Identification and expression analysis of peroxisome proliferator-activated receptors cDNA in a reptile, the leopard gecko (Eublepharis macularius).

PubMed

Kato, Keisuke; Oka, Yoshitaka; Park, Min Kyun

2008-05-01

Despite the physiological and evolutionary significance of lipid metabolism in amniotes, the molecular mechanisms involved have been unclear in reptiles. To elucidate this, we investigated peroxisome proliferators-activated receptors (PPARs) in the leopard gecko (Eublepharis macularius). PPARs belong to a nuclear hormone-receptor family mainly involved in lipid metabolism. Although PPARs have been widely studied in mammals, little information about them is yet available from reptiles. We identified in the leopard gecko partial cDNA sequences of PPARalpha and beta, and full sequences of two isoforms of PPARgamma. This is the first report of reptilian PPARgamma mRNA isoforms. We also evaluated the organ distribution of expression of these genes by using RT-PCR and competitive PCR. The expression level of PPARalpha mRNA was highest in the large intestine, and moderate in the liver and kidney. The expression level of PPARbeta mRNA was highest in the kidney and large intestine, and moderate in the liver. Similarly to the expression of human PPARgamma isoforms, PPARgammaa was expressed ubiquitously, whereas the expression of PPARgammab was restricted. The highest levels of their expression, however, were observed in the large intestine, rather than in the adipose tissue as in mammals. Taken together, these results showed that the profile of PPARbeta mRNA expression in the leopard gecko is similar to that in mammals, and that those of PPAR alpha and gamma are species specific. This may reflect adaptation to annual changes in lipid storage due to seasonal food availability.

Paraoxonase 2 (PON2) in the mouse central nervous system: A neuroprotective role?

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

Giordano, Gennaro; Cole, Toby B.; Dept. of Medicine

2011-11-15

The aims of this study were to characterize the expression of paraoxonase 2 (PON2) in mouse brain and to assess its antioxidant properties. PON2 levels were highest in the lung, intestine, heart and liver, and lower in the brain; in all tissues, PON2 expression was higher in female than in male mice. PON2 knockout [PON2{sup -/-}] mice did not express any PON2, as expected. In the brain, the highest levels of PON2 were found in the substantia nigra, the nucleus accumbens and the striatum, with lower levels in the cerebral cortex, hippocampus, cerebellum and brainstem. A similar regional distribution ofmore » PON2 activity (measured by dihydrocoumarin hydrolysis) was also found. PON3 was not detected in any brain area, while PON1 was expressed at very low levels, and did not show any regional difference. PON2 levels were higher in astrocytes than in neurons isolated from all brain regions, and were highest in cells from the striatum. PON2 activity and mRNA levels followed a similar pattern. Brain PON2 levels were highest around birth, and gradually declined. Subcellular distribution experiments indicated that PON2 is primarily expressed in microsomes and in mitochondria. The toxicity in neurons and astrocytes of agents known to cause oxidative stress (DMNQ and H{sub 2}O{sub 2}) was higher in cells from PON2{sup -/-} mice than in the same cells from wild-type mice, despite similar glutathione levels. These results indicate that PON2 is expressed in the brain, and that higher levels are found in dopaminergic regions such as the striatum, suggesting that this enzyme may provide protection against oxidative stress-mediated neurotoxicity.« less

Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

PubMed

Liu, Jing; Xiao, Zhijie; Wong, Sunny Kit-Man; Tin, Vicky Pui-Chi; Ho, Ka-Yan; Wang, Junwen; Sham, Mai-Har; Wong, Maria Pik

2013-10-01

Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

Bayesian Inference of Allele-Specific Gene Expression Indicates Abundant Cis-Regulatory Variation in Natural Flycatcher Populations

PubMed Central

Wang, Mi

2017-01-01

Abstract Polymorphism in cis-regulatory sequences can lead to different levels of expression for the two alleles of a gene, providing a starting point for the evolution of gene expression. Little is known about the genome-wide abundance of genetic variation in gene regulation in natural populations but analysis of allele-specific expression (ASE) provides a means for investigating such variation. We performed RNA-seq of multiple tissues from population samples of two closely related flycatcher species and developed a Bayesian algorithm that maximizes data usage by borrowing information from the whole data set and combines several SNPs per transcript to detect ASE. Of 2,576 transcripts analyzed in collared flycatcher, ASE was detected in 185 (7.2%) and a similar frequency was seen in the pied flycatcher. Transcripts with statistically significant ASE commonly showed the major allele in >90% of the reads, reflecting that power was highest when expression was heavily biased toward one of the alleles. This would suggest that the observed frequencies of ASE likely are underestimates. The proportion of ASE transcripts varied among tissues, being lowest in testis and highest in muscle. Individuals often showed ASE of particular transcripts in more than one tissue (73.4%), consistent with a genetic basis for regulation of gene expression. The results suggest that genetic variation in regulatory sequences commonly affects gene expression in natural populations and that it provides a seedbed for phenotypic evolution via divergence in gene expression. PMID:28453623

p53 and PCNA Expression in Keratocystic Odontogenic Tumors Compared with Selected Odontogenic Cysts

PubMed Central

Seyedmajidi, Maryam; Nafarzadeh, Shima; Siadati, Sepideh; Shafaee, Shahryar; Bijani, Ali; Keshmiri, Nazanin

2013-01-01

p53 and PCNA expression in keratocystic odontogenic tumors compared with selected odontogenic cysts Summary: The aim of this study was to evaluate p53 and PCNA expression in different odontogenic lesions regarding their different clinical behaviors. Slices prepared from 94 paraffin-embedded tissue blocks (25 radicular cysts (RC), 23 dentigerous cysts (DC), 23 keratocystic odontogenic tumors (KCOT) and 23 calcifying cystic odontogenic tumors (CCOT)) were stained with p53 and PCNA antibodies using immunohistochemistry procedure. The highest level of p53 expression was in the basal layer of RC, and the highest level of PCNA expression was in the suprabasal layer of KCOT. The differences of p53 expression in basal and suprabasal layers as well as PCNA expression in the suprabasal layer were significant but there was no significant difference in PCNA expression in the basal layer of these lesions. The expression of p53 in the basal layer of RC was higher than in other cysts. This may be due to intensive inflammatory infiltration. Also, the high level of PCNA expression in the suprabasal layer of KCOT may justify its neoplastic nature and tendency to recurrence. KCOT and calcifying cystic odontogenic tumors did not show similar expression of studied biomarkers. PMID:24551811

Collagenase IV plays an important role in regulating hair cycle by inducing VEGF, IGF-1, and TGF-β expression

PubMed Central

Hou, Chun; Miao, Yong; Wang, Jin; Wang, Xue; Chen, Chao-Yue; Hu, Zhi-Qi

2015-01-01

Background It has been reported that collagenases (matrix metalloproteinase 2 [MMP-2] and matrix metalloproteinase 9 [MMP-9]) are associated with hair cycle, whereas the mechanism of the association is largely unknown. Methods The mice were randomly allocated into four groups: saline, and 5, 10, and 15 nM SB-3CT. Immunohistochemical analysis was employed to examine MMP-2 and MMP-9 protein. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay were performed to determine mRNA and protein levels of VEGF, IGF-1, TGF-β, and GAPDH. Growing hair follicles from anagen phase III–IV were scored based on hematoxylin and eosin staining. Hair regrowth was also evaluated. Results Results showed that mRNA expressions of enzymes changed with a peak at late anagen and a trough at telogen after depilation. Immunostaining showed that the highest expression of MMP-2 was more than that of MMP-9, and the highest expression of enzymes changed during anagen. The localizations of MMP-2 changed from dermal papilla, keratinocyte strand, out of root sheath, and basal plate at early anagen, to hair bulb, inner root sheath, and outer root sheath at late anagen. The localization of MMP-9 changed from partial keratinocyte to dermal papilla at early anagen and to outer root sheath at late anagen. VEGF, IGF-1, and TGF-β have been shown to regulate hair growth. We found mRNA and protein expressions of VEGF and IGF-1 fluctuated with a peak at anagen and a decrease at catagen to telogen. In contrast, mRNA and protein expressions of TGF-β changed with highest and lowest levels at anagen and telogen, respectively. With selective inhibitor of collagenase IV, SB-3CT, mice showed significant suppressed hair growth and decreased expression of VEGF, IGF-1, and TGF-β. The MMPs agonist also significantly increased expression of VEGF, IGF-1, and TGF-β. Meanwhile, SB-3CT treatment significantly suppressed hair growth. Conclusion All these data suggest that the type IV collagenases, MMP-2 and MMP-9, play important roles in hair cycle, and this could be mediated by induced expression of VEGF, IGF-1, and TGF-β. PMID:26451090

Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b.

PubMed

Zhang, Tingting; Zhou, Jiti; Wang, Xiaowei; Zhang, Yu

2017-02-01

The coupled effects of nitrogen source and methane monooxygenase (MMO) on the growth and poly-β-hydroxybutyrate (PHB) accumulation capacity of methanotrophs were explored. The ammonia-supplied methanotrophs expressing soluble MMO (sMMO) grew at the highest rate, while N 2 -fixing bacteria expressing particulate MMO (pMMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing pMMO, which might cause their slightly lower growth rate. The highest PHB content (51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing pMMO. Ammonia-supplied bacteria also accumulated a higher content of PHB (45.2%) with the expression of pMMO, while N 2 -fixing bacteria containing pMMO only showed low PHB production capacity (32.1%). The maximal PHB contents of bacteria expressing sMMO were low, with no significant change under different nitrogen source conditions. The low MMO activity, low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N 2 with the expression of pMMO were greatly improved in the cyclic NO 3 - N 2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO. Copyright © 2016. Published by Elsevier B.V.

Transformation of Solanum tuberosum plastids allows high expression levels of β-glucuronidase both in leaves and microtubers developed in vitro.

PubMed

Segretin, María Eugenia; Lentz, Ezequiel Matías; Wirth, Sonia Alejandra; Morgenfeld, Mauro Miguel; Bravo-Almonacid, Fernando Félix

2012-04-01

Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of β-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of β-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on β-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8 days increased β-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.

RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

PubMed

Singh, Chabungbam Orville; Xin, Hu-hu; Chen, Rui-ting; Wang, Mei-xian; Liang, Shuang; Lu, Yan; Cai, Zi-zheng; Zhang, Deng-pan; Miao, Yun-gen

2015-06-01

Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L. © 2015 Wiley Periodicals, Inc.

Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle

PubMed Central

Nagler, James J.; Cavileer, Timothy D.; Verducci, Joseph S.; Schultz, Irvin R.; Hook, Sharon E.; Hayton, William L.

2012-01-01

Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (β), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erβ1, erβ2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17β, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erβ1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17β levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too. PMID:22732076

EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing’s Sarcoma

PubMed Central

Hossain, Mohammad Motarab; Ray, Swapan Kumar

2016-01-01

Ewing’s sarcoma is a pediatric tumor that mainly occurs in soft tissues and bones. Malignant characteristics of Ewing’s sarcoma are correlated with expression of EWS oncogene. We achieved knockdown of EWS expression using a plasmid vector encoding EWS short hairpin RNA (shRNA) to increase anti-tumor mechanisms of taxifolin (TFL), a new flavonoid, in human Ewing’s sarcoma cells in culture and animal models. Immunofluorescence microscopy and flow cytometric analysis showed high expression of EWS in human Ewing’s sarcoma SK-N-MC and RD-ES cell lines. EWS shRNA plus TFL inhibited 80% cell viability and caused the highest decreases in EWS expression at mRNA and protein levels in both cell lines. Knockdown of EWS expression induced morphological features of differentiation. EWS shRNA plus TFL caused more alterations in molecular markers of differentiation than either agent alone. EWS shRNA plus TFL caused the highest decreases in cell migration with inhibition of survival, angiogenic and invasive factors. Knockdown of EWS expression was associated with removal of DNA methylation from p53 promoter, promoting expression of p53, Puma, and Noxa. EWS shRNA plus TFL induced the highest amounts of apoptosis with activation of extrinsic and intrinsic pathways in both cell lines in culture. EWS shRNA plus TFL also inhibited growth of Ewing’s sarcoma tumors in animal models due to inhibition of differentiation inhibitors and angiogenic and invasive factors and also induction of activation of caspase-3 for apoptosis. Collectively, knockdown of EWS expression increased various anti-tumor mechanisms of TFL in human Ewing’s sarcoma in cell culture and animal models. PMID:27547487

EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing's Sarcoma.

PubMed

Hossain, Mohammad Motarab; Ray, Swapan Kumar

2014-10-01

Ewing's sarcoma is a pediatric tumor that mainly occurs in soft tissues and bones. Malignant characteristics of Ewing's sarcoma are correlated with expression of EWS oncogene. We achieved knockdown of EWS expression using a plasmid vector encoding EWS short hairpin RNA (shRNA) to increase anti-tumor mechanisms of taxifolin (TFL), a new flavonoid, in human Ewing's sarcoma cells in culture and animal models. Immunofluorescence microscopy and flow cytometric analysis showed high expression of EWS in human Ewing's sarcoma SK-N-MC and RD-ES cell lines. EWS shRNA plus TFL inhibited 80% cell viability and caused the highest decreases in EWS expression at mRNA and protein levels in both cell lines. Knockdown of EWS expression induced morphological features of differentiation. EWS shRNA plus TFL caused more alterations in molecular markers of differentiation than either agent alone. EWS shRNA plus TFL caused the highest decreases in cell migration with inhibition of survival, angiogenic and invasive factors. Knockdown of EWS expression was associated with removal of DNA methylation from p53 promoter, promoting expression of p53, Puma, and Noxa. EWS shRNA plus TFL induced the highest amounts of apoptosis with activation of extrinsic and intrinsic pathways in both cell lines in culture. EWS shRNA plus TFL also inhibited growth of Ewing's sarcoma tumors in animal models due to inhibition of differentiation inhibitors and angiogenic and invasive factors and also induction of activation of caspase-3 for apoptosis. Collectively, knockdown of EWS expression increased various anti-tumor mechanisms of TFL in human Ewing's sarcoma in cell culture and animal models.

