, sodium and pH, concentration, and potential inhibitors. Compared to wild-type animals, Pept1 null mice exhibited a 78% reduction of GlySar uptake at pH 6.0, 37°C. GlySar uptake showed pH dependence with peak values between pH 6.0-6.5 in wild-type animals, while no such tendency was observed in Pept1 null mice. GlySar exhibited Michaelis-Menten uptake kinetics and a minor nonsaturable component in wild-type animals. In contrast, GlySar uptake occurred by only a nonsaturable process in Pept1 null mice. GlySar uptake was significantly inhibited by dipeptides, aminocephalosporins, angiotensin-converting enzyme inhibitors, and the antiviral prodrug valacyclovir; these inhibitors had little, if any, effect on the uptake of GlySar in Pept1 null mice. The findings demonstrate that PEPT1 plays a critical role in the uptake of GlySar in jejunum, and suggest that PEPT1 is the major transporter responsible for the intestinal absorption of small peptides. PMID:20862774

Mimicry of erythropoietin and interleukin-6 signalling by an antibody/cytokine receptor chimera in murine myeloid 32D cells.

PubMed

Kawahara, Masahiro; Ueda, Hiroshi; Tsumoto, Kouhei; Kumagai, Izumi; Nagamune, Teruyuki

2007-04-01

We have previously designed antibody-cytokine receptor chimeras that could respond to a cognate antigen. While these chimeric receptors were functional, it has not been investigated exactly how they mimic signal transduction through corresponding wild-type receptors. In this study, we compared the growth properties and the phosphorylation status of intracellular signal transducers between the erythropoietin receptor (EpoR)- or gp130-based chimeric receptors and wild-type EpoR or EpoR-gp130 chimera, respectively. Expression plasmids, encoding V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2 domain of EpoR and transmembrane/cytoplasmic domains of either EpoR or gp130, were constructed, and pairs of chimeric receptor combinations (V(H)-EpoR and V(L)-EpoR, V(H)-gp130 and V(L)-gp130, V(H)-EpoR and V(L)-gp130, V(H)-gp130 and V(L)-EpoR) were expressed in an IL-3-dependent myeloid cell line, 32D. Growth assay revealed that the transfectants all grew in a HEL-dependent manner. As for phosphorylation of Stat3, Stat5, ERK and Akt, the chimeric receptors showed similar activation pattern of signalling molecules with wild-type receptors, although the chimeric receptors showed ligand-independency and a little lower maximal phosphorylation than the corresponding wild-type receptors. The results demonstrate that antibody-receptor chimeras could substantially mimic wild-type receptors.

Altered callose deposition during embryo sac formation of multi-pistil mutant (mp1) in Medicago sativa.

PubMed

Zhou, H C; Jin, L; Li, J; Wang, X J

2016-06-03

Whether callose deposition is the cause or result of ovule sterility in Medicago sativa remains controversial, because it is unclear when and where changes in callose deposition and dissolution occur during fertile and sterile embryo sac formation. Here, alfalfa spontaneous multi-pistil mutant (mp1) and wild-type plants were used to compare the dynamics of callose deposition during embryo sac formation using microscopy. The results showed that both mutant and wild-type plants experienced megasporogenesis and megagametogenesis, and there was no significant difference during megasporogenesis. In contrast to the wild-type plants, in which the mature embryo sac was observed after three continuous cycles of mitosis, functional megaspores of mutant plants developed abnormally after the second round of mitosis, leading to degeneration of synergid, central, and antipodal cells. Callose deposition in both mutant and wild-type plants was first observed in the walls of megasporocytes, and then in the megaspore tetrad walls. After meiosis, the callose wall began to degrade as the functional megaspore underwent mitosis, and almost no callose was observed in the mature embryo sac in wild-type plants. However, callose deposition was observed in mp1 plants around the synergid, and increased with the development of the embryo sac, and was mainly deposited at the micropylar end. Our results indicate that synergid, central, and antipodal cells, which are surrounded by callose, may degrade owing to lack of nutrition. Callose accumulation around the synergid and at the micropylar end may hinder signals required for the pollen tube to enter the embryo sac, leading to abortion.

Salt tolerance, salt accumulation, and ionic homeostasis in an epidermal bladder-cell-less mutant of the common ice plant Mesembryanthemum crystallinum.

PubMed

Agarie, Sakae; Shimoda, Toshifumi; Shimizu, Yumi; Baumann, Kathleen; Sunagawa, Haruki; Kondo, Ayumu; Ueno, Osamu; Nakahara, Teruhisa; Nose, Akihiro; Cushman, John C

2007-01-01

The aerial surfaces of the common or crystalline ice plant Mesembryanthemum crystallinum L., a halophytic, facultative crassulacean acid metabolism species, are covered with specialized trichome cells called epidermal bladder cells (EBCs). EBCs are thought to serve as a peripheral salinity and/or water storage organ to improve survival under high salinity or water deficit stress conditions. However, the exact contribution of EBCs to salt tolerance in the ice plant remains poorly understood. An M. crystallinum mutant lacking EBCs was isolated from plant collections mutagenized by fast neutron irradiation. Light and electron microscopy revealed that mutant plants lacked EBCs on all surfaces of leaves and stems. Dry weight gain of aerial parts of the mutant was almost half that of wild-type plants after 3 weeks of growth at 400 mM NaCl. The EBC mutant also showed reduced leaf succulence and leaf and stem water contents compared with wild-type plants. Aerial tissues of wild-type plants had approximately 1.5-fold higher Na(+) and Cl(-) content than the mutant grown under 400 mM NaCl for 2 weeks. Na(+) and Cl(-) partitioning into EBCs of wild-type plants resulted in lower concentrations of these ions in photosynthetically active leaf tissues than in leaves of the EBC-less mutant, particularly under conditions of high salt stress. Potassium, nitrate, and phosphate ion content decreased with incorporation of NaCl into tissues in both the wild type and the mutant, but the ratios of Na(+)/K(+) and Cl(-)/NO(3)(-)content were maintained only in the leaf and stem tissues of wild-type plants. The EBC mutant showed significant impairment in plant productivity under salt stress as evaluated by seed pod and seed number and average seed weight. These results clearly show that EBCs contribute to succulence by serving as a water storage reservoir and to salt tolerance by maintaining ion sequestration and homeostasis within photosynthetically active tissues of M. crystallinum.

Atp1a3-deficient heterozygous mice show lower rank in the hierarchy and altered social behavior.

PubMed

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.

Plant defence responses in oilseed rape MINELESS plants after attack by the cabbage moth Mamestra brassicae.

PubMed

Ahuja, Ishita; van Dam, Nicole Marie; Winge, Per; Trælnes, Marianne; Heydarova, Aysel; Rohloff, Jens; Langaas, Mette; Bones, Atle Magnar

2015-02-01

The Brassicaceae family is characterized by a unique defence mechanism known as the 'glucosinolate-myrosinase' system. When insect herbivores attack plant tissues, glucosinolates are hydrolysed by the enzyme myrosinase (EC 3.2.1.147) into a variety of degradation products, which can deter further herbivory. This process has been described as 'the mustard oil bomb'. Additionally, insect damage induces the production of glucosinolates, myrosinase, and other defences. Brassica napus seeds have been genetically modified to remove myrosinase-containing myrosin cells. These plants are termed MINELESS because they lack myrosin cells, the so-called toxic mustard oil mines. Here, we examined the interaction between B. napus wild-type and MINELESS plants and the larvae of the cabbage moth Mamestra brassicae. No-choice feeding experiments showed that M. brassicae larvae gained less weight and showed stunted growth when feeding on MINELESS plants compared to feeding on wild-type plants. M. brassicae feeding didn't affect myrosinase activity in MINELESS plants, but did reduce it in wild-type seedlings. M. brassicae feeding increased the levels of indol-3-yl-methyl, 1-methoxy-indol-3-yl-methyl, and total glucosinolates in both wild-type and MINELESS seedlings. M. brassicae feeding affected the levels of glucosinolate hydrolysis products in both wild-type and MINELESS plants. Transcriptional analysis showed that 494 and 159 genes were differentially regulated after M. brassicae feeding on wild-type and MINELESS seedlings, respectively. Taken together, the outcomes are very interesting in terms of analysing the role of myrosin cells and the glucosinolate-myrosinase defence system in response to a generalist cabbage moth, suggesting that similar studies with other generalist or specialist insect herbivores, including above- and below-ground herbivores, would be useful. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Methamphetamine- and 1-methyl-4-phenyl- 1,2,3, 6-tetrahydropyridine-induced dopaminergic neurotoxicity in inducible nitric oxide synthase-deficient mice.

PubMed

Itzhak, Y; Martin, J L; Ali, S F

1999-12-15

Previous studies have suggested a role for the retrograde messenger, nitric oxide (NO), in methamphetamine (METH)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced dopaminergic neurotoxicity. Since evidence supported the involvement of the neuronal nitric oxide synthase (nNOS) isoform in the dopaminergic neurotoxicity, the present study was undertaken to investigate whether the inducible nitric oxide synthase (iNOS) isoform is also associated with METH- and MPTP-induced neurotoxicity. The administration of METH (5mg/kg x 3) to iNOS deficient mice [homozygote iNOS(-/-)] and wild type mice (C57BL/6) resulted in significantly smaller depletion of striatal dopaminergic markers in the iNOS(-/-) mice compared with the wild-type mice. METH-induced hyperthermia was also significantly lower in the iNOS(-/-) mice than in wild-type mice. In contrast to the outcome of METH administration, MPTP injections (20 mg/kg x 3) resulted in a similar decrease in striatal dopaminergic markers in iNOS(-/-) and wild-type mice. In the set of behavioral experiments, METH-induced locomotor sensitization was investigated. The acute administration of METH (1.0 mg/kg) resulted in the same intensity of locomotor activity in iNOS(-/-) and wild-type mice. Moreover, 68 to 72 h after the exposure to the high-dose METH regimen (5 mg/kg x 3), a marked sensitized response to a challenge injection of METH (1.0 mg/kg) was observed in both the iNOS(-/-) and wild-type mice. The finding that iNOS(-/-) mice were unprotected from MPTP-induced neurotoxicity suggests that the partial protection against METH-induced neurotoxicity observed was primarily associated with the diminished hyperthermic effect of METH seen in the iNOS(-/-) mice. Moreover, in contrast to nNOS deficiency, iNOS deficiency did not affect METH-induced behavioral sensitization. Copyright 1999 Wiley-Liss, Inc.

Review on comparative efficacy of bevacizumab, panitumumab and cetuximab antibody therapy with combination of FOLFOX-4 in KRAS-mutated colorectal cancer patients.

PubMed

Pathak, Surajit; S, Sushmitha; Banerjee, Antara; Marotta, Francesco; Gopinath, Madhumala; Murugesan, Ramachandran; Zhang, Hong; B, Bhavani; Girigoswami, Agnishwar; Sollano, Jose; Sun, Xiao-Feng

2018-01-26

Colorectal cancer, fourth leading form of cancer worldwide and is increasing in alarming rate in the developing countries. Treating colorectal cancer has become a big challenge worldwide and several antibody therapies such as bevacizumab, panitumumab and cetuximab are being used with limited success. Moreover, mutation in KRAS gene which is linked with the colorectal cancer initiation and progression further interferes with the antibody therapies. Considering median progression free survival and overall survival in account, this review focuses to identify the most efficient antibody therapy in combination with chemotherapy (FOLFOX-4) in KRAS mutated colorectal cancer patients. The bevacizumab plus FOLFOX-4 therapy shows about 9.3 months and 8.7 months of progression free survival for KRAS wild and mutant type, respectively. The overall survival is about 34.8 months for wild type whereas for the mutant it is inconclusive for the same therapy. In comparison, panitumumab results in better progression-free survival which is about (9.6 months) and overall survival is about (23.9 months) for the wild type KRAS and the overall survival is about 15.5 months for the mutant KRAS . Cetuximab plus FOLFOX-4 therapy shows about 7.7 months and 5.5 months of progression-free survival for wild type KRAS and mutant type, respectively. Thus, panitumumab shows significant improvement in overall survival rate for wild type KRAS , validating as a cost effective therapeutic for colorectal cancer therapy. This review depicts that panitumumab along with FOLFOX-4 has a higher response in colorectal cancer patients than the either of the two monoclonal antibodies plus FOLFOX-4.

Metabolic rescue in pluripotent cells from patients with mtDNA disease.

PubMed

Ma, Hong; Folmes, Clifford D L; Wu, Jun; Morey, Robert; Mora-Castilla, Sergio; Ocampo, Alejandro; Ma, Li; Poulton, Joanna; Wang, Xinjian; Ahmed, Riffat; Kang, Eunju; Lee, Yeonmi; Hayama, Tomonari; Li, Ying; Van Dyken, Crystal; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Koski, Amy; Mitalipov, Nargiz; Amato, Paula; Wolf, Don P; Huang, Taosheng; Terzic, Andre; Laurent, Louise C; Izpisua Belmonte, Juan Carlos; Mitalipov, Shoukhrat

2015-08-13

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Inhibition of apoptosis by the intrinsic but not the extrinsic apoptotic pathway in myocardial ischemia-reperfusion.

PubMed

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.

Abscisic acid deficiency increases defence responses against Myzus persicae in Arabidopsis.

PubMed

Hillwig, Melissa S; Chiozza, Mariana; Casteel, Clare L; Lau, Siau Ting; Hohenstein, Jessica; Hernández, Enrique; Jander, Georg; MacIntosh, Gustavo C

2016-02-01

Comparison of Arabidopsis thaliana (Arabidopsis) gene expression induced by Myzus persicae (green peach aphid) feeding, aphid saliva infiltration and abscisic acid (ABA) treatment showed a significant positive correlation. In particular, ABA-regulated genes are over-represented among genes that are induced by M. persicae saliva infiltration into Arabidopsis leaves. This suggests that the induction of ABA-related gene expression could be an important component of the Arabidopsis-aphid interaction. Consistent with this hypothesis, M. persicae populations induced ABA production in wild-type plants. Furthermore, aphid populations were smaller on Arabidopsis aba1-1 mutants, which cannot synthesize ABA, and showed a significant preference for wild-type plants compared with the mutant. Total free amino acids, which play an important role in aphid nutrition, were not altered in the aba1-1 mutant line, but the levels of isoleucine (Ile) and tryptophan (Trp) were differentially affected by aphids in wild-type and mutant plants. Recently, indole glucosinolates have been shown to promote aphid resistance in Arabidopsis. In this study, 4-methoxyindol-3-ylmethylglucosinolate was more abundant in the aba1-1 mutant than in wild-type Arabidopsis, suggesting that the induction of ABA signals that decrease the accumulation of defence compounds may be beneficial for aphids. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Proteomic analysis of the flooding tolerance mechanism in mutant soybean.

PubMed

Komatsu, Setsuko; Nanjo, Yohei; Nishimura, Minoru

2013-02-21

Flooding stress of soybean is a serious problem because it reduces growth; however, flooding-tolerant cultivars have not been identified. To analyze the flooding tolerance mechanism of soybean, the flooding-tolerant mutant was isolated and analyzed using a proteomic technique. Flooding-tolerance tests were repeated five times using gamma-ray irradiated soybeans, whose root growth (M6 stage) was not suppressed even under flooding stress. Two-day-old wild-type and mutant plants were subjected to flooding stress for 2days, and proteins were identified using a gel-based proteomic technique. In wild-type under flooding stress, levels of proteins related to development, protein synthesis/degradation, secondary metabolism, and the cell wall changed; however, these proteins did not markedly differ in the mutant. In contrast, an increased number of fermentation-related proteins were identified in the mutant under flooding stress. The root tips of mutant plants were not affected by flooding stress, even though the wild-type plants had damaged root. Alcohol dehydrogenase activity in the mutant increased at an early stage of flooding stress compared with that of the wild-type. Taken together, these results suggest that activation of the fermentation system in the early stages of flooding may be an important factor for the acquisition of flooding tolerance in soybean. Copyright © 2012 Elsevier B.V. All rights reserved.

Role of RpoS in virulence and stress tolerance of the plant pathogen Erwinia carotovora subsp. carotovora.

PubMed

Andersson, R A; Kõiv, V; Norman-Setterblad, C; Pirhonen, M

1999-12-01

The plant-pathogenic bacterium Erwinia carotovora subsp. carotovora causes plant disease mainly through a number of extracellular plant-cell-wall-degrading enzymes. In this study, the ability of an rpoS mutant of the Er. carotovora subsp. carotovora strain SCC3193 to infect plants and withstand environmental stress was characterized. This mutant was found to be sensitive to osmotic and oxidative stresses in vitro and to be deficient in glycogen accumulation. The production of extracellular enzymes in vitro was similar in the mutant and in the wild-type strains. However, the rpoS mutant caused more severe symptoms than the wild-type strain on tobacco plants and also produced more extracellular enzymes in planta, but did not grow to higher cell density in planta compared to the wild-type strain. When tested on plants with reduced catalase activities, which show higher levels of reactive oxygen species, the rpoS mutant was found to cause lower symptom levels and to have impaired growth. In addition, the mutant was unable to compete with the wild-type strain in planta and in vitro. These results suggest that a functional rpoS gene is needed mainly for survival in a competitive environment and during stress conditions, and not for effective infection of plants.

Effect of impaired twitching motility and biofilm dispersion on performance of Pseudomonas aeruginosa-powered microbial fuel cells.

PubMed

Shreeram, Devesh D; Panmanee, Warunya; McDaniel, Cameron T; Daniel, Susan; Schaefer, Dale W; Hassett, Daniel J

2018-02-01

Pseudomonas aeruginosa is a metabolically voracious bacterium that is easily manipulated genetically. We have previously shown that the organism is also highly electrogenic in microbial fuel cells (MFCs). Polarization studies were performed in MFCs with wild-type strain PAO1 and three mutant strains (pilT, bdlA and pilT bdlA). The pilT mutant was hyperpiliated, while the bdlA mutant was suppressed in biofilm dispersion chemotaxis. The double pilT bdlA mutant was expected to have properties of both mutations. Polarization data indicate that the pilT mutant showed 5.0- and 3.2-fold increases in peak power compared to the wild type and the pilT bdlA mutant, respectively. The performance of the bdlA mutant was surprisingly the lowest, while the pilT bdlA electrogenic performance fell between the pilT mutant and wild-type bacteria. Measurements of biofilm thickness and bacterial viability showed equal viability among the different strains. The thickness of the bdlA mutant, however, was twice that of wild-type strain PAO1. This observation implicates the presence of dead or dormant bacteria in the bdlA mutant MFCs, which increases biofilm internal resistance as confirmed by electrochemical measurements.

Immune mechanisms induced by an HSV-1 mutant strain: Discrepancy analysis of the immune system gene profile in comparison with a wild-type strain.

PubMed

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.

Surface changes and polymyxin interactions with a resistant strain of Klebsiella pneumoniae.

PubMed

Velkov, Tony; Deris, Zakuan Z; Huang, Johnny X; Azad, Mohammad A K; Butler, Mark; Sivanesan, Sivashangarie; Kaminskas, Lisa M; Dong, Yao-Da; Boyd, Ben; Baker, Mark A; Cooper, Matthew A; Nation, Roger L; Li, Jian

2014-05-01

This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiella pneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by (1)H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential.

Antagonism of scavenger receptor CD36 by 5A peptide prevents chronic kidney disease progression in mice independent of blood pressure regulation

PubMed Central

Souza, Ana Carolina P.; Bocharov, Alexander V.; Baranova, Irina; Vishnyakova, Tatyana; Huang, Yuning G.; Wilkins, Kenneth J.; Hu, Xuzhen; Street, Jonathan M.; Alvarez-Prats, Alejandro; Mullick, Adam E.; Patterson, Amy P.; Remaley, Alan; Eggerman, Thomas L.; Yuen, Peter S.T.; Star, Robert A.

2016-01-01

Scavenger receptor CD36 participates in lipid metabolism and inflammatory pathways important for cardiovascular disease and chronic kidney disease (CKD). Few pharmacological agents are available to slow the progression of CKD. However, apolipoprotein AI-mimetic peptide 5A antagonizes CD36 in vitro. To test the efficacy of 5A, and to test the role of CD36 during CKD, we compared wild type to CD36 knockout mice and wild type mice treated with 5A, in a progressive CKD model that resembles human disease. Knockout and 5A-treated wild type mice were protected from CKD progression without changes in blood pressure and had reductions in cardiovascular risk surrogate markers that are associated with CKD. Treatment with 5A did not further protect CD36 knockout mice from CKD progression, implicating CD36 as its main site of action. In a separate model of kidney fibrosis, 5A-treated wild type mice had less macrophage infiltration and interstitial fibrosis. Peptide 5A exerted anti-inflammatory effects in the kidney and decreases renal expression of inflammasome genes. Thus, CD36 is a new therapeutic target for CKD and its associated cardiovascular risk factors. Peptide 5A may be a promising new agent to slow CKD progression. PMID:26994575

Colonization of fish skin is vital for Vibrio anguillarum to cause disease.

PubMed

Weber, Barbara; Chen, Chang; Milton, Debra L

2010-02-01

Vibrio anguillarum causes a fatal haemorrhagic septicaemia in marine fish. During initial stages of infection, host surfaces are colonized; however, few virulence factors required for colonization of the host are identified. In this study, in vivo bioluminescent imaging was used to analyse directly the colonization of the whole rainbow trout animal by V. anguillarum. The wild type rapidly colonized both the skin and the intestines by 24 h; however, the bacterial numbers on the skin were significantly higher than in the intestines indicating that skin colonization may be important for disease to occur. Mutants defective for the anguibactin iron uptake system, exopolysaccharide transport, or Hfq, an RNA chaperone, were attenuated for virulence, did not colonize the skin, and penetrated skin mucus less efficiently than the wild type. These mutants, however, did colonize the intestines and were as resistant to 2% bile salts as is the wild type. Moreover, exopolysaccharide mutants were significantly more sensitive to lysozyme and antimicrobial peptides, while the Hfq and anguibactin mutants were sensitive to lysozyme compared with the wild type. Vibrio anguillarum encodes several mechanisms to protect against antimicrobial components of skin mucus enabling an amazingly abundant growth on the skin enhancing its disease opportunities. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae

PubMed Central

Davis, Carrie Anne; Ares, Manuel

2006-01-01

Mutations in RRP6 result in the accumulation of aberrant polyadenylated transcripts from small nucleolar RNA genes. We exploited this observation to search for novel noncoding RNA genes in the yeast genome. When RNA from rrp6Δ yeast is compared with wild-type on whole-genome microarrays, numerous intergenic loci exhibit an increased mutant/wild type signal ratio. Among these loci, we found one encoding a new C/D box small nucleolar RNA, as well as a surprising number that gave rise to heterogeneous Trf4p-polyadenylated RNAs with lengths of ≈250–500 nt. This class of RNAs is not easily detected in wild-type cells and appears associated with promoters. Fine mapping of several such transcripts shows they originate near known promoter elements but do not usually extend far enough to act as mRNAs, and may regulate the transcription of downstream mRNAs. Rather than being uninformative transcriptional “noise,” we hypothesize that these transcripts reflect important features of RNA polymerase activity at the promoter. This activity is normally undetectable in wild-type cells because the transcripts are somehow distinguished from true mRNAs and are degraded in an Rrp6p-dependent fashion in the nucleus. PMID:16484372

Early nodule senescence is activated in symbiotic mutants of pea (Pisum sativum L.) forming ineffective nodules blocked at different nodule developmental stages.

PubMed

Serova, Tatiana A; Tsyganova, Anna V; Tsyganov, Viktor E

2018-04-03

Plant symbiotic mutants are useful tool to uncover the molecular-genetic mechanisms of nodule senescence. The pea (Pisum sativum L.) mutants SGEFix - -1 (sym40), SGEFix - -3 (sym26), and SGEFix - -7 (sym27) display an early nodule senescence phenotype, whereas the mutant SGEFix - -2 (sym33) does not show premature degradation of symbiotic structures, but its nodules show an enhanced immune response. The nodules of these mutants were compared with each other and with those of the wild-type SGE line using seven marker genes that are known to be activated during nodule senescence. In wild-type SGE nodules, transcript levels of all of the senescence-associated genes were highest at 6 weeks after inoculation (WAI). The senescence-associated genes showed higher transcript abundance in mutant nodules than in wild-type nodules at 2 WAI and attained maximum levels in the mutant nodules at 4 WAI. Immunolocalization analyses showed that the ethylene precursor 1-aminocyclopropane-1-carboxylate accumulated earlier in the mutant nodules than in wild-type nodules. Together, these results showed that nodule senescence was activated in ineffective nodules blocked at different developmental stages in pea lines that harbor mutations in four symbiotic genes.

Genetic diversity and antibiotic susceptibility of Staphylococcus aureus isolates from wild boars.

PubMed

Seinige, D; Von Altrock, A; Kehrenberg, C

2017-10-01

We here report the occurrence of S. aureus in wild boars and characterize isolates genotypically and phenotypically in order to get knowledge about the occurrence of clonal lineages and genotypes in free-living wild animals. Forty-one S. aureus isolates obtained from 111 wild boars hunted in Lower Saxony, Germany, were investigated and compared to human and livestock isolates. The S. aureus belonged to multilocus sequence types ST1, ST7, ST30, ST133, ST425, ST804, ST890 and to the new ST3237, ST3238, ST3255 and ST3369. The livestock associated CC398-MRSA lineage, however, was not found. In addition to well-known spa types, the new types t14999, t15000, t15001 and t15002 were detected. Macrorestriction analysis revealed a variety of different SmaI fragment patterns. Most isolates were susceptible to all antimicrobials tested, including methicillin, and resistance was detected only to ampicillin, penicillin and erythromycin. PCR analysis confirmed the presence of staphylococcal enterotoxin genes (seh) in all t127-ST1 isolates. A high degree of genetic diversity was detected with many spa types and clonal lineages previously reported in humans and livestock animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Voltage-dependent ion channels in the mouse RPE: comparison with Norrie disease mice.

PubMed

Wollmann, Guido; Lenzner, Steffen; Berger, Wolfgang; Rosenthal, Rita; Karl, Mike O; Strauss, Olaf

2006-03-01

We studied electrophysiological properties of cultured retinal pigment epithelial (RPE) cells from mouse and a mouse model for Norrie disease. Wild-type RPE cells revealed the expression of ion channels known from other species: delayed-rectifier K(+) channels composed of Kv1.3 subunits, inward rectifier K(+) channels, Ca(V)1.3 L-type Ca(2+) channels and outwardly rectifying Cl(-) channels. Expression pattern and the ion channel characteristics current density, blocker sensitivity, kinetics and voltage-dependence were compared in cells from wild-type and Norrie mice. Although no significant differences were observed, our study provides a base for future studies on ion channel function and dysfunction in transgenic mouse models.

Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair.

PubMed

Ihara, Makoto; Takeshita, Satoshi; Okaichi, Kumio; Okumura, Yutaka; Ohnishi, Takeo

2014-03-01

From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37 °C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.

Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication

PubMed Central

Wu, G. Albert; Prochnik, Simon; Jenkins, Jerry; Salse, Jerome; Hellsten, Uffe; Murat, Florent; Perrier, Xavier; Ruiz, Manuel; Scalabrin, Simone; Terol, Javier; Takita, Marco Aurélio; Labadie, Karine; Poulain, Julie; Couloux, Arnaud; Jabbari, Kamel; Cattonaro, Federica; Del Fabbro, Cristian; Pinosio, Sara; Zuccolo, Andrea; Chapman, Jarrod; Grimwood, Jane; Tadeo, Francisco R.; Estornell, Leandro H.; Muñoz-Sanz, Juan V.; Ibanez, Victoria; Herrero-Ortega, Amparo; Aleza, Pablo; Pérez-Pérez, Julián; Ramón, Daniel; Brunel, Dominique; Luro, François; Chen, Chunxian; Farmerie, William G.; Desany, Brian; Kodira, Chinnappa; Mohiuddin, Mohammed; Harkins, Tim; Fredrikson, Karin; Burns, Paul; Lomsadze, Alexandre; Borodovsky, Mark; Reforgiato, Giuseppe; Freitas-Astúa, Juliana; Quetier, Francis; Navarro, Luis; Roose, Mikeal; Wincker, Patrick; Schmutz, Jeremy; Morgante, Michele; Machado, Marcos Antonio; Talon, Manuel; Jaillon, Olivier; Ollitrault, Patrick; Gmitter, Frederick; Rokhsar, Daniel

2014-01-01

The domestication of citrus, is poorly understood. Cultivated types are selections from, or hybrids of, wild progenitor species, whose identities and contributions remain controversial. By comparative analysis of a collection of citrus genomes, including a high quality haploid reference, we show that cultivated types were derived from two progenitor species. Though cultivated pummelos represent selections from a single progenitor species, C. maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species, C. reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, implying that wild mandarins were part of the early breeding germplasm. A wild “mandarin” from China exhibited substantial divergence from C. reticulata, suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and enables sequence-directed genetic improvement. PMID:24908277

Comparison of KRAS genotype: therascreen assay vs. LNA-mediated qPCR clamping assay.

PubMed

Chang, Shao-Chun; Denne, Jonathan; Zhao, Luping; Horak, Christine; Green, George; Khambata-Ford, Shirin; Bray, Christopher; Celik, Ilhan; Van Cutsem, Eric; Harbison, Christopher

2013-09-01

Kirsten rat sarcoma virus (KRAS) wild-type status determined using a locked nucleic acid (LNA)-mediated quantitative polymerase chain reaction (qPCR) clamping assay (LNA assay) predicted response to therapy in the CRYSTAL (Cetuximab Combined With Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) study. A companion KRAS diagnostic tool has been developed for routine clinical use (QIAGEN therascreen kit) (QIAGEN Manchester Ltd, Manchester, UK). We wanted to assess the concordance between the validated US Food and Drug Administration (FDA)-approved therascreen assay and the LNA assay in determining the KRAS status of a subset of patients enrolled in the CRYSTAL study. DNA extracted from paraffin-embedded tumor sections was tested for KRAS status using the therascreen assay. Efficacy data from the CRYSTAL study were assessed to determine if the overall survival (OS) hazard ratio for cetuximab in patients identified as having KRAS wild-type status using the therascreen assay was equivalent to that in patients identified as KRAS wild-type using the LNA assay. This was determined by assessing if the concordance between the therascreen assay and the LNA assay met the minimum threshold (prespecified as 0.8) to achieve a significant difference in the OS hazard ratio in favor of the cetuximab + FOLFIRI (5-fluorouracil, leucovorin [folinic acid], irinotecan) arm in the KRAS wild-type population as identified using the therascreen assay. Of the 148 samples determined to be KRAS wild-type (therascreen assay), 141 (95.3%) samples were also KRAS wild-type (LNA assay) and 7 samples (4.7%) were KRAS mutant (LNA assay). The prespecified primary concordance measure p was 141/148 = 0.953 (95% confidence interval [CI], 0.905-0.981). The concordance was statistically significantly higher than the prespecified threshold of 0.8 for concordance between the therascreen assay and the LNA assay. Consistent with the concordance exceeding the prespecified threshold, the OS hazard ratio (cetuximab + FOLFIRI arm vs. FOLFIRI arm) in the KRAS wild-type population, determined by the therascreen assay, supported a significant benefit for cetuximab (ie, the 95% CI excluded 1) and was comparable to the OS hazard ratio observed in the CRYSTAL study KRAS wild-type population (LNA assay) even after adjustment for potentially confounding baseline variables. These results support the utility of the therascreen assay for identifying patients who may benefit from cetuximab therapy for metastatic colorectal cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Survival of irradiated recipient mice after transplantation of bone marrow from young, old and "early aging" mice.

PubMed

Guest, Ian; Ilic, Zoran; Scrable, Heidi; Sell, Stewart

2015-12-01

Bone marrow transplantation is used to examine survival, hematopoietic stem cell function and pathology in recipients of young and old wild type bone marrow derived stem cells (BMDSCs) as well as cells from p53-based models of premature aging. There is no difference in the long term survival of recipients of 8 week-old p53+/m donor cells compared to recipients of 8 week-old wild-type (WT) donor cells (70 weeks) or of recipients of 16-18 weeks-old donor cells from either p53+/m or WT mice. There is shorter survival in recipients of older versus younger WT donor bone marrow, but the difference is only significant when comparing 8 and 18 week-old donors. In the p44-based model, short term survival/engraftment is significantly reduced in recipients of 11 month-old p44 donor cells compared to 4 week-old p44 or wild type donor cells of either age; mid-life survival at 40 weeks is also significantly less in recipients of p44 cells. BMDSCs are readily detectable within recipient bone marrow, lymph node, intestinal villi and liver sinusoids, but not in epithelial derived cells. These results indicate that recipients of young BMDSCs may survive longer than recipients of old bone marrow, but the difference is marginal at best.

Survival of irradiated recipient mice after transplantation of bone marrow from young, old and “early aging” mice

PubMed Central

Guest, Ian; Ilic, Zoran; Sell, Stewart

2015-01-01

Bone marrow transplantation is used to examine survival, hematopoietic stem cell function and pathology in recipients of young and old wild type bone marrow derived stem cells (BMDSCs) as well as cells from p53-based models of premature aging. There is no difference in the long term survival of recipients of 8 week-old p53+/m donor cells compared to recipients of 8 week-old wild-type (WT) donor cells (70 weeks) or of recipients of 16–18 weeks-old donor cells from either p53+/m or WT mice. There is shorter survival in recipients of older versus younger WT donor bone marrow, but the difference is only significant when comparing 8 and 18 week-old donors. In the p44-based model, short term survival/engraftment is significantly reduced in recipients of 11 month-old p44 donor cells compared to 4 week-old p44 or wild type donor cells of either age; mid-life survival at 40 weeks is also significantly less in recipients of p44 cells. BMDSCs are readily detectable within recipient bone marrow, lymph node, intestinal villi and liver sinusoids, but not in epithelial derived cells. These results indicate that recipients of young BMDSCs may survive longer than recipients of old bone marrow, but the difference is marginal at best. PMID:26796640

Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure in Platelets

PubMed Central

Dasgupta, Swapan K.; Le, Anhquyen; Haudek, Sandra B.; Entman, Mark L.; Rumbaut, Rolando E.; Thiagarajan, Perumal

2013-01-01

Background The transbilayer movement of phosphatidylserine mediates the platelet procoagulant activity during collagen stimulation. The Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 inhibits senescence induced but not activation induced phosphatidylserine exposure. To investigate further the specific mechanisms, we now utilized mice with genetic deletion of the ROCK1 isoform. Methods and Results ROCK1-deficient mouse platelets expose significantly more phosphatidylserine and generate more thrombin upon activation with collagen compared to wild-type platelets. There were no significant defects in platelet shape change, aggregation, or calcium response compared to wild-type platelets. Collagen-stimulated ROCK1-deficient platelets also displayed decreased phosphorylation levels of Lim Kinase-1 and cofilin-1. However, there was no reduction in phosphorylation levels of myosin phosphatase subunit-1 (MYPT1) or myosin light chain (MLC). In an in vivo light/dye-induced endothelial injury/thrombosis model, ROCK1-deficient mice presented a shorter occlusion time in cremasteric venules when compared to wild-type littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). Conclusions These studies define ROCK1 as a new regulator for collagen-induced phosphatidylserine exposure in platelets with functional consequences on thrombosis. This effect was downstream of calcium signaling and was mediated by Lim Kinase-1 / cofilin-1-induced cytoskeletal changes. PMID:24358370

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses

DOE PAGES

Nafisi, Majse; Stranne, Maria; Fimognari, Lorenzo; ...

2015-07-22

Here we report that the epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed andmore » surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.« less

Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli.

PubMed

Pobre, Vânia; Arraiano, Cecília M

2015-02-14

The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome. In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms. In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia.

PubMed

Lerman, Oren Z; Galiano, Robert D; Armour, Mary; Levine, Jamie P; Gurtner, Geoffrey C

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.

Cellular Dysfunction in the Diabetic Fibroblast

PubMed Central

Lerman, Oren Z.; Galiano, Robert D.; Armour, Mary; Levine, Jamie P.; Gurtner, Geoffrey C.

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O2), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 ± 1.3 pg/ml versus 34.8 ± 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients. PMID:12507913

Draft genome sequence of Cicer reticulatum L., the wild progenitor of chickpea provides a resource for agronomic trait improvement.

PubMed

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.

Virulence Effects and Signaling Partners Modulated by Brucella melitensis Light-sensing Histidine Kinase

NASA Astrophysics Data System (ADS)

Gourley, Christopher R.

The facultative intracellular pathogen Brucella melitensis utilizes diverse virulence factors. A Brucella light sensing histidine kinase can influence in vitro virulence of the bacteria during intracellular infection. First, we demonstrated that the B. melitensis light sensing kinase (BM-LOV-HK) affects virulence in an IRF-1-/- mouse model of infection. Infection with a Δ BM-LOV-HK strain resulted in less bacterial colonization of IRF-1-/- spleens and extended survivorship compared to mice infected with wild type B. melitensis 16M. Second, using PCR arrays, we observed less expression of innate and adaptive immune system activation markers in ΔBM-LOV-HK infected mouse spleens than wild type B. melitensis 16M infected mouse spleens 6 days after infection. Third, we demonstrated by microarray analysis of B. melitensis that deletion of BM-LOV-HK alters bacterial gene expression. Downregulation of genes involved in control of the general stress response system included the alternative sigma factor RpoE1 and its anti-anti sigma factor PhyR. Conversely, genes involved in flagella production, quorum sensing, and the type IV secretion system (VirB operon) were upregulated in the Δ BM-LOV-HK strain compared to the wild type B. melitensis 16M. Analysis of genes differentially regulated in Δ BM-LOV-HK versus the wild type strain indicated an overlap of 110 genes with data from previous quorum sensing regulator studies of Δ vjbR and/ΔblxR(babR) strains. Also, several predicted RpoE1 binding sites located upstream of genes were differentially regulated in the ΔBM-LOV-HK strain. Our results suggest BM-LOV-HK is important for in vivo Brucella virulence, and reveals that BM-LOV-HK directly or indirect regulates members of the Brucella quorum sensing, type IV secretion, and general stress systems.

Prognostic significance of IDH 1 mutation in patients with glioblastoma multiforme.

PubMed

Khan, Inamullah; Waqas, Muhammad; Shamim, Muhammad Shahzad

2017-05-01

Focus of brain tumour research is shifting towards tumour genesis and genetics, and possible development of individualized treatment plans. Genetic analysis shows recurrent mutation in isocitrate dehydrogenase (IDH1) gene in most Glioblastoma multiforme (GBM) cells. In this review we evaluated the prognostic significance of IDH 1 mutation on the basis of published evidence. Multiple retrospective clinical analyses correlate the presence of IDH1 mutation in GBM with good prognostic outcomes compared to wild-type IDH1. A systematic review reported similar results. Based on the review of current literature IDH1 mutation is an independent factor for longer overall survival (OS) and progression free survival (PFS) in GBM patients when compared to wild-type IDH1. The prognostic significance opens up new avenues for treatment.

FOLFIRI plus panitumumab in the treatment of wild-type KRAS and wild-type NRAS metastatic colorectal cancer.

PubMed

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.

Association of MYCN copy number with clinical features, tumor biology, and outcomes in neuroblastoma: A report from the Children's Oncology Group.

PubMed

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.

Control of blood pressure, appetite, and glucose by leptin in mice lacking leptin receptors in proopiomelanocortin neurons.

PubMed

do Carmo, Jussara M; da Silva, Alexandre A; Cai, Zhengwei; Lin, Shuying; Dubinion, John H; Hall, John E

2011-05-01

Although the central nervous system melanocortin system is an important regulator of energy balance, the role of proopiomelanocortin (POMC) neurons in mediating the chronic effects of leptin on appetite, blood pressure, and glucose regulation is unknown. Using Cre/loxP technology we tested whether leptin receptor deletion in POMC neurons (LepR(flox/flox)/POMC-Cre mice) attenuates the chronic effects of leptin to increase mean arterial pressure (MAP), enhance glucose use and oxygen consumption, and reduce appetite. LepR(flox/flox)/POMC-Cre, wild-type, LepR(flox/flox), and POMC-Cre mice were instrumented for MAP and heart rate measurement by telemetry and venous catheters for infusions. LepR(flox/flox)/POMC-Cre mice were heavier, hyperglycemic, hyperinsulinemic, and hyperleptinemic compared with wild-type, LepR(flox/flox), and POMC-Cre mice. Despite exhibiting features of metabolic syndrome, LepR(flox/flox)/POMC-Cre mice had normal MAP and heart rate compared with LepR(flox/flox) but lower MAP and heart rate compared with wild-type mice. After a 5-day control period, leptin was infused (2 μg/kg per minute, IV) for 7 days. In control mice, leptin increased MAP by ≈5 mm Hg despite decreasing food intake by ≈35%. In contrast, leptin infusion in LepR(flox/flox)/POMC-Cre mice reduced MAP by ≈3 mm Hg and food intake by ≈28%. Leptin significantly decreased insulin and glucose levels in control mice but not in LepR(flox/flox)/POMC-Cre mice. Leptin increased oxygen consumption in LepR(flox/flox)/POMC-Cre and wild-type mice. Activation of POMC neurons is necessary for the chronic effects of leptin to raise MAP and reduce insulin and glucose levels, whereas leptin receptors in other areas of the brain other than POMC neurons appear to play a key role in mediating the chronic effects of leptin on appetite and oxygen consumption.

Rapid, Accurate, and Non-Invasive Measurement of Zebrafish Axial Length and Other Eye Dimensions Using SD-OCT Allows Longitudinal Analysis of Myopia and Emmetropization

PubMed Central

Collery, Ross F.; Veth, Kerry N.; Dubis, Adam M.; Carroll, Joseph; Link, Brian A.

2014-01-01

Refractive errors in vision can be caused by aberrant axial length of the eye, irregular corneal shape, or lens abnormalities. Causes of eye length overgrowth include multiple genetic loci, and visual parameters. We evaluate zebrafish as a potential animal model for studies of the genetic, cellular, and signaling basis of emmetropization and myopia. Axial length and other eye dimensions of zebrafish were measured using spectral domain-optical coherence tomography (SD-OCT). We used ocular lens and body metrics to normalize and compare eye size and relative refractive error (difference between observed retinal radial length and controls) in wild-type and lrp2 zebrafish. Zebrafish were dark-reared to assess effects of visual deprivation on eye size. Two relative measurements, ocular axial length to body length and axial length to lens diameter, were found to accurately normalize comparisons of eye sizes between different sized fish (R2 = 0.9548, R2 = 0.9921). Ray-traced focal lengths of wild-type zebrafish lenses were equal to their retinal radii, while lrp2 eyes had longer retinal radii than focal lengths. Both genetic mutation (lrp2) and environmental manipulation (dark-rearing) caused elongated eye axes. lrp2 mutants had relative refractive errors of −0.327 compared to wild-types, and dark-reared wild-type fish had relative refractive errors of −0.132 compared to light-reared siblings. Therefore, zebrafish eye anatomy (axial length, lens radius, retinal radius) can be rapidly and accurately measured by SD-OCT, facilitating longitudinal studies of regulated eye growth and emmetropization. Specifically, genes homologous to human myopia candidates may be modified, inactivated or overexpressed in zebrafish, and myopia-sensitizing conditions used to probe gene-environment interactions. Our studies provide foundation for such investigations into genetic contributions that control eye size and impact refractive errors. PMID:25334040

NF-κB1, NF-κB2 and c-Rel differentially regulate susceptibility to colitis-associated adenoma development in C57BL/6 mice.

PubMed

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.

Laccase SilA from Streptomyces ipomoeae CECT 3341, a key enzyme for the degradation of lignin from agricultural residues?

PubMed Central

Blánquez, Alba; Ball, Andrew S.; González-Pérez, José Antonio; Jiménez-Morillo, Nicasio T.; González-Vila, Francisco; Arias, M. Enriqueta

2017-01-01

The role of laccase SilA produced by Streptomyces ipomoeae CECT 3341 in lignocellulose degradation was investigated. A comparison of the properties and activities of a laccase-negative mutant strain (SilA−) with that of the wild-type was studied in terms of their ability to degrade lignin from grass lignocellulose. The yields of solubilized lignin (acid precipitable polymeric lignin, APPL) obtained from wheat straw by both strains in Solid State Fermentation (SSF) conditions demonstrated the importance of SilA laccase in lignin degradation with the wild-type showing 5-fold more APPL produced compared with the mutant strain (SilA−). Analytical pyrolysis and FT-IR (Fourier Transform Infrared Spectroscopy) confirmed that the APPL obtained from the substrate fermented by wild-type strain was dominated by lignin derived methoxyphenols whereas those from SilA− and control APPLs were composed mainly of polysaccharides. This is the first report highlighting the role of this laccase in lignin degradation. PMID:29112957

Laccase SilA from Streptomyces ipomoeae CECT 3341, a key enzyme for the degradation of lignin from agricultural residues?

PubMed

Blánquez, Alba; Ball, Andrew S; González-Pérez, José Antonio; Jiménez-Morillo, Nicasio T; González-Vila, Francisco; Arias, M Enriqueta; Hernández, Manuel

2017-01-01

The role of laccase SilA produced by Streptomyces ipomoeae CECT 3341 in lignocellulose degradation was investigated. A comparison of the properties and activities of a laccase-negative mutant strain (SilA-) with that of the wild-type was studied in terms of their ability to degrade lignin from grass lignocellulose. The yields of solubilized lignin (acid precipitable polymeric lignin, APPL) obtained from wheat straw by both strains in Solid State Fermentation (SSF) conditions demonstrated the importance of SilA laccase in lignin degradation with the wild-type showing 5-fold more APPL produced compared with the mutant strain (SilA-). Analytical pyrolysis and FT-IR (Fourier Transform Infrared Spectroscopy) confirmed that the APPL obtained from the substrate fermented by wild-type strain was dominated by lignin derived methoxyphenols whereas those from SilA- and control APPLs were composed mainly of polysaccharides. This is the first report highlighting the role of this laccase in lignin degradation.

Flavonoid production in transgenic hop (Humulus lupulus L.) altered by PAP1/MYB75 from Arabidopsis thaliana L.

PubMed

Gatica-Arias, A; Farag, M A; Stanke, M; Matoušek, J; Wessjohann, L; Weber, G

2012-01-01

Hop is an important source of secondary metabolites, such as flavonoids. Some of these are pharmacologically active. Nevertheless, the concentration of some classes as flavonoids in wild-type plants is rather low. To enhance the production in hop, it would be interesting to modify the regulation of genes in the flavonoid biosynthetic pathway. For this purpose, the regulatory factor PAP1/AtMYB75 from Arabidopsis thaliana L. was introduced into hop plants cv. Tettnanger by Agrobacterium-mediated genetic transformation. Twenty kanamycin-resistant transgenic plants were obtained. It was shown that PAP1/AtMYB75 was stably incorporated and expressed in the hop genome. In comparison to the wild-type plants, the color of female flowers and cones of transgenic plants was reddish to pink. Chemical analysis revealed higher levels of anthocyanins, rutin, isoquercitin, kaempferol-glucoside, kaempferol-glucoside-malonate, desmethylxanthohumol, xanthohumol, α-acids and β-acids in transgenic plants compared to wild-type plants.

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

PubMed

Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

2007-01-01

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

Lipoprotein lipase gene-deficient mice with hypertriglyceridaemia associated with acute pancreatitis.

PubMed

Tang, Maochun; Zong, Pengfei; Zhang, Ting; Wang, Dongyan; Wang, Yuhui; Zhao, Yan

2016-10-01

To investigate the severity of pancreatitis in lipoprotein lipase (LPL)-deficient hypertriglyceridaemic (HTG) heterozygous mice and to establish an experimental animal model for HTG pancreatitis study. LPL-deficient HTG heterozygous mice were rescued by somatic gene transfer and mated with wild-type mice. The plasma amylase, triglyceride, and pathologic changes in the pancreas of the LPL-deficient HTG heterozygous mice were compared with those of wild-type mice to assess the severity of pancreatitis. In addition, acute pancreatitis (AP) was induced by caerulein (50 µg/kg) for further assessment. The levels of plasma amylase and triglyceride were significantly higher in the LPL-deficient HTG heterozygous mice. According to the pancreatic histopathologic scores, the LPL-deficient HTG heterozygous mice showed more severe pathologic damage than the wild-type mice. Lipoprotein lipase deficient heterozygous mice developed severe caerulein-induced pancreatitis. In addition, their high triglyceride levels were stable. Therefore, LPL-deficient HTG heterozygous mice are a useful experimental model for studying HTG pancreatitis.

Sleep apneas are increased in mice lacking monoamine oxidase A.

PubMed

Real, Caroline; Popa, Daniela; Seif, Isabelle; Callebert, Jacques; Launay, Jean-Marie; Adrien, Joëlle; Escourrou, Pierre

2007-10-01

Alterations in the serotonin (5-HT) system have been suggested as a mechanism of sleep apnea in humans and rodents. The objective is to evaluate the contribution of 5-HT to this disorder. We studied sleep and breathing (whole-body plethysmography) in mutant mice that lack monoamine oxidase A (MAOA) and have increased concentrations of monoamines, including 5-HT. Compared to wild-type mice, the mutants showed similar amounts of slow wave sleep (SWS) and rapid eye movement sleep (REMS), but exhibited a 3-fold increase in SWS and REMS apnea indices. Acute administration of the MAOA inhibitor clorgyline decreased REMS amounts and increased the apnea index in wild-type but not mutant mice. Parachlorophenylalanine, a 5-HT synthesis inhibitor, reduced whole brain concentrations of 5-HT in both strains, and induced a decrease in apnea index in mutant but not wild-type mice. Our results show that MAOA deficiency is associated with increased sleep apnea in mice and suggest that an acute or chronic excess of 5-HT contributes to this phenotype.

Effects of gene deletion of the tissue inhibitor of the matrix metalloproteinase-type 1 (TIMP-1) on left ventricular geometry and function in mice

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.

Taxonomic and functional trait diversity of wild bees in different urban settings.

PubMed

Normandin, Étienne; Vereecken, Nicolas J; Buddle, Christopher M; Fournier, Valérie

2017-01-01

Urbanization is one of the major anthropogenic processes contributing to local habitat loss and extirpation of numerous species, including wild bees, the most widespread pollinators. Little is known about the mechanisms through which urbanization impacts wild bee communities, or the types of urban green spaces that best promote their conservation in cities. The main objective of this study was to describe and compare wild bee community diversity, structure, and dynamics in two Canadian cities, Montreal and Quebec City. A second objective was to compare functional trait diversity among three habitat types (cemeteries, community gardens and urban parks) within each city. Bees were collected using pan traps and netting on the same 46 sites, multiple times, over the active season in 2012 and 2013. A total of 32,237 specimens were identified, representing 200 species and 6 families, including two new continental records, Hylaeus communis Nylander (1852) and Anthidium florentinum (Fabricius, 1775). Despite high community evenness, we found significant abundance of diverse species, including exotic ones. Spatio-temporal analysis showed higher stability in the most urbanized city (Montreal) but low nestedness of species assemblages among the three urban habitats in both cities. Our study demonstrates that cities are home to diverse communities of wild bees, but in turn affect bee community structure and dynamics. We also found that community gardens harbour high levels of functional trait diversity. Urban agriculture therefore contributes substantially to the provision of functionally diverse bee communities and possibly to urban pollination services.

Taxonomic and functional trait diversity of wild bees in different urban settings

PubMed Central

Buddle, Christopher M.; Fournier, Valérie

2017-01-01

Urbanization is one of the major anthropogenic processes contributing to local habitat loss and extirpation of numerous species, including wild bees, the most widespread pollinators. Little is known about the mechanisms through which urbanization impacts wild bee communities, or the types of urban green spaces that best promote their conservation in cities. The main objective of this study was to describe and compare wild bee community diversity, structure, and dynamics in two Canadian cities, Montreal and Quebec City. A second objective was to compare functional trait diversity among three habitat types (cemeteries, community gardens and urban parks) within each city. Bees were collected using pan traps and netting on the same 46 sites, multiple times, over the active season in 2012 and 2013. A total of 32,237 specimens were identified, representing 200 species and 6 families, including two new continental records, Hylaeus communis Nylander (1852) and Anthidium florentinum (Fabricius, 1775). Despite high community evenness, we found significant abundance of diverse species, including exotic ones. Spatio-temporal analysis showed higher stability in the most urbanized city (Montreal) but low nestedness of species assemblages among the three urban habitats in both cities. Our study demonstrates that cities are home to diverse communities of wild bees, but in turn affect bee community structure and dynamics. We also found that community gardens harbour high levels of functional trait diversity. Urban agriculture therefore contributes substantially to the provision of functionally diverse bee communities and possibly to urban pollination services. PMID:28286711

Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants

NASA Astrophysics Data System (ADS)

Tosatto, Laura; Horrocks, Mathew H.; Dear, Alexander J.; Knowles, Tuomas P. J.; Dalla Serra, Mauro; Cremades, Nunilo; Dobson, Christopher M.; Klenerman, David

2015-11-01

Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson’s disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.

The role of nicotinic receptor alpha 7 subunits in nicotine discrimination.

PubMed

Stolerman, I P; Chamberlain, S; Bizarro, L; Fernandes, C; Schalkwyk, L

2004-03-01

The subtypes of nicotinic receptors at which the behavioural effects of nicotine originate are not fully understood. The experiments described here use mice lacking the alpha7 subunit of nicotinic receptors to investigate the role of alpha7-containing receptors in nicotine discrimination. Wild-type and alpha7-knockout mice were trained in a two-lever nicotine discrimination procedure using a tandem schedule of food reinforcement. Mutant mice exhibited baseline rates of lever-pressing as low as 52.2% of rates in wild-type controls (n=21-24). Mutant and wild-type mice acquired discrimination of nicotine (0.4 or 0.8 mg/kg) at a similar rate (n=10-12) and reached similar final levels of accuracy (71.9 +/- 4.4% and 90.8 +/- 3.1% after 60 training sessions for 0.4 and 0.8 mg/kg training doses, respectively, in mutant mice, as compared with 75.0 +/- 6.5% and 87.6 +/- 4.8% for wild types). The genotypes exhibited similar steep dose-response curves for nicotine discrimination. In both genotypes, dose-response curves for mice trained with 0.8 mg/kg of nicotine were displaced three- to four-fold to the right as compared with those for the mice trained with the smaller dose. The predominant effect of nicotine on the overall rate of responding was a reduction at the largest doses tested and there was no difference between the genotypes. The results suggest that nicotinic receptors containing the alpha7 subunit do not contribute to the discriminative stimulus or response-rate-depressant effects of nicotine, although they may regulate baseline rates of operant responding.

Long-term high-fat feeding induces greater fat storage in mice lacking UCP3.