In vitro resistance to 5-nitroimidazoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression.

PubMed

Argüello-García, Raúl; Cruz-Soto, Maricela; Romero-Montoya, Lydia; Ortega-Pierres, Guadalupe

2009-12-01

The susceptibility of Giardia duodenalis trophozoites exposed in vitro to sublethal concentrations of metronidazole (MTZ) and albendazole (ABZ) may exhibit inter-culture (variability) and intra-culture (variation) differences in drug susceptibility. It was previously reported that MTZ-resistant trophozoites may display changes in pyruvate:ferredoxin oxidoreductase (PFOR) expression while changes at the beta-tubulin molecule are apparently absent in ABZ-resistant cultures. To assess the levels of gene expression of these molecules, we obtained cloned cultures growing at concentrations up to 23 microM MTZ (WBRM23) and up to 8muM ABZ (WBRA8) and gene sequence and expression of pfor and beta-tubulin loci were compared with these of drug-susceptible clone WB1. Neither the pfor nor the beta-tubulin genes showed changes at sequence level but the MTZ-resistant clones WBRM21 and WBRM23 showed up-regulation of the pfor RNA using the gdh gene as reference. By using WB1 and WBRA8 clones in representational difference analyses of gene expression (RDA) an insert referred to as ARR-VSP was selected and sequenced. It showed the highest homology to one VSP molecule in the Giardia Genome Database (orf GL50803_101765). This isogene was up-regulated in five ABZ-resistant clones and the clone WBRA8 exhibited the highest RNA expression level. When successive progenies of clones WB1, WBRM23 and WBRA8 were analyzed in Northern blot assays to detect pfor and ARR-VSP RNAs respectively, the expression patterns showed variation for both genes but it was much lower in the clone WBRA8. These results suggest that G. duodenalis cultures either susceptible or resistant to MTZ and ABZ may display variability and variation at RNA expression levels albeit these were more marked in the MTZ-resistant parasites. These data might have further implications defining major mechanisms involved in drug resistance of Giardia.

Human Endometriosis Tissue Microarray Reveals Site-specific Expression of Estrogen Receptors, Progesterone Receptor, and Ki67.

PubMed

Colón-Caraballo, Mariano; García, Miosotis; Mendoza, Adalberto; Flores, Idhaliz

2018-04-07

Most available therapies for endometriosis are hormone-based and generally broadly used without taking into consideration the ovarian hormone receptor expression status. This contrasts strikingly with the standard of care for other hormone-based conditions such as breast cancer. We therefore aimed to characterize the expression of ovarian steroid hormone receptors for estrogen alpha (ESR1), estrogen beta (ESR2), and progesterone (PGR) in different types of endometriotic lesions and eutopic endometrium from women with endometriosis and controls using a tissue microarray (TMA). Nuclear expression levels of the receptors were analyzed by tissue (ie, ectopic vs. eutopic endometrium) and cell type (ie, glands vs. stroma). Ovarian lesions showed the lowest expression of ESR1 and PGR, and the highest expression of ESR2, whereas the fallopian tube lesions showed high expression of the 3 receptors. Differences among endometria included lower expression of ESR1 and higher expression of ESR2 in stroma of proliferative endometrium from patients versus patients, and a trend towards loss of PGR nuclear positivity in proliferative endometrium from patients. The largest ESR2:ESR1 ratios were observed in ovarian lesions and secretory endometrium. The highest proportion of samples with >10% Ki67 positive nuclei was in glands of fallopian tube (54%) and extrapelvic lesions (75%); 60% of glands of secretory endometrium from patients had >10% Ki67 positivity compared with only 15% in controls. Our results provide a better understanding of endometriosis heterogeneity by revealing lesion type-specific differences and case-by-case variability in the expression of ovarian hormone receptors. This knowledge could potentially predict individual responses to hormone therapies, and set the basis for the application of personalized medicine approaches for women with endometriosis.

Expression of PAT and NPT II proteins during the developmental stages of a genetically modified pepper developed in Korea.

PubMed

Kim, Hyo Jin; Lee, Si Myung; Kim, Jae Kwang; Ryu, Tae Hun; Suh, Seok Cheol; Cho, Hyun Suk

2010-10-27

Estimation of the protein levels introduced in a biotechnology-derived product is conducted as part of an overall safety assessment. An enzyme-linked immunosorbent assay (ELISA) was used to analyze phosphinothricin acetyltransferase (PAT) and neomycin phosphotransferase II (NPT II) protein expression in a genetically modified (GM) pepper plant developed in Korea. PAT and NPT II expression levels, based on both dry weight and fresh weight, were variable among different plant generations and plant sections from isolated genetically modified organism (GMO) fields at four developmental stages. PAT expression was highest in leaves at anthesis (11.44 μg/gdw and 2.17 μg/gfw) and lowest in roots (0.12 μg/gdw and 0.01 μg/gfw). NPT II expression was also highest in leaves at anthesis (17.31 μg/gdw and 3.41 μg/gfw) and lowest in red pepper (0.65 μg/gdw and 0.12 μg/gfw). In pollen, PAT expression was 0.59-0.62 μg/gdw, while NPT II was not detected. Both PAT and NPT II showed a general pattern of decreased expression with progression of the growing season. As expected, PAT and NPT II protein expression was not detectable in control pepper plants.

Differences of Cd uptake and expression of OAS and IRT genes in two varieties of ryegrasses.

PubMed

Chi, Sunlin; Qin, Yuli; Xu, Weihong; Chai, Yourong; Feng, Deyu; Li, Yanhua; Li, Tao; Yang, Mei; He, Zhangmi

2018-06-16

Pot experiment was conducted to study the difference of cadmium uptake and OAS and IRT genes' expression between the two ryegrass varieties under cadmium stress. The results showed that with the increase of cadmium levels, the dry weights of roots of the two ryegrass varieties, and the dry weights of shoots and plants of Abbott first increased and then decreased. When exposed to 75 mg kg -1 Cd, the dry weights of shoot and plant of Abbott reached the maximum, which increased by 11.13 and 10.67% compared with the control. At 75 mg kg -1 Cd, cadmium concentrations in shoot of the two ryegrass varieties were higher than the critical value of Cd hyperaccumulator (100 mg kg -1 ), 111.19 mg kg -1 (Bond), and 133.69 mg kg -1 (Abbott), respectively. The OAS gene expression in the leaves of the two ryegrass varieties showed a unimodal curve, which was up to the highest at the cadmium level of 150 mg kg -1 , but fell back at high cadmium levels of 300 and 600 mg kg -1 . The OAS gene expression in Bond and Abbott roots showed a bimodal curve. The OAS gene expression in Bond root and Abbott stem mainly showed a unimodal curve. The expression of IRT genes family in the leaves of ryegrass varieties was basically in line with the characteristics of unimodal curve, which was up to the highest at cadmium level of 75 or 150 mg kg -1 , respectively. The IRT expression in the ryegrass stems showed characteristics of bimodal and unimodal curves, while that in the roots was mainly unimodal. The expression of OAS and IRT genes was higher in Bond than that in Abbott due to genotype difference between the two varieties. The expression of OAS and IRT was greater in leaves than that in roots and stems. Ryegrass tolerance to cadmium can be increased by increasing the expression of OAS and IRT genes in roots and stems, and transfer of cadmium from roots and stems to the leaves can be enhanced by increasing expression OAS and IRT in leaves.

Expression characteristics of BMP2, BMPR-IA and Noggin in different stages of hair follicle in yak skin.

PubMed

Song, Liang-Li; Cui, Yan; Yu, Si-Jiu; Liu, Peng-Gang; Liu, Jun; Yang, Xue; He, Jun-Feng; Zhang, Qian

2018-05-01

Bone morphogenetic protein 2 (BMP2), BMP receptor-IA (BMPR-IA), and the BMP2 antagonist Noggin are important proteins involved in regulating the hair follicle (HF) cycle in skin. In order to explore the expression profiles of BMP2, BMPR-IA, and Noggin in the HF cycle of yak skin, we collected adult yak skin in the telogen, proanagen, and midanagen phases of HFs and evaluated gene and protein expression by real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. qRT-PCR and western blotting results showed that BMP2 and BMPR-IA expression levels were highest in the telogen of HFs and higher than that of Noggin in the same phase. The expression of Noggin was significantly higher in proanagen and midanagen phases of HFs than in the telogen phase, with the highest expression observed in the proanagen phase. Moreover, the expression of Noggin in the proanagen phase was significantly higher than those of BMP2 and BMPR-IA during the same phase. Immunohistochemistry results showed that BMP2, BMPR-IA, and Noggin were expressed in the skin epidermis, sweat glands, sebaceous glands, HF outer root sheath, and hair matrix. In summary, the characteristic expression profiles of BMP2, BMPR-IA, and Noggin suggested that BMP2 and BMPR-IA had inhibitory effects on the growth of HFs in yaks, whereas Noggin promoted the growth of yak HFs, mainly by affecting skin epithelial cell activity. These results provide a basis for further studies of HF development and cycle transition in yak skin. Copyright © 2017. Published by Elsevier Inc.

A Novel igf3 Gene in Common Carp (Cyprinus carpio): Evidence for Its Role in Regulating Gonadal Development.

PubMed

Song, Feibiao; Wang, Lanmei; Zhu, Wenbin; Fu, Jianjun; Dong, Juanjuan; Dong, Zaijie

2016-01-01

Since the insulin-like growth factor 3 (igf3) gene was recently discovered in fish ovary, its function in the gonads has received much attention. In this study, we isolated two igf3 subtypes from common carp (Cyprinus carpio), which comprised full-length cDNA of 707 and 1153 nucleotides encoding 205 and 198 amino acids (aa), respectively. The Igf3 aa sequence had the highest gene homology of 72% with the corresponding sequence in zebrafish (Danio rerio). Phylogenetic tree construction revealed that the C. carpio igf3 gene was first clustered with D. rerio and then with other teleost species. Igf3 mRNA was widely expressed, with expression being highest in the gonads and blood. In the gonad development stage, igf3a mRNA expression was highest in the maturity and recession stage of the ovary, and decline phase of the testis, while igf3b was highest in the recession and fully mature periods of the ovaries and testes, respectively. Western blotting of testis protein samples showed two bands of approximately 21 kDa and 34 kDa corresponding to the calculated molecular mass of the two Igf3 subtypes; no signal was detected in the ovary. The Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids. 17β-Ethinylestradiol treatment increased both ovary and testis igf3 mRNA expression. These findings suggest that Igf3 may play an important role in C. carpio gonadal development.

A Novel igf3 Gene in Common Carp (Cyprinus carpio): Evidence for Its Role in Regulating Gonadal Development

PubMed Central

Zhu, Wenbin; Fu, Jianjun; Dong, Juanjuan; Dong, Zaijie

2016-01-01

Since the insulin-like growth factor 3 (igf3) gene was recently discovered in fish ovary, its function in the gonads has received much attention. In this study, we isolated two igf3 subtypes from common carp (Cyprinus carpio), which comprised full-length cDNA of 707 and 1153 nucleotides encoding 205 and 198 amino acids (aa), respectively. The Igf3 aa sequence had the highest gene homology of 72% with the corresponding sequence in zebrafish (Danio rerio). Phylogenetic tree construction revealed that the C. carpio igf3 gene was first clustered with D. rerio and then with other teleost species. Igf3 mRNA was widely expressed, with expression being highest in the gonads and blood. In the gonad development stage, igf3a mRNA expression was highest in the maturity and recession stage of the ovary, and decline phase of the testis, while igf3b was highest in the recession and fully mature periods of the ovaries and testes, respectively. Western blotting of testis protein samples showed two bands of approximately 21 kDa and 34 kDa corresponding to the calculated molecular mass of the two Igf3 subtypes; no signal was detected in the ovary. The Igf3 protein was localized in the ovary granulosa cells and testis spermatogonium and spermatids. 17β-Ethinylestradiol treatment increased both ovary and testis igf3 mRNA expression. These findings suggest that Igf3 may play an important role in C. carpio gonadal development. PMID:28002497

Molecular characterization of Myf5 and comparative expression patterns of myogenic regulatory factors in Siniperca chuatsi.

PubMed

Zhu, Xin; Li, Yu-Long; Liu, Li; Wang, Jian-Hua; Li, Hong-Hui; Wu, Ping; Chu, Wu-Ying; Zhang, Jian-She

2016-01-01

Myogenic regulatory factors (MRFs) are muscle-specific basic helix-loop-helix (bHLH) transcription factor that plays an essential role in regulating skeletal muscle development and growth. To investigate molecular characterization of Myf5 and compare the expressional patterns of the four MRFs, we cloned the Myf5 cDNA sequence and analyzed the MRFs expressional patterns using quantitative real-time polymerase chain reaction in Chinese perch (Siniperca chuatsi). Sequence analysis indicated that Chinese perch Myf5 and other MRFs shared a highly conserved bHLH domain with those of other vertebrates. Sequence alignment and phylogenetic tree showed that Chinese perch MRFs had the highest identity with the MRFs of Epinephelus coioides. Spatio-temporal expressional patterns revealed that the MRFs were primarily expressed in muscle, especially in white muscle. During embryonic development period, Myf5, MyoD and MyoG mRNAs had a steep increase at neurula stage, and their highest expressional level was predominantly observed at hatching period. Whereas the highest expressional level of the MRF4 was observed at the muscular effect stage. The expressional patterns of post-embryonic development showed that the Myf5, MyoD and MyoG mRNAs were highest at 90 days post-hatching (dph). Furthermore, starvation and refeeding results showed that the transcription of the MRFs in the fast skeletal muscle of Chinese perch responded quickly to a single meal after 7 days of fasting. It indicated that the MRFs might contribute to muscle recovery after refeeding in Chinese perch. Copyright © 2015 Elsevier B.V. All rights reserved.

Computational Systems Biology Approach Predicts Regulators and Targets of microRNAs and Their Genomic Hotspots in Apoptosis Process.

PubMed

Alanazi, Ibrahim O; Ebrahimie, Esmaeil

2016-07-01

Novel computational systems biology tools such as common targets analysis, common regulators analysis, pathway discovery, and transcriptomic-based hotspot discovery provide new opportunities in understanding of apoptosis molecular mechanisms. In this study, after measuring the global contribution of microRNAs in the course of apoptosis by Affymetrix platform, systems biology tools were utilized to obtain a comprehensive view on the role of microRNAs in apoptosis process. Network analysis and pathway discovery highlighted the crosstalk between transcription factors and microRNAs in apoptosis. Within the transcription factors, PRDM1 showed the highest upregulation during the course of apoptosis, with more than 9-fold expression increase compared to non-apoptotic condition. Within the microRNAs, MIR1208 showed the highest expression in non-apoptotic condition and downregulated by more than 6 fold during apoptosis. Common regulators algorithm showed that TNF receptor is the key upstream regulator with a high number of regulatory interactions with the differentially expressed microRNAs. BCL2 and AKT1 were the key downstream targets of differentially expressed microRNAs. Enrichment analysis of the genomic locations of differentially expressed microRNAs led us to the discovery of chromosome bands which were highly enriched (p < 0.01) with the apoptosis-related microRNAs, such as 13q31.3, 19p13.13, and Xq27.3 This study opens a new avenue in understanding regulatory mechanisms and downstream functions in the course of apoptosis as well as distinguishing genomic-enriched hotspots for apoptosis process.

Comparative transcriptome analysis of a lowly virulent strain of Erwinia amylovora in shoots of two apple cultivars - susceptible and resistant to fire blight.

PubMed

Puławska, Joanna; Kałużna, Monika; Warabieda, Wojciech; Mikiciński, Artur

2017-11-13

Erwinia amylovora is generally considered to be a homogeneous species in terms of phenotypic and genetic features. However, strains show variation in their virulence, particularly on hosts with different susceptibility to fire blight. We applied the RNA-seq technique to elucidate transcriptome-level changes of the lowly virulent E. amylovora 650 strain during infection of shoots of susceptible (Idared) and resistant (Free Redstar) apple cultivars. The highest number of differentially expressed E. amylovora genes between the two apple genotypes was observed at 24 h after inoculation. Six days after inoculation, only a few bacterial genes were differentially expressed in the susceptible and resistant apple cultivars. The analysis of differentially expressed gene functions showed that generally, higher expression of genes related to stress response and defence against toxic compounds was observed in Free Redstar. Also in this cultivar, higher expression of flagellar genes (FlaI), which are recognized as PAMP (pathogen-associated molecular pattern) by the innate immune systems of plants, was noted. Additionally, several genes that have not yet been proven to play a role in the pathogenic abilities of E. amylovora were found to be differentially expressed in the two apple cultivars. This RNA-seq analysis generated a novel dataset describing the transcriptional response of the lowly virulent strain of E. amylovora in susceptible and resistant apple cultivar. Most genes were regulated in the same way in both apple cultivars, but there were also some cultivar-specific responses suggesting that the environment in Free Redstar is more stressful for bacteria what can be the reason of their inability to infect of this cultivar. Among genes with the highest fold change in expression between experimental combinations or with the highest transcript abundance, there are many genes without ascribed functions, which have never been tested for their role in pathogenicity. Overall, this study provides the first transcriptional profile by RNA-seq of E. amylovora during infection of a host plant and insights into the transcriptional response of this pathogen in the environments of susceptible and resistant apple plants.

Toll-like receptors 2, 4, and 9 expressions over the entire clinical and immunopathological spectrum of American cutaneous leishmaniasis due to Leishmania (V.) braziliensis and Leishmania (L.) amazonensis

PubMed Central

Campos, Marliane Batista; Lima, Luciana Vieira do Rêgo; de Lima, Ana Carolina Stocco; Vasconcelos dos Santos, Thiago; Ramos, Patrícia Karla Santos; Gomes, Claudia Maria de Castro

2018-01-01

Leishmania (V.) braziliensis and Leishmania(L.) amazonensis are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCLDTH+/++), borderline disseminated cutaneous leishmaniasis (BDCLDTH±), anergic diffuse cutaneous leishmaniasis (ADCLDTH-), and mucosal leishmaniasis (MLDTH++++). It has recently been demonstrated, however, that while L. (V.) braziliensis shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), L. (L.) amazonensis drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of Toll-like receptors (TLRs) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-β cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused byL. (L.) amazonensis; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused byL. (V.) braziliensis. CD4+ T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8+ T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α+showed increased expression from ADCL to ML, while IL-10+and TGF-β+ showed increased expression in the opposite direction, from ML to ADCL. With regards to TLR2, 4, and 9 expressions, strong interactions of TLR2 and 4 with clinical forms associated with L. (V.) braziliensis were observed, while TLR9, in contrast, showed a strong interaction with clinical forms linked to L. (L.) amazonensis. These findings strongly suggest the ability of L. (V.) braziliensis and L. (L.) amazonensis to interact with those TLRs to promote a dichotomous T-cell immune response in ACL. PMID:29543867

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

PubMed

Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

2015-11-20

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

The histological characteristics, age-related thickness change of skin, and expression of the HSPs in the skin during hair cycle in yak (Bos grunniens)

PubMed Central

Yang, Xue; Cui, Yan; Yue, Jing; He, Honghong; Yu, Chuan; Liu, Penggang; Liu, Jun; Ren, Xiandong; Meng, Yun

2017-01-01

Objective This experiment was conducted to study the histological characteristics, age-related thickness changes, and expression of HSPs in the skin of yak. Methods A total of 20 yaks (10 males and 10 females) were used. Different regions of the normal skin of three different ages (newborn, half-year-old and adult) of yaks were harvested for histological study and thickness measurement. Biopsy samples were taken from the scapula regions of the skin from the same five approximately 1-year-old yaks during the hair cycle (telogen, anagen and catagen). RT-PCR, western blot and immunohistochemistry methods using the mRNA and protein levels were used to detect the expression of HSP27, HSP70 and HSP90. RT-PCR method was used to detect the mRNA expression of CGI-58 and KDF1. The IPP6.0 software was used to analyze the immunohistochemistry and measure the thickness of the skin. Results The general histological structure of hairy yak skin was similar to other domestic mammals. The unique features included prominent cutaneous vascular plexuses, underdeveloped sweat glands, a large number of nasolabial glands in the nasolabial plate, and hair follicle groups composed of one primary follicle and several secondary follicles. The skin, epidermis and dermis thickness did vary significantly between different body regions and different ages. The thickness of the skin, epidermis and dermis increased from newborn to adult in yaks. Yak skin thickness decreased from dorsally to ventrally on the trunk. The skin on the lateral surface was thicker than the skin on the medial surface on the limbs. HSP27, HSP70 and HSP90 showed different expression patterns during the hair cycle using RT-PCR, western blot and immunohistochemistry methods. The expression of HSP27 mRNA and protein in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of HSP70 mRNA and protein in the telogen stage was the highest, followed by the anagen stage, and the expression in the catagen stage was the lowest. The expression of HSP90 mRNA and protein in the anagen stage was the highest, followed by the telogen stage, and the expression in the catagen stage was the lowest. HSPs were mainly expressed in the outer root sheath of hair follicle during the hair cycle, also expressed in epidermis, sebaceous gland and sweat gland in the skin of Yak. The expression of CGI-58 mRNA in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of KDF1 mRNA in the telogen stage was the highest, followed by the catagen stage, and the expression in the anagen stage was the lowest. Meaning In this study, we examined and fully described the histology of normal skin in Yak and measured the skin thickness of different ages and different regions in Yak. These data may be useful to better understand and appreciate the adaptability features of yak skin. Our investigation reports the expression patterns of HSPs in yak skin for the first time. The different expression pattern of HSPs during the hair cycle suggests they may play different roles in yak hair follicle biology. PMID:28463974

The histological characteristics, age-related thickness change of skin, and expression of the HSPs in the skin during hair cycle in yak (Bos grunniens).

PubMed

Yang, Xue; Cui, Yan; Yue, Jing; He, Honghong; Yu, Chuan; Liu, Penggang; Liu, Jun; Ren, Xiandong; Meng, Yun

2017-01-01

This experiment was conducted to study the histological characteristics, age-related thickness changes, and expression of HSPs in the skin of yak. A total of 20 yaks (10 males and 10 females) were used. Different regions of the normal skin of three different ages (newborn, half-year-old and adult) of yaks were harvested for histological study and thickness measurement. Biopsy samples were taken from the scapula regions of the skin from the same five approximately 1-year-old yaks during the hair cycle (telogen, anagen and catagen). RT-PCR, western blot and immunohistochemistry methods using the mRNA and protein levels were used to detect the expression of HSP27, HSP70 and HSP90. RT-PCR method was used to detect the mRNA expression of CGI-58 and KDF1. The IPP6.0 software was used to analyze the immunohistochemistry and measure the thickness of the skin. The general histological structure of hairy yak skin was similar to other domestic mammals. The unique features included prominent cutaneous vascular plexuses, underdeveloped sweat glands, a large number of nasolabial glands in the nasolabial plate, and hair follicle groups composed of one primary follicle and several secondary follicles. The skin, epidermis and dermis thickness did vary significantly between different body regions and different ages. The thickness of the skin, epidermis and dermis increased from newborn to adult in yaks. Yak skin thickness decreased from dorsally to ventrally on the trunk. The skin on the lateral surface was thicker than the skin on the medial surface on the limbs. HSP27, HSP70 and HSP90 showed different expression patterns during the hair cycle using RT-PCR, western blot and immunohistochemistry methods. The expression of HSP27 mRNA and protein in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of HSP70 mRNA and protein in the telogen stage was the highest, followed by the anagen stage, and the expression in the catagen stage was the lowest. The expression of HSP90 mRNA and protein in the anagen stage was the highest, followed by the telogen stage, and the expression in the catagen stage was the lowest. HSPs were mainly expressed in the outer root sheath of hair follicle during the hair cycle, also expressed in epidermis, sebaceous gland and sweat gland in the skin of Yak. The expression of CGI-58 mRNA in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of KDF1 mRNA in the telogen stage was the highest, followed by the catagen stage, and the expression in the anagen stage was the lowest. In this study, we examined and fully described the histology of normal skin in Yak and measured the skin thickness of different ages and different regions in Yak. These data may be useful to better understand and appreciate the adaptability features of yak skin. Our investigation reports the expression patterns of HSPs in yak skin for the first time. The different expression pattern of HSPs during the hair cycle suggests they may play different roles in yak hair follicle biology.

Molecular cloning and expression analysis of jasmonic acid dependent but salicylic acid independent LeWRKY1.

PubMed

Lu, M; Wang, L F; Du, X H; Yu, Y K; Pan, J B; Nan, Z J; Han, J; Wang, W X; Zhang, Q Z; Sun, Q P

2015-11-30

Various plant genes can be activated or inhibited by phytohormones under conditions of biotic and abiotic stress, especially in response to jasmonic acid (JA) and salicylic acid (SA). Interactions between JA and SA may be synergistic or antagonistic, depending on the stress condition. In this study, we cloned a full-length cDNA (LeWRKY1, GenBank accession No. FJ654265) from Lycopersicon esculentum by rapid amplification of cDNA ends. Sequence analysis showed that this gene is a group II WRKY transcription factor. Analysis of LeWRKY1 mRNA expression in various tissues by qRT-PCR showed that the highest and lowest expression occurred in the leaves and stems, respectively. In addition, LeWRKY1 expression was induced by JA and Botrytis cinerea Pers., but not by SA.

Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation.

PubMed

Troncoso-Ponce, M A; Rivoal, J; Dorion, S; Moisan, M-C; Garcés, R; Martínez-Force, E

2011-03-01

A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation. Copyright © 2010 Elsevier GmbH. All rights reserved.

Differential expression and regulation of vitamin D hydroxylases and inflammatory genes in prostate stroma and epithelium by 1,25-dihydroxyvitamin D in men with prostate cancer and an in vitro model.