PubMed

Costford, Sheila R; Chaudhry, Shehla N; Crawford, Sean A; Salkhordeh, Mahmoud; Harper, Mary-Ellen

2008-11-01

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein highly expressed in skeletal muscle. While UCP3's function is still unknown, it has been hypothesized to act as a fatty acid (FA) anion exporter, protecting mitochondria against lipid peroxidation and/or facilitating FA oxidation. The aim of this study was to determine the effects of long-term feeding of a 45% fat diet on whole body indicators of muscle metabolism in congenic C57BL/6 mice that were either lacking UCP3 (Ucp3(-/-)) or had a transgenically induced approximately twofold increase in UCP3 levels (UCP3tg). Mice were fed the high-fat (HF) diet for a period of either 4 or 8 mo immediately following weaning. After long-term HF feeding, UCP3tg mice weighed an average of 15% less than wild-type mice (P < 0.05) and were 20% less metabolically efficient than both wild-type and Ucp3(-/-) mice (P < 0.01). Additionally, wild-type mice had 21% lower, whereas UCP3tg mice had 36% lower, levels of adiposity compared with Ucp3(-/-) mice (P < 0.05 and P < 0.001, respectively), indicating a protective effect of UCP3 against fat gain. No differences in whole body oxygen consumption were detected following long-term HF feeding. Glucose and insulin tolerance tests revealed that both the UCP3tg and Ucp3(-/-) mice were more glucose tolerant and insulin sensitive compared with wild-type mice after short-term HF feeding, but this protection was not maintained in the long term. Findings indicate that UCP3 is involved in protection from fat gain induced by long-term HF feeding, but not in protection from insulin resistance.

Regulation of Drought Tolerance by the F-Box Protein MAX2 in Arabidopsis1[C][W][OPEN

PubMed Central

Bu, Qingyun; Lv, Tianxiao; Shen, Hui; Luong, Phi; Wang, Jimmy; Wang, Zhenyu; Huang, Zhigang; Xiao, Langtao; Engineer, Cawas; Kim, Tae Houn; Schroeder, Julian I.; Huq, Enamul

2014-01-01

MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions. PMID:24198318

InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals.

PubMed

Luo, Yihui; Liu, Yan; Sun, Dexter; Ojcius, David M; Zhao, Jinfang; Lin, Xuai; Wu, Dong; Zhang, Rongguang; Chen, Ming; Li, Lanjuan; Yan, Jie

2011-10-21

Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.

Significance and Regional Dependency of Peptide Transporter (PEPT) 1 in the Intestinal Permeability of Glycylsarcosine: In Situ Single-Pass Perfusion Studies in Wild-Type and Pept1 Knockout Mice

PubMed Central

Jappar, Dilara; Wu, Shu-Pei; Hu, Yongjun

2010-01-01

The purpose of this study was to evaluate the role, relevance, and regional dependence of peptide transporter (PEPT) 1 expression and function in mouse intestines using the model dipeptide glycylsarcosine (GlySar). After isolating specific intestinal segments, in situ single-pass perfusions were performed in wild-type and Pept1 knockout mice. The permeability of [3H]GlySar was measured as a function of perfusate pH, dipeptide concentration, potential inhibitors, and intestinal segment, along with PEPT1 mRNA and protein. We found the permeability of GlySar to be saturable (Km = 5.7 mM), pH-dependent (maximal value at pH 5.5), and specific for PEPT1; other peptide transporters, such as PHT1 and PHT2, were not involved, as judged by the lack of GlySar inhibition by excess concentrations of histidine. GlySar permeabilities were comparable in the duodenum and jejunum of wild-type mice but were much larger than that in ileum (approximately 2-fold). A PEPT1-mediated permeability was not observed for GlySar in the colon of wild-type mice (<10% residual uptake compared to proximal small intestine). Moreover, GlySar permeabilities were very low and not different in the duodenum, jejunum, ileum, and colon of Pept1 knockout mice. Functional activity of intestinal PEPT1 was confirmed by real-time polymerase chain reaction and immunoblot analyses. Our findings suggest that a loss of PEPT1 activity (e.g., due to polymorphisms, disease, or drug interactions) should have a major effect in reducing the intestinal absorption of di-/tripeptides, peptidomimetics, and peptide-like drugs. PMID:20660104

Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast.

PubMed

Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H Ekkehard

2005-03-01

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH(-) to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis.

Impaired pH Homeostasis in Arabidopsis Lacking the Vacuolar Dicarboxylate Transporter and Analysis of Carboxylic Acid Transport across the Tonoplast1

PubMed Central

Hurth, Marco Alois; Suh, Su Jeoung; Kretzschmar, Tobias; Geis, Tina; Bregante, Monica; Gambale, Franco; Martinoia, Enrico; Neuhaus, H. Ekkehard

2005-01-01

Arabidopsis (Arabidopsis thaliana) mutants lacking the tonoplastic malate transporter AttDT (A. thaliana tonoplast dicarboxylate transporter) and wild-type plants showed no phenotypic differences when grown under standard conditions. To identify putative metabolic changes in AttDT knock-out plants, we provoked a metabolic scenario connected to an increased consumption of dicarboxylates. Acidification of leaf discs stimulated dicarboxylate consumption and led to extremely low levels of dicarboxylates in mutants. To investigate whether reduced dicarboxylate concentrations in mutant leaf cells and, hence, reduced capacity to produce OH− to overcome acidification might affect metabolism, we measured photosynthetic oxygen evolution under conditions where the cytosol is acidified. AttDT::tDNA protoplasts showed a much stronger inhibition of oxygen evolution at low pH values when compared to wild-type protoplasts. Apparently citrate, which is present in higher amounts in knock-out plants, is not able to replace dicarboxylates to overcome acidification. To raise more information on the cellular level, we performed localization studies of carboxylates. Although the total pool of carboxylates in mutant vacuoles was nearly unaltered, these organelles contained a lower proportion of malate and fumarate and a higher proportion of citrate when compared to wild-type vacuoles. These alterations concur with the observation that radioactively labeled malate and citrate are transported into Arabidopsis vacuoles by different carriers. In addition, wild-type vacuoles and corresponding organelles from AttDT::tDNA mutants exhibited similar malate channel activities. In conclusion, these results show that Arabidopsis vacuoles contain at least two transporters and a channel for dicarboxylates and citrate and that the activity of AttDT is critical for regulation of pH homeostasis. PMID:15728336

Induction of a dwarf phenotype with IBH1 may enable increased production of plant-made pharmaceuticals in plant factory conditions.

PubMed

Nagatoshi, Yukari; Ikeda, Miho; Kishi, Hiroyuki; Hiratsu, Keiichiro; Muraguchi, Atsushi; Ohme-Takagi, Masaru

2016-03-01

Year-round production in a contained, environmentally controlled 'plant factory' may provide a cost-effective method to produce pharmaceuticals and other high-value products. However, cost-effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size. We estimate that, in a given volume of cultivation space, we can grow five times more AtIBH1SRDX plants than wild-type plants. Although, the AtIBH1SRDX plants also showed reduced biomass compared with wild-type plants, they produced about four times more biomass per unit of cultivation volume. To test whether the dwarf phenotype affects the production of recombinant proteins, we expressed the genes for anti-hepatitis B virus antibodies (anti-HBs) in tobacco plants and found that the production of anti-HBs per unit fresh weight did not significantly differ between wild-type and AtIBH1SRDX plants. These data indicate that P35S:AtIBH1SRDX plants produced about fourfold more antibody per unit of cultivation volume, compared with wild type. Our results indicate that AtIBH1SRDX provides a useful tool for the modification of plant phenotype for cost-effective production of high-value products by stably transformed plants in plant factory conditions. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Both germ line and somatic genetics of the p53 pathway affect ovarian cancer incidence and survival.

PubMed

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.

NADPH Oxidase Inhibition Improves Neurological Outcomes in Surgically-Induced Brain Injury

PubMed Central

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

Overexpression of a cotton (Gossypium hirsutum) WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants.

PubMed

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.

Responses of hybrid aspen over-expressing a PIP2;5 aquaporin to low root temperature.

PubMed

Ranganathan, Kapilan; El Kayal, Walid; Cooke, Janice E K; Zwiazek, Janusz J

2016-03-15

Aquaporins mediate the movement of water across cell membranes. Plasma membrane intrinsic protein 2;5 from Populus trichocarpa×deltoides (PtdPIP2;5) was previously demonstrated to be a functionally important water conducting aquaporin. To study the relevance of aquaporin-mediated root water transport at low temperatures, we generated transgenic Populus tremula×alba over-expressing PtdPIP2;5 under control of the maize ubiquitin promoter, and compared the physiological responses and water transport properties of the PtdPIP2;5 over-expressing lines (PtdPIP2;5ox) with wild-type plants. We hypothesized that over-expression of PtdPIP2;5 would reduce temperature sensitivity of root water transport and gas exchange. Decreasing root temperatures to 10 and 5°C significantly decreased hydraulic conductivities (Lp) in wild-type plants, but had no significant effect on Lp in PtdPIP2;5ox plants. Recovery of Lp in the transgenic lines returned to 20°C from 5°C was faster than in the wild-type plants. Low root temperature did not induce major changes in transcript levels for other PIPs. When roots were exposed to 5°C in solution culture and shoots were exposed to 20°C, wild-type plants had significantly lower net photosynthetic and transpiration rates compared to PtdPIP2;5ox plants. Taken together, our results demonstrate that over-expression of PtdPIP2;5 in P. tremula×alba was effective in alleviating the effects of low root temperature on Lp and gas exchange. Copyright © 2016 Elsevier GmbH. All rights reserved.

The Structural Role of Elastic Fibers in the Cornea Investigated Using a Mouse Model for Marfan Syndrome

PubMed Central

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

Salicylic acid deficiency in NahG transgenic lines and sid2 mutants increases seed yield in the annual plant Arabidopsis thaliana

PubMed Central

Abreu, Maria Elizabeth; Munné-Bosch, Sergi

2009-01-01

Salicylic acid-deficient NahG transgenic lines and sid2 mutants were used to evaluate the role of this compound in the development of the short-lived, annual plant Arabidopsis thaliana, with a particular focus on the interplay between salicylic acid and other phytohormones. Low salicylic acid levels led to increased growth, as well as to smaller abscisic acid levels and reduced damage to PSII (as indicated by Fv/Fm ratios) during the reproductive stages in rosette leaves of NahG transgenic lines and sid2 mutants, compared with wild-type plants. Furthermore, salicylic acid deficiency highly influenced seed yield and composition. Seed production increased by 4.4-fold and 3.5-fold in NahG transgenic lines and sid2 mutants, respectively, compared to the wild type. Salicylic acid deficiency also improved seed composition in terms of antioxidant vitamin concentrations, seeds of salicylic acid-deficient plants showing higher levels of α- and γ-tocopherol (vitamin E) and β-carotene (pro-vitamin A) than seeds of wild-type plants. Seeds of salicylic acid-deficient plants also showed higher nitrogen concentrations than seeds of wild-type plants. It is concluded that (i) the sid2 gene, which encodes for isochorismate synthase, plays a central role in salicylic acid biosynthesis during plant development in A. thaliana, (ii) salicylic acid plays a role in the regulation of growth, senescence, and seed production, (iii) there is a cross-talk between salicylic acid and other phytohormones during plant development, and (iv) the concentrations of antioxidant vitamins in seeds may be influenced by the endogenous levels of salicylic acid in plants. PMID:19188277

Pseudomonas fluorescens F113 Mutant with Enhanced Competitive Colonization Ability and Improved Biocontrol Activity against Fungal Root Pathogens ▿

PubMed Central

Barahona, Emma; Navazo, Ana; Martínez-Granero, Francisco; Zea-Bonilla, Teresa; Pérez-Jiménez, Rosa María; Martín, Marta; Rivilla, Rafael

2011-01-01

Motility is one of the most important traits for efficient rhizosphere colonization by Pseudomonas fluorescens F113rif (F113). In this bacterium, motility is a polygenic trait that is repressed by at least three independent pathways, including the Gac posttranscriptional system, the Wsp chemotaxis-like pathway, and the SadB pathway. Here we show that the kinB gene, which encodes a signal transduction protein that together with AlgB has been implicated in alginate production, participates in swimming motility repression through the Gac pathway, acting downstream of the GacAS two-component system. Gac mutants are impaired in secondary metabolite production and are unsuitable as biocontrol agents. However, the kinB mutant and a triple mutant affected in kinB, sadB, and wspR (KSW) possess a wild-type phenotype for secondary metabolism. The KSW strain is hypermotile and more competitive for rhizosphere colonization than the wild-type strain. We have compared the biocontrol activity of KSW with those of the wild-type strain and a phenotypic variant (F113v35 [V35]) which is hypermotile and hypercompetitive but is affected in secondary metabolism since it harbors a gacS mutation. Biocontrol experiments in the Fusarium oxysporum f. sp. radicis-lycopersici/Lycopersicum esculentum (tomato) and Phytophthora cactorum/Fragaria vesca (strawberry) pathosystems have shown that the three strains possess biocontrol activity. Biocontrol activity was consistently lower for V35, indicating that the production of secondary metabolites was the most important trait for biocontrol. Strain KSW showed improved biocontrol compared with the wild-type strain, indicating that an increase in competitive colonization ability resulted in improved biocontrol and that the rational design of biocontrol agents by mutation is feasible. PMID:21685161

Pseudomonas fluorescens F113 mutant with enhanced competitive colonization ability and improved biocontrol activity against fungal root pathogens.

PubMed

Barahona, Emma; Navazo, Ana; Martínez-Granero, Francisco; Zea-Bonilla, Teresa; Pérez-Jiménez, Rosa María; Martín, Marta; Rivilla, Rafael

2011-08-01

Motility is one of the most important traits for efficient rhizosphere colonization by Pseudomonas fluorescens F113rif (F113). In this bacterium, motility is a polygenic trait that is repressed by at least three independent pathways, including the Gac posttranscriptional system, the Wsp chemotaxis-like pathway, and the SadB pathway. Here we show that the kinB gene, which encodes a signal transduction protein that together with AlgB has been implicated in alginate production, participates in swimming motility repression through the Gac pathway, acting downstream of the GacAS two-component system. Gac mutants are impaired in secondary metabolite production and are unsuitable as biocontrol agents. However, the kinB mutant and a triple mutant affected in kinB, sadB, and wspR (KSW) possess a wild-type phenotype for secondary metabolism. The KSW strain is hypermotile and more competitive for rhizosphere colonization than the wild-type strain. We have compared the biocontrol activity of KSW with those of the wild-type strain and a phenotypic variant (F113v35 [V35]) which is hypermotile and hypercompetitive but is affected in secondary metabolism since it harbors a gacS mutation. Biocontrol experiments in the Fusarium oxysporum f. sp. radicis-lycopersici/Lycopersicum esculentum (tomato) and Phytophthora cactorum/Fragaria vesca (strawberry) pathosystems have shown that the three strains possess biocontrol activity. Biocontrol activity was consistently lower for V35, indicating that the production of secondary metabolites was the most important trait for biocontrol. Strain KSW showed improved biocontrol compared with the wild-type strain, indicating that an increase in competitive colonization ability resulted in improved biocontrol and that the rational design of biocontrol agents by mutation is feasible.

Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.

PubMed

Weng, MeiZhi; Zheng, ZhongLiang; Bao, Wei; Cai, YongJun; Yin, Yan; Zou, GuoLin; Zou, GouLin

2009-11-01

Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.

Aluminum-Resistant Arabidopsis Mutants That Exhibit Altered Patterns of Aluminum Accumulation and Organic Acid Release from Roots1

PubMed Central

Larsen, Paul B.; Degenhardt, Jörg; Tai, Chin-Yin; Stenzler, Laura M.; Howell, Stephen H.; Kochian, Leon V.

1998-01-01

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L.V. Kochian [1998] Plant Physiol 117: 19–27). PMID:9576769

The Tomato Transcription Factor Pti4 Regulates Defense-Related Gene Expression via GCC Box and Non-GCC Box cis ElementsW⃞

PubMed Central

Chakravarthy, Suma; Tuori, Robert P.; D'Ascenzo, Mark D.; Fobert, Pierre R.; Després, Charles; Martin, Gregory B.

2003-01-01

The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box–containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs. PMID:14630974

Native Alanine Substitution in the Glycine Hinge Modulates Conformational Flexibility of Heme Nitric Oxide/Oxygen (H-NOX) Sensing Proteins.

PubMed

Hespen, Charles W; Bruegger, Joel J; Guo, Yirui; Marletta, Michael A

2018-06-15

Heme nitric oxide/oxygen sensing (H-NOX) domains are direct NO sensors that regulate a variety of biological functions in both bacteria and eukaryotes. Previous work on H-NOX proteins has shown that upon NO binding, a conformational change occurs along two glycine residues on adjacent helices (termed the glycine hinge). Despite the apparent importance of the glycine hinge, it is not fully conserved in all H-NOX domains. Several H-NOX sensors from the family Flavobacteriaceae contain a native alanine substitution in one of the hinge residues. In this work, the effect of the increased steric bulk within the Ala-Gly hinge on H-NOX function was investigated. The hinge in Kordia algicida OT-1 ( Ka H-NOX) is composed of A71 and G145. Ligand-binding properties and signaling function for this H-NOX were characterized. The variant A71G was designed to convert the hinge region of Ka H-NOX to the typical Gly-Gly motif. In activity assays with its cognate histidine kinase (HnoK), the wild type displayed increased signal specificity compared to A71G. Increasing titrations of unliganded A71G gradually inhibits HnoK autophosphorylation, while increasing titrations of unliganded wild type H-NOX does not inhibit HnoK. Crystal structures of both wild type and A71G Ka H-NOX were solved to 1.9 and 1.6 Å, respectively. Regions of H-NOX domains previously identified as involved in protein-protein interactions with HnoK display significantly higher b-factors in A71G compared to wild-type H-NOX. Both biochemical and structural data indicate that the hinge region controls overall conformational flexibility of the H-NOX, affecting NO complex formation and regulation of its HnoK.

Comparative Transcriptome Analyses between a Spontaneous Late-Ripening Sweet Orange Mutant and Its Wild Type Suggest the Functions of ABA, Sucrose and JA during Citrus Fruit Ripening

PubMed Central

Zhang, Ya-Jian; Wang, Xing-Jian; Wu, Ju-Xun; Chen, Shan-Yan; Chen, Hong; Chai, Li-Jun; Yi, Hua-Lin

2014-01-01

A spontaneous late-ripening mutant of ‘Jincheng’ (C. sinensis L. Osbeck) sweet orange exhibited a delay of fruit pigmentation and harvesting. In this work, we studied the processes of orange fruit ripening through the comparative analysis between the Jincheng mutant and its wild type. This study revealed that the fruit quality began to differ on 166th days after anthesis. At this stage, fruits were subjected to transcriptome analysis by RNA sequencing. 13,412 differentially expressed unigenes (DEGs) were found. Of these unigenes, 75.8% were down-regulated in the wild type, suggesting that the transcription level of wild type was lower than that of the mutant during this stage. These DEGs were mainly clustered into five pathways: metabolic pathways, plant-pathogen interaction, spliceosome, biosynthesis of plant hormones and biosynthesis of phenylpropanoids. Therefore, the expression profiles of the genes that are involved in abscisic acid, sucrose, and jasmonic acid metabolism and signal transduction pathways were analyzed during the six fruit ripening stages. The results revealed the regulation mechanism of sweet orange fruit ripening metabolism in the following four aspects: First, the more mature orange fruits were, the lower the transcription levels were. Second, the expression level of PME boosted with the maturity of the citrus fruit. Therefore, the expression level of PME might represent the degree of the orange fruit ripeness. Third, the interaction of PP2C, PYR/PYL, and SnRK2 was peculiar to the orange fruit ripening process. Fourth, abscisic acid, sucrose, and jasmonic acid all took part in orange fruit ripening process and might interact with each other. These findings provide an insight into the intricate process of sweet orange fruit ripening. PMID:25551568

Mutational analysis of photosystem I polypeptides in the cyanobacterium Synechocystis sp. PCC 6803. Targeted inactivation of psaI reveals the function of psaI in the structural organization of psaL

NASA Technical Reports Server (NTRS)

Xu, Q.; Hoppe, D.; Chitnis, V. P.; Odom, W. R.; Guikema, J. A.; Chitnis, P. R.; Spooner, B. S. (Principal Investigator)

1995-01-01

We cloned, characterized, and inactivated the psaI gene encoding a 4-kDa hydrophobic subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. The psaI gene is located 90 base pairs downstream from psaL, and is transcribed on 0.94- and 0.32-kilobase transcripts. To identify the function of PsaI, we generated a cyanobacterial strain in which psaI has been interrupted by a gene for chloramphenicol resistance. The wild-type and the mutant cells showed comparable rates of photoautotrophic growth at 25 degrees C. However, the mutant cells grew slower and contained less chlorophyll than the wild-type cells, when grown at 40 degrees C. The PsaI-less membranes from cells grown at either temperature showed a small decrease in NADP+ photoreduction rate when compared to the wild-type membranes. Inactivation of psaI led to an 80% decrease in the PsaL level in the photosynthetic membranes and to a complete loss of PsaL in the purified photosystem I preparations, but had little effect on the accumulation of other photosystem I subunits. Upon solubilization with nonionic detergents, photosystem I trimers could be obtained from the wild-type, but not from the PsaI-less membranes. The PsaI-less photosystem I monomers did not contain detectable levels of PsaL. Therefore, a structural interaction between PsaL and PsaI may stabilize the association of PsaL with the photosystem I core. PsaL in the wild-type and PsaI-less membranes showed equal resistance to removal by chaotropic agents. However, PsaL in the PsaI-less strain exhibited an increased susceptibility to proteolysis. From these data, we conclude that PsaI has a crucial role in aiding normal structural organization of PsaL within the photosystem I complex and the absence of PsaI alters PsaL organization, leading to a small, but physiologically significant, defect in photosystem I function.

The role of protease-activated receptor-2 on pulmonary neutrophils in the innate immune response to cockroach allergen

PubMed Central

2012-01-01

Background Serine proteases in German cockroach (GC) have been shown to mediate allergic airway inflammation through the activation of protease activated receptor (PAR)-2. Neutrophils play an important role in regulating the innate immune response, and are recruited into the airways following GC frass exposure. As such, we investigated the role of PAR-2 in airway neutrophil recruitment, activation and cytokine production following allergen exposure. Methods Wild type and PAR-2-deficient mice were administered a single intratracheal instillation of PBS or GC frass and neutrophil recruitment, expression of PAR-2, CD80, CD86, and MHC class II were assessed by flow cytometry and levels of tumor necrosis factor (TNF)α was assessed by ELISA. Uptake of AlexaFluor 405-labeled GC frass by neutrophils was performed by flow cytometry. Results Neutrophil recruitment in the lung and airways following GC frass exposure was significantly decreased in PAR-2-deficient mice compared to wild type mice. GC frass exposure increased the level of PAR-2 on pulmonary neutrophils and increased numbers of PAR-2-positive neutrophils were found in the lungs; however PAR-2 did not play a role in meditating allergen uptake. Comparing wild type and PAR-2-deficient mice, we found that a single exposure to GC frass increased levels of CD80 and CD86 on pulmonary neutrophils, an effect which was independent of PAR-2 expression. Neutrophils isolated from the whole lungs of naïve PAR-2-deficient mice treated ex vivo with GC frass produced significantly less TNFα than in similarly treated wild type neutrophils. Lastly, neutrophils were isolated from the bronchoalveolar lavage fluid of wild type and PAR-2-deficient mice following a single intratracheal exposure to GC frass. Airway neutrophils from PAR-2-deficient mice released substantially decreased levels of TNFα, suggesting a role for PAR-2 in neutrophil-derived cytokine production. Conclusions Together these data suggest PAR-2 expression can be upregulated on lung neutrophils following allergen exposure and the consequence is altered release of TNFα which could drive the early innate immune response. PMID:22954301

An rfaH Mutant of Salmonella enterica Serovar Typhimurium is Attenuated in Swine and Reduces Intestinal Colonization, Fecal Shedding, and Disease Severity Due to Virulent Salmonella Typhimurium

PubMed Central

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

The refractive state of the eye in Icelandic horses with the Silver mutation.

PubMed

Johansson, Maria K; Jäderkvist Fegraeus, Kim; Lindgren, Gabriella; Ekesten, Björn

2017-06-02

The syndrome Multiple Congenital Ocular Anomalies (MCOA) is a congenital eye disorder in horses. Both the MCOA syndrome and the Silver coat colour in horses are caused by the same missense mutation in the premelanosome protein (PMEL) gene. Horses homozygous for the Silver mutation (TT) are affected by multiple ocular defects causing visual impairment or blindness. Horses heterozygous for the Silver mutation (CT) have less severe clinical signs, usually cysts arising from the ciliary body iris or retina temporally. It is still unknown if the vision is impaired in horses heterozygous for the Silver mutation. A recent study reported that Comtois horses carrying the Silver mutation had significantly deeper anterior chambers of the eye compared to wild-type horses. This could potentially cause refractive errors. The purpose of the present study was to investigate if Icelandic horses with the Silver mutation have refractive errors compared to wild-type horses. One hundred and fifty-two Icelandic horses were included in the study, 71 CT horses and five TT horses. All horses were genotyped for the missense mutation in PMEL. Each CT and TT horse was matched by a wild-type (CC) horse of the same age ± 1 year. Skiascopy and a brief ophthalmic examination were performed in all horses. Association between refraction and age, eye, genotype and sex was tested by linear mixed-effect model analysis. TT horses with controls were not included in the statistical analyses as they were too few. The interaction between age and genotype had a significant impact on the refractive state (P = 0.0001). CT horses older than 16 years were on average more myopic than wild-type horses of the same age. No difference in the refractive state could be observed between genotypes (CT and CC) in horses younger than 16 years. TT horses were myopic (-2 D or more) in one or both eyes regardless of age. Our results indicate that an elderly Icelandic horse (older than 16 years) carrying the Silver mutation is more likely to be myopic than a wild-type horse of the same age.

Thalidomide ameliorates portal hypertension via nitric oxide synthase independent reduced systolic blood pressure

PubMed Central

Theodorakis, Nicholas G; Wang, Yining N; Korshunov, Vyacheslav A; Maluccio, Mary A; Skill, Nicholas J

2015-01-01

AIM: Portal hypertension is a common complication of liver cirrhosis and significantly increases mortality and morbidity. Previous reports have suggested that the compound thalidomide attenuates portal hypertension (PHT). However, the mechanism for this action is not fully elucidated. One hypothesis is that thalidomide destabilizes tumor necrosis factor α (TNFα) mRNA and therefore diminishes TNFα induction of nitric oxide synthase (NOS) and the production of nitric oxide (NO). To examine this hypothesis, we utilized the murine partial portal vein ligation (PVL) PHT model in combination with endothelial or inducible NOS isoform gene knockout mice. METHODS: Wild type, inducible nitric oxide synthase (iNOS)-/- and endothelial nitric oxide synthase (eNOS)-/- mice received either PVL or sham surgery and were given either thalidomide or vehicle. Serum nitrate (total nitrate, NOx) was measured daily for 7 d as a surrogate of NO synthesis. Serum TNFα level was quantified by enzyme-linked immunosorbent assay. TNFα mRNA was quantified in liver and aorta tissue by reverse transcription-polymerase chain reaction. PHT was determined by recording splenic pulp pressure (SPP) and abdominal aortic flow after 0-7 d. Response to thalidomide was determined by measurement of SPP and mean arterial pressure (MAP). RESULTS: SPP, abdominal aortic flow (Qao) and plasma NOx were increased in wild type and iNOS-/- PVL mice when compared to sham operated control mice. In contrast, SPP, Qao and plasma NOx were not increased in eNOS-/- PVL mice when compared to sham controls. Serum TNFα level in both sham and PVL mice was below the detection limit of the commercial ELISA used. Therefore, the effect of thalidomide on serum TNFα levels was undetermined in wild type, eNOS-/- or iNOS-/- mice. Thalidomide acutely increased plasma NOx in wild type and eNOS-/- mice but not iNOS-/- mice. Moreover, thalidomide temporarily (0-90 min) decreased mean arterial pressure, SPP and Qao in wild type, eNOS-/- and iNOS-/- PVL mice, after which time levels returned to the respective baseline. CONCLUSION: Thalidomide does not reduce portal pressure in the murine PVL model by modulation of NO biosynthesis. Rather, thalidomide reduces PHT by decreasing MAP by an undetermined mechanism. PMID:25892862

Characterization and proteomic analysis of the Pseudomonas sp. HK-6 xenB knockout mutant under RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) stress.

PubMed

Lee, Bheong-Uk; Choi, Moon-Seop; Oh, Kye-Heon

2015-01-01

Pseudomonas sp. HK-6 is able to utilize RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) as its sole nitrogen source. The role of the xenB gene, encoding xenobiotic reductase B, was investigated using HK-6 xenB knockout mutants. The xenB mutant degraded RDX to a level that was 10-fold less than that obtained with the wild-type HK-6 strain. After 60 days of culture with 25 or 50 μM RDX, no residual RDX was detected in the supernatants of the wild-type aerobically grown cultures, whereas approximately 90 % of the RDX remained in the xenB mutant cultures. The xenB mutant bacteria exhibited a 10(2)-10(4)-fold decrease in survival rate compared to the wild-type. The expression of DnaK and GroEL proteins, two typical stress shock proteins (SSPs), in the xenB mutant increased after immediate exposure to RDX, yet dramatically decreased after 4 h of exposure. In addition, DnaK and GroEL were more highly expressed in the cultures with 25 μM RDX in the medium but showed low expression in the cultures with 50 or 75 μM RDX. The expression levels of the dnaK and groEL genes measured by RT-qPCR were also much lower in the xenB genetic background. Analyses of the proteomes of the HK-6 and xenB mutant cells grown under conditions of RDX stress showed increased induction of several proteins, such as Alg8, alginate biosynthesis sensor histidine kinase, and OprH in the xenB mutants when compared to wild-type. However, many proteins, including two SSPs (DnaK and GroEL) and proteins involved in metabolism, exhibited lower expression levels in the xenB mutant than in the wild-type HK-6 strain. The xenB knockout mutation leads to reduced RDX degradation ability, which renders the mutant more sensitive to RDX stress and results in a lower survival rate and an altered proteomic profile under RDX stress.

Thalidomide ameliorates portal hypertension via nitric oxide synthase independent reduced systolic blood pressure.

PubMed

Theodorakis, Nicholas G; Wang, Yining N; Korshunov, Vyacheslav A; Maluccio, Mary A; Skill, Nicholas J

2015-04-14

Portal hypertension is a common complication of liver cirrhosis and significantly increases mortality and morbidity. Previous reports have suggested that the compound thalidomide attenuates portal hypertension (PHT). However, the mechanism for this action is not fully elucidated. One hypothesis is that thalidomide destabilizes tumor necrosis factor α (TNFα) mRNA and therefore diminishes TNFα induction of nitric oxide synthase (NOS) and the production of nitric oxide (NO). To examine this hypothesis, we utilized the murine partial portal vein ligation (PVL) PHT model in combination with endothelial or inducible NOS isoform gene knockout mice. Wild type, inducible nitric oxide synthase (iNOS)(-/-) and endothelial nitric oxide synthase (eNOS)(-/-) mice received either PVL or sham surgery and were given either thalidomide or vehicle. Serum nitrate (total nitrate, NOx) was measured daily for 7 d as a surrogate of NO synthesis. Serum TNFα level was quantified by enzyme-linked immunosorbent assay. TNFα mRNA was quantified in liver and aorta tissue by reverse transcription-polymerase chain reaction. PHT was determined by recording splenic pulp pressure (SPP) and abdominal aortic flow after 0-7 d. Response to thalidomide was determined by measurement of SPP and mean arterial pressure (MAP). SPP, abdominal aortic flow (Qao) and plasma NOx were increased in wild type and iNOS(-/-) PVL mice when compared to sham operated control mice. In contrast, SPP, Qao and plasma NOx were not increased in eNOS(-/-) PVL mice when compared to sham controls. Serum TNFα level in both sham and PVL mice was below the detection limit of the commercial ELISA used. Therefore, the effect of thalidomide on serum TNFα levels was undetermined in wild type, eNOS(-/-) or iNOS(-/-) mice. Thalidomide acutely increased plasma NOx in wild type and eNOS(-/-) mice but not iNOS(-/-) mice. Moreover, thalidomide temporarily (0-90 min) decreased mean arterial pressure, SPP and Qao in wild type, eNOS(-/-) and iNOS(-/-) PVL mice, after which time levels returned to the respective baseline. Thalidomide does not reduce portal pressure in the murine PVL model by modulation of NO biosynthesis. Rather, thalidomide reduces PHT by decreasing MAP by an undetermined mechanism.

Epithelial V-Like Antigen Mediates Efficacy of Anti-Alpha4 Integrin Treatment in a Mouse Model of Multiple Sclerosis

PubMed Central

Wright, Erik; Rahgozar, Kusha; Hallworth, Nicholas; Lanker, Stefan; Carrithers, Michael D.

2013-01-01

Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha4 integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA+ lymphocytes following immunization. Following active induction of EAE using the MOG35–55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19+, CD21+, sIgG+), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5+IgG+ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19+, CD21+, sIgG+ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19+, CD21+, sIgG+ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that these findings could optimize the selection of treatment responders. PMID:23951051

'Who's who' in two different flower types of Calluna vulgaris (Ericaceae): morphological and molecular analyses of flower organ identity

PubMed Central

2009-01-01

Background The ornamental crop Calluna vulgaris is of increasing importance to the horticultural industry in the northern hemisphere due to a flower organ mutation: the flowers of the 'bud-flowering' phenotype remain closed i.e. as buds throughout the total flowering period and thereby maintain more colorful flowers for a longer period of time than the wild-type. This feature is accompanied and presumably caused by the complete lack of stamens. Descriptions of this botanical particularity are inconsistent and partially conflicting. In order to clarify basic questions of flower organ identity in general and stamen loss in detail, a study of the wild-type and the 'bud-flowering' flower type of C. vulgaris was initiated. Results Flowers were examined by macro- and microscopic techniques. Organ development was investigated comparatively in both the wild-type and the 'bud-flowering' type by histological analyses. Analysis of epidermal cell surface structure of vegetative tissues and perianth organs using scanning electron microscopy revealed that in wild-type flowers the outer whorls of colored organs may be identified as sepals, while the inner ones may be identified as petals. In the 'bud-flowering' type, two whorls of sepals are directly followed by the gynoecium. Both, petals and stamens, are completely missing in this flower type. The uppermost whorl of green leaves represents bracts in both flower types. In addition, two MADS-box genes (homologs of AP3/DEF and SEP1/2) were identified in C. vulgaris using RACE-PCR. Expression analysis by qRT-PCR was conducted for both genes in leaves, bracts, sepals and petals. These experiments revealed an expression pattern supporting the organ classification based on morphological characteristics. Conclusions Organ identity in both wild-type and 'bud-flowering' C. vulgaris was clarified using a combination of microscopic and molecular methods. Our results for bract, sepal and petal organ identity are supported by the 'ABCDE model'. However, loss of stamens in the 'bud-flowering' phenotype is an exceptional flower organ modification that cannot be explained by modified spatial expression of known organ identity genes. PMID:20003430

Role of cytochrome P450 2E1 in protein nitration and ubiquitin-mediated degradation during acetaminophen toxicity.

PubMed

Abdelmegeed, Mohamed A; Moon, Kwan-Hoon; Chen, Chi; Gonzalez, Frank J; Song, Byoung-Joon

2010-01-01

It is well established that following a toxic dose of acetaminophen (APAP), nitrotyrosine protein adducts (3-NT), a hallmark of peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where cytochrome P450 2E1 (CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in APAP-mediated toxicity. This study was aimed at investigating the relationship between CYP2E1 and nitration (3-NT formation) followed by ubiquitin-mediated degradation of proteins in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400mg/kg) for 4 and 24h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of nitric oxide synthase (NOS), was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible than the native proteins for ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity.

Local and distal effects of arbuscular mycorrhizal colonization on direct pathway Pi uptake and root growth in Medicago truncatula

PubMed Central

Watts-Williams, Stephanie J.; Jakobsen, Iver; Cavagnaro, Timothy R.; Grønlund, Mette

2015-01-01

Two pathways exist for plant Pi uptake from soil: via root epidermal cells (direct pathway) or via associations with arbuscular mycorrhizal (AM) fungi, and the two pathways interact in a complex manner. This study investigated distal and local effects of AM colonization on direct root Pi uptake and root growth, at different soil P levels. Medicago truncatula was grown at three soil P levels in split-pots with or without AM fungal inoculation and where one root half grew into soil labelled with 33P. Plant genotypes included the A17 wild type and the mtpt4 mutant. The mtpt4 mutant, colonized by AM fungi, but with no functional mycorrhizal pathway for Pi uptake, was included to better understand effects of AM colonization per se. Colonization by AM fungi decreased expression of direct Pi transporter genes locally, but not distally in the wild type. In mtpt4 mutant plants, direct Pi transporter genes and the Pi starvation-induced gene Mt4 were more highly expressed than in wild-type roots. In wild-type plants, less Pi was taken up via the direct pathway by non-colonized roots when the other root half was colonized by AM fungi, compared with non-mycorrhizal plants. Colonization by AM fungi strongly influenced root growth locally and distally, and direct root Pi uptake activity locally, but had only a weak influence on distal direct pathway activity. The responses to AM colonization in the mtpt4 mutant suggested that in the wild type, the increased P concentration of colonized roots was a major factor driving the effects of AM colonization on direct root Pi uptake. PMID:25944927

The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus: the role of Glu232 in catalysis.

PubMed

Hall, James; Reschke, Stefan; Cao, Hongnan; Leimkühler, Silke; Hille, Russ

2014-11-14

The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of various DNA repair pathways in chromosomal inversion formation in CHO mutants.

PubMed

Cartwright, Ian M; Kato, Takamitsu A

2015-01-01

In an effort to better understand the formation of chromosomal inversions, we investigated the role of various DNA repair pathways, including the non-homologous end joining (NHEJ), homologous recombination (HR), and Fanconi Anemia (FA) repair pathways for the formation of radiation induced chromosomal inversions. CHO10B2 wild type, CHO DNA repair-deficient, and CHO DNA repair-deficient corrected mutant cells were synchronized into G1 phase and exposed to gamma-rays. First post-irradiation metaphase cells were analyzed for chromosomal inversions by a differential chromatid staining technique involving a single cycle pre-irradiation ethynyl-uridine treatment and statistic calculations. It was observed that inhibition of the NHEJ pathway resulted in an overall decrease in the number of radiation-induced inversions, roughly a 50% decrease when compared to the CHO wild type. Interestingly, inhibition of the FA pathway resulted in an increase in both the number of spontaneous inversions and the number of radiation-induced inversions observed after exposure to 2 Gy of ionizing radiation. It was observed that FA-deficient cells contained roughly 330% (1.24 inversions per cell) more spontaneous inversions and 20% (0.4 inversions per cell) more radiation-induced inversions than the wild-type CHO cell lines. The HR mutants, defective in Rad51 foci, showed similar number of spontaneous and radiation-induced inversion as the wild-type cells. Gene complementation resulted in both spontaneous and radiation-induced inversions resembling the CHO wild-type cells. We have concluded that the NHEJ repair pathway contributes to the formation of radiation-induced inversions. Additionally, through an unknown molecular mechanism it appears that the FA signal pathway prevents the formation of both spontaneous and radiation induced inversions.

Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Responses to Sorghum bicolor (Poales: Poaceae) Tissues From Lowered Lignin Lines

PubMed Central

Dowd, Patrick F.; Sattler, Scott E.

2015-01-01

The presence of lignin within biomass impedes the production of liquid fuels. Plants with altered lignin content and composition are more amenable to lignocellulosic conversion to ethanol and other biofuels but may be more susceptible to insect damage where lignin is an important resistance factor. However, reduced lignin lines of switchgrasses still retained insect resistance in prior studies. Therefore, we hypothesized that sorghum lines with lowered lignin content will also retain insect resistance. Sorghum excised leaves and stalk pith Sorghum bicolor (L.) Moench (Poales: Poaceae) from near isogenic brown midrib (bmr) 6 and 12 mutants lines, which have lowered lignin content and increased lignocellulosic ethanol conversion efficiency, were examined for insect resistance relative to wild-type (normal BTx623). Greenhouse and growth chamber grown plant tissues were fed to first-instar larvae of corn earworms, Helicoverpa zea (Boddie) and fall armyworms Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), two sorghum major pests. Younger bmr leaves had significantly greater feeding damage in some assays than wild-type leaves, but older bmr6 leaves generally had significantly less damage than wild-type leaves. Caterpillars feeding on the bmr6 leaves often weighed significantly less than those feeding on wild-type leaves, especially in the S. frugiperda assays. Larvae fed the pith from bmr stalks had significantly higher mortality compared with those larvae fed on wild-type pith, which suggested that bmr pith was more toxic. Thus, reducing lignin content or changing subunit composition of bioenergy grasses does not necessarily increase their susceptibility to insects and may result in increased resistance, which would contribute to sustainable production. PMID:25601946

Spontaneous mutation reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.

PubMed

Horn, Nikki; Wegmann, Udo; Dertli, Enes; Mulholland, Francis; Collins, Samuel R A; Waldron, Keith W; Bongaerts, Roy J; Mayer, Melinda J; Narbad, Arjan

2013-01-01

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

Deficiency of intestinal mucin-2 ameliorates experimental alcoholic liver disease in mice

PubMed Central

Hartmann, Phillipp; Chen, Peng; Wang, Hui J.; Wang, Lirui; McCole, Declan F.; Brandl, Katharina; Stärkel, Peter; Belzer, Clara; Hellerbrand, Claus; Tsukamoto, Hidekazu; Ho, Samuel B.; Schnabl, Bernd

2013-01-01

The intestinal mucus layer protects the epithelium from noxious agents, viruses, and pathogenic bacteria present in the gastrointestinal tract. It is composed of mucins, predominantly mucin-2 (Muc2), secreted by goblet cells of the intestine. Experimental alcoholic liver disease requires translocation of bacterial products across the intestinal barrier into the systemic circulation, which induces an inflammatory response in the liver and contributes to steatohepatitis. We investigated the roles of the intestinal mucus layer, and in particular Muc2, in development of experimental alcohol-associated liver disease in mice. We studied experimental alcohol-induced liver disease, induced by the Tsukamoto-French method (which involves continuous intragastric feeding of an isocaloric diet or alcohol) in wild-type and Muc2−/− mice. Muc2−/− mice showed less alcohol-induced liver injury and steatosis that developed in wild-type mice. Most notably, Muc2−/− mice had significantly lower plasma levels of lipopolysaccharide than wild-type mice after alcohol feeding. In contrast to wild-type mice, Muc2−/− mice were protected from alcohol-associated microbiome changes that are dependent on intestinal mucins. The anti-microbial proteins Reg3b and Reg3g were expressed at significantly higher levels in the jejunum of Muc2−/− mice fed the isocaloric diet or alcohol, compared with wild-type mice. Consequently, Muc2−/− mice showed increased killing of commensal bacteria and prevented intestinal bacterial overgrowth. Conclusion: Muc2−/− mice are protected from intestinal bacterial overgrowth and dysbiosis in response to alcohol feeding. Subsequently, lower amounts of bacterial products such as endotoxin translocate into the systemic circulation, decreasing liver disease. PMID:23408358

The aldo-keto reductase AKR1B7 coexpresses with renin without influencing renin production and secretion.

PubMed

Machura, Katharina; Iankilevitch, Elina; Neubauer, Björn; Theuring, Franz; Kurtz, Armin

2013-03-01

On the basis of evidence that within the adult kidney, the aldo-keto reductase AKR1B7 (aldo-keto reductase family 1, member 7, also known as mouse vas deferens protein, MVDP) is selectively expressed in renin-producing cells, we aimed to define a possible role of AKR1B7 for the regulation and function of renin cells in the kidney. We could confirm colocalization and corecruitment of renin and of AKR1B7 in wild-type kidneys. Renin cells in AKR1B7-deficient kidneys showed normal morphology, numbers, and intrarenal distribution. Plasma renin concentration (PRC) and renin mRNA levels of AKR1B7-deficient mice were normal at standard chow and were lowered by a high-salt diet directly comparable to wild-type mice. Treatment with a low-salt diet in combination with an angiotensin-converting enzyme inhibitor strongly increased PRC and renin mRNA in a similar fashion both in AKR1B7-deficient and wild-type mice. Under this condition, we also observed a strong retrograde recruitment of renin-expressing cell along the preglomerular vessels, however, without a difference between AKR1B7-deficient and wild-type mice. The isolated perfused mouse kidney model was used to study the acute regulation of renin secretion by ANG II and by perfusion pressure. Regarding these parameters, no differences were observed between AKR1B7-deficient and wild-type kidneys. In summary, our data suggest that AKR1B7 is not of major relevance for the regulation of renin production and secretion in spite of its striking coregulation with renin expression.

The R292K Mutation That Confers Resistance to Neuraminidase Inhibitors Leads to Competitive Fitness Loss of A/Shanghai/1/2013 (H7N9) Influenza Virus in Ferrets

PubMed Central

Yen, Hui-Ling; Zhou, Jie; Choy, Ka-Tim; Sia, Sin Fun; Teng, Ooiean; Ng, Iris H.; Fang, Vicky J.; Hu, Yunwen; Wang, Wei; Cowling, Benjamin J.; Nicholls, John M.; Guan, Yi; Peiris, Joseph Sriyal Malik

2014-01-01

Background Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment. Methods We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo. Results Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs. Conclusions The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus. PMID:24951824

The R292K mutation that confers resistance to neuraminidase inhibitors leads to competitive fitness loss of A/Shanghai/1/2013 (H7N9) influenza virus in ferrets.

PubMed

Yen, Hui-Ling; Zhou, Jie; Choy, Ka-Tim; Sia, Sin Fun; Teng, Ooiean; Ng, Iris H; Fang, Vicky J; Hu, Yunwen; Wang, Wei; Cowling, Benjamin J; Nicholls, John M; Guan, Yi; Peiris, Joseph Sriyal Malik

2014-12-15

Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment. We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo. Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs. The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus. © 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.

High-fat hyperphagia in neurotrophin-4 deficient mice reveals potential role of vagal intestinal sensory innervation in long-term controls of food intake.

PubMed

Byerly, Mardi S; Fox, Edward A

2006-06-12

Neurotrophin-4 (NT-4) deficient mice exhibit substantial loss of intestinal vagal afferent innervation and short-term deficits in feeding behavior, suggesting reduced satiation. However, they do not show long-term changes in feeding or body weight because of compensatory behaviors. The present study examined whether high-fat hyperphagia induction would overcome compensation and reveal long-term effects associated with the reduced vagal sensory innervation of NT-4 mutants. First, modifications of a feeding schedule previously developed in rats were examined in wild-type mice to identify the regimen most effective at producing hyperphagia. The most successful schedule, which was run for 26 days, included access to a 43%-fat diet and pelleted chow every other day and access to only powdered chow on the alternate days. On high-fat access days mice consumed 25% more calories than mice with continuous daily access to the same high-fat diet and pelleted chow. This feeding regimen also induced hyperphagia in NT-4 deficient mice and their wild-type controls: on high-fat exposure days mutants consumed 35% more calories relative to continuous-access mutants, and wild types ate 25% more than continuous-access wild types. Moreover, on high-fat access days the alternating NT-4 mutants significantly increased caloric intake by 9% compared to alternating wild types. Thus, high-fat hyperphagia appeared to override compensation, permitting short-term changes in meal consumption by mutants that accrued into long-term changes in total daily food intake. This raises the possibility that intestinal vagal sensory innervation contributes to long-term, as well as to short-term regulation of food intake.

Major role for FeoB in Campylobacter jejuni ferrous iron acquisition, gut colonization, and intracellular survival.

PubMed

Naikare, Hemant; Palyada, Kiran; Panciera, Roger; Marlow, Denver; Stintzi, Alain

2006-10-01

To assess the importance of ferrous iron acquisition in Campylobacter physiology and pathogenesis, we disrupted and characterized the Fe2+ iron transporter, FeoB, in Campylobacter jejuni NCTC 11168, 81-176, and ATCC 43431. The feoB mutant was significantly affected in its ability to transport 55Fe2+. It accumulated half the amount of iron than the wild-type strain during growth in an iron-containing medium. The intracellular iron of the feoB mutant was localized in the periplasmic space versus the cytoplasm for the wild-type strain. These results indicate that the feoB gene of C. jejuni encodes a functional ferrous iron transport system. Reverse transcriptase PCR analysis revealed the cotranscription of feoB and Cj1397, which encodes a homolog of Escherichia coli feoA. C. jejuni 81-176 feoB mutants exhibited reduced ability to persist in human INT-407 embryonic intestinal cells and porcine IPEC-1 small intestinal epithelial cells compared to the wild type. C. jejuni NCTC 11168 feoB mutant was outcompeted by the wild type for colonization and/or survival in the rabbit ileal loop. The feoB mutants of the three C. jejuni strains were significantly affected in their ability to colonize the chick cecum. And finally, the three feoB mutants were outcompeted by their respective wild-type strains for infection of the intestinal tracts of colostrum-deprived piglets. Taken together, these results demonstrate that FeoB-mediated ferrous iron acquisition contributes significantly to colonization of the gastrointestinal tract during both commensal and infectious relationship, and thus it plays an important role in Campylobacter pathogenesis.

CXCR6 plays a critical role in angiotensin II-induced renal injury and fibrosis.

PubMed

Xia, Yunfeng; Jin, Xiaogao; Yan, Jingyin; Entman, Mark L; Wang, Yanlin

2014-07-01

Recent studies have shown that angiotensin II (Ang II) plays a critical role in the pathogenesis and progression of hypertensive kidney disease. However, the signaling mechanisms are poorly understood. In this study, we investigated the role of CXCR6 in Ang II-induced renal injury and fibrosis. Wild-type and CXCR6-green fluorescent protein (GFP) knockin mice were treated with Ang II via subcutaneous osmotic minipumps at 1500 ng/kg per minute after unilateral nephrectomy for ≤ 4 weeks. Wild-type and CXCR6-GFP knockin mice had virtually identical blood pressure at baseline. Ang II treatment led to an increase in blood pressure that was similar between wild-type and CXCR6-GFP knockin mice. CXCR6-GFP knockin mice were protected from Ang II-induced renal dysfunction, proteinuria, and fibrosis. CXCR6-GFP knockin mice accumulated fewer bone marrow-derived fibroblasts and myofibroblasts and produced less extracellular matrix protein in the kidneys after Ang II treatment. Furthermore, CXCR6-GFP knockin mice exhibited fewer F4/80(+) macrophages and CD3(+) T cells and expressed less proinflammatory cytokines in the kidneys after Ang II treatment. Finally, wild-type mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts, macrophages, and T cells in the kidney after Ang II treatment when compared with wild-type mice engrafted with CXCR6(+/+) bone marrow cells. Our results indicate that CXCR6 plays a pivotal role in the development of Ang II-induced renal injury and fibrosis through regulation of macrophage and T-cell infiltration and bone marrow-derived fibroblast accumulation. © 2014 American Heart Association, Inc.

Expression of γ-tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal-induced oxidative stress by reducing reactive oxygen species

PubMed Central

Jin, Shuangxia; Daniell, Henry

2014-01-01

Summary The γ-tocopherol methyltransferase (γ-TMT) is an important enzyme regulating synthesis of four tocopherols (α, γ, β and δ). In this report, we investigated the role of γ-TMT in regulating abiotic stress within chloroplasts. The At γ-tmt overexpressed via the tobacco chloroplast genome accumulated up to 7.7% of the total leaf protein, resulting in massive proliferation of the inner envelope membrane (IEM, up to 8 layers). Such high level expression of γ-TMT converted most of γ-tocopherol to α-tocopherol in transplastomic seeds (~10 fold higher) in the absence of abiotic stress. When grown in 400 mM NaCl, α-tocopherol content in transplastomic TMT leaves increased up to 8.2-fold and 2.4-fold higher than wild-type leaves. Likewise, under heavy metal stress α-tocopherol content in the TMT leaves increased up to 7.5-fold, twice higher than in the wild-type. Under extreme salt stress, the wild-type accumulated higher starch and total soluble sugars but TMT plants were able to regulate sugar transport. Hydrogen peroxide and superoxide content in wild-type increased up to 3-fold within 48 hours of NaCl stress when compared to TMT plants. The ion leakage from TMT leaves was significantly less than wild-type plants under abiotic stress and with less malondialdehyde, indicating lower lipid peroxidation. Taken together, these studies show that α-tocopherol plays a crucial role in the alleviation of salt and heavy metal stresses by decreasing ROS, lipid peroxidation and ion leakage, in addition to enhancing vitamin E conversion. Increased proliferation of the IEM should facilitate studies on retrograde signaling from chloroplast to the nucleus. PMID:25051898

Enhanced susceptibility to acute pneumococcal otitis media in mice deficient in complement C1qa, factor B, and factor B/C2.

PubMed

Tong, Hua Hua; Li, Yong Xing; Stahl, Gregory L; Thurman, Joshua M

2010-03-01

To define the roles of specific complement activation pathways in host defense against Streptococcus pneumoniae in acute otitis media (AOM), we investigated the susceptibility to AOM in mice deficient in complement factor B and C2 (Bf/C2(-/)(-)), C1qa (C1qa(-/)(-)), and factor B (Bf(-)(/)(-)). Bacterial titers of both S. pneumoniae serotype 6A and 14 in the middle ear lavage fluid samples from Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice were significantly higher than in samples from wild-type mice 24 h after transtympanical infection (P < 0.05) and remained persistently higher in samples from Bf/C2(-/)(-) mice than in samples from wild-type mice. Bacteremia occurred in Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice infected with both strains, but not in wild-type mice. Recruitment of inflammatory cells was paralleled by enhanced production of inflammatory mediators in the middle ear lavage samples from Bf/C2(-/)(-) mice. C3b deposition on both strains was greatest for sera obtained from wild-type mice, followed by C1qa(-)(/)(-) and Bf(-)(/)(-) mice, and least for Bf/C2(-)(/)(-) mice. Opsonophagocytosis and whole-blood killing capacity of both strains were significantly decreased in the presence of sera or whole blood from complement-deficient mice compared to wild-type mice. These findings indicate that both the classical and alternative complement pathways are critical for middle ear immune defense against S. pneumoniae. The reduced capacity of complement-mediated opsonization and phagocytosis in the complement-deficient mice appears to be responsible for the impaired clearance of S. pneumoniae from the middle ear and dissemination to the bloodstream during AOM.

Tenofovir alafenamide demonstrates broad cross-genotype activity against wild-type HBV clinical isolates and maintains susceptibility to drug-resistant HBV isolates in vitro.

PubMed

Liu, Yang; Miller, Michael D; Kitrinos, Kathryn M

2017-03-01

Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV). This study evaluated the antiviral activity of TAF against wild-type genotype A-H HBV clinical isolates as well as adefovir-resistant, lamivudine-resistant, and entecavir-resistant HBV isolates. Full length HBV genomes or the polymerase/reverse transcriptase (pol/RT) region from treatment-naïve patients infected with HBV genotypes A-H were amplified and cloned into an expression vector under the control of a CMV promoter. In addition, 11 drug resistant HBV constructs were created by site-directed mutagenesis of a full length genotype D construct. Activity of TAF was measured by transfection of each construct into HepG2 cells and assessment of HBV DNA levels following treatment across a range of TAF concentrations. TAF activity in vitro was similar against wild-type genotype A-H HBV clinical isolates. All lamivudine- and entecavir-resistant isolates and 4/5 adefovir-resistant isolates were found to be sensitive to inhibition by TAF in vitro as compared to the wild-type isolate. The adefovir-resistant isolate rtA181V + rtN236T exhibited low-level reduced susceptibility to TAF. TAF is similarly active in vitro against wild-type genotype A-H HBV clinical isolates. The TAF sensitivity results for all drug-resistant isolates are consistent with what has been observed with the parent drug TFV. The in vitro cell-based HBV phenotyping assay results support the use of TAF in treatment of HBV infected subjects with diverse HBV genotypes, in both treatment-naive and treatment-experienced HBV infected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Production of the Plant Hormone Auxin by Salmonella and Its Role in the Interactions with Plants and Animals.