PubMed

Giangreco, Angeline A; Dambal, Shweta; Wagner, Dennis; Van der Kwast, Theodorus; Vieth, Reinhold; Prins, Gail S; Nonn, Larisa

2015-04-01

Previous work on vitamin D in the prostate has focused on the prostatic epithelium, from which prostate cancer arises. Prostatic epithelial cells are surrounded by stroma, which has well-established regulatory control over epithelial proliferation, differentiation, and the inflammatory response. Here we examined the regulation of vitamin D-related genes and inflammatory genes by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D) in laser-capture microdissected prostate tissue from a vitamin D3 clinical trial and in an in vitro model that facilitates stromal-epithelial crosstalk. Analysis of the trial tissues showed that VDR was present in both cell types, whereas expression of the hydroxylases was the highest in the epithelium. Examination of gene expression by prostatic (1,25(OH)2D) concentrations showed that VDR was significantly lower in prostate tissues with the highest concentration of 1,25(OH)2D, and down-regulation of VDR by 1,25(OH) 2D was confirmed in the primary cell cultures. Analysis of inflammatory genes in the patient tissues revealed that IL-6 expression was the highest in the prostate stroma while PTGS2 (COX2) levels were lowest in the prostate cancer tissues from men in the highest tertile of prostatic 1,25(OH)2D. In vitro, TNF-α, IL-6 and IL-8 were suppressed by 1,25 (OH)2D in the primary epithelial cells, whereas TNF-α and PTGS2 were suppressed by 1,25(OH) 2D in the stromal cells. Importantly, the ability of 1,25(OH)2D to alter pro-inflammatory-induced changes in epithelial cell growth were dependent on the presence of the stromal cells. In summary, whereas both stromal and epithelial cells of the prostate express VDR and can presumably respond to 1,25(OH)2D, the prostatic epithelium appears to be the main producer of 1,25(OH)2D. Further, while the prostate epithelium was more responsive to the anti-inflammatory activity of 1,25 (OH)2D than stromal cells, stroma-epithelial crosstalk enhanced the phenotypic effects of 1,25(OH)2D and the inflammatory process in the prostate gland. Copyright © 2014 Elsevier Ltd. All rights reserved.

Tricellulin, occludin and claudin-3 expression in salmon intestine and kidney during salinity adaptation.

PubMed

Tipsmark, C K; Madsen, S S

2012-08-01

Molecular regulation of tight junctions in osmoregulatory epithelia of euryhaline fishes must be extensive during ontogeny and acclimation to salinity changes. In this study, five tight junction proteins were examined in Atlantic salmon (Salmo salar): tight junction associated tricellulin, occludin and claudin-3 isoforms (a, b, c). A survey of tissue distribution in freshwater (FW) salmon showed that tricellulin expression was highest in the intestine. Occludin was detected in tissues with importance for epithelial transport and the order of expression was gill>intestine>kidney. The three claudin-3 isoforms were expressed at highest level in kidney tissue. Transfer of juvenile FW salmon to seawater (SW) elevated intestinal tricellulin and occludin mRNA, and these transcripts were also elevated at the time of best SW-tolerance during the course of smoltification. In the kidney, expression of tricellulin and claudin-3 isoforms was elevated after SW-transfer and tricellulin, occludin, claudin-3a and -3b increased in March before the peak smolt stage. In the gill, none of the examined tight junction proteins were impacted by SW-transfer. The data suggest that expression of tricellulin and occludin is dynamically involved in reorganization of intestinal epithelium and possibly changed paracellular permeability during SW-acclimation. The increased renal tricellulin and claudin-3 expression in SW suggests a role in remodeling of the kidney during SW-acclimation. Copyright © 2012 Elsevier Inc. All rights reserved.

Functionally distinct roles for different miR-155 expression levels through contrasting effects on gene expression, in acute myeloid leukaemia.

PubMed

Narayan, N; Morenos, L; Phipson, B; Willis, S N; Brumatti, G; Eggers, S; Lalaoui, N; Brown, L M; Kosasih, H J; Bartolo, R C; Zhou, L; Catchpoole, D; Saffery, R; Oshlack, A; Goodall, G J; Ekert, P G

2017-04-01

Enforced expression of microRNA-155 (miR-155) in myeloid cells has been shown to have both oncogenic or tumour-suppressor functions in acute myeloid leukaemia (AML). We sought to resolve these contrasting effects of miR-155 overexpression using murine models of AML and human paediatric AML data sets. We show that the highest miR-155 expression levels inhibited proliferation in murine AML models. Over time, enforced miR-155 expression in AML in vitro and in vivo, however, favours selection of intermediate miR-155 expression levels that results in increased tumour burden in mice, without accelerating the onset of disease. Strikingly, we show that intermediate and high miR-155 expression also regulate very different subsets of miR-155 targets and have contrasting downstream effects on the transcriptional environments of AML cells, including genes involved in haematopoiesis and leukaemia. Furthermore, we show that elevated miR-155 expression detected in paediatric AML correlates with intermediate and not high miR-155 expression identified in our experimental models. These findings collectively describe a novel dose-dependent role for miR-155 in the regulation of AML, which may have important therapeutic implications.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles.

PubMed

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.

Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles

PubMed Central

Huang, Shi-Ming; Zhao, Xia; Zhao, Xue-Mei; Wang, Xiao-Ying; Li, Shan-Shan; Zhu, Yu-Hui

2014-01-01

Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection. PMID:25664019

HOXA9 gene expression in the chronic myeloid leukemia progression.

PubMed

Tedeschi, Fabian A; Zalazar, Fabian E

2006-11-01

In the present work we study the HOXA9 expression in sequential samples of patients with CML using RT-PCR. To obtain a semi-quantitative value, the HOXA9 expression was referred to the ABL gene in the same sample. The relative HOXA9 expression was higher in patients in the accelerated phase of the disease (p<0.005). Interestingly, a patient with poorer prognosis (high Sokal's score), showing the highest HOXA9/ABL ratio, quickly entered a blast crisis and died 5 months later. These first results could be considered as an evidence of an actual biological phenomenon that could provide additional information about the HOXA9 role in the CML progression.

Thymidylate synthase (TS) gene expression in primary lung cancer patients: a large-scale study in Japanese population.

PubMed

Tanaka, F; Wada, H; Fukui, Y; Fukushima, M

2011-08-01

Previous small-sized studies showed lower thymidylate synthase (TS) expression in adenocarcinoma of the lung, which may explain higher antitumor activity of TS-inhibiting agents such as pemetrexed. To quantitatively measure TS gene expression in a large-scale Japanese population (n = 2621) with primary lung cancer, laser-captured microdissected sections were cut from primary tumors, surrounding normal lung tissues and involved nodes. TS gene expression level in primary tumor was significantly higher than that in normal lung tissue (mean TS/β-actin, 3.4 and 1.0, respectively; P < 0.01), and TS gene expression level was further higher in involved node (mean TS/β-actin, 7.7; P < 0.01). Analyses of TS gene expression levels in primary tumor according to histologic cell type revealed that small-cell carcinoma showed highest TS expression (mean TS/β-actin, 13.8) and that squamous cell carcinoma showed higher TS expression as compared with adenocarcinoma (mean TS/β-actin, 4.3 and 2.3, respectively; P < 0.01); TS gene expression was significantly increased along with a decrease in the grade of tumor cell differentiation. There was no significant difference in TS gene expression according to any other patient characteristics including tumor progression. Lower TS expression in adenocarcinoma of the lung was confirmed in a large-scale study.

Flp and Cre expressed from Flp–2A–Cre and Flp–IRES–Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange

PubMed Central

Anderson, Rachelle P.; Voziyanova, Eugenia; Voziyanov, Yuri

2012-01-01

Recombinase-mediated cassette exchange (RMCE) is a powerful tool for unidirectional integration of DNA fragments of interest into a pre-determined genome locale. In this report, we examined how the efficiency of dual RMCE catalyzed by Flp and Cre depends on the nature of transcription units that express the recombinases. The following recombinase transcription units were analyzed: (i) Flp and Cre genes expressed as individual transcription units located on different vectors, (ii) Flp and Cre genes expressed as individual transcription units located on the same vector, (iii) Flp and Cre genes expressed from a single promoter and separated by internal ribosome entry sequence and (iv) Flp and Cre coding sequences separated by the 2A peptide and expressed as a single gene. We found that the highest level of dual RMCE (35–45% of the transfected cells) can be achieved when Flp and Cre recombinases are expressed as Flp–2A–Cre and Flp–IRES–Cre transcription units. In contrast, the lowest level of dual RMCE (∼1% of the transfected cells) is achieved when Flp and Cre are expressed as individual transcription units. The analysis shows that it is the relative Flp–to–Cre ratio that critically affects the efficiency of dual RMCE. Our results will be helpful for maximizing the efficiency of dual RMCE aimed to engineer and re-engineer genomes. PMID:22270085

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions.

PubMed

Haigler, Candace H; Singh, Bir; Zhang, Deshui; Hwang, Sangjoon; Wu, Chunfa; Cai, Wendy X; Hozain, Mohamed; Kang, Wonhee; Kiedaisch, Brett; Strauss, Richard E; Hequet, Eric F; Wyatt, Bobby G; Jividen, Gay M; Holaday, A Scott

2007-04-01

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V (max) SPS activity in leaf and fiber. Lines with the highest V (max) SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of (14)C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO(2) concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.

Cloning and expression of hepatic synaptotagmin 1 in mouse.

PubMed

Sancho-Knapik, Sara; Guillén, Natalia; Osada, Jesús

2015-05-15

Mouse hepatic synaptotagmin 1 (SYT1) cDNA was cloned, characterized and compared to the brain one. The hepatic transcript was 1807 bp in length, smaller than the brain, and only encoded by 9 of 11 gene exons. In this regard, 5'-and 3'-untranslated regions were 66 and 476 bp, respectively; the open reading frame of 1266 bp codified for a protein of 421 amino acids, identical to the brain, with a predicted molecular mass of 47.4 kDa and highly conserved across different species. Immunoblotting of protein showed two isoforms of higher molecular masses than the theoretical prediction based on amino acid sequence suggesting posttranslational modifications. Subcellular distribution of protein isoforms corresponded to plasma membrane, lysosomes and microsomes and was identical between the brain and liver. Nonetheless, the highest molecular weight isoform was smaller in the liver, irrespective of subcellular location. Quantitative mRNA tissue distribution showed that it was widely expressed and that the highest values corresponded to the brain, followed by the liver, spleen, abdominal fat, intestine and skeletal muscle. These findings indicate tissue-specific splicing of the gene and posttranslational modification and the variation in expression in the different tissues might suggest a different requirement of SYT1 for the specific function in each organ. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative study of SOS2 and a novel PMP3-1 gene expression in two sunflower (Helianthus annuus L.) lines differing in salt tolerance.

PubMed

Saadia, Mubshara; Jamil, Amer; Ashraf, Muhammad; Akram, Nudrat Aisha

2013-06-01

Gene expression pattern of two important regulatory proteins, salt overly sensitive 2 (SOS2) and plasma membrane protein 3-1 (PMP3-1), involved in ion homeostasis, was analyzed in two salinity-contrasting sunflower (Helianthus annuus L.) lines, Hysun-38 (salt tolerant) and S-278 (moderately salt tolerant). The pattern was studied at selected time intervals (24 h) under 150 mM NaCl treatment. Using reverse transcription PCR, SOS2 gene fragment was obtained from young leaf and root tissues of opposing lines while that for PMP3-1 was obtained only from young root tissues. Both tolerant and moderately tolerant lines showed a gradual increase in SOS2 expression in sunflower root tissues. Leaf tissues showed the gradually increasing pattern of SOS2 expression in tolerant plants as compared to that for moderately tolerant ones that showed a relatively lower level of expression for this gene. We found the highest level of PMP 3-1 expression in the roots of tolerant sunflower line at 6 and 12 h postsalinity treatment. The moderately tolerant line showed higher expression of PMP3-1 at 12 and 24 h after salt treatment. Overall, the expression of genes for both the regulator proteins varied significantly in the two sunflower lines differing in salinity tolerance.

Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

PubMed

Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

2012-01-01

Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

Cellular dissection of psoriasis for transcriptome analyses and the post-GWAS era

PubMed Central

2014-01-01

Background Genome-scale studies of psoriasis have been used to identify genes of potential relevance to disease mechanisms. For many identified genes, however, the cell type mediating disease activity is uncertain, which has limited our ability to design gene functional studies based on genomic findings. Methods We identified differentially expressed genes (DEGs) with altered expression in psoriasis lesions (n = 216 patients), as well as candidate genes near susceptibility loci from psoriasis GWAS studies. These gene sets were characterized based upon their expression across 10 cell types present in psoriasis lesions. Susceptibility-associated variation at intergenic (non-coding) loci was evaluated to identify sites of allele-specific transcription factor binding. Results Half of DEGs showed highest expression in skin cells, although the dominant cell type differed between psoriasis-increased DEGs (keratinocytes, 35%) and psoriasis-decreased DEGs (fibroblasts, 33%). In contrast, psoriasis GWAS candidates tended to have highest expression in immune cells (71%), with a significant fraction showing maximal expression in neutrophils (24%, P < 0.001). By identifying candidate cell types for genes near susceptibility loci, we could identify and prioritize SNPs at which susceptibility variants are predicted to influence transcription factor binding. This led to the identification of potentially causal (non-coding) SNPs for which susceptibility variants influence binding of AP-1, NF-κB, IRF1, STAT3 and STAT4. Conclusions These findings underscore the role of innate immunity in psoriasis and highlight neutrophils as a cell type linked with pathogenetic mechanisms. Assignment of candidate cell types to genes emerging from GWAS studies provides a first step towards functional analysis, and we have proposed an approach for generating hypotheses to explain GWAS hits at intergenic loci. PMID:24885462

[Expression of OPG/RANK/RANKL in the rat dental pulp tissue of periodontitis combined with vascular calcification and its clinical significance].