PubMed

Cox, Clayton E; Brandl, Maria T; de Moraes, Marcos H; Gunasekera, Sarath; Teplitski, Max

2017-01-01

The ability of human enteric pathogens to colonize plants and use them as alternate hosts is now well established. Salmonella , similarly to phytobacteria, appears to be capable of producing the plant hormone auxin via an indole-3-pyruvate decarboxylase (IpdC), a key enzyme of the IPyA pathway. A deletion of the Salmonella ipdC significantly reduced auxin synthesis in laboratory culture. The Salmonella ipdC gene was expressed on root surfaces of Medicago truncatula . M. truncatula auxin-responsive GH3::GUS reporter was activated by the wild type Salmonella , and not but the ipdC mutant, implying that the bacterially produced IAA (Indole Acetic Acid) was detected by the seedlings. Seedling infections with the wild type Salmonella caused an increase in secondary root formation, which was not observed in the ipdC mutant. The wild type Salmonella cells were detected as aggregates at the sites of lateral root emergence, whereas the ipdC mutant cells were evenly distributed in the rhizosphere. However, both strains appeared to colonize seedlings well in growth pouch experiments. The ipdC mutant was also less virulent in a murine model of infection. When mice were infected by oral gavage, the ipdC mutant was as proficient as the wild type strain in colonization of the intestine, but it was defective in the ability to cross the intestinal barrier. Fewer cells of the ipdC mutant, compared with the wild type strain, were detected in Peyer's patches, spleen and in the liver. Orthologs of ipdC are found in all Salmonella genomes and are distributed among many animal pathogens and plant-associated bacteria of the Enterobacteriaceae , suggesting a broad ecological role of the IpdC-catalyzed pathway.

Hepatitis B virus x gene and cyanobacterial toxins promote aflatoxin B1-induced hepatotumorigenesis in mice

PubMed Central

Lian, Min; Liu, Ying; Yu, Shun-Zhang; Qian, Geng-Sun; Wan, Shu-Guang; Dixon, Kenneth R

2006-01-01

AIM: To assess the combinative role of aflatoxin B1 (AFB1), cyanobacterial toxins (cyanotoxins), and hepatitis B virus (HBV) x gene in hepatotumorigenicity. METHODS: One-week-old animals carrying HBV x gene and their wild-type littermates were intraperitoneally (ip) injected with either single-dose AFB1 [6 mg/kg body weight (bw)], repeated-dose cyanotoxins (microcystin-LR or nodularin, 10 μg/kg bw once a week for 15 wk), DMSO (vehicle control) alone, or AFB1 followed by cyanotoxins a week later, and were sacrificed at 24 and 52 wk post-treatment. RESULTS: AFB1 induced liver tumors in 13 of 29 (44.8%) transgenic mice at 52 wk post-treatment, significantly more frequent than in wild-type mice (13.3%). This significant difference was not shown in the 24-wk study. Compared with AFB1 exposure alone, MC-LR and nodularin yielded approximately 3-fold and 6-fold increases in the incidence of AFB1-induced liver tumors in wild-type animals at 24 wk, respectively. HBV x gene did not further elevate the risk associated with co-exposure to AFB1 and cyanotoxins. With the exception of an MC-LR-dosed wild-type mouse, no liver tumor was observed in mice treated with cyanotoxins alone at 24 wk. Neither DMSO-treated transgenic mice nor their wild-type littermates had pathologic alterations relevant to hepatotumorigenesis in even up to 52 wk. CONCLUSION: HBV x gene and nodularin promote the development of AFB1-induced liver tumors. Co-exposure to AFB1 and MC-LR tends to elevate the risk of liver tumors at 24 wk relative to exposure to one of them. The combinative effect of AFB1, cyanotoxins and HBVx on hepatotumorigenesis is weak at 24 wk. PMID:16718789

Computational investigation of the human SOD1 mutant, Cys146Arg, that directs familial amyotrophic lateral sclerosis.

PubMed

Srinivasan, E; Rajasekaran, R

2017-07-25

The genetic substitution mutation of Cys146Arg in the SOD1 protein is predominantly found in the Japanese population suffering from familial amyotrophic lateral sclerosis (FALS). A complete study of the biophysical aspects of this particular missense mutation through conformational analysis and producing free energy landscapes could provide an insight into the pathogenic mechanism of ALS disease. In this study, we utilized general molecular dynamics simulations along with computational predictions to assess the structural characterization of the protein as well as the conformational preferences of monomeric wild type and mutant SOD1. Our static analysis, accomplished through multiple programs, predicted the deleterious and destabilizing effect of mutant SOD1. Subsequently, comparative molecular dynamic studies performed on the wild type and mutant SOD1 indicated a loss in the protein conformational stability and flexibility. We observed the mutational consequences not only in local but also in long-range variations in the structural properties of the SOD1 protein. Long-range intramolecular protein interactions decrease upon mutation, resulting in less compact structures in the mutant protein rather than in the wild type, suggesting that the mutant structures are less stable than the wild type SOD1. We also presented the free energy landscape to study the collective motion of protein conformations through principal component analysis for the wild type and mutant SOD1. Overall, the study assisted in revealing the cause of the structural destabilization and protein misfolding via structural characterization, secondary structure composition and free energy landscapes. Hence, the computational framework in our study provides a valuable direction for the search for the cure against fatal FALS.

Diagnostic Limitation of Fine-Needle Aspiration (FNA) on Indeterminate Thyroid Nodules Can Be Partially Overcome by Preoperative Molecular Analysis: Assessment of RET/PTC1 Rearrangement in BRAF and RAS Wild-Type Routine Air-Dried FNA Specimens.

PubMed

Ko, Young Sin; Hwang, Tae Sook; Kim, Ja Yeon; Choi, Yoon-La; Lee, Seung Eun; Han, Hye Seung; Kim, Wan Seop; Kim, Suk Kyeong; Park, Kyoung Sik

2017-04-12

Molecular markers are helpful diagnostic tools, particularly for cytologically indeterminate thyroid nodules. Preoperative RET/PTC1 rearrangement analysis in BRAF and RAS wild-type indeterminate thyroid nodules would permit the formulation of an unambiguous surgical plan. Cycle threshold values according to the cell count for detection of the RET/PTC1 rearrangement by real-time reverse transcription-polymerase chain reaction (RT-PCR) using fresh and routine air-dried TPC1 cells were evaluated. The correlation of RET/PTC1 rearrangement between fine-needle aspiration (FNA) and paired formalin-fixed paraffin-embedded (FFPE) specimens was analyzed. RET/PTC1 rearrangements of 76 resected BRAF and RAS wild-type classical PTCs were also analyzed. Results of RT-PCR and the Nanostring were compared. When 100 fresh and air-dried TPC1 cells were used, expression of RET/PTC1 rearrangement was detectable after 35 and 33 PCR cycles, respectively. The results of RET/PTC1 rearrangement in 10 FNA and paired FFPE papillary thyroid carcinoma (PTC) specimens showed complete correlation. Twenty-nine (38.2%) of 76 BRAF and RAS wild-type classical PTCs had RET/PTC1 rearrangement. Comparison of RET/PTC1 rearrangement analysis between RT-PCR and the Nanostring showed moderate agreement with a κ value of 0.56 ( p = 0.002). The RET/PTC1 rearrangement analysis by RT-PCR using routine air-dried FNA specimen was confirmed to be technically applicable. A significant proportion (38.2%) of the BRAF and RAS wild-type PTCs harbored RET/PTC1 rearrangements.

Differential Inhibition of Ex-Vivo Tumor Kinase Activity by Vemurafenib in BRAF(V600E) and BRAF Wild-Type Metastatic Malignant Melanoma

PubMed Central

Tahiri, Andliena; Røe, Kathrine; Ree, Anne H.; de Wijn, Rik; Risberg, Karianne; Busch, Christian; Lønning, Per E.; Kristensen, Vessela; Geisler, Jürgen

2013-01-01

Background Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed. Methodology In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status. Results Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro. Conclusions Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers. PMID:24023633

Strain-specific reverse transcriptase PCR assay: means to distinguish candidate vaccine from wild-type strains of respiratory syncytial virus.

PubMed Central

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

The relative absorption cross-sections of photosystem I and photosystem II in chloroplasts from three types of Nicotiana tabacum.

PubMed

Melis, A; Thielen, A P

1980-02-08

In the present study we used three types of Nicotiana tabacum, cv John William's Broad Leaf (the wild type and two mutants, the yellow-green Su/su and the yellow Su/su var. Aurea) in order to correlat functional properties of Photosystem II and Photosystem I with the structural organization of their chloroplasts. The effective absorption cross-section of Photosystem II and Photosystem I centers was measured by means of the rate constant of their photoconversion under light-limiting conditions. In agreement with earlier results (Okabe, K., Schmid, G.H. and Straub, J. (1977) Plant Physiol. 60, 150--156) the photosynthetic unit size for both System II and System I in the two mutants was considerably smaller as compared to the wild type. We observed biphasic kinetics in the photoconversion of System II in all three types of N. tabacum. However, the photoconversion of System I occurred with monophasic and exponential kinetics. Under our experimental conditions, the effective cross-section of Photosystem I was comparable to that of the fast System II component (alpha centers). The relative amplitude of the slow System II component (beta centers) varied between 30% in the wild type to 70% in the Su/su var. Aurea mutant. The increased fraction of beta centers is correlated with the decreased fraction of appressed photosynthetic membranes in the chloroplasts of the two mutants. As a working hypothesis, it is suggested that beta centers are located on photosynthetic membranes directly exposed to the stroma medium.

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B.

PubMed

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.

Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahmad, Md. Faiz; Kaushal, Prem Singh; Wan, Qun

2012-11-01

Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 ({alpha}) that contains the catalytic site and RR2 ({beta}) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-typemore » enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-({beta},{gamma}-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.« less

Structure change of β-hairpin induced by turn optimization: an enhanced sampling molecular dynamics simulation study.

PubMed

Shao, Qiang; Yang, Lijiang; Gao, Yi Qin

2011-12-21

Our previous study showed that for the tested polypeptides which have similar β-hairpin structures but different sequences, their folding free energy pathways are dominantly determined by the turn conformational propensity. In this study, we study how the turn conformational propensity affects the structure of hairpins. The folding of two mutants of GB1p peptide (GB1m2 and GB1m3), which have the optimized turn sequence ((6)DDATK(11)T → (6)NPATG(11)K) with native structures unsolved, were simulated using integrated tempering sampling molecular dynamics simulations and the predicted stable structures were compared to wild-type GB1p. It was observed that the turn optimization of GB1p generates a more favored 5-residue type I(') turn in addition to the 6-residue type I turn in wild-type GB1p. As a result two distinctly different hairpin structures are formed corresponding to the "misfolded" (M) and the "folded" (F) states. M state is a one-residue-shifted asymmetric β-hairpin structure whereas F state has the similar symmetric hairpin structure as wild-type GB1p. The formation of the favored type I(') turn has a small free energy barrier and leads to the shifted β-hairpin structure, following the modified "zipping" model. The presence of disfavored type I turn structure makes the folding of a β-hairpin consistent with the "hydrophobic-core-centric" model. On the other hand, the folding simulations on other two GB1p mutants (GB1r1 and GBr2), which have the position of the hydrophobic core cluster further away from the turn compared to wild-type GB1p, showed that moving the hydrophobic core cluster away from the turn region destabilizes but does not change the hairpin structure. Therefore, the present study showed that the turn conformational propensity is a key factor in affecting not only the folding pathways but also the stable structure of β-hairpins, and the turn conformational change induced by the turn optimization leads to significant changes of β-hairpin structure.

Reductions in expression of growth regulating genes in skeletal muscle with age in wild type and myostatin null mice.

PubMed

Jones, Jennifer C; Kroscher, Kellie A; Dilger, Anna C

2014-03-28

Genes that decline in expression with age and are thought to coordinate growth cessation have been identified in various organs, but their expression in skeletal muscle is unknown. Therefore, our objective was to determine expression of these genes (Ezh2, Gpc3, Mdk, Mest, Mycn, Peg3, and Plagl1) in skeletal muscle from birth to maturity. We hypothesized that expression of these genes would decline with age in skeletal muscle but differ between sexes and between wild type and myostatin null mice. Female and male wild type and myostatin null mice (C57BL/6J background) were sacrificed by carbon dioxide asphyxiation followed by decapitation at d -7, 0, 21, 42, and 70 days of age. Whole bodies at d -7, all muscles from both hind limbs at d 0, and bicep femoris muscle from d 21, 42 and 70 were collected. Gene expression was determined by quantitative real-time PCR. In general, expression of these growth-regulating genes was reduced at d 21 compared with day 0 and d -7. Expression of Gpc3, Mest, and Peg3 was further reduced at d 42 and 70 compared with d 21, however the expression of Mycn increased from d 21 to d 42 and 70. Myostatin null mice, as expected, were heavier with increased biceps femoris weight at d 70. However, with respect to sex and genotype, there were few differences in expression. Expression of Ezh2 was increased at d 70 and expression of Mdk was increased at d 21 in myostatin null mice compared with wild type, but no other genotype effects were present. Expression of Mdk was increased in females compared to males at d 70, but no other sex effects were present. Overall, these data suggest the downregulation of these growth-regulating genes with age might play a role in the coordinated cessation of muscle growth similar to organ growth but likely have a limited role in the differences between sexes or genotypes.

Supersensitive detection and discrimination of enantiomers by dorsal olfactory receptors: evidence for hierarchical odour coding.

PubMed

Sato, Takaaki; Kobayakawa, Reiko; Kobayakawa, Ko; Emura, Makoto; Itohara, Shigeyoshi; Kizumi, Miwako; Hamana, Hiroshi; Tsuboi, Akio; Hirono, Junzo

2015-09-11

Enantiomeric pairs of mirror-image molecular structures are difficult to resolve by instrumental analyses. The human olfactory system, however, discriminates (-)-wine lactone from its (+)-form rapidly within seconds. To gain insight into receptor coding of enantiomers, we compared behavioural detection and discrimination thresholds of wild-type mice with those of ΔD mice in which all dorsal olfactory receptors are genetically ablated. Surprisingly, wild-type mice displayed an exquisite "supersensitivity" to enantiomeric pairs of wine lactones and carvones. They were capable of supersensitive discrimination of enantiomers, consistent with their high detection sensitivity. In contrast, ΔD mice showed selective major loss of sensitivity to the (+)-enantiomers. The resulting 10(8)-fold differential sensitivity of ΔD mice to (-)- vs. (+)-wine lactone matched that observed in humans. This suggests that humans lack highly sensitive orthologous dorsal receptors for the (+)-enantiomer, similarly to ΔD mice. Moreover, ΔD mice showed >10(10)-fold reductions in enantiomer discrimination sensitivity compared to wild-type mice. ΔD mice detected one or both of the (-)- and (+)-enantiomers over a wide concentration range, but were unable to discriminate them. This "enantiomer odour discrimination paradox" indicates that the most sensitive dorsal receptors play a critical role in hierarchical odour coding for enantiomer identification.

Supersensitive detection and discrimination of enantiomers by dorsal olfactory receptors: evidence for hierarchical odour coding

PubMed Central

Sato, Takaaki; Kobayakawa, Reiko; Kobayakawa, Ko; Emura, Makoto; Itohara, Shigeyoshi; Kizumi, Miwako; Hamana, Hiroshi; Tsuboi, Akio; Hirono, Junzo

2015-01-01

Enantiomeric pairs of mirror-image molecular structures are difficult to resolve by instrumental analyses. The human olfactory system, however, discriminates (−)-wine lactone from its (+)-form rapidly within seconds. To gain insight into receptor coding of enantiomers, we compared behavioural detection and discrimination thresholds of wild-type mice with those of ΔD mice in which all dorsal olfactory receptors are genetically ablated. Surprisingly, wild-type mice displayed an exquisite “supersensitivity” to enantiomeric pairs of wine lactones and carvones. They were capable of supersensitive discrimination of enantiomers, consistent with their high detection sensitivity. In contrast, ΔD mice showed selective major loss of sensitivity to the (+)-enantiomers. The resulting 108-fold differential sensitivity of ΔD mice to (−)- vs. (+)-wine lactone matched that observed in humans. This suggests that humans lack highly sensitive orthologous dorsal receptors for the (+)-enantiomer, similarly to ΔD mice. Moreover, ΔD mice showed >1010-fold reductions in enantiomer discrimination sensitivity compared to wild-type mice. ΔD mice detected one or both of the (−)- and (+)-enantiomers over a wide concentration range, but were unable to discriminate them. This “enantiomer odour discrimination paradox” indicates that the most sensitive dorsal receptors play a critical role in hierarchical odour coding for enantiomer identification. PMID:26361056

Leading Process Branch Instability in Lis1+/− Nonradially Migrating Interneurons

PubMed Central

Gopal, Pallavi P.; Simonet, Jacqueline C.; Shapiro, William

2010-01-01

Mammalian forebrain development requires extensive migration, yet the mechanisms through which migrating neurons sense and respond to guidance cues are not well understood. Similar to the axon growth cone, the leading process and branches of neurons may guide migration, but the cytoskeletal events that regulate branching are unknown. We have previously shown that loss of microtubule-associated protein Lis1 reduces branching during migration compared with wild-type neurons. Using time-lapse imaging of Lis1+/− and Lis1+/+ cells migrating from medial ganglionic eminence explant cultures, we show that the branching defect is not due to a failure to initiate branches but a defect in the stabilization of new branches. The leading processes of Lis1+/− neurons have reduced expression of stabilized, acetylated microtubules compared with Lis1+/+ neurons. To determine whether Lis1 modulates branch stability through its role as the noncatalytic β regulatory subunit of platelet-activating factor (PAF) acetylhydrolase 1b, exogenous PAF was applied to wild-type cells. Excess PAF added to wild-type neurons phenocopies the branch instability observed in Lis1+/− neurons, and a PAF antagonist rescues leading process branching in Lis1+/− neurons. These data highlight a role for Lis1, acting through the PAF pathway, in leading process branching and microtubule stabilization. PMID:19861636

Comparative Metabolite Profiling of Triterpenoid Saponins and Flavonoids in Flower Color Mutations of Primula veris L.

PubMed Central

Apel, Lysanne; Kammerer, Dietmar R.; Stintzing, Florian C.; Spring, Otmar

2017-01-01

Primula veris L. is an important medicinal plant with documented use for the treatment of gout, headache and migraine reaching back to the Middle Ages. Triterpenoid saponins from roots and flowers are used in up-to-date phytotherapeutic treatment of bronchitis and colds due to their expectorant and secretolytic effects. In addition to the wild type plants with yellow petals, a red variant and an intermediate orange form of Primula veris L. have recently been found in a natural habitat. The secondary metabolite profiles of roots, leaves and flowers of these rare variants were investigated and compared with the wild type metabolome. Two flavonoids, six flavonoid glycosides, four novel methylated flavonoid glycosides, five anthocyanins and three triterpenoid saponins were identified in alcoholic extracts from the petals, leaves and roots of the three variants by high performance liquid chromatography (HPLC)-diode array detection (DAD)/mass spectrometry (MSn) analyses. Anthocyanins were detected in the petals of the red and orange variety, but not in the wild type. No other effects on the metabolite profiles of the three varieties have been observed. The possibility is discussed that a regulatory step of the anthocyanin biosynthetic pathway may have been affected by mutation thus triggering color polymorphism in the petals. PMID:28098796

Mutations in CypA Binding Region of HIV-1 Capsid Affect Capsid Stability and Viral Replication in Primary Macrophages.

PubMed

Setiawan, Laurentia C; van Dort, Karel A; Rits, Maarten A N; Kootstra, Neeltje A

2016-04-01

Mutations in the cyclophilin A (CypA) binding region in the HIV-1 capsid affect their dependency on the known HIV-1 cofactor CypA and allow escape from the HIV-1 restriction factor Trim5α in human and simian cells. Here we study the effect of these mutations in the CypA binding region of capsid on cofactor binding, capsid destabilization, and viral replication in primary cells. We showed that the viral capsid with mutations in the CypA binding region (CypA-BR) interacted efficiently with CypA, but had an increased stability upon infection as compared to the wild-type capsid. Interestingly, the wild-type virus was able to infect monocyte-derived macrophages (MDM) more efficiently as compared to the CypA-BR mutant variant. The lower infectivity of the CypA-BR mutant virus in MDM was associated with lower levels of reverse transcription products. Similar to the wild-type virus, the CypA-BR mutant variant was unable to induce a strong innate response in primary macrophages. These data demonstrate that mutations in the CypA binding site of the capsid resulted in higher capsid stability and hampered infectivity in macrophages.

Comparison of the Live Attenuated Yellow Fever Vaccine 17D-204 Strain to Its Virulent Parental Strain Asibi by Deep Sequencing

PubMed Central

Beck, Andrew; Tesh, Robert B.; Wood, Thomas G.; Widen, Steven G.; Ryman, Kate D.; Barrett, Alan D. T.

2014-01-01

Background. The first comparison of a live RNA viral vaccine strain to its wild-type parental strain by deep sequencing is presented using as a model the yellow fever virus (YFV) live vaccine strain 17D-204 and its wild-type parental strain, Asibi. Methods. The YFV 17D-204 vaccine genome was compared to that of the parental strain Asibi by massively parallel methods. Variability was compared on multiple scales of the viral genomes. A modeled exploration of small-frequency variants was performed to reconstruct plausible regions of mutational plasticity. Results. Overt quasispecies diversity is a feature of the parental strain, whereas the live vaccine strain lacks diversity according to multiple independent measurements. A lack of attenuating mutations in the Asibi population relative to that of 17D-204 was observed, demonstrating that the vaccine strain was derived by discrete mutation of Asibi and not by selection of genomes in the wild-type population. Conclusions. Relative quasispecies structure is a plausible correlate of attenuation for live viral vaccines. Analyses such as these of attenuated viruses improve our understanding of the molecular basis of vaccine attenuation and provide critical information on the stability of live vaccines and the risk of reversion to virulence. PMID:24141982

Platelet-Derived Growth Factor Promotes Repair of Chronically Demyelinated White Matter

PubMed Central

Vana, Adam C.; Flint, Nicole C.; Harwood, Norah E.; Le, Tuan Q.; Fruttiger, Marcus; Armstrong, Regina C.

2009-01-01

In multiple sclerosis, remyelination becomes limited after repeated or prolonged episodes of demyelination. To test the effect of platelet-derived growth factor-A (PDGF-A) in recovery from chronic demyelination we induced corpus callosum demyelination using cuprizone treatment in hPDGF-A transgenic (tg) mice with the human PDGF-A gene under control of an astrocyte-specific promoter. After chronic demyelination and removal of cuprizone from the diet, remyelination and oligodendrocyte density improved significantly in hPDGF-A tg mice compared with wild-type mice. In hPDGF-A tg mice, oligodendrocyte progenitor density and proliferation values were increased in the corpus callosum during acute demyelination but not during chronic demyelination or the subsequent recovery period, compared with hPDGF-A tg mice without cuprizone or to treatment-matched wild-type mice. Proliferation within the subventricular zone and subcallosal zone was elevated throughout cuprizone treatment but was not different between hPDGF-A tg and wild-type mice. Importantly, hPDGF-A tg mice had reduced apoptosis in the corpus callosum during the recovery period after chronic demyelination. Therefore, PDGF-A may support oligodendrocyte generation and survival to promote remyelination of chronic lesions. Furthermore, preventing oligodendrocyte apoptosis may be important not only during active demyelination but also for supporting the generation of new oligodendrocytes to remyelinate chronic lesions. PMID:17984680

Comparison of the live attenuated yellow fever vaccine 17D-204 strain to its virulent parental strain Asibi by deep sequencing.

PubMed

Beck, Andrew; Tesh, Robert B; Wood, Thomas G; Widen, Steven G; Ryman, Kate D; Barrett, Alan D T

2014-02-01

The first comparison of a live RNA viral vaccine strain to its wild-type parental strain by deep sequencing is presented using as a model the yellow fever virus (YFV) live vaccine strain 17D-204 and its wild-type parental strain, Asibi. The YFV 17D-204 vaccine genome was compared to that of the parental strain Asibi by massively parallel methods. Variability was compared on multiple scales of the viral genomes. A modeled exploration of small-frequency variants was performed to reconstruct plausible regions of mutational plasticity. Overt quasispecies diversity is a feature of the parental strain, whereas the live vaccine strain lacks diversity according to multiple independent measurements. A lack of attenuating mutations in the Asibi population relative to that of 17D-204 was observed, demonstrating that the vaccine strain was derived by discrete mutation of Asibi and not by selection of genomes in the wild-type population. Relative quasispecies structure is a plausible correlate of attenuation for live viral vaccines. Analyses such as these of attenuated viruses improve our understanding of the molecular basis of vaccine attenuation and provide critical information on the stability of live vaccines and the risk of reversion to virulence.

Improved Salinity Tolerance in Carrizo Citrange Rootstock through Overexpression of Glyoxalase System Genes

PubMed Central

Alvarez-Gerding, Ximena; Cortés-Bullemore, Rowena; Medina, Consuelo; Romero-Romero, Jesús L.; Inostroza-Blancheteau, Claudio; Aquea, Felipe; Arce-Johnson, Patricio

2015-01-01

Citrus plants are widely cultivated around the world and, however, are one of the most salt stress sensitive crops. To improve salinity tolerance, transgenic Carrizo citrange rootstocks that overexpress glyoxalase I and glyoxalase II genes were obtained and their salt stress tolerance was evaluated. Molecular analysis showed high expression for both glyoxalase genes (BjGlyI and PgGlyII) in 5H03 and 5H04 lines. Under control conditions, transgenic and wild type plants presented normal morphology. In salinity treatments, the transgenic plants showed less yellowing, marginal burn in lower leaves and showed less than 40% of leaf damage compared with wild type plants. The transgenic plants showed a significant increase in the dry weight of shoot but there are no differences in the root and complete plant dry weight. In addition, a higher accumulation of chlorine is observed in the roots in transgenic line 5H03 but in shoot it was lower. Also, the wild type plant accumulated around 20% more chlorine in the shoot compared to roots. These results suggest that heterologous expression of glyoxalase system genes could enhance salt stress tolerance in Carrizo citrange rootstock and could be a good biotechnological approach to improve the abiotic stress tolerance in woody plant species. PMID:26236739

Finding gene clusters for a replicated time course study

PubMed Central

2014-01-01

Background Finding genes that share similar expression patterns across samples is an important question that is frequently asked in high-throughput microarray studies. Traditional clustering algorithms such as K-means clustering and hierarchical clustering base gene clustering directly on the observed measurements and do not take into account the specific experimental design under which the microarray data were collected. A new model-based clustering method, the clustering of regression models method, takes into account the specific design of the microarray study and bases the clustering on how genes are related to sample covariates. It can find useful gene clusters for studies from complicated study designs such as replicated time course studies. Findings In this paper, we applied the clustering of regression models method to data from a time course study of yeast on two genotypes, wild type and YOX1 mutant, each with two technical replicates, and compared the clustering results with K-means clustering. We identified gene clusters that have similar expression patterns in wild type yeast, two of which were missed by K-means clustering. We further identified gene clusters whose expression patterns were changed in YOX1 mutant yeast compared to wild type yeast. Conclusions The clustering of regression models method can be a valuable tool for identifying genes that are coordinately transcribed by a common mechanism. PMID:24460656

Functional Analysis of the Trichoderma harzianum nox1 Gene, Encoding an NADPH Oxidase, Relates Production of Reactive Oxygen Species to Specific Biocontrol Activity against Pythium ultimum▿†

PubMed Central

Montero-Barrientos, M.; Hermosa, R.; Cardoza, R. E.; Gutiérrez, S.; Monte, E.

2011-01-01

The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The nox1 gene of Trichoderma harzianum was cloned, and its role in antagonism against phytopathogens was analyzed in nox1-overexpressed transformants. The increased levels of nox1 expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with Pythium ultimum. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against P. ultimum the nox1-overexpressed transformants were more effective than the wild type, but not in assays against Botrytis cinerea or Rhizoctonia solani. A transcriptomic analysis using a Trichoderma high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type T. harzianum and P. ultimum, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a nox1-overexpressed transformant with this pathogen. Our results show that nox1 is involved in T. harzianum ROS production and antagonism against P. ultimum. PMID:21421791

Characterization of Enteroviruses from Non-Human Primates in Cameroon Revealed Virus Types Widespread in Humans along with Candidate New Types and Species

PubMed Central

Sadeuh-Mba, Serge Alain; Bessaud, Maël; Joffret, Marie-Line; Endegue Zanga, Marie-Claire; Balanant, Jean; Mpoudi Ngole, Eitel; Njouom, Richard; Reynes, Jean-Marc; Delpeyroux, Francis; Rousset, Dominique

2014-01-01

Enteroviruses (EVs) infecting African Non-Human Primates (NHP) are still poorly documented. This study was designed to characterize the genetic diversity of EVs among captive and wild NHP in Cameroon and to compare this diversity with that found in humans. Stool specimens were collected in April 2008 in NHP housed in sanctuaries in Yaounde and neighborhoods. Moreover, stool specimens collected from wild NHP from June 2006 to October 2008 in the southern rain forest of Cameroon were considered. RNAs purified directly from stool samples were screened for EVs using a sensitive RT-nested PCR targeting the VP1 capsid coding gene whose nucleotide sequence was used for molecular typing. Captive chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) were primarily infected by EV types already reported in humans in Cameroon and elsewhere: Coxsackievirus A13 and A24, Echovirus 15 and 29, and EV-B82. Moreover EV-A119, a novel virus type recently described in humans in central and west Africa, was also found in a captive Chimpanzee. EV-A76, which is a widespread virus in humans, was identified in wild chimpanzees, thus suggesting its adaptation and parallel circulation in human and NHP populations in Cameroon. Interestingly, some EVs harbored by wild NHP were genetically distinct from all existing types and were thus assigned as new types. One chimpanzee-derived virus was tentatively assigned as EV-J121 in the EV-J species. In addition, two EVs from wild monkeys provisionally registered as EV-122 and EV-123 were found to belong to a candidate new species. Overall, this study indicates that the genetic diversity of EVs among NHP is more important than previously known and could be the source of future new emerging human viral diseases. PMID:25079078

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

DTIC Science & Technology

2012-07-01

compared between wild type and mutant plants via chromatin immunoprecipitation (ChIP). Additionally, differences in centromere structure between wild...specific focus on non-CpG contexts. The proposed work is ongoing, and so far the major accomplishments include creation of relevant plant lines...laboratories that study topics related to breast cancer and epigenetics 1. Monthly journal club meetings at the Center for Vertebrate Genomics (CVG) which

Differences in pathogenicity, response to vaccination, and innate immune responses in different types of ducks infected with a virulent H5N1 highly pathogenic avian influenza virus from Vietnam

USDA-ARS?s Scientific Manuscript database

Wild ducks are reservoirs of avian influenza viruses in nature, and usually don’t show signs of disease. However, some Asian lineage H5N1 highly pathogenic avian influenza (HPAI) viruses can cause disease and death in both wild and domestic ducks. The objective of this study was to compare the cli...

Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media

PubMed Central

Murrah, Kyle A.; Turner, Roberta L.; Pang, Bing; Perez, Antonia C.; Reimche, Jennifer L.; King, Lauren B.; Wren, John; Gandhi, Uma; Swords, W. Edward; Ornelles, David A.

2015-01-01

Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease. PMID:25251686

Mechanics of membrane-cytoskeleton attachment in Paramecium

NASA Astrophysics Data System (ADS)

Campillo, C.; Jerber, J.; Fisch, C.; Simoes-Betbeder, M.; Dupuis-Williams, P.; Nassoy, P.; Sykes, C.

2012-12-01

In this paper we assess the role of the protein MKS1 (Meckel syndrome type 1) in the cortical membrane mechanics of the ciliated protist Paramecium. This protein is known to be crucial in the process of cilium formation, and we investigate its putative role in membrane-cytoskeleton attachment. Therefore, we compare cells where the gene coding for MKS1 is silenced to wild-type cells. We found that scanning electron microscopy observation of the cell surface reveals a cup-like structure in wild-type cells that is lost in silenced cells. Since this structure is based on the underlying cytoskeleton, one hypothesis to explain this observation is a disruption of membrane attachment to the cytoskeleton in the absence of MKS1 that should affect plasma membrane mechanics. We test this by probing the mechanics of wild-type and silenced cells by micropipette aspiration. Strikingly, we observe that, at the same aspiration pressure, the membrane of silenced cells is easily aspirated by the micropipette whereas that of wild-type cells enters only at a moderate velocity, an effect that suggests a detachment of the membrane from the underlying cytoskeleton in silenced cells. We quantify this detachment by measuring the deformation of the cell cortex and the rate of cell membrane entry in the micropipette. This study offers a new perspective for the characterization of membrane-cytoskeleton attachment in protists and paves the way for a better understanding of the role of membrane-cortex attachment in cilium formation.

The RAS mutation status predicts survival in patients undergoing hepatic resection for colorectal liver metastases: The results from a genetic analysis of all-RAS.

PubMed

Amikura, Katsumi; Akagi, Kiwamu; Ogura, Toshiro; Takahashi, Amane; Sakamoto, Hirohiko

2018-03-01

We investigated the impact of mutations in KRAS exons 3-4 and NRAS exons 2-3 in addition to KRAS exon 2, so-called all-RAS mutations, in patients with colorectal liver metastasis (CLM) undergoing hepatic resection. We analyzed 421 samples from CLM patients for their all-RAS mutation status to compare the overall survival rate (OS), recurrence-free survival rate (RFS), and the pattern of recurrence between the patients with and without RAS mutations. RAS mutations were detected in 191 (43.8%). Thirty-two rare mutations (12.2%) were detected in 262 patients with KRAS exon 2 wild-type. After excluding 79 patients who received anti-EGFR antibody therapy, 168 were classified as all-RAS wild-type, and 174 as RAS mutant-type. A multivariate analysis of factors associated with OS and RFS identified the RAS status as an independent factor (OS; hazard ratio [HR] = 1.672, P = 0.0031, RFS; HR = 1.703, P = 0.0024). Recurrence with lung metastasis was observed significantly more frequent in patients with RAS mutations than in patients with RAS wild-type (P = 0.0005). Approximately half of CLM patients may have a RAS mutation. CLM patients with RAS mutations had a significantly worse survival rate in comparison to patients with RAS wild-type, regardless of the administration of anti-EGFR antibody therapy. © 2017 Wiley Periodicals, Inc.

Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

PubMed Central

Azencott, Harold R.; Peter, Gary F.; Prausnitz, Mark R.

2007-01-01

To assess the cell wall’s role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare the effects of electroporation and sonication in a direct fashion in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it non-specifically disrupts cell-surface barriers. PMID:17602827

Increased ethanol preference and serotonin 1A receptor-dependent attenuation of ethanol-induced hypothermia in PACAP-deficient mice.

PubMed

Tanaka, Kazuhiro; Kunishige-Yamamoto, Akiko; Hashimoto, Hitoshi; Shintani, Norihito; Hayata, Atsuko; Baba, Akemichi

2010-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice display remarkable behavioral changes including increased novelty-seeking behavior and reduced hypothermia induced by either serotonin (5-HT)(1A) receptor agonists or ethanol. Because 5-HT(1A) receptors have been implicated in the development of alcohol dependence, we have examined ethanol preference in PACAP-deficient mice using a two-bottle choice and a conditioned place preference test, as well as additive effects of ethanol and 5-HT(1A) receptor agents on hypothermia. PACAP-deficient mice showed an increased preference towards ethanol compared with wild-type mice. However, they showed no preference for the ethanol compartment after conditioning and neither preference nor aversion to sucrose or quinine. The 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) restored the attenuated hypothermic response to ethanol in the mutants to similar levels in wild-type mice, with no effect in wild-types. In contrast, the 5-HT(1A) receptor antagonist WAY-100635 attenuated the ethanol-induced hypothermia in wild-type mice, with no effect in the mutants. These results demonstrate increased ethanol preference in PACAP-deficient mice that may be mediated by 5-HT(1A) receptor-dependent attenuation of ethanol-induced central inhibition. Copyright 2009 Elsevier Inc. All rights reserved.

The skeletal structure of insulin-like growth factor I-deficient mice

NASA Technical Reports Server (NTRS)

Bikle, D.; Majumdar, S.; Laib, A.; Powell-Braxton, L.; Rosen, C.; Beamer, W.; Nauman, E.; Leary, C.; Halloran, B.

2001-01-01

The importance of insulin-like growth factor I (IGF-I) for growth is well established. However, the lack of IGF-I on the skeleton has not been examined thoroughly. Therefore, we analyzed the structural properties of bone from mice rendered IGF-I deficient by homologous recombination (knockout [k/o]) using histomorphometry, peripheral quantitative computerized tomography (pQCT), and microcomputerized tomography (muCT). The k/o mice were 24% the size of their wild-type littermates at the time of study (4 months). The k/o tibias were 28% and L1 vertebrae were 26% the size of wild-type bones. Bone formation rates (BFR) of k/o tibias were 27% that of the wild-type littermates. The k/o bones responded normally to growth hormone (GH; 1.7-fold increase) and supranormally to IGF-I (5.2-fold increase) with respect to BFR. Cortical thickness of the proximal tibia was reduced 17% in the k/o mouse. However, trabecular bone volume (bone volume/total volume [BV/TV]) was increased 23% (male mice) and 88% (female mice) in the k/o mice compared with wild-type controls as a result of increased connectivity, increased number, and decreased spacing of the trabeculae. These changes were either less or not found in L1. Thus, lack of IGF-I leads to the development of a bone structure, which, although smaller, appears more compact.

The ability to entrain to long photoperiods differs between 3 Drosophila melanogaster wild-type strains and is modified by twilight simulation.

PubMed

Rieger, Dirk; Peschel, Nicolai; Dusik, Verena; Glotz, Silvia; Helfrich-Förster, Charlotte

2012-02-01

The ability to adapt to different environmental conditions including seasonal changes is a key feature of the circadian clock. Here, we compared the ability of 3 Drosophila melanogaster wild-type strains to adapt rhythmic activity to long photoperiods simulated in the laboratory. Fruit flies are predominantly crepuscular with activity bouts in the morning (M) and evening (E). The M peak follows dawn and the E peak follows dusk when the photoperiod is extended. We show that this ability is restricted to a certain extension of the phase angle between M and E peaks, such that the E peak does not delay beyond a certain phase under long days. We demonstrate that this ability is significantly improved by simulated twilight and that it depends additionally on the genetic background and the ambient temperature. At 20 °C, the laboratory strain CantonS had the most flexible phase angle between M and E peaks, a Northern wild-type strain had an intermediate one, and a Southern wild-type strain had the lowest flexibility. Furthermore, we found that the 3 strains differed in clock light sensitivity, with the CantonS and the Northern strains more light sensitive than the Southern strain. These results are generally in accord with the recently discovered polymorphisms in the timeless gene (tim) that affect clock light sensitivity.

Gravitropism of inflorescence stems in starch-deficient mutants of Arabidopsis

NASA Technical Reports Server (NTRS)

Weise, S. E.; Kiss, J. Z.

1999-01-01

Previous studies have assayed the gravitropic response of roots and hypocotyls of wild type Arabidopsis thaliana, two reduced-starch strains, and a starchless strain. Because there have been few reports on inflorescence gravitropism, in this article, we use microscopic analyses and time-course studies of these mutants and their wild type to study gravitropism in these stems. Sedimentation of plastids was observed in endodermal cells of the wild type and reduced-starch mutants but not in the starchless mutant. In all of these strains, the short inflorescence stems (1.0-2.9 cm) were less responsive to the gravistimulus compared with the long stems (3.0-6.0 cm). In both long and short inflorescence stems, the wild type initially had the greatest response; the starchless mutant had the least response; and the reduced starch mutants exhibited an intermediate response. Furthermore, growth rates among all four strains were approximately equal. At about 6 h after reorientation, inflorescences of all strains returned to a position parallel to the gravity vector. Thus, in inflorescence stems, sedimentation of plastids may act as an accelerator but is not required to elicit a gravitropic response. Furthermore, the site of perception appears to be diffuse throughout the inflorescence stem. These results are consistent with both a plastid-based statolith model and the protoplast pressure hypothesis, and it is possible that multiple systems for gravity perception occur in plant cells.

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol

PubMed Central

Blednov, Y.A.; Harris, R.A.

2009-01-01

The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol. PMID:19705551

Identification of the recA (tif) gene product of Escherichia coli

PubMed Central

Gudas, Lorraine J.; Mount, David W.

1977-01-01

Treatments that inhibit DNA synthesis in recA+lexA+Escherichia coli stimulate synthesis of a 40,000 molecular weight protein species (protein X). The protein X molecules produced by wild-type and mutant E. coli strains have been compared by two-dimensional gel electrophoresis. One recA mutant (DM1415 spr recA1) produced a protein X with a more acidic isoelectric point than protein X from the wild type, demonstrating that protein X is probably the product of the recA gene. Additional mutants carrying the recA-linked tif-1 mutation yielded a protein X that was more basic than the wild-type protein, indicating that the tif-1 mutation also alters the recA protein. Protein X molecules from the above mutants and wild-type E. coli have been shown to yield similar partial products upon limited proteolysis in sodium dodecyl sulfate, indicating they are the same protein species. These and additional studies suggest that (i) the tif-1 mutation alters a site on the recA protein that is sensitive to DNA synthesis inhibition, (ii) synthesis of recA protein is self-regulated, and (iii) synthesis of recA protein is also regulated by the lexA product with lexA-suppressor mutations such as spr resulting in constitutive synthesis of recA protein. Images PMID:341152

RLIP76 Protein Knockdown Attenuates Obesity Due to a High-fat Diet*

PubMed Central

Singhal, Sharad S.; Figarola, James; Singhal, Jyotsana; Reddy, Marpadga A.; Liu, Xueli; Berz, David; Natarajan, Rama; Awasthi, Sanjay

2013-01-01

Feeding a Western high-fat diet (HFD) to C57BL/6 mice induces obesity, associated with a chronic inflammatory state, lipid transport, and metabolic derangements, and organ system effects that particularly prominent in the kidneys. Here, we report that RLIP76 homozygous knock-out (RLIP76−/−) mice are highly resistant to obesity as well as these other features of metabolic syndrome caused by HFD. The normal increase in pro-inflammatory and fibrotic markers associated with HFD induced obesity in wild-type C57B mice was broadly and nearly completely abrogated in RLIP76−/− mice. This is a particularly striking finding because chemical markers of oxidative stress including lipid hydroperoxides and alkenals were significantly higher in RLIP76−/− mice. Whereas HFD caused marked suppression of AMPK in wild-type C57B mice, RLIP76−/− mice had baseline activation of AMP-activated protein kinase, which was not further affected by HFD. The baseline renal function was reduced in RLIP76−/− mice as compared with wild-type, but was unaffected by HFD, in marked contrast to severe renal impairment and glomerulopathy in the wild-type mice given HFD. Our findings confirm a fundamental role of RLIP76 in regulating the function of obesity-promoting pro-inflammatory cytokines, and provide a novel mechanism for targeted therapy of obesity and metabolic syndrome. PMID:23821548

Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

PubMed

Li, Yafan; Wheeler, Deric L; Ananthaswamy, Honnavara N; Verma, Ajit K; Oberley, Terry D

2007-12-01

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

The Candida albicans Pho4 Transcription Factor Mediates Susceptibility to Stress and Influences Fitness in a Mouse Commensalism Model

PubMed Central

Urrialde, Verónica; Prieto, Daniel; Pla, Jesús; Alonso-Monge, Rebeca

2016-01-01

The Pho4 transcription factor is required for growth under low environmental phosphate concentrations in Saccharomyces cerevisiae. A characterization of Candida albicans pho4 mutants revealed that these cells are more susceptible to both osmotic and oxidative stress and that this effect is diminished in the presence of 5% CO2 or anaerobiosis, reflecting the relevance of oxygen metabolism in the Pho4-mediated response. A pho4 mutant was as virulent as wild type strain when assayed in the Galleria mellonella infection model and was even more resistant to murine macrophages in ex vivo killing assays. The lack of Pho4 neither impairs the ability to colonize the murine gut nor alters the localization in the gastrointestinal tract. However, we found that Pho4 influenced the colonization of C. albicans in the mouse gut in competition assays; pho4 mutants were unable to attain high colonization levels when inoculated simultaneously with an isogenic wild type strain. Moreover, pho4 mutants displayed a reduced adherence to the intestinal mucosa in a competitive ex vivo assays with wild type cells. In vitro competitive assays also revealed defects in fitness for this mutant compared to the wild type strain. Thus, Pho4, a transcription factor involved in phosphate metabolism, is required for adaptation to stress and fitness in C. albicans. PMID:27458452

Contribution of Bordetella bronchiseptica Filamentous Hemagglutinin and Pertactin to Respiratory Disease in Swine▿ †

PubMed Central

Nicholson, Tracy L.; Brockmeier, Susan L.; Loving, Crystal L.

2009-01-01

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Two well-studied virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). We hypothesized that both FHA and PRN would serve critical roles in the adhesion process and be necessary for colonization of the swine respiratory tract. To investigate the role of FHA and PRN in Bordetella pathogenesis in swine, we constructed mutants containing an in-frame deletion of the FHA or the PRN structural gene in a virulent B. bronchiseptica swine isolate. Both mutants were compared to the wild-type swine isolate for their ability to colonize and cause disease in swine. Colonization of the FHA mutant was lower than that of the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, the PRN mutant caused similar disease severity relative to the wild type; however, colonization of the PRN mutant was reduced relative to the wild type during early and late infection and induced higher anti-Bordetella antibody titers. Together, our results indicate that despite inducing different pathologies and antibody responses, both FHA and PRN are necessary for optimal colonization of the swine respiratory tract. PMID:19237531

[Clinical Observation of Icotinib Hydrochloride for Advanced Non-small Cell Lung Cancer Patients with EGFR Status Identified].

PubMed

Li, Xi; Qin, Na; Wang, Jinghui; Yang, Xinjie; Zhang, Xinyong; Lv, Jialin; Wu, Yuhua; Zhang, Hui; Nong, Jingying; Zhang, Quan; Zhang, Shucai

2015-12-01

Icotinib is the first self-developed small molecular drug in China for targeted therapy of lung cancer. Compared to the other two commercially available epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib, icotinib is similar to them in chemical structure, mechanism of activity and therapeutic effects. To explore the efficacy and side effects of icotinib hydrochloride in the treatment of the advanced non-small cell lung cancer (NSCLC) patients with EGFR mutation and wild-type. Patients with advanced NSCLC who were treated with icotinib hydrochloride in Beijing Chest Hospital were retrospective analyzed from March 2009 to December 2014. The clinical data of 124 patients (99 with EGFR mutation and 25 with wild type) with advanced NSCLC were enrolled in this study. The patients' overall objective response rate (ORR) was 51.6 % and the disease control rate (DCR) was 79.8%; The patients with EGFR mutation, ORR was 63.6%, DCR was 93.9%. The ORR was 4.0% and the DCR was 24.0% in the wild-type patients. Median progression-free survival (PFS) with icotinib treatment in EGFR mutation patients was 10.5 months and 1.0 month in wild-type patients. The major adverse events were mild skin rash (30.6%) and diarrhea (16.1%). Monotherapy with icotinib hydrochloride is effective and tolerable for the advanced NSCLC EGFR mutation patients.


Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

PubMed Central

da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

2013-01-01

Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant. PMID:24688492

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

PubMed

Francis, Brian R; White, Karen H; Thorsness, Peter E

2007-04-01

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

Wild-type cells rescue genotypically Math1-null hair cells in the inner ears of chimeric mice.

PubMed

Du, Xiaoping; Jensen, Patricia; Goldowitz, Daniel; Hamre, Kristin M

2007-05-15

The transcription factor Math1 has been shown to be critical in the formation of hair cells (HCs) in the inner ear. However, the influence of environmental factors in HC specification suggests that cell extrinsic factors are also crucial to their development. To test whether extrinsic factors impact development of Math1-null (Math1(beta-Gal/beta-Gal)) HCs, we examined neonatal (postnatal ages P0-P4.5) Math1-null chimeric mice in which genotypically mutant and wild-type cells intermingle to form the inner ear. We provide the first direct evidence that Math1-null HCs are able to be generated and survive in the conducive chimeric environment. beta-Galactosidase expression was used to identify genetically mutant cells while cells were phenotypically defined as HCs by morphological characteristics notably the expression of HC-specific markers. Genotypically mutant HCs were found in all sensory epithelia of the inner ear at all ages examined. Comparable results were obtained irrespective of the wild-type component of the chimeric mice. Thus, genotypically mutant cells retain the competence to differentiate into HCs. The implication is that the lack of the Math1 gene in HC precursors can be overcome by environmental influences, such as cell-cell interactions with wild-type cells, to ultimately result in the formation of HCs.

Mutation of the RESURRECTION1 Locus of Arabidopsis Reveals an Association of Cuticular Wax with Embryo Development1

PubMed Central

Chen, Xinbo; Goodwin, S. Mark; Liu, Xionglun; Chen, Xinlu; Bressan, Ray A.; Jenks, Matthew A.

2005-01-01

Insertional mutagenesis of Arabidopsis (Arabidopsis thaliana) was used to identify a novel recessive mutant, designated resurrection1 (rst1), which possesses a dramatic alteration in its cuticular waxes and produces shrunken nonviable seeds due to arrested embryo development. The RST1 gene sequence associated with these phenotypes was verified by three independent, allelic, insertion mutants, designated rst1-1, rst1-2, and rst1-3, with inserts in the first exon, 12th intron, and fourth exon, respectively. These three rst1 allelic mutants have nearly identical alterations in their wax profiles and embryo development. Compared to wild type, the wax on rst1 inflorescence stems is reduced nearly 60% in total amount, has a proportional reduction in aldehydes and aldehyde metabolites, and has a proportional increase in acids, primary alcohols, and esters. Compared to wild type, the C29 alkanes on rst1 are nearly 6-fold lower, and the C30 primary alcohols are 4-fold higher. These results indicate that rst1 causes shunting of most wax precursors away from alkane synthesis and into the primary-alcohol-producing branch of the pathway. In contrast to stems, the wax on rst1 mutant leaves increased roughly 43% in amount relative to the wild type, with the major increase occurring in the C31 and C33 alkanes. Unique among known wax mutants, approximately 70% of rst1 seeds are shrunken and nonviable, with these being randomly distributed within both inflorescence and silique. Viable seeds of rst1 are slightly larger than those of wild type, and although the viable rst1 seeds contain more total triacylglycerol-derived fatty acids, the proportions of these fatty acids are not significantly different from wild type. Shrunken seeds contain 34% of the fatty acids of wild-type seeds, with proportionally more palmitic, stearic, and oleic acids, and less of the longer and more desaturated homologs. Histological analysis of aborted rst1 seeds revealed that embryo development terminates at the approximate heart-shaped stage, whereas viable rst1 and wild-type embryos develop similarly. The RST1 gene encodes a predicted 1,841-amino acid novel protein with a molecular mass of 203.6 kD and a theoretical pI of 6.21. The RST1 transcript was found in all tissues examined including leaves, flowers, roots, stems, and siliques, but accumulation levels were not correlated with the degree to which different organs appeared affected by the mutation. The new RST1 gene reveals a novel genetic connection between lipid synthesis and embryo development; however, RST1's exact role is still quite unknown. The degree to which RST1 is associated with lipid signaling in development is an important focus of ongoing studies. PMID:16183838

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.

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

PubMed Central

Li, Fang; Ryu, Byoung Y.; Krueger, Robin L.; Heldt, Scott A.

2012-01-01

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 106 transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins. PMID:22013043

Crosstalk between stromal components and tumor cells of TNBC via secreted factors enhances tumor growth and metastasis

PubMed Central

Jin, Kideok; Pandey, Niranjan B.; Popel, Aleksander S.

2017-01-01

Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Reliable and reproducible methods for testing drugs against metastasis are not available. Stromal cells may play a critical role in tumor progression and metastasis. In this study, we determined that fibroblasts and macrophages secreted IL-8 upon induction by tumor cell-conditioned media (TCM) from MDA-MB-231 cancer cells. Our data showed that the proliferation of MDA-MB-231 cells co-cultured with fibroblasts or macrophages was enhanced compared to the monoculture. Furthermore, TNBC cell migration, a key step in tumor metastasis, was promoted by conditioned media (CM) from TCM-induced fibroblasts or macrophages. Knockdown of the IL-8 receptor CXCR2 by CRISPR-Cas9 reduces MDA-MB-231 cell proliferation and migration compared to wild type. In a mouse xenograft tumor model, the growth of MDA-MB-231-CXCR2−/− tumor was significantly decreased compared to the growth of tumors from wild-type cells. In addition, the incidence of thoracic metastasis of MDA-MB-231-CXCR2−/− tumors was reduced compared to wild type. We found that the auto- and paracrine loop exists between TNBC cells and stroma, which results in enhanced IL-8 secretion from the stromal components. Significantly, inhibition of the IL-8 signaling pathway by reparixin, an inhibitor of the IL-8 receptor, CXCR1/2, reduced MDA-MB-231 tumor growth and metastasis. Taken together, these findings implicate IL-8 signaling as a critical event in TNBC tumor growth and metastasis via crosstalk with stromal components. PMID:28947965

Altered Astrocyte-Neuron Interactions and Epileptogenesis in Tuberous Sclerosis Complex Disorder

DTIC Science & Technology

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

Monitoring Rates and Heterogeneity of High-Pressure Germination of Bacillus Spores by Phase-Contrast Microscopy of Individual Spores

DTIC Science & Technology

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

Differentially enhanced insect resistance, at a cost, in Arabidopsis thaliana constitutively expressing a transcription factor of defensive metabolites.

PubMed

Johnson, Eric T; Dowd, Patrick F

2004-08-11

A transgenic line of Arabidopsis thaliana constitutively expressing a conserved MYB transcription factor of phenylpropanoid biosynthesis resulting in solid-purple leaves had significantly increased resistance to leaf feeding by first instar fall armyworms (Spodoptera frugiperda), but no enhanced resistance to cabbage looper (Trichoplusia ni) larvae, when compared to wild type plants. However, inflorescence and silique (seed pod) production were significantly reduced by 22 and 52%, respectively, in the transgenic line compared to wild type plants. Reduction in feeding by S. frugiperda was significantly positively correlated with reduction in weights of survivors, but both were negatively correlated with the concentration of anthocyanins. These results indicate that a single gene regulator can activate a defensive pathway sufficient to produce increased resistance to insects but that this activation confers a cost in plant productivity.

GAS6/TAM Pathway Signaling in Hemostasis and Thrombosis.