PubMed

Pan, Ke-Qing; Zhang, Peng-Mei; Deng, Jing; Lou, Xiu-Xiu; Meng, Yun; Liu, Gui-Rong

2016-08-01

To study the expression and possible role of OPG/RANK/RANKLin the rat dental pulp of periodontitis combined with vascular calcification. Thirty-six male Wister rats were randomly divided into 4 groups: control group(group C), periodontitis group(group CP), vascular calcification group(group VDN) and compound group(group CP+VDN). Each group underwent corresponding management to establish animal model. When the model was successful, the maxillae including molars were sectioned, pulp tissue was examined by H-E staining; Immunohistochemical staining method was used to evaluate the expression and ratio of OPG and RANKL in pulp tissues. Statistical analysis was carried out using SPSS 19.0 software package. The pulp tissue of group CP, VDN, CP+VDN showed varied degrees of damage, neutrophil infiltration, pulp vascular congestion, odontoblasts vacuolar changes, pulp necrosis by H-E staining, and the changes in CP+VDN group was the most significant, followed by CP group, VDN group. Immunohistochemistry showed OPG in pulp tissues in group CP, VDN, CP+VDN were significantly lower than that in normal group (P<0.05), and the expression in group CP+VDN was the least;Expression of RANKL in pulp tissues in group CP, VDN, CP+VDN were significantly higher than that in normal group(P<0.05)，and the expression in group CP+VDN was the highest. The ratio of OPG/RANKL in normal group was the highest, and the ratio in CP+VDN group was the lowest. Periodontitis and vascular calcification can damage the pulp tissue, periodontitis compound with vascular calcification may aggravate the injury; OPG/RANKL/RANK system may play an important role in pulp tissue injury.

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli

PubMed Central

Elkhatib, Walid F.

2016-01-01

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed “ghost” cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli.

PubMed

Wasfi, Reham; Elkhatib, Walid F; Khairalla, Ahmed S

2016-01-01

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets.

Low temperature stress on the hematological parameters and HSP gene expression in the turbot Scophthalmus maximus

NASA Astrophysics Data System (ADS)

Ji, Liqin; Jiang, Keyong; Liu, Mei; Wang, Baojie; Han, Longjiang; Zhang, Mingming; Wang, Lei

2016-05-01

To study the effect of low temperature stress on hematological parameters and HSP gene expression in the turbot ( Scophthalmus maximus), water temperature was lowered rapidly from 18 to 1°C. During the cooling process, three individuals were removed from culture tanks at 18, 13, 8, 5, 3, and 1°C. Blood samples and tissues were taken from each individual, hematological indices and HSP gene expression in tissues were measured. The red blood cell count, white blood cell count, and hemoglobin concentration decreased significantly ( P < 0.05) as temperature decreased. Enzyme activities of plasma alanine transaminase and creatine kinase increased as temperature decreased, whereas aspartic transaminase and γ-glutamyl transpeptidase activities displayed no obvious changes above 1°C and lactate dehydrogenase activity increased first and then decreased. Blood urea nitrogen and uric acid levels were highest at 8°C, and creatinine concentration was highest at 3°C. The concentrations of plasma cortisol, cholesterol, and triglyceride all increased significantly ( P < 0.05) as temperature decreased. The serum glucose concentration increased first and then decreased to the initial level. The HSP70 mRNA expression showed various patterns in different tissues, whereas HSP90 mRNA expression showed the same tendency in all tissues. Overall, these results indicate that temperature decreases in the range of 8 to 5°C may induce a stress response in S. maximus and that temperature should be kept above 8°C in the aquaculture setting to avoid damage to the fish.

[Effect of GPER on the activation of PI3K/Akt induced by 17β-estradiol in endometrial carcinoma cells].

PubMed

Zhang, Yan-Cai; Guo, Rui-Xia; Ge, Xin; Qiao, Yu-Huan

2012-04-01

To investigate the expression of G protein-coupled ER (GPER) and ER in the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) induced by 17β-estradiol (17β-E(2))in endometrial carcinoma cells, Ishikawa and HEC-1A. Expressions of GPER, ERα and ERβ protein in Ishikawa and HEC-1A cells were detected by immunohistochemical SP method. Levels of GPER, ERα and ERβ were examined by western blot in Ishikawa and HEC-1A cells after treated with 1×10(-6) mol/L 17β-E(2) at different time (0, 15, 30, 60, 120 minutes). GPER was positive expressed in Ishikawa and HEC-1A cells. ERα and ERβ were both positive expressed in Ishikawa cells. While, ERα was weakly expressed and ERβ was almost negatively expressed in HEC-1A cells. Western blot analysis showed that 1×10(-6) mol/L 17β-E(2) treatment, the Ishikawa and HEC-1A cells GPER protein level for 15 minutes markedly increased (P < 0.05), which Ishikawa 30 minutes, when cells reached the highest level (0.192 ± 0.004), HEC-1A cells for 15 minutes and reached the highest level (0.184 ± 0.006); Ishikawa and HEC-1A cells, Akt, activation of 15 minutes from the treatment start was significantly increased (P < 0.05), which Ishikawa cells for 30 minutes and reached the highest level (0.666 ± 0.021), HEC-1A cells for 15 minutes and reached maximum (0.788 ± 0.035); Ishikawa and HEC-1A cells, ERα and ERβ protein expression did not change significantly (P > 0.05). GPER likely involved in non-nuclear activation of PI3K/Akt signaling pathways in endometrial carcinoma cells, Ishikawa and HEC-1A.

cDNA cloning and characterization of the antibacterial peptide cecropin 1 from the diamondback moth, Plutella xylostella L.

PubMed

Jin, Fengliang; Sun, Qiang; Xu, Xiaoxia; Li, Linmiao; Gao, Gang; Xu, Yingjie; Yu, Xiaoqiang; Ren, Shunxiang

2012-10-01

Cecropins are linear cationic antibacterial peptides that have potent activities against microorganisms. In the present study, a 480bp full-length cDNA encoding diamondback moth (Plutella xylostella) cecropin 1 (designated as Px-cec1) was obtained using RT-PCR. A Northern blot analysis showed that the Px-cec1 transcript was predominantly expressed in fat bodies, hemocytes, midgut and epidermis with the highest expression level in fat bodies. The expression of Px-cec1 mRNA in fat bodies was significantly increased 24h after microbial challenge, with the highest induced expression by Staphylococcus aureus. A circular dichroism (CD) analysis revealed that the recombinant Px-cec1 mainly contained α-helixes. Antimicrobial assays demonstrated that recombinant Px-cec1 exhibited a broad spectrum of anti-microbial properties against fungi, Gram-positive and Gram-negative bacteria, but it did not exhibit hemolytic activity against human erythrocytes. Furthermore, Px-cec1 caused significant morphological alterations of S. aureus, as shown by scanning electron microscopy and transmission electron microscopy. These results demonstrated that Px-cec1 exerts its antibacterial activity by acting on the cell membrane to disrupt bacterial cell structures. Copyright © 2012 Elsevier Inc. All rights reserved.

Identification and expression patterns of adipokine genes during adipocyte differentiation in the Tibetan goat (Capra hircus).

PubMed

Li, Xueying; Wang, Yan; Guo, Jiazhong; Zhong, Tao; Li, Li; Zhang, Hongping; Wang, Linjie

2018-02-15

Adipokines are secreted by adipose tissue and play an important role in the regulation of lipid metabolism. However, the information regarding adipokines in goats is limited. PPARγ is a key gene in adipocyte differentiation and activates adipokine genes. Rosiglitazone is a PPARγ agonist and can promote the expression of PPARγ to increase the expression of lipogenesis-related genes. Therefore, investigation of the relationship between rosiglitazone and adipokines will help us to better understand the function of PPARγ in lipid metabolism in Tibetan goats. In this study, we cloned the resistin (RETN), apelin (APLN), fibroblast growth factor 21 (FGF21), and visfatin (NAMPT) genes from non-pregnant female Tibetan goat adipose tissue. APLN and NAMPT were predominantly expressed in the kidney, and FGF21 was expressed at the highest levels in the liver in vivo. In fat tissues, the highest expression levels of FGF21 and RETN were detected in omental fat, whereas their expression in perirenal and subcutaneous fat was extremely weak. APLN and NAMPT were abundantly expressed in omental and subcutaneous fat in vivo. In addition, the four adipokines had different expression profiles during goat adipocyte differentiation in vitro. Oil red O staining showed that rosiglitazone could promote adipocyte differentiation and lipid droplet formation. In addition, rosiglitazone significantly increased the expression of FGF21 and RETN (p<0.05) but decreased the expression of APLN and NAMPT (p<0.05). These results suggest that the four adipocytokine genes may have different roles during goat adipocyte differentiation. And PPARγ could regulate the expression of the four adipokines, but the detailed regulatory mechanism still needs to be elucidated. Copyright © 2017 Elsevier B.V. All rights reserved.

Energy sources and levels influenced on performance parameters, thyroid hormones, and HSP70 gene expression of broiler chickens under heat stress.

PubMed

Raghebian, Majid; Sadeghi, Ali Asghar; Aminafshar, Mehdi

2016-12-01

The present study was conducted to evaluate the effects of energy sources and levels on body and organs weights, thyroid hormones, and heat shock protein (HSP70) gene expression in broilers under heat stress. In a completely randomized design, 600 1-day-old Cobb chickens were assigned to five dietary treatments and four replicates. The chickens were fed diet based on corn as main energy source and energy level based on Cobb standard considered as control (C), corn-based diet with 3 % lesser energy than the control (T1), corn-based diet with 6 % lesser energy than the control (T2), corn and soybean oil-based diet according to Cobb standard (T3), and corn and soybean oil-based diet with 3 % upper energy than the control (T4). Temperature was increased to 34 °C for 8 h daily from days 12 to 41 of age to induce heat stress. The chickens in T1 and T2 had lower thyroid hormones and corticosterone levels than those in C, T3, and T4. The highest liver weight was for C and the lowest one was for T4. The highest gene expression was found in chickens fed T4 diet, and the lowest gene expression was for those in T2 group. The highest feed intake and worse feed conversion ratio was related to chickens in T2. The chickens in T3 and T4 had higher feed intake and weight gain than those in C. The results showed that the higher energy level supplied from soybean oil could enhance gene expression of HSP70 and decline the level of corticosterone and thyroid hormones and consequently improved performance.

Mitogen-activated protein kinase 1 from disk abalone (Haliotis discus discus): Roles in early development and immunity-related transcriptional responses.

PubMed

Perera, N C N; Godahewa, G I; Lee, Jehee

2016-12-01

Mitogen-activated protein kinase (MAPK) is involved in the regulation of cellular events by mediating signal transduction pathways. MAPK1 is a member of the extracellular-signal regulated kinases (ERKs), playing roles in cell proliferation, differentiation, and development. This is mainly in response to growth factors, mitogens, and many environmental stresses. In the current study, we have characterized the structural features of a homolog of MAPK1 from disk abalone (AbMAPK1). Further, we have unraveled its expressional kinetics against different experimental pathogenic infections or related chemical stimulants. AbMAPK1 harbors a 5' untranslated region (UTR) of 23 bps, a coding sequence of 1104 bps, and a 3' UTR of 448 bp. The putative peptide comprises a predicted molecular mass of 42.2 kDa, with a theoretical pI of 6.28. Based on the in silico analysis, AbMAPK1 possesses two N-glycosylation sites, one S_TK catalytic domain, and a conserved His-Arg-Asp domain (HRD). In addition, a conservative glycine rich ATP-phosphate-binding loop and a threonine-x-tyrosine motif (TEY) important for the autophosphorylation were also identified in the protein. Homology assessment of AbMAPK1 showed several conserved regions, and ark clam (Aplysia californica) showed the highest sequence identity (87.9%). The phylogenetic analysis supported close evolutionary kinship with molluscan orthologs. Constitutive expression of AbMAPK1 was observed in six different tissues of disk abalone, with the highest expression in the digestive tract, followed by the gills and hemocytes. Highest AbMAPK1 mRNA expression level was detected at the trochophore developmental stage, suggesting its role in abalone cell differentiation and proliferation. Significant modulation of AbMAPK1 expression under pathogenic stress suggested its putative involvement in the immune defense mechanism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transient transfection of intraerythrocytic Babesia gibsoni using elongation factor-1 alpha promoter.

PubMed

Liu, Mingming; Asada, Masahito; Cao, Shinuo; Adjou Moumouni, Paul Franck; Vudriko, Patrick; Efstratiou, Artemis; Hakimi, Hassan; Masatani, Tatsunori; Sunaga, Fujiko; Kawazu, Shin-Ichiro; Yamagishi, Junya; Xuan, Xuenan

2017-09-01

The development of gene manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not yet been established for Babesia gibsoni. Here, we report for the first time, the successful transient transfection of B. gibsoni. The plasmid containing the firefly luciferase reporter gene (pBS-ELA) was transfected into B. gibsoni by an AMAXA 4D Nucleofector™ device. Transfection using program FA113 and Lonza buffer SF showed the highest luciferase expression. Twenty micrograms of plasmid produced the highest relative transfection efficiency. The fluorescent protein-expressing parasites were determined by GFP-containing plasmid (pBS-EGA) at 48 and 72h post transfection. This finding is the first step towards a stable transfection method for B. gibsoni, which may contribute to a better understanding of the biology of the parasite. Copyright © 2017 Elsevier B.V. All rights reserved.

Circadian expression of clock and putative clock-controlled genes in skeletal muscle of the zebrafish.