PubMed

Law, Luke A; Graham, Douglas K; Di Paola, Jorge; Branchford, Brian R

2018-01-01

The GAS6/TYRO3-AXL-MERTK (TAM) signaling pathway is essential for full and sustained platelet activation, as well as thrombus stabilization. Inhibition of this pathway decreases platelet aggregation, shape change, clot retraction, aggregate formation under flow conditions, and surface expression of activation markers. Transgenic mice deficient in GAS6, or any of the TAM family of receptors that engage this ligand, exhibit in vivo protection against arterial and venous thrombosis but do not demonstrate either spontaneous or prolonged bleeding compared to their wild-type counterparts. Comparable results are observed in wild-type mice treated with pharmacological inhibitors of the GAS6-TAM pathway. Thus, GAS6/TAM inhibition offers an attractive novel therapeutic option that may allow for a moderate reduction in platelet activation and decreased thrombosis while still permitting the primary hemostatic function of platelet plug formation.

Overexpression of Arabidopsis VIT1 increases accumulation of iron in cassava roots and stems.

PubMed

Narayanan, Narayanan; Beyene, Getu; Chauhan, Raj Deepika; Gaitán-Solis, Eliana; Grusak, Michael A; Taylor, Nigel; Anderson, Paul

2015-11-01

Iron is extremely abundant in the soil, but its uptake in plants is limited due to low solubility in neutral or alkaline soils. Plants can rely on rhizosphere acidification to increase iron solubility. AtVIT1 was previously found to be involved in mediating vacuolar sequestration of iron, which indicates a potential application for iron biofortification in crop plants. Here, we have overexpressed AtVIT1 in the starchy root crop cassava using a patatin promoter. Under greenhouse conditions, iron levels in mature cassava storage roots showed 3-4 times higher values when compared with wild-type plants. Significantly, the expression of AtVIT1 showed a positive correlation with the increase in iron concentration of storage roots. Conversely, young leaves of AtVIT1 transgenic plants exhibit characteristics of iron deficiency such as interveinal chlorosis of leaves (yellowing) and lower iron concentration when compared with the wild type plants. Interestingly, the AtVIT1 transgenic plants showed 4 and 16 times higher values of iron concentration in the young stem and stem base tissues, respectively. AtVIT1 transgenic plants also showed 2-4 times higher values of iron content when compared with wild-type plants, with altered partitioning of iron between source and sink tissues. These results demonstrate vacuolar iron sequestration as a viable transgenic strategy to biofortify crops and to help eliminate micronutrient malnutrition in at-risk human populations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Genome-wide screen of Saccharomyces cerevisiae for killer toxin HM-1 resistance.

PubMed

Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

2011-01-01

We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM-1, which kills susceptible yeasts through inhibiting 1,3-beta-glucan synthase. By using HM-1 plate assay, we found that eight gene-deletion mutants had higher HM-1-resistance compared with the wild-type. Among these eight genes, five--ALG3, CAX4, MNS1, OST6 and YBL083C--were associated with N-glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM-1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2-alpha-mannosidase showed with a 13-fold higher HM-1 resistance compared with the wild-type. By HM-1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild-type cells. These results indicate that the presence of the terminal 1,3-alpha-linked mannose residue of the B-chain of the N-glycan structure is essential for interaction with HM-1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM-1 resistance. A deletion mutant of osmoregulatory mitogen-activated protein kinase Hog1p was more sensitive to HM-1, suggesting that high-osmolarity glycerol pathways plays an important role in the compensatory response to HM-1 action. Copyright © 2010 John Wiley & Sons, Ltd.

Dietary restriction ameliorates haematopoietic ageing independent of telomerase, whilst lack of telomerase and short telomeres exacerbates the ageing phenotype.

PubMed

Al-Ajmi, Nouf; Saretzki, Gabriele; Miles, Colin; Spyridopoulos, Ioakim

2014-10-01

Ageing is associated with an overall decline in the functional capacity of tissues and stem cells, including haematopoietic stem and progenitor cells (HSPCs), as well as telomere dysfunction. Dietary restriction (DR) is a recognised anti-ageing intervention that extends lifespan and improves health in several organisms. To investigate the role of telomeres and telomerase in haematopoietic ageing, we compared the HSPC profile and clonogenic capacity of bone marrow cells from wild type with telomerase-deficient mice and the effect of DR on these parameters. Compared with young mice, aged wild type mice demonstrated a significant accumulation of HSPCs (1.3% vs 0.2%, P=0.002) and elevated numbers of granulocyte/macrophage colony forming units (CFU-GM, 26.4 vs 17.3, P=0.0037) consistent with myeloid "skewing" of haematopoiesis. DR was able to restrict the increase in HSPC number as well as the myeloid "skewing" in aged wild type mice. In order to analyse the influence of short telomeres on the ageing phenotype we examined mice lacking the RNA template for telomerase, TERC(-/-). Telomere shortening resulted in a similar bone marrow phenotype to that seen in aged mice, with significantly increased HSPC numbers and an increased formation of all myeloid colony types but at a younger age than wild type mice. However, an additional increase in erythroid colonies (BFU-E) was also evident. Mice lacking telomerase reverse transcriptase without shortened telomeres, TERT(-/-), also presented with augmented haematopoietic ageing which was ameliorated by DR, demonstrating that the effect of DR was not dependent on the presence of telomerase in HSPCs. We conclude that whilst shortened telomeres mimic some aspects of haematopoietic ageing, both shortened telomeres and the lack of telomerase produce specific phenotypes, some of which can be prevented by dietary restriction. Copyright © 2014 Elsevier Inc. All rights reserved.

A transcriptionally active estrogen receptor mutant is a novel type of dominant negative inhibitor of estrogen action.

PubMed

McInerney, E M; Ince, B A; Shapiro, D J; Katzenellenbogen, B S

1996-12-01

We have characterized a human estrogen receptor (ER) mutant, V364E, which has a single amino acid substitution in its hormone-binding domain. This ER mutant is fully active or even superactive at saturating levels of estradiol (10(-8) M E2) yet has the capacity to act as a strong dominant negative inhibitor of the wild type ER. In transient transfection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter reporter genes, V364E treated with 10(-8) M E2 exhibited approximately 250% and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when present alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity. On the TATA promoter, where V364E was more inhibitory, estrogen-stimulated activity was reduced by approximately 50% at a 1:1 ratio of mutant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a approximately 40-fold lower affinity for E2 compared with wild type ER. In promoter interference assays, V364E exhibited a strict dependence upon E2 for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E2 concentration or by mutation of its DNA-binding domain), this mutant retained full dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild type ER are present together in cells, even without DNA binding. Since competition for ERE binding and the formation of inactive heterodimers cannot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in ER-mediated transcription play a key role in the repression of transcription by V364E. The properties and probable mechanism of action of V364E distinguish it from other previously described dominant negative inhibitors, in which competition for cis-acting DNA elements by transcriptionally inactive receptors played a large role in the resultant dominant negative phenotype.

Microarray analysis of retinal gene expression in Egr-1 knockout mice

PubMed Central

Schippert, Ruth; Schaeffel, Frank

2009-01-01

Purpose We found earlier that 42 day-old Egr-1 knockout mice had longer eyes and a more myopic refractive error compared to their wild-types. To identify genes that could be responsible for the temporarily enhanced axial eye growth, a microarray analysis was performed in knockout and wild-type mice at the postnatal ages of 30 and 42 days. Methods The retinas of homozygous and wild-type Egr-1 knockout mice (Taconic, Ry, Denmark) were prepared for RNA isolation (RNeasy Mini Kit, Qiagen) at the age of 30 or 42 days, respectively (n=12 each). Three retinas were pooled and labeled cRNA was made. The samples were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Hybridization signals were calculated using GC-RMA normalization. Genes were identified as differentially expressed if they showed a fold-change (FC) of at least 1.5 and a p-value <0.05. A false-discovery rate of 5% was applied. Ten genes with potential biologic relevance were examined further with semiquantitative real-time RT–PCR. Results Comparing mRNA expression levels between wild-type and homozygous Egr-1 knockout mice, we found 73 differentially expressed genes at the age of 30 days and 135 genes at the age of 42 days. Testing for differences in gene expression between the two ages (30 versus 42 days), 54 genes were differently expressed in wild-type mice and 215 genes in homozygous animals. Based on three networks proposed by Ingenuity pathway analysis software, nine differently expressed genes in the homozygous Egr-1 knockout mice were chosen for further validation by real-time RT–PCR, three genes in each network. In addition, the gene that was most prominently regulated in the knockout mice, compared to wild-type, at both 30 days and 42 days of age (protocadherin beta-9 [Pcdhb9]), was tested with real-time RT–PCR. Changes in four of the ten genes could be confirmed by real-time RT–PCR: nuclear prelamin A recognition factor (Narf), oxoglutarate dehydrogenase (Ogdh), selenium binding protein 1 (Selenbp1), and Pcdhb9. Except for Pcdhb9, the genes whose mRNA expression levels were validated were listed in one of the networks proposed by Ingenuity pathway analysis software. In addition to these genes, the software proposed several key-regulators which did not change in our study: retinoic acid, vascular endothelial growth factor A (VEGF-A), FBJ murine osteosarcoma viral oncogene homolog (cFos), and others. Conclusions Identification of genes that are differentially regulated during the development period between postnatal day 30 (when both homozygous and wild-type mice still have the same axial length) and day 42 (where the difference in eye length is apparent) could improve the understanding of mechanisms for the control of axial eye growth and may lead to potential targets for pharmacological intervention. With the aid of pathway-analysis software, a coarse picture of possible biochemical pathways could be generated. Although the mRNA expression levels of proteins proposed by the software, like VEGF, FOS, retinoic acid (RA) receptors, or cellular RA binding protein, did not show any changes in our experiment, these molecules have previously been implicated in the signaling cascades controlling axial eye growth. According to the pathway-analysis software, they represent links between several proteins whose mRNA expression was changed in our study. PMID:20019881

Microarray analysis of retinal gene expression in Egr-1 knockout mice.

PubMed

Schippert, Ruth; Schaeffel, Frank; Feldkaemper, Marita Pauline

2009-12-10

We found earlier that 42 day-old Egr-1 knockout mice had longer eyes and a more myopic refractive error compared to their wild-types. To identify genes that could be responsible for the temporarily enhanced axial eye growth, a microarray analysis was performed in knockout and wild-type mice at the postnatal ages of 30 and 42 days. The retinas of homozygous and wild-type Egr-1 knockout mice (Taconic, Ry, Denmark) were prepared for RNA isolation (RNeasy Mini Kit, Qiagen) at the age of 30 or 42 days, respectively (n=12 each). Three retinas were pooled and labeled cRNA was made. The samples were hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Hybridization signals were calculated using GC-RMA normalization. Genes were identified as differentially expressed if they showed a fold-change (FC) of at least 1.5 and a p-value <0.05. A false-discovery rate of 5% was applied. Ten genes with potential biologic relevance were examined further with semiquantitative real-time RT-PCR. Comparing mRNA expression levels between wild-type and homozygous Egr-1 knockout mice, we found 73 differentially expressed genes at the age of 30 days and 135 genes at the age of 42 days. Testing for differences in gene expression between the two ages (30 versus 42 days), 54 genes were differently expressed in wild-type mice and 215 genes in homozygous animals. Based on three networks proposed by Ingenuity pathway analysis software, nine differently expressed genes in the homozygous Egr-1 knockout mice were chosen for further validation by real-time RT-PCR, three genes in each network. In addition, the gene that was most prominently regulated in the knockout mice, compared to wild-type, at both 30 days and 42 days of age (protocadherin beta-9 [Pcdhb9]), was tested with real-time RT-PCR. Changes in four of the ten genes could be confirmed by real-time RT-PCR: nuclear prelamin A recognition factor (Narf), oxoglutarate dehydrogenase (Ogdh), selenium binding protein 1 (Selenbp1), and Pcdhb9. Except for Pcdhb9, the genes whose mRNA expression levels were validated were listed in one of the networks proposed by Ingenuity pathway analysis software. In addition to these genes, the software proposed several key-regulators which did not change in our study: retinoic acid, vascular endothelial growth factor A (VEGF-A), FBJ murine osteosarcoma viral oncogene homolog (cFos), and others. Identification of genes that are differentially regulated during the development period between postnatal day 30 (when both homozygous and wild-type mice still have the same axial length) and day 42 (where the difference in eye length is apparent) could improve the understanding of mechanisms for the control of axial eye growth and may lead to potential targets for pharmacological intervention. With the aid of pathway-analysis software, a coarse picture of possible biochemical pathways could be generated. Although the mRNA expression levels of proteins proposed by the software, like VEGF, FOS, retinoic acid (RA) receptors, or cellular RA binding protein, did not show any changes in our experiment, these molecules have previously been implicated in the signaling cascades controlling axial eye growth. According to the pathway-analysis software, they represent links between several proteins whose mRNA expression was changed in our study.

Role of P-glycoprotein and breast cancer resistance protein-1 in the brain penetration and brain pharmacodynamic activity of the novel phosphatidylinositol 3-kinase inhibitor GDC-0941.

PubMed

Salphati, Laurent; Lee, Leslie B; Pang, Jodie; Plise, Emile G; Zhang, Xiaolin

2010-09-01

2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) is a novel small molecule inhibitor of the phosphatidylinositol 3-kinase (PI3K) pathway currently evaluated in the clinic as an anticancer agent. The objectives of this study were to determine in vitro whether GDC-0941 was a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp1) and to investigate the impact of these transporters on the pharmacokinetics, brain penetration, and activity of GDC-0941 in FVBn mice (wild-type) and Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) knockout mice. Studies with Madin-Darby canine kidney cells transfected with P-gp or Bcrp1 established that this compound was a substrate of both transporters. After administrations to mice, GDC-0941 brain-to-plasma ratio ranged from 0.02 to 0.06 in the wild-type and Bcrp1(-/-) mice and was modestly higher in the Mdr1a/b(-/-) mice, ranging from 0.08 to 0.11. In contrast, GDC-0941 brain-to-plasma ratio in Mdr1a/b(-/-)/Bcrp1(-/-) triple knockout mice was 30-fold higher than in the wild-type mice. The plasma clearance of GDC-0941 was similar in wild-type and all knockout mice, ranging from 15 to 25 ml/(min . kg) in the wild-type mice and from 18 to 35 ml/(min . kg) in the knockout mice. Exposure after oral administration was comparable in the four strains of mice. The PI3K pathway was markedly inhibited in the brain of Mdr1a/b(-/-)/Bcrp1(-/-) mice for up to 6 h postdose, as evidenced by a 60% suppression of the phosphorylated Akt signal, whereas no inhibition was detected in the brain of wild-type mice. The concerted effects of P-gp and Bcrp1 in restricting GDC-0941 access and pathway modulation in mouse brain may have implications for the treatment of patients with brain tumors.

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation.

PubMed Central

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

Interferon-Induced Ifit2/ISG54 Protects Mice from Lethal VSV Neuropathogenesis

PubMed Central

Fensterl, Volker; Wetzel, Jaime L.; Ramachandran, Srividya; Ogino, Tomoaki; Stohlman, Stephen A.; Bergmann, Cornelia C.; Diamond, Michael S.; Virgin, Herbert W.; Sen, Ganes C.

2012-01-01

Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2 −/−) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1 −/− mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2−/− mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2 −/− mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2 −/− mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2 −/− mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2−/− mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2 −/− mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon. PMID:22615570

Ectopically expressing MdPIP1;3, an aquaporin gene, increased fruit size and enhanced drought tolerance of transgenic tomatoes.

PubMed

Wang, Lin; Li, Qing-Tian; Lei, Qiong; Feng, Chao; Zheng, Xiaodong; Zhou, Fangfang; Li, Lingzi; Liu, Xuan; Wang, Zhi; Kong, Jin

2017-12-19

Water deficit severely reduces apple growth and production, is detrimental to fruit quality and size. This problem is exacerbated as global warming is implicated in producing more severe drought stress. Thus water-efficiency has becomes the major target for apple breeding. A desired apple tree can absorb and transport water efficiently, which not only confers improved drought tolerance, but also guarantees fruit size for higher income returns. Aquaporins, as water channels, control water transportation across membranes and can regulate water flow by changing their amount and activity. The exploration of molecular mechanism of water efficiency and the gene wealth will pave a way for molecular breeding of drought tolerant apple tree. In the current study, we screened out a drought inducible aquaporin gene MdPIP1;3, which specifically enhanced its expression during fruit expansion in 'Fuji' apple (Malus domestica Borkh. cv. Red Fuji). It localized on plasma membranes and belonged to PIP1 subfamily. The tolerance to drought stress enhanced in transgenic tomato plants ectopically expressing MdPIP1;3, showing that the rate of losing water in isolated transgenic leaves was slower than wild type, and stomata of transgenic plants closed sensitively to respond to drought compared with wild type. Besides, length and diameter of transgenic tomato fruits increased faster than wild type, and in final, fruit sizes and fresh weights of transgenic tomatoes were bigger than wild type. Specially, in cell levels, fruit cell size from transgenic tomatoes was larger than wild type, showing that cell number per mm 2 in transgenic fruits was less than wild type. Altogether, ectopically expressing MdPIP1;3 enhanced drought tolerance of transgenic tomatoes partially via reduced water loss controlled by stomata closure in leaves. In addition, the transgenic tomato fruits are larger and heavier with larger cells via more efficient water transportation across membranes. Our research will contribute to apple production, by engineering apples with big fruits via efficient water transportation when well watered and enhanced drought tolerance in transgenic apples under water deficit.

Cell-to-cell signaling in Xylella fastidiosa suppresses movement and xylem vessel colonization in grape.

PubMed

Chatterjee, Subhadeep; Newman, Karyn L; Lindow, Steven E

2008-10-01

Cell-to-cell signaling mediated by a fatty acid diffusible signaling factor (DSF) is central to the regulation of the virulence of Xylella fastidiosa. DSF production by X. fastidiosa is dependent on rpfF and, although required for insect colonization, appears to reduce its virulence to grape. To understand what aspects of colonization of grape are controlled by DSF in X. fastidiosa and, thus, those factors that contribute to virulence, we assessed the colonization of grape by a green fluorescent protein-marked rpfF-deficient mutant. The rpfF-deficient mutant was detected at a greater distance from the point of inoculation than the wild-type strain at a given sampling time, and also attained a population size that was up to 100-fold larger than that of the wild-type strain at a given distance from the point of inoculation. Confocal laser-scanning microscopy revealed that approximately 10-fold more vessels in petioles of symptomatic leaves harbored at least some cells of either the wild type or rpfF mutant when compared with asymptomatic leaves and, thus, that disease symptoms were associated with the extent of vessel colonization. Importantly, the rpfF mutant colonized approximately threefold more vessels than the wild-type strain. Although a wide range of colony sizes were observed in vessels colonized by both the wild type and rpfF mutant, the proportion of colonized vessels harboring large numbers of cells was significantly higher in plants inoculated with the rpfF mutant than with the wild-type strain. These studies indicated that the hypervirulence phenotype of the rpfF mutant is due to both a more extensive spread of the pathogen to xylem vessels and unrestrained multiplication within vessels leading to blockage. These results suggest that movement and multiplication of X. fastidiosa in plants are linked, perhaps because cell wall degradation products are a major source of nutrients. Thus, DSF-mediated cell-to-cell signaling, which restricts movement and colonization of X. fastidiosa, may be an adaptation to endophytic growth of the pathogen that prevents the excessive growth of cells in vessels.

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

Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (Fragaria vesca).

PubMed

Osorio, Sonia; Castillejo, Cristina; Quesada, Miguel A; Medina-Escobar, Nieves; Brownsey, Geoff J; Suau, Rafael; Heredia, Antonio; Botella, Miguel A; Valpuesta, Victoriano

2008-04-01

In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria x ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca. Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea. Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.

Stomatal control in tomato with ABA-deficient roots: response of grafted plants to soil drying.

PubMed

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.

Contractile dysfunctions in ATP-dependent K+ channel-deficient mouse muscle during fatigue involve excessive depolarization and Ca2+ influx through L-type Ca2+ channels.

PubMed

Cifelli, Carlo; Boudreault, Louise; Gong, Bing; Bercier, Jean-Philippe; Renaud, Jean-Marc

2008-10-01

Muscles deficient in ATP-dependent potassium (KATP) channels develop contractile dysfunctions during fatigue that may explain their apparently faster rate of fatigue compared with wild-type muscles. The objectives of this study were to determine: (1) whether the contractile dysfunctions, namely unstimulated force and depressed force recovery, result from excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) whether reducing the magnitude of these two contractile dysfunctions reduces the rate of fatigue in KATP channel-deficient muscles. To reduce Ca2+ influx, we lowered the extracellular Ca2+ concentration ([Ca2+]o) from 2.4 to 0.6 mM or added 1 microM verapamil, an L-type Ca2+ channel blocker. Flexor digitorum brevis (FDB) muscles deficient in KATP channels were obtained by exposing wild-type muscles to 10 microM glibenclamide or by using FDB from Kir6.2-/- mice. Fatigue was elicited with one contraction per second for 3 min at 37 degrees C. In wild-type FDB, lowered [Ca2+]o or verapamil did not affect the decrease in peak tetanic force and unstimulated force during fatigue and force recovery following fatigue. In KATP channel-deficient FDB, lowered [Ca2+]o or verapamil slowed down the decrease in peak tetanic force recovery, reduced unstimulated force and improved force recovery. In Kir6.2-/- FDB, the rate of fatigue became slower than in wild-type FDB in the presence of verapamil. The cell membrane depolarized from -83 to -57 mV in normal wild-type FDB. The depolarizations in some glibenclamide-exposed fibres were similar to those of normal FDB, while in other fibres the cell membrane depolarized to -31 mV in 80 s, which was also the time when these fibres supercontracted. It is concluded that: (1) KATP channels are crucial in preventing excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) they contribute to the decrease in force during fatigue.

Effect of understory management on phenological responses of eastern black walnut on an alluvial Arkansas soil

USDA-ARS?s Scientific Manuscript database

Black walnut (Juglans nigra L.) is commonly grown in agroforestry practices for nuts and/or timber with little knowledge of how understory herbage management might affect tree phenology. We compared black walnut plant type (variety and wild-type) for phenological response in date of budburst, leaf ...

Unlike type 2 diabetes, type 1 does not interact with the codon 54 polymorphism of the fatty acid binding protein 2 gene.

PubMed

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.

Comparative proteomic analysis of virulent and rifampicin attenuated Flavobacterium psychrophilum

USDA-ARS?s Scientific Manuscript database

Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease and rainbow trout fry syndrome. In this study we compared a wild-type strain (CSF 259.93) with a rifampicin resistant and virulence attenuated strain of F. psychrophilum (CSF 259.93B.17). The attenuated strain harbour...

Strain H2-419-4 of Haematococcus pluvialis induced by ethyl methanesulphonate and ultraviolet radiation

NASA Astrophysics Data System (ADS)

Sun, Yanhong; Liu, Jianguo; Zhang, Xiaoli; Lin, Wei

2008-05-01

Two strains H2-410 and H2-419 were obtained from the chemically mutated survivors of wild Haematococcus pluvialis 2 by using ethyl methanesulphonate (EMS). Strains H2-410 and H2-419 showed a fast cell growth with 13% and 20% increase in biomass compared to wild type, respectively. Then H2-419-4, a fast cell growth and high astaxanthin accumulation strain, was obtained by exposing the strain H2-419 to ultraviolet radiation (UV) further. The total biomass, the astaxanthin content per cell, astaxanthin production of H2-419-4 showed 68%, 28%, and 120% increase compared to wild H. pluvialis 2, respectively. HPLC (High Performance Liquid Chromatography) data showed also an obvious proportional variation of different carotenoid compositions in the extracts of H2-419-4 and the wild type, although no peak of carotenoids appeared or disappeared. Therefore, the main compositions in strain H2-419-4, like its wild one, were free of astaxanthin, monoester, and diester of astaxanthin. The asexual reproduction in survivors after exposed to UV was not synchronous, and different from the normal synchronous asexual reproduction as the mother cells were motile instead of non-motile. Interestingly, some survivors from UV irradiation produced many mini-spores (or gamete?), the spores moved away from the mother cell gradually 4 or 5 days later. This is quite similar to sexual reproduction described by Elliot in 1934. However, whether this was sexual reproduction remains questionable, as no mating process has been observed.

DSCAM Localization and Function at the Mouse Cone Synapse

PubMed Central

de Andrade, Gabriel Belem; Long, Samuel S.; Fleming, Harrison; Li, Wei; Fuerst, Peter G.

2014-01-01

The Down Syndrome Cell Adhesion Molecule (DSCAM) is required for regulation of cell number, soma spacing and cell type specific dendrite avoidance in many types of retinal ganglion and amacrine cells. In this study we assay the organization of cells making up the outer plexiform layer of the retina in the absence of Dscam. Some types of OFF bipolar cells, type 3b and type 4 bipolar cells, had defects in dendrite arborization in the Dscam mutant retina, while other cell types appeared similar to wild type. The cone synapses that these cells project their dendrites to were intact, as visualized by electron microscopy, and had a distribution and density that was not significantly different than wild type. The spacing of type 3b bipolar cell dendrites was further analyzed by Voronoi domain analysis, Density Recovery Profiling (DRP) analysis and Nearest Neighbor Analysis (NNA). Spacing was found to be significantly different when comparing wild type and mutant type 3b bipolar cell dendrites. Defects in arborization of these bipolar cells could not be attributed to the disorganization of inner plexiform layer cells that occurs in the Dscam mutant retina or an increase in cell number, as they arborized when Dscam was targeted in retinal ganglion cells only or in the bax null retina. Localization of DSCAM was assayed and the protein was localized near to cone synapses in mouse, macaque and ground squirrel retinas. DSCAM protein was detected in several types of bipolar cells, including type 3b and type 4 bipolar cells. PMID:24477985

Retrospective study of RAS/PIK3CA/BRAF tumor mutations as predictors of response to first-line chemotherapy with bevacizumab in metastatic colorectal cancer patients.

PubMed

Nakayama, Izuma; Shinozaki, Eiji; Matsushima, Tomohiro; Wakatsuki, Takeru; Ogura, Mariko; Ichimura, Takashi; Ozaka, Masato; Takahari, Daisuke; Suenaga, Mitsukuni; Chin, Keisho; Mizunuma, Nobuyuki; Yamaguchi, Kensei

2017-01-09

After analysis of minor RAS mutations (KRAS exon 3, 4/NRAS) in the FIRE-3 and PRIME studies, an expanded range of RAS mutations were established as a negative predictive marker for the efficacy of anti-EGFR antibody treatment. BRAF and PIK3CA mutations may be candidate biomarkers for anti-EGFR targeted therapies. However, it remains unknown whether RAS/PIK3CA/BRAF tumor mutations can predict the efficacy of bevacizumab in metastatic colorectal cancer. We assessed whether selection according to RAS/PIK3CA/BRAF mutational status could be beneficial for patients treated with bevacizumab as first-line treatment for metastatic colorectal cancer. Of the 1001 consecutive colorectal cancer patients examined for RAS, PIK3CA, and BRAF tumor mutations using a multiplex kit (Luminex®), we studied 90 patients who received combination chemotherapy with bevacizumab as first-line treatment for metastatic colorectal cancer. The objective response rate (ORR) and progression-free survival (PFS) were evaluated according to mutational status. The ORR was higher among patients with wild-type tumors (64.3%) compared to those with tumors that were only wild type with respect to KRAS exon 2 (54.8%), and the differences in ORR between patients with wild-type and mutant-type tumors were greater when considering only KRAS exon 2 mutations (6.8%) rather than RAS/PIK3CA/BRAF mutations (18.4%). There were no statistically significant differences in ORR or PFS between all wild-type tumors and tumors carrying any of the mutations. Multivariate analysis revealed that liver metastasis and RAS and BRAF mutations were independent negative factors for disease progression after first-line treatment with bevacizumab. Patient selection according to RAS/PIK3CA/BRAF mutations could help select patients who will achieve a better response to bevacizumab treatment. We found no clinical benefit of restricting combination therapy with bevacizumab for metastatic colorectal cancer patients with EGFR-wild type tumors.