PubMed

Amaral, Ian P G; Johnston, Ian A

2012-01-01

To identify circadian patterns of gene expression in skeletal muscle, adult male zebrafish were acclimated for 2 wk to a 12:12-h light-dark photoperiod and then exposed to continuous darkness for 86 h with ad libitum feeding. The increase in gut food content associated with the subjective light period was much diminished by the third cycle, enabling feeding and circadian rhythms to be distinguished. Expression of zebrafish paralogs of mammalian transcriptional activators of the circadian mechanism (bmal1, clock1, and rora) followed a rhythmic pattern with a ∼24-h periodicity. Peak expression of rora paralogs occurred at the beginning of the subjective light period [Zeitgeber time (ZT)07 and ZT02 for roraa and rorab], whereas the highest expression of bmal1 and clock paralogs occurred 12 h later (ZT13-15 and ZT16 for bmal and clock paralogs). Expression of the transcriptional repressors cry1a, per1a/1b, per2, per3, nr1d2a/2b, and nr1d1 also followed a circadian pattern with peak expression at ZT0-02. Expression of the two paralogs of cry2 occurred in phase with clock1a/1b. Duplicated genes had a high correlation of expression except for paralogs of clock1, nr1d2, and per1, with cry1b showing no circadian pattern. The highest expression difference was 9.2-fold for the activator bmal1b and 51.7-fold for the repressor per1a. Out of 32 candidate clock-controlled genes, only myf6, igfbp3, igfbp5b, and hsf2 showed circadian expression patterns. Igfbp3, igfbp5b, and myf6 were expressed in phase with clock1a/1b and had an average of twofold change in expression from peak to trough, whereas hsf2 transcripts were expressed in phase with cry1a and had a 7.2-fold-change in expression. The changes in expression of clock and clock-controlled genes observed during continuous darkness were also observed at similar ZTs in fish exposed to a normal photoperiod in a separate control experiment. The role of circadian clocks in regulating muscle maintenance and growth are discussed.

Cloning of peroxisome proliferators activated receptors in the cobia (Rachycentron canadum) and their expression at different life-cycle stages under cage aquaculture.

PubMed

Tsai, Mei-Ling; Chen, Houng-Yung; Tseng, Mei-Cheuh; Chang, Rey-Chang

2008-12-01

We present the cDNA sequences and tissue mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, beta and gamma isotypes in the cobia (Rachycentron canadum), a warm water pelagic fish that is becoming a fish of choice for offshore cage farming. RT-PCR and real-time PCR showed that PPARalpha mRNA predominated in red muscle, heart and liver whereas PPARbeta was expressed mainly in liver and pyloric caeca. In contrast, PPARgamma transcripts were detected in all of the tissues examined, with the highest level occurring in visceral fat depot. Our 52-wk time-series investigation showed that while the mRNA expression of PPARgamma in the cobia was positively (P < 0.05) related to its body lipid deposition, a negative (P < 0.05) relationship was found between PPARalpha expression in the liver and body lipid deposition. There was a significant increase in body lipid deposition and hepatic PPARgamma expression as the fish grew. The hepatic PPARgamma expression could be a sufficient parameter describing the bodily expression of PPARgamma because of its positive correlation with PPARgamma expressions in all other tissues. These results showed that PPARgamma and alpha played a pivotal role in the control of lipid metabolic and storage functions in the liver, muscle and visceral fat depot of the cobia.

Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

PubMed

Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

2016-05-01

Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

Different response of human glioma tumor-initiating cells to epidermal growth factor receptor kinase inhibitors.

PubMed

Griffero, Fabrizio; Daga, Antonio; Marubbi, Daniela; Capra, Maria Cristina; Melotti, Alice; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adriana; Porcile, Carola; Barbieri, Federica; Favoni, Roberto E; Lo Casto, Michele; Zona, Gianluigi; Spaziante, Renato; Florio, Tullio; Corte, Giorgio

2009-03-13

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

Size dependent biodistribution and toxicokinetics of iron oxide magnetic nanoparticles in mice

NASA Astrophysics Data System (ADS)

Yang, Lin; Kuang, Huijuan; Zhang, Wanyi; Aguilar, Zoraida P.; Xiong, Yonghua; Lai, Weihua; Xu, Hengyi; Wei, Hua

2014-12-01

In spite of the immense benefits from iron oxide magnetic nanoparticles (IOMNs), there is scanty information regarding their metabolic activities and toxicity in vivo. In this study, we investigated the size dependent in vivo biodistribution, toxicokinetics, and toxicity and gene expression changes of various sizes of carboxyl coated IOMNs (diameters of 10, 20, 30, and 40 nm). Our findings demonstrated that the various sizes of IOMNs accumulated primarily in the liver and spleen on the first day post-injection. Interestingly, size dependent biodistribution and transport were observed: the smallest IOMNs (10 nm) showed the highest uptake by the liver, whereas the largest IOMNs (40 nm) showed the highest uptake by the spleen. Moreover, the IOMNs with the smallest size (10 nm) were cleared faster from the liver and kidneys, but more readily entered the brain and the uterus. IOMNs with the largest size (40 nm) accumulated more readily but were easily eliminated in the spleen. However, the level of iron in the heart decreased in all IOMN exposed groups. In addition, blood biochemistry, hematological analyses and histological examination demonstrated that there was no apparent acute toxicity caused by IOMNs in mice. However, smaller IOMNs (10 nm and 20 nm) more effectively changed the expression level of sensitive genes related to oxidant stress, iron transport, metabolic process, apoptosis, and others.In spite of the immense benefits from iron oxide magnetic nanoparticles (IOMNs), there is scanty information regarding their metabolic activities and toxicity in vivo. In this study, we investigated the size dependent in vivo biodistribution, toxicokinetics, and toxicity and gene expression changes of various sizes of carboxyl coated IOMNs (diameters of 10, 20, 30, and 40 nm). Our findings demonstrated that the various sizes of IOMNs accumulated primarily in the liver and spleen on the first day post-injection. Interestingly, size dependent biodistribution and transport were observed: the smallest IOMNs (10 nm) showed the highest uptake by the liver, whereas the largest IOMNs (40 nm) showed the highest uptake by the spleen. Moreover, the IOMNs with the smallest size (10 nm) were cleared faster from the liver and kidneys, but more readily entered the brain and the uterus. IOMNs with the largest size (40 nm) accumulated more readily but were easily eliminated in the spleen. However, the level of iron in the heart decreased in all IOMN exposed groups. In addition, blood biochemistry, hematological analyses and histological examination demonstrated that there was no apparent acute toxicity caused by IOMNs in mice. However, smaller IOMNs (10 nm and 20 nm) more effectively changed the expression level of sensitive genes related to oxidant stress, iron transport, metabolic process, apoptosis, and others. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05061d

Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines.

PubMed

Okumura, Fumihiko; Kameda, Hiroyuki; Ojima, Takao; Hatakeyama, Shigetsugu

2010-05-07

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. Copyright (c) 2010 Elsevier Inc. All rights reserved.

[Detection and analysis of the characteristic expression of microRNAs of anal fistula patients].

PubMed

Qiu, Jianming; Yu, Jiping; Yang, Guangen; Xu, Kan; Tao, Yong; Lin, Ali; Wang, Dong

2016-07-01

To detect and analyze the characteristic miRNAs profile of anal fistula and explore their possible target genes and potential clinical significance. The anal mucosa close to the hemorrhoids were collected from three patients undergoing fistulectomy and hemorrhoidectomy (fistula group) as well as three patients receiving only hemorroidectomy(hemorrhoids group), matching with fistula group in age, gender and body weight. miRNA microarray was used to compare the expression of 1 285 human miRNAs of the anal mucosa between two groups. Cluster analysis was adopted to analyze the accumulation of the differentially expressed miRNAs(P<0.05, fold≥2.0 or ≤0.5) and their target genes were predicted with 10 softwares such as DIANAmT, miRanda, miRDB, miRWalk etc. Comprehensive scoring was performed to identify genes with highest predictive score. Gene ontology (GO) concentration technique was used to analyze the target gene-associated biological process. Immunohistochemistry was used to examine protein expression of genes with the highest score. Among 1285 miRNAs in fistula group, 13 miRNAs were differentially expressed with those in hemorrhoid group, including 2 of up-regulation and 11 of down-regulation. Paired t test showed that in fistula group, miRNA-3609 up-regulation was 5.98 folds(P=0.0231) and miR-181a-2-3p down-regulation was 0.13 folds(P=0.0067) compared to those in hemorrhoid group, which had the greatest differential expression. Cluster analysis suggested that up-regulated miR-3609 and miR-6086 had similar change trend in both groups. Among 11 down-regulated miRNAs, miR-125bp-1-3p and miR-548q had similar expression and other 9 miRNAs had similar expression as well, including miR-1185-1-3p, miR-532-3p, miR-1233-5p, miR-769-5p, miR-149-5p, miR-99b-3p, miR-141-3p, miR-138-5p, and miR-181a-2-3p. Target gene prediction analysis of above 13 genes showed that 7 miRNAs(53.8%) were eligible to predict their potential target genes, yielding totally 104 possible target genes. The rest of 6 miRNAs(46.2%) failed to predict any target gene. The highest score in prediction of target gene was chitinase 1(ChIT1) and its corresponding differential miRNA was miR-769-5p(r=-0.94286, P=0.0167). Gene ontology analysis showed that the most associated biological process related with these 104 target genes was keratinization, immune response and signal transduction. Immunohistochemistry revealed ChiT1 expression of anal mucosa in fistula group was significantly higher compared to hemorrhoid group(P<0.01). There is a characteristic miRNAs profile in anal fistula patients, which may play a role in the occurrence and development of anal fistula.

MUC Expression in Gallbladder Epithelial Tissues in Cholesterol-Associated Gallbladder Disease

PubMed Central

Yoo, Kyo-Sang; Choi, Ho Soon; Jun, Dae Won; Lee, Hang Lak; Lee, Oh Young; Yoon, Byung Chul; Lee, Kyeong Geun; Paik, Seung Sam; Kim, Yong Seok; Lee, Jin

2016-01-01

Background/Aims Gallstone pathogenesis is linked to mucin hypersecretion and bacterial infection. Several mucin genes have been identified in gallbladder epithelial cells (GBECs). We investigated MUC expression in cholesterol-associated gallbladder disease and evaluated the relationship between mucin and bacterial infection. Methods The present study involved 20 patients with cholesterol stones with cholecystitis, five with cholesterol stones with cholesterolosis, six with cholesterol polyps, two with gallbladder cancer, and six controls. Canine GBECs treated with lipopolysaccharide were also studied. MUC3, MUC5AC, MUC5B, and MUC6 antibodies were used for dot/slot immunoblotting and immunohistochemical studies of the gallbladder epithelial tissues, canine GBECs, and bile. Reverse-transcription polymerase chain reaction was performed to evaluate MUC3 and MUC5B expression. Results MUC3, MUC5AC, MUC5B, and MUC6 were expressed in the normal gallbladder epithelium, and of those, MUC3 and MUC5B exhibited the highest expression levels. Greatly increased levels of MUC3 and MUC5B expression were observed in the cholesterol stone group, and slightly increased levels were observed in the cholesterol polyp group; MUC3 and MUC5B mRNA was also upregulated in those groups. Canine GBECs treated with lipopolysaccharide also showed upregulation of MUC3 and MUC5B. Conclusions The mucin genes with the highest expression levels in gallbladder tissue in cholesterol-associated diseases were MUC3 and MUC5B. Cholesterol stones and gallbladder infections were associated with increased MUC3 and MUC5B expression. PMID:27563024

Fibroblast growth factor 8 is expressed at higher levels in lactating human breast and in breast cancer.

PubMed

Zammit, C; Coope, R; Gomm, J J; Shousha, S; Johnston, C L; Coombes, R C

2002-04-08

Fibroblast growth factor 8 can transform NIH3T3 cells and its expression has been found to be associated with breast and prostate cancer. Following our finding that fibroblast growth factor 8 mRNA expression is increased in breast cancer, we have undertaken an immunohistochemistry study of fibroblast growth factor 8 expression in a series of human breast tissues and other normal tissues. Our findings confirm increased expression of fibroblast growth factor 8 in malignant breast tissue but also show significant fibroblast growth factor 8 expression in non-malignant breast epithelial cells. No significant difference in fibroblast growth factor 8 expression was found between different grades of ductal carcinoma, lobular carcinoma and ductal carcinoma in-situ or cancer of different oestrogen receptor, progesterone receptor or nodal status. The highest levels of fibroblast growth factor 8 expression were found in lactating breast tissues and fibroblast growth factor 8 was also detected in human milk. A survey of other normal tissues showed that fibroblast growth factor 8 is expressed in the proliferative cells of the dermis and epithelial cells in colon, ovary fallopian tube and uterus. Fibroblast growth factor 8 appears to be expressed in several organs in man and appears to have an importance in lactation.

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin

PubMed Central

Ciocca, Daniel R.; Rozados, Viviana R.; Carrión, F. Darío Cuello; Gervasoni, Silvia I.; Matar, Pablo; Scharovsky, O. Graciela

2003-01-01

Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in resistance to certain cytotoxic drugs. PMID:12820652

Role of microRNAs in senescence and its contribution to peripheral neuropathy in the arsenic exposed population of West Bengal, India.

PubMed

Chatterjee, Debmita; Bandyopadhyay, Apurba; Sarma, Nilendu; Basu, Santanu; Roychowdhury, Tarit; Roy, Sib Sankar; Giri, Ashok K

2018-02-01

Arsenic induced senescence (AIS) has been identified in the population of West Bengal, India very recently. Also there is a high incidence of arsenic induced peripheral neuropathy (PN) throughout India. However, the epigenetic regulation of AIS and its contribution in arsenic induced PN remains unexplored. We recruited seventy two arsenic exposed and forty unexposed individuals from West Bengal to evaluate the role of senescence associated miRNAs (SA-miRs) in AIS and their involvement if any, in PN. The downstream molecules of the miRNA associated with the disease outcome, was also checked by immuoblotting. In vitro studies were conducted with HEK 293 cells and sodium arsenite exposure. Our results show that all the SA-miRs were upregulated in comparison to unexposed controls. miR-29a was the most significantly altered, highest expression being in the arsenic exposed group with PN, suggesting its association with the occurrence of PN. We looked for the expression of peripheral myelin protein 22 (PMP22), a specific target of miR-29a associated with myelination and found that both in vitro and in vivo results showed over-expression of the protein. Since this was quite contrary to miRNA regulation, we checked for intermediate players β-catenin and GSK-3β upon arsenic exposure which affects PMP22 expression. We found that β-catenin was upregulated in vitro and was also highest in the arsenic exposed group with PN while GSK-3β followed the reverse pattern. Our findings suggest that arsenic exposure alters the expression of SA-miRs and the mir-29a/beta catenin/PMP22 axis might be responsible for arsenic induced PN. Copyright © 2017. Published by Elsevier Ltd.

The effect of dietary bacterial organic selenium on growth performance, antioxidant capacity, and Selenoproteins gene expression in broiler chickens.

PubMed

Dalia, A M; Loh, T C; Sazili, A Q; Jahromi, M F; Samsudin, A A

2017-08-18

Selenium (Se) is an essential trace mineral in broilers, which has several important roles in biological processes. Organic forms of Se are more efficient than inorganic forms and can be produced biologically via Se microbial reduction. Hence, the possibility of using Se-enriched bacteria as feed supplement may provide an interesting source of organic Se, and benefit broiler antioxidant system and other biological processes. The objective of this study was to examine the impacts of inorganic Se and different bacterial organic Se sources on the performance, serum and tissues Se status, antioxidant capacity, and liver mRNA expression of selenoproteins in broilers. Results indicated that different Se sources did not significantly (P ≤ 0.05) affect broiler growth performance. However, bacterial organic Se of T5 (basal diet +0.3 mg /kg feed ADS18 Se), T4 (basal diet +0.3 mg /kg feed ADS2 Se), and T3 (basal diet +0.3 mg /kg feed ADS1 Se) exhibited significantly (P ≤ 0.05) highest Se concentration in serum, liver, and kidney respectively. Dietary inorganic Se and bacterial organic Se were observed to significantly affect broiler serum ALT, AST, LDH activities and serum creatinine level. ADS18 supplemented Se of (Stenotrophomonas maltophilia) bacterial strain showed the highest GSH-Px activity with the lowest MDA content in serum, and the highest GSH-Px and catalase activity in the kidney, while bacterial Se of ADS2 (Klebsiella pneumoniae) resulted in a higher level of GSH-Px1 and catalase in liver. Moreover, our study showed that in comparison with sodium selenite, only ADS18 bacterial Se showed a significantly higher mRNA level in GSH-Px1, GSH-Px4, DIO1, and TXNDR1, while both ADS18 and ADS2 showed high level of mRNA of DIO2 compared to sodium selenite. The supplementation of bacterial organic Se in broiler chicken, improved tissue Se deposition, antioxidant status, and selenoproteins gene expression, and can be considered as an effective alternative source of Se in broiler chickens.

Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions.

PubMed

Juhas, Mario; Ajioka, James W

2015-07-01

The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Complete mitochondrial genome of Concholepas concholepas inferred by 454 pyrosequencing and mtDNA expression in two mollusc populations.

PubMed

Núñez-Acuña, Gustavo; Aguilar-Espinoza, Andrea; Gallardo-Escárate, Cristian

2013-03-01

Despite the great relevance of mitochondrial genome analysis in evolutionary studies, there is scarce information on how the transcripts associated with the mitogenome are expressed and their role in the genetic structuring of populations. This work reports the complete mitochondrial genome of the marine gastropod Concholepas concholepas, obtained by 454 pryosequencing, and an analysis of mitochondrial transcripts of two populations 1000 km apart along the Chilean coast. The mitochondrion of C. concholepas is 15,495 base pairs (bp) in size and contains the 37 subunits characteristic of metazoans, as well as a non-coding region of 330 bp. In silico analysis of mitochondrial gene variability showed significant differences among populations. In terms of levels of relative abundance of transcripts associated with mitochondrion in the two populations (assessed by qPCR), the genes associated with complexes III and IV of the mitochondrial genome had the highest levels of expression in the northern population while transcripts associated with the ATP synthase complex had the highest levels of expression in the southern population. Moreover, fifteen polymorphic SNPs were identified in silico between the mitogenomes of the two populations. Four of these markers implied different amino acid substitutions (non-synonymous SNPs). This work contributes novel information regarding the mitochondrial genome structure and mRNA expression levels of C. concholepas. Copyright © 2012 Elsevier Inc. All rights reserved.

Isolation of chicken embryonic stem cell and preparation of chicken chimeric model.

PubMed

Zhang, Yani; Yang, Haiyan; Zhang, Zhentao; Shi, Qingqing; Wang, Dan; Zheng, Mengmeng; Li, Bichun; Song, Jiuzhou

2013-03-01

Chicken embryonic stem cells (ESCs) were separated from blastoderms at stage-X and cultured in vitro. Alkaline phosphatase activity and stage-specific embryonic antigen-1 staining was conducted to detect ESCs. Then, chicken ESCs were transfected with linearized plasmid pEGFP-N1 in order to produce chimeric chicken. Firstly, the optimal electrotransfection condition was compared; the results showed the highest transfection efficiency was obtained when the field strength and pulse duration was 280 V and 75 μs, respectively. Secondly, the hatchability of shedding methods, drilling a window at the blunt end of egg and drilling a window at the lateral shell of egg was compared, the results showed that the hatchability was the highest for drilling a window at the lateral shell of egg. Thirdly, the hatchability of microinjection (ESCs was microinjected into chick embryo cavity) was compared too, the results showed there were significant difference between the injection group transfected with ESCs and that of other two groups. In addition, five chimeric chickens were obtained in this study and EGFP gene was expressed in some organs, but only two chimeric chicken expressed EGFP gene in the gonad, indicating that the chimeric chicken could be obtained through chick embryo cavity injection by drilling a window at the lateral shell of egg.

The E2F3—Oncomir 1 axis is activated in Wilms Tumor

PubMed Central

Kort, Eric J.; Farber, Leslie; Tretiakova, Maria; Petillo, David; Furge, Kyle A.; Yang, Ximing J.; Cornelius, Albert; Teh, Bin T.

2008-01-01

Oncomir-1 is an oncogenic cluster of microRNAs located on chromosome 13. Previous in vitro studies demonstrated that it is transcriptionally regulated by the transcription factor E2F3. In this report we combine expression profiling of both messenger RNA (mRNA) and micro RNAs (miRNA) in Wilms tumor (WT) samples to provide the first evidence that the E2F3—Oncomir 1 axis, previously identified in cell culture, is deregulated in primary human tumors. Analysis of RNA expression signatures demonstrated that an E2F3 gene signature was activated in all Wilms tumor samples analyzed, in contrast to other kidney tumors. This finding was validated by immunohistochemistry (IHC) on the protein level. Expression of E2F3 was lowest in early stage tumors, and highest in metastatic tissue. Expression profiling of miRNAs in WT showed that expression of each measured member of the Oncomir-1 family was highest in WT relative to other kidney tumor subtypes. Quantitative polymerase chain reaction (PCR) confirmed that these microRNAs were overexpressed in Wilms tumor relative to normal kidney tissue. These results suggest that the E2F3—Oncomir-1 axis is activated in Wilms tumor. Our study also demonstrates the utility of integrated genomics combining gene signature analysis with miRNA expression profiling to identify protein-miRNA interactions that are perturbed in disease states. PMID:18519660

[Alkaline-adapted beta-mannanase of Bacillus pumilus: gene heterologous expression and enzyme characterization].

PubMed

Tang, Jiajie; Guo, Su; Wang, Wei; Wei, Wei; Wei, Dongzhi

2015-11-04

We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications. We obtained a mannanase gene (named man(B)) from Bacillus pumilus Nsic2 and expressed the gene man(B) in Escherichia coli and Bacillus subtilis. Furthermore, we characterized the enzyme. The gene man(B) had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid beta-mannanase (Man(B)). The protein sequence showed the highest identity with the beta-mannanase from B. pumilus CCAM080065. We expressed the gene man(B) in E. coli BL21 (DE3) with the enzyme activity of 11021.3 U/mL. Compared with other mannanases, Man(B) showed higher stability under alkaline conditions and was stable at pH6.0 -9.0. The specific activity of purified Man(B) was 4191 ± 107 U/mg. The K(m) and V(max) values of purified Man(B) were 35.7 mg/mL and 14.9 μmol/(mL x min), respectively. Meanwhile, we achieved recombinant protein secretion expression in B. subtilis WB800N. We achieved heterologous expression of the gene man(B) and characterized its enzyme. The alkaline-adapted Man(B) showed potential value in industrial applications due to its pH stability.

YAP expression in normal and neoplastic breast tissue: an immunohistochemical study.

PubMed

Jaramillo-Rodríguez, Yolanda; Cerda-Flores, Ricardo M; Ruiz-Ramos, Ruben; López-Márquez, Francisco C; Calderón-Garcidueñas, Ana Laura

2014-04-01

Yes-associated protein (YAP) is a transcriptional factor involved in normal cell proliferation, apoptosis and carcinogenesis; however, its contribution to breast cancer (BC) is still controversial. We undertook this study to compare the expression of YAP by immunohistochemistry (IHC) in normal breast tissue of women without breast cancer (BC) (controls), non-neoplastic breast tissue in women with cancer (internal controls) and in four different subtypes of invasive ductal carcinoma. There were 17 controls and 105 tumor cases (53 luminal A, 15 luminal B, 20 overexpression of HER2 and 17 triple negative cases) studied by IHC. Statistical analysis included χ(2) for linear trend (Extended Mantel-Haenszel). There were 40% of internal controls that showed expression of YAP in myoepithelial cells, whereas in controls expression was 100%. In controls, 3/17 (17.6%) showed cytoplasmic staining in luminal cells. There was a significant difference in nuclear expression between the ductal BC subtypes. Luminal A had 4% of positive cases with <10% of cells affected in each case; in contrast, there were 17-20% of positive cases in the other groups with 50% or more of stained cells. YAP expression in stromal cells was not observed in controls or in triple-negative cases, and luminal B pattern had the highest YAP nuclear expression (20%). YAP showed decreased expression in tumor cells compared with normal breast tissue. These findings are consistent with a role of YAP as a suppressor gene in BC and show differences in YAP expression in different patterns of ductal BC. Copyright © 2014 IMSS. Published by Elsevier Inc. All rights reserved.

Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

PubMed

Kim, Yong-Kyoung; Kim, Yeon Bok; Uddin, Md Romij; Lee, Sanghyun; Kim, Soo-Un; Park, Sang Un

2014-10-17

To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 μg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4-fold higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.

Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia).

PubMed

Tuan, Pham Anh; Kim, Jae Kwang; Lee, Sanghyun; Chae, Soo Cheon; Park, Sang Un

2012-12-05

Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.

Anti-Inflammatory Effect of Angelica gigas via Heme Oxygenase (HO)-1 Expression.

PubMed

Cho, Joon Hyeong; Kwon, Jung Eun; Cho, Youngmi; Kim, Inhye; Kang, Se Chan

2015-06-15

Angelica gigas (AG) is effective against various medical conditions such as bacterial infection, inflammation, and cancer. It contains a number of coumarin compounds and the group of interest is the pyranocoumarin, which comprises decursin and decursinol angelate. This group has an effect on controlling inflammation, which is caused by excessive nitric oxide (NO) production. Heme oxygenases (HOs), particularly HO-1, play a role in regulating the production of NO. Thus, this study aimed to investigate the anti-inflammatory effects of AG by measuring HO-1 expression. Treatments with CH2Cl2 layer and Angelica gigas extract (AGE) showed the highest NO inhibition effects. Decursin, decursinol angelate, and nodakenin were isolated from the CH2Cl2 layer of AGE. Decursin also demonstrated the highest anti-oxidative effect among the coumarins. Although decursin had the best NO inhibition and anti-oxidative effects, the effects of AGE treatment far surpassed that of decursin. This is owing to the combination effect of the coumarins present within AGE, which is a solvent extract of AG. The expression of HO-1 is an effective indicator of the anti-inflammatory effects of AG. Based on the results of the coumarin compounds, HO-1 expression was found to be dose dependent and specific to decursin.

Anti-Inflammatory Effect of Angelica gigas via Heme Oxygenase (HO)-1 Expression

PubMed Central

Cho, Joon Hyeong; Kwon, Jung Eun; Cho, Youngmi; Kim, Inhye; Kang, Se Chan

2015-01-01

Angelica gigas (AG) is effective against various medical conditions such as bacterial infection, inflammation, and cancer. It contains a number of coumarin compounds and the group of interest is the pyranocoumarin, which comprises decursin and decursinol angelate. This group has an effect on controlling inflammation, which is caused by excessive nitric oxide (NO) production. Heme oxygenases (HOs), particularly HO-1, play a role in regulating the production of NO. Thus, this study aimed to investigate the anti-inflammatory effects of AG by measuring HO-1 expression. Treatments with CH2Cl2 layer and Angelica gigas extract (AGE) showed the highest NO inhibition effects. Decursin, decursinol angelate, and nodakenin were isolated from the CH2Cl2 layer of AGE. Decursin also demonstrated the highest anti-oxidative effect among the coumarins. Although decursin had the best NO inhibition and anti-oxidative effects, the effects of AGE treatment far surpassed that of decursin. This is owing to the combination effect of the coumarins present within AGE, which is a solvent extract of AG. The expression of HO-1 is an effective indicator of the anti-inflammatory effects of AG. Based on the results of the coumarin compounds, HO-1 expression was found to be dose dependent and specific to decursin. PMID:26083119

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder (Paralichthys olivaceus)

NASA Astrophysics Data System (ADS)

Huang, Yajuan; Hu, Nan; Si, Yufeng; Li, Siping; Wu, Shuxian; Zhang, Meizhao; Wen, Haishen; Li, Jifang; Li, Yun; He, Feng

2018-06-01

Follistatin (Fst) is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns of Fst gene at five different development stages of Japanese flounder (stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months). The muscle tissue of Japanese flounder was obtained at different development stages in this experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

Larval hemolymph of rhinoceros beetle, Allomyrina dichotoma, enhances insulin secretion through ATF3 gene expression in INS-1 pancreatic β-cells.