New Interest in Wild Forest Products in Europe as an Expression of Biocultural Dynamics.

PubMed

Wiersum, K F

2017-01-01

In Europe, interest in wild forest products is increasing. Such products may be interpreted in a biological sense as deriving from autonomously growing forest species or in a biocultural sense as reflecting dynamics in human living with biodiversity through re-wilding of earlier domesticated species. In this article I elaborate the idea that the new interests reflect biocultural dynamics. First, I identify these dynamics as involving both domestication and re-wilding and characterize these processes as involving biological, environmental, and cultural dimensions. Next, I present a comparative review of two approaches to re-wilding forest production in the Netherlands: meat production from new types of natural grazing systems, and food production from plants re-introduced to the wild. The first approach is based on the stimulation of naturally occurring ecological processes and the second on the stimulation of new forms of experiencing bio-cultural heritage. The examples demonstrate that the new interests in wild forest products involve both a return to earlier stages of domestication in an ecological sense and a new phase of acculturation to evolving socio-cultural conditions.

The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

PubMed

Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

2014-04-01

Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation. Copyright © 2014 Elsevier B.V. All rights reserved.

Seed fates in crop-wild hybrid sunflower: crop allele and maternal effects.

PubMed

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.

Murine study of portal hypertension associated endothelin-1 hypo-response.

PubMed

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.

Occult infection related hepatitis B surface antigen variants showing lowered secretion capacity

PubMed Central

Kim, Hong; Lee, Seoung-Ae; Won, You-Sub; Lee, HyunJoo; Kim, Bum-Joon

2015-01-01

AIM: To elucidate the molecular mechanisms underlying hepatitis B virus (HBV) occult infection of genotype C. METHODS: A total of 10 types of hepatitis B surface antigen (HBsAg) variants from a Korean occult cohort were used. After a complete HBV genome plasmid mutated such that it does not express HBsAg and plasmid encoding, each HBsAg variant was transiently co-transfected into HuH-7 cells. The secretion capacity and intracellular expression of the HBV virions and HBsAgs in their respective variants were analyzed using real-time quantitative polymerase chain reaction assays and commercial HBsAg enzyme-linked immunosorbent assays, respectively. RESULTS: All variants exhibited lower levels of HBsAg secretion into the medium compared with the wild type. In particular, in eight of the ten variants, very low levels of HBsAg secretion that were similar to the negative control were detected. In contrast, most variants (9/10) exhibited normal virion secretion capacities comparable with, or even higher than, the wild type. This provided new insight into the intrinsic nature of occult HBV infection, which leads to HBsAg sero-negativeness but has horizontal infectivity. Furthermore, most variants generated higher reactive oxidative species production than the wild type. This finding provides potential links between occult HBV infection and liver disease progression. CONCLUSION: The presently obtained data indicate that deficiency in the secretion capacity of HBsAg variants may have a pivotal function in the occult infections of HBV genotype C. PMID:25684944

Introgression from cultivated rice alters genetic structures of wild relative populations: implications for in situ conservation

PubMed Central

Jin, Xin; Chen, Yu; Liu, Ping; Li, Chen; Cai, Xingxing; Rong, Jun

2018-01-01

Abstract Maintaining genetic integrity is essential for in situ and ex situ conservation of crop wild relative (CWR) species. However, introgression of crop alleles into CWR species/populations may change their genetic structure and diversity, resulting in more invasive weeds or, in contrast, the extinction of endangered populations. To determine crop-wild introgression and its consequences, we examined the genetic structure and diversity of six wild rice (Oryza rufipogon) populations under in situ conservation in China. Thirty-four simple sequence repeat (SSR) and 34 insertion/deletion markers were used to genotype the wild rice populations and two sets of rice cultivars (O. sativa), corresponding to the two types of molecular markers. Shared alleles and STRUCTURE analyses suggested a variable level of crop-wild introgression and admixture. Principal coordinates and cluster analyses indicated differentiation of wild rice populations, which was associated with the spatial distances to cultivated rice fields. The level of overall genetic diversity was comparable between wild rice populations and rice cultivars, but a great number of wild-specific alleles was detected in the wild populations. We conclude based on the results that crop-wild introgression can considerably alter the pattern of genetic structure and relationships of CWR populations. Appropriate measures should be taken for effective in situ conservation of CWR species under the scenario of crop-wild introgression. PMID:29308123

Introgression from cultivated rice alters genetic structures of wild relative populations: implications for in situ conservation.

PubMed

Jin, Xin; Chen, Yu; Liu, Ping; Li, Chen; Cai, Xingxing; Rong, Jun; Lu, Bao-Rong

2018-02-01

Maintaining genetic integrity is essential for in situ and ex situ conservation of crop wild relative (CWR) species. However, introgression of crop alleles into CWR species/populations may change their genetic structure and diversity, resulting in more invasive weeds or, in contrast, the extinction of endangered populations. To determine crop-wild introgression and its consequences, we examined the genetic structure and diversity of six wild rice ( Oryza rufipogon ) populations under in situ conservation in China. Thirty-four simple sequence repeat (SSR) and 34 insertion/deletion markers were used to genotype the wild rice populations and two sets of rice cultivars ( O. sativa ), corresponding to the two types of molecular markers. Shared alleles and STRUCTURE analyses suggested a variable level of crop-wild introgression and admixture. Principal coordinates and cluster analyses indicated differentiation of wild rice populations, which was associated with the spatial distances to cultivated rice fields. The level of overall genetic diversity was comparable between wild rice populations and rice cultivars, but a great number of wild-specific alleles was detected in the wild populations. We conclude based on the results that crop-wild introgression can considerably alter the pattern of genetic structure and relationships of CWR populations. Appropriate measures should be taken for effective in situ conservation of CWR species under the scenario of crop-wild introgression.

Cellobiohydrolase I enzymes

DOEpatents

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.

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

Hfe Deficiency Impairs Pulmonary Neutrophil Recruitment in Response to Inflammation

PubMed Central

Benesova, Karolina; Vujić Spasić, Maja; Schaefer, Sebastian M.; Stolte, Jens; Baehr-Ivacevic, Tomi; Waldow, Katharina; Zhou, Zhe; Klingmueller, Ursula; Benes, Vladimir; Mall, Marcus A.; Muckenthaler, Martina U.

2012-01-01

Regulation of iron homeostasis and the inflammatory response are tightly linked to protect the host from infection. Here we investigate how imbalanced systemic iron homeostasis in a murine disease model of hereditary hemochromatosis (Hfe−/− mice) affects the inflammatory responses of the lung. We induced acute pulmonary inflammation in Hfe−/− and wild-type mice by intratracheal instillation of 20 µg of lipopolysaccharide (LPS) and analyzed local and systemic inflammatory responses and iron-related parameters. We show that in Hfe−/− mice neutrophil recruitment to the bronchoalveolar space is attenuated compared to wild-type mice although circulating neutrophil numbers in the bloodstream were elevated to similar levels in Hfe−/− and wild-type mice. The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types. As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections. In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis. PMID:22745741

Transplantation of bone marrow-derived mesenchymal stem cells rescues partially rachitic phenotypes induced by 1,25-Dihydroxyvitamin D deficiency in mice

PubMed Central

Zhang, Zengli; Yin, Shaomeng; Xue, Xian; Ji, Ji; Tong, Jian; Goltzman, David; Miao, Dengshun

2016-01-01

To determine whether the transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) can improve the 1,25(OH)2D deficiency-induced rachitic phenotype, 2×106 BM-MSCs from wild-type mice or vehicle were transplanted by tail vein injection into mice deficient in 1,25(OH)2D due to targeted deletion of 1α(OH)ase (1α(OH)ase-/-). Our results show that 1α(OH)ase mRNA was expressed in the BM-MSCs derived from wild-type mice, and was detected in long bone, kidney and intestine from BM-MSC-transplanted 1α(OH)ase-/- recipients. Serum calcium, 1,25(OH)2D3 levels and body weight were significantly increased in BM-MSC-transplanted 1α(OH)ase-/- recipients compared to vehicle-treated 1α(OH)ase-/- mice. Skeletal mineralization improved in 1α(OH)ase-/- recipients as demonstrated by BMD measurement, micro-CT analysis and von Kossa staining of undecalcified sections. Expression levels of type I collagen, osteocalcin, bone sialoprotein and vitronectin and the size of calcified nodules were decreased in BM-MSC cultures from 1α(OH)ase-/- mice compared with those from wild-type mice, however, these parameters were increased in those from BM-MSCs-transplanted 1α(OH)ase-/- recipients compared with those from vehicle-treated 1α(OH)ase-/- mice. This study indicates that donor BM-MSCs cells can relocate to multiple tissues where they synthesize 1α(OH)ase and produce 1,25(OH)2D that contributes to the improvement of serum calcium and skeletal mineralization. Results from this study suggest that BM-MSC transplantation may provide a therapeutic approach to treatment of pseudovitamin D-deficiency rickets. PMID:27830022

Transplantation of bone marrow-derived mesenchymal stem cells rescues partially rachitic phenotypes induced by 1,25-Dihydroxyvitamin D deficiency in mice.

PubMed

Zhang, Zengli; Yin, Shaomeng; Xue, Xian; Ji, Ji; Tong, Jian; Goltzman, David; Miao, Dengshun

2016-01-01

To determine whether the transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) can improve the 1,25(OH) 2 D deficiency-induced rachitic phenotype, 2×10 6 BM-MSCs from wild-type mice or vehicle were transplanted by tail vein injection into mice deficient in 1,25(OH) 2 D due to targeted deletion of 1α(OH)ase (1α(OH)ase -/- ). Our results show that 1α(OH)ase mRNA was expressed in the BM-MSCs derived from wild-type mice, and was detected in long bone, kidney and intestine from BM-MSC-transplanted 1α(OH)ase -/- recipients. Serum calcium, 1,25(OH) 2 D 3 levels and body weight were significantly increased in BM-MSC-transplanted 1α(OH)ase -/- recipients compared to vehicle-treated 1α(OH)ase -/- mice. Skeletal mineralization improved in 1α(OH)ase -/- recipients as demonstrated by BMD measurement, micro-CT analysis and von Kossa staining of undecalcified sections. Expression levels of type I collagen, osteocalcin, bone sialoprotein and vitronectin and the size of calcified nodules were decreased in BM-MSC cultures from 1α(OH)ase -/- mice compared with those from wild-type mice, however, these parameters were increased in those from BM-MSCs-transplanted 1α(OH)ase -/- recipients compared with those from vehicle-treated 1α(OH)ase -/- mice. This study indicates that donor BM-MSCs cells can relocate to multiple tissues where they synthesize 1α(OH)ase and produce 1,25(OH) 2 D that contributes to the improvement of serum calcium and skeletal mineralization. Results from this study suggest that BM-MSC transplantation may provide a therapeutic approach to treatment of pseudovitamin D-deficiency rickets.

Whole-exome sequencing in a single proband reveals a mutation in the CHST8 gene in autosomal recessive peeling skin syndrome

PubMed Central

Cabral, Rita M.; Kurban, Mazen; Wajid, Muhammad; Shimomura, Yutaka; Petukhova, Lynn; Christiano, Angela M.

2015-01-01

Generalized peeling skin syndrome (PSS) is an autosomal recessive genodermatosis characterized by lifelong, continuous shedding of the upper epidermis. Using whole-genome homozygozity mapping and whole-exome sequencing, we identified a novel homozygous missense mutation (c.229C>T, R77W) within the CHST8 gene, in a large consanguineous family with non-inflammatory PSS type A. CHST8 encodes a Golgi transmembrane N-acetylgalactosamine-4-O-sulfotransferase (GalNAc4-ST1), which we show by immunofluorescence staining to be expressed throughout normal epidermis. A colorimetric assay for total sulfated glycosaminoglycan (GAG) quantification, comparing human keratinocytes (CCD1106 KERTr) expressing wild type and mutant recombinant GalNAc4-ST1, revealed decreased levels of total sulfated GAGs in cells expressing mutant GalNAc4-ST1, suggesting loss of function. Western blotting revealed lower expression levels of mutant recombinant GalNAc4-ST1 compared to wild type, suggesting that accelerated degradation may result in loss of function, leading to PSS type A. This is the first report describing a mutation as the cause of PSS type A. PMID:22289416

Whole-exome sequencing in a single proband reveals a mutation in the CHST8 gene in autosomal recessive peeling skin syndrome.

PubMed

Cabral, Rita M; Kurban, Mazen; Wajid, Muhammad; Shimomura, Yutaka; Petukhova, Lynn; Christiano, Angela M

2012-04-01

Generalized peeling skin syndrome (PSS) is an autosomal recessive genodermatosis characterized by lifelong, continuous shedding of the upper epidermis. Using whole-genome homozygozity mapping and whole-exome sequencing, we identified a novel homozygous missense mutation (c.229C>T, R77W) within the CHST8 gene, in a large consanguineous family with non-inflammatory PSS type A. CHST8 encodes a Golgi transmembrane N-acetylgalactosamine-4-O-sulfotransferase (GalNAc4-ST1), which we show by immunofluorescence staining to be expressed throughout normal epidermis. A colorimetric assay for total sulfated glycosaminoglycan (GAG) quantification, comparing human keratinocytes (CCD1106 KERTr) expressing wild type and mutant recombinant GalNAc4-ST1, revealed decreased levels of total sulfated GAGs in cells expressing mutant GalNAc4-ST1, suggesting loss of function. Western blotting revealed lower expression levels of mutant recombinant GalNAc4-ST1 compared to wild type, suggesting that accelerated degradation may result in loss of function, leading to PSS type A. This is the first report describing a mutation as the cause of PSS type A. Copyright © 2012 Elsevier Inc. All rights reserved.

Ultrasonic vocalizations of adult male Foxp2-mutant mice: behavioral contexts of arousal and emotion.

PubMed

Gaub, S; Fisher, S E; Ehret, G

2016-02-01

Adult mouse ultrasonic vocalizations (USVs) occur in multiple behavioral and stimulus contexts associated with various levels of arousal, emotion and social interaction. Here, in three experiments of increasing stimulus intensity (water; female urine; male interacting with adult female), we tested the hypothesis that USVs of adult males express the strength of arousal and emotion via different USV parameters (18 parameters analyzed). Furthermore, we analyzed two mouse lines with heterozygous Foxp2 mutations (R552H missense, S321X nonsense), known to produce severe speech and language disorders in humans. These experiments allowed us to test whether intact Foxp2 function is necessary for developing full adult USV repertoires, and whether mutations of this gene influence instinctive vocal expressions based on arousal and emotion. The results suggest that USV calling rate characterizes the arousal level, while sound pressure and spectrotemporal call complexity (overtones/harmonics, type of frequency jumps) may provide indices of levels of positive emotion. The presence of Foxp2 mutations did not qualitatively affect the USVs; all USV types that were found in wild-type animals also occurred in heterozygous mutants. However, mice with Foxp2 mutations displayed quantitative differences in USVs as compared to wild-types, and these changes were context dependent. Compared to wild-type animals, heterozygous mutants emitted mainly longer and louder USVs at higher minimum frequencies with a higher occurrence rate of overtones/harmonics and complex frequency jump types. We discuss possible hypotheses about Foxp2 influence on emotional vocal expressions, which can be investigated in future experiments using selective knockdown of Foxp2 in specific brain circuits. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

A new strategy for strain improvement of Aurantiochytrium sp. based on heavy-ions mutagenesis and synergistic effects of cold stress and inhibitors of enoyl-ACP reductase.

PubMed

Cheng, Yu-Rong; Sun, Zhi-Jie; Cui, Gu-Zhen; Song, Xiaojin; Cui, Qiu

2016-11-01

Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp. strains with heavy ion mutagenesis and pre-selection by synergistic usage of cold stress (4°C) and FAS inhibitors (triclosan and isoniazid). Results showed that (1) isoniazid and triclosan have positive effects on improving DHA level of cells; (2) mutants from irradiation dosage of 120Gy yielded more DHA compared with cells from 40Gy, 80Gy treatment and wild type; (3) DHA contents of mutants pre-selected by inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan)at 4°C, were significantly higher than that of wild type; (4) compared to the wild type, the DHA productivity and yield of a mutant (T-99) obtained from Aurantiochytrium sp. CGMCC 6208 by the proposed method increased by 50% from 0.18 to 0.27g/Lh and 30% from 21 to 27g/L, respectively. In conclusion, this study developed a feasible method to screen Aurantiochytrium sp. with high DHA yield by a combination of heavy-ion mutagenesis and mutant-preselection by FAS inhibitors and cold stress. Copyright © 2016 Elsevier Inc. All rights reserved.

AMP deaminase 3 deficiency enhanced 5'-AMP induction of hypometabolism.

PubMed

Daniels, Isadora Susan; O Brien, William G; Nath, Vinay; Zhao, Zhaoyang; Lee, Cheng Chi

2013-01-01

A hypometabolic state can be induced in mice by 5'-AMP administration. Previously we proposed that an underlying mechanism for this hypometabolism is linked to reduced erythrocyte oxygen transport function due to 5'-AMP uptake altering the cellular adenylate equilibrium. To test this hypothesis, we generated mice deficient in adenosine monophosphate deaminase 3 (AMPD3), the key catabolic enzyme for 5'-AMP in erythrocytes. Mice deficient in AMPD3 maintained AMPD activities in all tissues except erythrocytes. Developmentally and morphologically, the Ampd3(-/-) mice were indistinguishable from their wild type siblings. The levels of ATP, ADP but not 5'-AMP in erythrocytes of Ampd3(-/-) mice were significantly elevated. Fasting blood glucose levels of the Ampd3(-/-) mice were comparable to wild type siblings. In comparison to wild type mice, the Ampd3(-/-) mice displayed a deeper hypometabolism with a significantly delayed average arousal time in response to 5'-AMP administration. Together, these findings demonstrate a central role of AMPD3 in the regulation of 5'-AMP mediated hypometabolism and further implicate erythrocytes in this behavioral response.

Role of Proteus mirabilis MR/P fimbriae and flagella in adhesion, cytotoxicity and genotoxicity induction in T24 and Vero cells.

PubMed

Scavone, Paola; Villar, Silvia; Umpiérrez, Ana; Zunino, Pablo

2015-06-01

Proteus mirabilis is frequently associated with complicated urinary tract infections (UTI). It is proposed that several virulence factors are associated with P. mirabilis uropathogenicity. The aim of this work was to elucidate genotoxic and cytotoxic effects mediated by MR/P fimbriae and flagella in eukaryotic cells in vitro. Two cell lines (kidney- and bladder-derived) were infected with a clinical wild-type P. mirabilis strain and an MR/P and a flagellar mutant. We evaluated adhesion, genotoxicity and cytotoxicity by microscopy, comet assay and triple staining technique, respectively. Mutant strains displayed lower adhesion rates than the P. mirabilis wild-type strain and were significantly less effective to induce genotoxic and cytotoxic effects compared to the wild type. We report for the first time that P. mirabilis MR/P fimbriae and flagella mediate genotoxic and cytotoxic effects on eukaryotic cells, at least in in vitro conditions. These results could contribute to design new strategies for the control of UTI. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Thioredoxin reverses age-related hypertension by chronically improving vascular redox and restoring eNOS function

PubMed Central

Hilgers, Rob H. P.; Kundumani-Sridharan, Venkatesh; Subramani, Jaganathan; Chen, Leon C.; Cuello, Luis G.; Rusch, Nancy J.; Das, Kumuda C.

2017-01-01

The incidence of high blood pressure with advancing age is notably high, and it is an independent prognostic factor for the onset or progression of a variety of cardiovascular disorders. Although age-related hypertension is an established phenomenon, current treatments are only palliative but not curative. Thus, there is a critical need for a curative therapy against age-related hypertension, which could greatly decrease the incidence of cardiovascular disorders. We show that overexpression of human thioredoxin (TRX), a redox protein, in mice prevents age-related hypertension. Further, injection of recombinant human TRX (rhTRX) for three consecutive days reversed hypertension in aged wild-type mice, and this effect lasted for at least 20 days. Arteries of wild-type mice injected with rhTRX or mice with TRX overexpression exhibited decreased arterial stiffness, greater endothelium-dependent relaxation, increased nitric oxide production, and decreased superoxide anion (O2•−) generation compared to either saline-injected aged wild-type mice or mice with TRX deficiency. Our study demonstrates a potential translational role of rhTRX in reversing age-related hypertension with long-lasting efficacy. PMID:28179506

The global regulator CodY is required for the fitness of Bacillus cereus in various laboratory media and certain beverages.

PubMed

Kovács, Ákos T

2016-07-01

The impact of gene mutations on the growth of the cells can be studied using pure cultures. However, the importance of certain proteins and pathways can be also examined via co-culturing wild type and its mutant derivative. Here, the relative fitness of a mutant strain that lacks the global nitrogen regulator, CodY, was examined in Bacillus cereus, a food poisoning Gram-positive bacterium. Fitness measurements revealed that the ΔcodY strain was outcompeted when cocultured with the wild-type ATCC 14579 under various rich laboratory medium, and also when inoculated in certain beverages. In nutrient-poor minimal medium, the ΔcodY mutant had comparable fitness to the wild-type strain. Interestingly, the relative fitness of the ΔcodY strain was antagonistic when it was cultivated in apple or orange juices due to unknown properties of these beverages, highlighting the importance of chemical composition of the test medium during the bacterial fitness measurements. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Reduced Infectivity in Cattle for an Outer Membrane Protein Mutant of Anaplasma marginale

PubMed Central

Brayton, Kelly A.; Magunda, Forgivemore; Munderloh, Ulrike G.; Kelley, Karen L.; Barbet, Anthony F.

2015-01-01

Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10::himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species. PMID:25595772

A mutant of barley lacking NADH-hydroxypyruvate reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blackwell, R.; Lea, P.

1989-04-01

A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used tomore » show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.« less

Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection

PubMed Central

Kumar, Meera Ajeet; Christensen, Kendra; Woods, Benjamin; Dettlaff, Ashley; Perley, Danielle; Scheidegger, Adam; Balakrishnan, Lata; Milavetz, Barry

2017-01-01

The location of nucleosomes in SV40 virions and minichromosomes isolated during infection were determined by next generation sequencing (NGS). The patterns of reads within the regulatory region of chromatin from wild-type virions indicated that micrococcal nuclease-resistant nucleosomes were specifically positioned at nt 5223 and nt 363, while in minichromosomes isolated 48 h post-infection we observed nuclease-resistant nucleosomes at nt 5119 and nt 212. The nucleosomes at nt 5223 and nt 363 in virion chromatin would be expected to repress early and late transcription, respectively. In virions from the mutant cs1085, which does not repress early transcription, we found that these two nucleosomes were significantly reduced compared to wild-type virions confirming a repressive role for them. In chromatin from cells infected for only 30 min with wild-type virus, we observed a significant reduction in the nucleosomes at nt 5223 and nt 363 indicating that the potential repression by these nucleosomes appeared to be relieved very early in infection. PMID:28126638

Hepatitis B "e" antigen-mediated inhibition of HBV replication fitness and transcription efficiency in vitro.

PubMed

Samal, Jasmine; Kandpal, Manish; Vivekanandan, Perumal

2015-10-01

A mutation at nucleotide 1896 (G1896A) is the most common cause for the loss of HBeAg. In contrast to clinical data, cell culture studies report a high-replicating phenotype for the G1896A mutant. Differences between the wild-type and the G1896A mutant in early steps of HBV replication including the synthesis of pre-genomic RNA and transcripts have not been investigated. The G1896A mutant is associated with higher replication fitness, transcription efficiency and higher levels of secreted HBsAg than the wild-type. Interestingly, trans-complementation of the G1896A mutant with HBeAg lowers the replication fitness and transcriptionefficiency to levels comparable to that of the wild-type. Our results highlight the role of HBeAg in modulating the early steps in HBV replication. In sum, our findings highlight the role of HBeAg in regulating hepatitis B virus replication fitness and transcription efficiency and new insights on the early steps of replication in the G1896A mutant. Copyright © 2015 Elsevier Inc. All rights reserved.

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

PubMed

Sanz Sanz, Arturo; Niranjan, Yashavanthi; Hammarén, Henrik; Ungureanu, Daniela; Ruijtenbeek, Rob; Touw, Ivo P; Silvennoinen, Olli; Hilhorst, Riet

2014-10-01

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP. Copyright © 2014 Elsevier B.V. All rights reserved.

Paraoxonase 1 192 and 55 polymorphisms in osteosarcoma.

PubMed

Ergen, Arzu; Kılıcoglu, Onder; Ozger, Harzem; Agachan, Bedia; Isbir, Turgay

2011-08-01

Paraoxonase is an HDL-associated enzyme that plays a preventive role against oxidative stres. Previous studies suggested that involved an amino acid substitution at position 192 gives rise to two alloenzymes with a low activity (Q allele) and a high activity (R allele) towards paraoxon. There also exists a second polymorphism of the human PON1 gene affecting amino acid 55, giving rise to a leucine (L-allele) substitution for methionine (M-allele). PON1 gene polymorphisms were studied in 50 patients with osteosarcoma and 50 healthy controls. Paraoxonase genotypes were determined by PCR-RFLP. We found a reduction in the frequency of PON1 192 R allele in patients (P=0.015). Besides, PON1 192 wild type QQ genotype (P=0.015) and PON1 55 wild type L allele (P=0.001) were higher in patients compared to healthy controls. PON1 192 QQ genotype was associated with osteosarcoma in multivariate logistic regression analysis. Our findings have suggested that PON1 192 wild type genotypes may be associated with a risk of developing osteosarcoma.

Wild corvid birds colonized with vancomycin-resistant Enterococcus faecium of human origin harbor epidemic vanA plasmids.

PubMed

Oravcová, Veronika; Peixe, Luísa; Coque, Teresa M; Novais, Carla; Francia, Maria V; Literák, Ivan; Freitas, Ana R

2018-06-02

The most prevalent type of acquired vancomycin resistance in Enterococcus faecium (VREfm) is encoded by the vanA transposon Tn1546, mainly located on transferable plasmids. vanA plasmids have been characterized in VREfm from a variety of sources but not wild birds. The aim of this study was to analyse the genetic context of VREfm strains recovered from wild corvid birds and to compare their plasmid and strain characteristics with human strains. To achieve that, 75 VREfm isolates, including strains from wild birds recovered during wide surveillance studies performed in Europe, Canada and the United States (2010-2013), and clinical and wastewater strains from Czech Republic, a region lacking data about vanA plasmids, were analysed. Their population structure, presence of major putative virulence markers and characterization of vanA transposons and plasmids were established. VREfm from wild birds were mainly associated with major human lineages (ST18 and ST78) circulating in hospitals worldwide and were enriched in putative virulence markers that are highly associated with clinical E. faecium from human infections. They also carried plasmids of the same families usually found in the clinical setting [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18]. The clinically widespread IS1251-carrying Tn1546 type "F" was predominant and Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18- or pLG1-like (Europe) plasmids. VREfm from hospitals and wastewaters carried Tn1546-vanA in different plasmid types including mosaic pRUM-Inc18 plasmids, not identified in wild birds. This is the first characterization of vanA plasmids obtained from wild birds. A similar plasmid pool seems to exist in different clonal E. faecium backgrounds of humans and wild birds. The isolation of VREfm strains from wild birds that belong to human E. faecium adapted lineages and carry virulence genes, Tn1546 and plasmid variants widespread in the clinical setting is of concern and highlight their role as potential drivers of the global dissemination of vancomycin resistance. Copyright © 2018 Elsevier Ltd. All rights reserved.