PubMed

Kim, Seung-Whan; Suh, Hyun-Woo; Yoo, Bo-Kyung; Kwon, Kisang; Yu, Kweon; Choi, Ji-Young; Kwon, O-Yu

2018-05-22

In this study, we show that INS-1 pancreatic β-cells treated for 2 h with hemolymph of larvae of rhinoceros beetle, Allomyrina dichotoma, secreted about twice as much insulin compared to control cells without such treatment. Activating transcription factor 3 (ATF3) was the highest upregulated gene in DNA chip analysis. The A. dichotoma hemolymph dose-dependently induced increased expression levels of genes encoding ATF3 and insulin. Conversely, treatment with ATF3 siRNA inhibited expression levels of both genes and curbed insulin secretion. These results suggest that the A. dichotoma hemolymph has potential for treating and preventing diabetes or diabetes-related complications.

Digital gene expression analysis of the zebra finch genome

PubMed Central

2010-01-01

Background In order to understand patterns of adaptation and molecular evolution it is important to quantify both variation in gene expression and nucleotide sequence divergence. Gene expression profiling in non-model organisms has recently been facilitated by the advent of massively parallel sequencing technology. Here we investigate tissue specific gene expression patterns in the zebra finch (Taeniopygia guttata) with special emphasis on the genes of the major histocompatibility complex (MHC). Results Almost 2 million 454-sequencing reads from cDNA of six different tissues were assembled and analysed. A total of 11,793 zebra finch transcripts were represented in this EST data, indicating a transcriptome coverage of about 65%. There was a positive correlation between the tissue specificity of gene expression and non-synonymous to synonymous nucleotide substitution ratio of genes, suggesting that genes with a specialised function are evolving at a higher rate (or with less constraint) than genes with a more general function. In line with this, there was also a negative correlation between overall expression levels and expression specificity of contigs. We found evidence for expression of 10 different genes related to the MHC. MHC genes showed relatively tissue specific expression levels and were in general primarily expressed in spleen. Several MHC genes, including MHC class I also showed expression in brain. Furthermore, for all genes with highest levels of expression in spleen there was an overrepresentation of several gene ontology terms related to immune function. Conclusions Our study highlights the usefulness of next-generation sequence data for quantifying gene expression in the genome as a whole as well as in specific candidate genes. Overall, the data show predicted patterns of gene expression profiles and molecular evolution in the zebra finch genome. Expression of MHC genes in particular, corresponds well with expression patterns in other vertebrates. PMID:20359325

K-Lysine acetyltransferase 2a regulates a hippocampal gene expression network linked to memory formation

PubMed Central

Stilling, Roman M; Rönicke, Raik; Benito, Eva; Urbanke, Hendrik; Capece, Vincenzo; Burkhardt, Susanne; Bahari-Javan, Sanaz; Barth, Jonas; Sananbenesi, Farahnaz; Schütz, Anna L; Dyczkowski, Jerzy; Martinez-Hernandez, Ana; Kerimoglu, Cemil; Dent, Sharon YR; Bonn, Stefan; Reymann, Klaus G; Fischer, Andre

2014-01-01

Neuronal histone acetylation has been linked to memory consolidation, and targeting histone acetylation has emerged as a promising therapeutic strategy for neuropsychiatric diseases. However, the role of histone-modifying enzymes in the adult brain is still far from being understood. Here we use RNA sequencing to screen the levels of all known histone acetyltransferases (HATs) in the hippocampal CA1 region and find that K-acetyltransferase 2a (Kat2a)—a HAT that has not been studied for its role in memory function so far—shows highest expression. Mice that lack Kat2a show impaired hippocampal synaptic plasticity and long-term memory consolidation. We furthermore show that Kat2a regulates a highly interconnected hippocampal gene expression network linked to neuroactive receptor signaling via a mechanism that involves nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In conclusion, our data establish Kat2a as a novel and essential regulator of hippocampal memory consolidation. PMID:25024434

Cloning and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum

PubMed Central

Shibuya, Hajime; Nagasaki, Hiroaki; Kaneko, Satoshi; Yoshida, Shigeki; Park, Gwi Gun; Kusakabe, Isao; Kobayashi, Hideyuki

1998-01-01

The cDNA coding for Penicillium purpurogenum α-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides. PMID:9797312

Bone morphogenetic protein 2 and decorin expression in old fracture fragments and surrounding tissues.

PubMed

Han, X G; Wang, D K; Gao, F; Liu, R H; Bi, Z G

2015-09-21

Bone morphogenetic protein 2 (BMP-2) can promote fracture healing. Although the complex role BMP-2 in bone formation is increasingly understood, the role of endogenous BMP-2 in nonunion remains unclear. Decorin (DCN) can promote the formation of bone matrix and calcium deposition to control bone morphogenesis. In this study, tissue composition and expression of BMP-2 and DCN were detected in different parts of old fracture zones to explore inherent anti-fibrotic ability and osteogenesis. Twenty-three patients were selected, including eight cases of delayed union and 15 cases of nonunion. Average duration of delayed union or nonunion was 15 months. Fracture fragments and surrounding tissues, including bone grafts, marrow cavity contents, and sticking scars, were categorically sampled during surgery. Through observation and histological testing, component comparisons were made between fracture fragments and surrounding tissue. The expression levels of DCN and BMP-2 in different tissues were detected by immunohistochemical staining and real-time polymerase chain reaction. The expression of DCN and BMP- 2 in different parts of the nonunion area showed that, compared with bone graft and marrow cavity contents, sticking scars had the highest expression of BMP-2. Compared with the marrow cavity contents and sticking scars, bone grafts had the highest expression of DCN. The low antifibrotic and osteogenic activity of the nonunion area was associated with non-co-expression of BMP-2 and DCN. Therefore, the co-injection of osteogenic factor BMP and DCN into the nonunion area can improve the induction of bone formation and enhance the conversion of the old scar, thereby achieving better nonunion treatment.

Expression profiling of various genes during the fruit development and ripening of mango.

PubMed

Pandit, Sagar S; Kulkarni, Ram S; Giri, Ashok P; Köllner, Tobias G; Degenhardt, Jörg; Gershenzon, Jonathan; Gupta, Vidya S

2010-06-01

Mango (Mangifera indica L. cv. Alphonso) development and ripening are the programmed processes; conventional indices and volatile markers help to determine agronomically important stages of fruit life (fruit-setting, harvesting maturity and ripening climacteric). However, more and precise markers are required to understand this programming; apparently, fruit's transcriptome can be a good source of such markers. Therefore, we isolated 18 genes related to the physiology and biochemistry of the fruit and profiled their expression in developing and ripening fruits, flowers and leaves of mango using relative quantitation PCR. In most of the tissues, genes related to primary metabolism, abiotic stress, ethylene response and protein turnover showed high expression as compared to that of the genes related to flavor production. Metallothionin and/or ethylene-response transcription factor showed highest level of transcript abundance in all the tissues. Expressions of mono- and sesquiterpene synthases and 14-3-3 lowered during ripening; whereas, that of lipoxygenase, ethylene-response factor and ubiquitin-protein ligase increased during ripening. Based on these expression profiles, flower showed better positive correlation with developing and ripening fruits than leaf. Most of the genes showed their least expression on the second day of harvest, suggesting that harvesting signals significantly affect the fruit metabolism. Important stages in the fruit life were clearly indicated by the significant changes in the expression levels of various genes. These indications complemented those from the previous analyses of fruit development, ripening and volatile emission, revealing the harmony between physiological, biochemical and molecular activities of the fruit.

Effects of subchronic benzo(a)pyrene exposure on neurotransmitter receptor gene expression in the rat hippocampus related with spatial learning and memory change.

PubMed

Qiu, Chongying; Cheng, Shuqun; Xia, Yinyin; Peng, Bin; Tang, Qian; Tu, Baijie

2011-11-18

Exposure of laboratory rats to Benzo(a)pyrene (BaP), an environmental contaminant with its high lipophilicify which is widely dispersed in the environment and can easily cross the blood brain barrier presenting in the central nervous system, is associated with impaired learning and memory. The purpose of the research was to examine whether subchronic exposure to BaP affects spatial learning and memory, and how it alters normal gene expression in hippocampus, as well as selection of candidate genes involving neurotransmitter receptor attributed to learning and memory. Morris water maze (MWM) was used to evaluate behavioral differences between BaP-treated and vehicle-treated groups. To gain a better insight into the mechanism of BaP-induced neurotoxicity on learning and memory, we used whole genome oligo microarrays as well as Polymerase Chain Reaction (PCR) to assess the global impact of gene expression. Male Sprague-Dawley rats were intraperitoneally injected with 6.25mg/kg of BaP or vehicle for 14 weeks. The results from the Morris water maze (MWM) test showed that rats treated with BaP exhibited significantly higher mean latencies as compared to vehicle controls. BaP exposure significantly decreased the number of crossing the platform and the time spent in the target area. After the hippocampus was collected from each rat, total RNA was isolated. Microarray and PCR revealed that exposure to BaP affected mRNA expression of neurotransmitter receptors. The web tool DAVID was used to analyze the significantly enriched gene ontology (GO) and KEGG pathways in the differentially expressed genes. Analysis showed that the most significantly affected gene ontology category was behavior. Furthermore, the fourth highest significantly affected gene ontology category was learning and memory. KEGG molecular pathway analysis showed that "neuroactive ligand-receptor interaction" was affected by BaP with highest statistical significance, and 9 candidate neurotransmitter receptor genes involving learning and memory were selected out. Our results revealed a close link between behavioral changes and altered neurotransmitter receptor gene expression in BaP-treated rats. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Cloning and Expression of Yak Active Chymosin in Pichia pastoris

PubMed Central

Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

2016-01-01

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production. PMID:27004812

Cloning and Expression of Yak Active Chymosin in Pichia pastoris.

PubMed

Luo, Fan; Jiang, Wei Hua; Yang, Yuan Xiao; Li, Jiang; Jiang, Ming Feng

2016-09-01

Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.

The novel putative bile acid transporter SLC10A5 is highly expressed in liver and kidney

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

Fernandes, Carla F.; Godoy, Jose R.; Doering, Barbara

2007-09-14

Here we report the identification, cloning, and characterization of SLC10A5, which is a new member of Solute Carrier Family 10 (SLC10), also known as the 'sodium/bile acid cotransporter family'. Expression of SLC10A5/Slc10a5 was examined by quantitative real-time PCR and revealed its highest expression levels in liver and kidney in humans, rat and mouse. In rat liver and kidney, Slc10a5 expression was localized by in situ hybridization to hepatocytes and proximal tubules, respectively. A SLC10A5-FLAG fusion protein was expressed in HEK293 cells and showed an apparent molecular weight of 42 kDa after immunoprecipitation. When expressed in Xenopus laevis oocytes, the SLC10A5-FLAGmore » protein was detected in the oocyte's plasma membrane but showed no transport activity for taurocholate, cholate, estrone-3-sulfate, or dehydroepiandrosterone sulfate. As bile acid carriers are the most related carriers to SLC10A5 though, we strongly suppose that SLC10A5 is an orphan carrier with yet non-identified substrates.« less

Synthesis and in vitro transfection efficiency of spermine-based cationic lipids with different central core structures and lipophilic tails.

PubMed

Niyomtham, Nattisa; Apiratikul, Nuttapon; Suksen, Kanoknetr; Opanasopit, Praneet; Yingyongnarongkul, Boon-Ek

2015-02-01

Twelve spermine-based cationic lipids with four different central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) and three hydrophobic tails (lauric acid, myristic acid and palmitic acid) were synthesized. The liposomes containing lipids and DOPE showed moderate to good in vitro DNA delivery into HeLa cells. GFP expression experiments revealed that liposomes composed of lipids with 3-amino-1,2-dioxypropyl as a central core structure exhibited highest transfection efficiency under serum-free condition. Whereas, lipid with 2-amino-1,3-dioxypropyl core structure showed highest transfection under 10% serum condition. Moreover, the liposomes and lipoplexes composted of these cationic lipids exhibited low cytotoxicity. Copyright © 2015. Published by Elsevier Ltd.

[Expressions of Ras and Sos1 in epithelial ovarian cancer tissues and their clinical significance].

PubMed

Xiao, Zheng-Hua; Linghu, Hua; Liu, Qian-Fen

2016-11-20

To detect the expressions of Ras and Sos1 proteins in human epithelial ovarian cancer (EOC) tissues and explore their correlation with the clinicopathological features of the patients. The expressions of Ras and Sos1 proteins were detected immunohistochemically in 62 EOC tissues, 5 borderline ovarian cancer tissues, 15 benign epithelial ovarian neoplasm tissues, and 18 normal ovarian tissues. The EOC tissues showed significantly higher expression levels of both Ras and Sos1 than the other tissues tested (P<0.05). In EOC tissues, Ras and Sos1 proteins were expressed mostly on the cell membrane and in the cytoplasm. The expression level of Ras was correlated with pathological types of the tumor (P<0.05) and was the highest in serous cystadenomcarcinoma; Sos1 expression did not show significant correlation with the clinicopathological indexes of the patients. High expressions of both Ras and Sos1 proteins were associated with shorter progression-free survival of the patients, but this association was not statistically significant. Ras and Sos1 protein may participate in in the occurrence and development of EOC. The tissue-specific variation of Ras expression can lend support to a specific diagnosis of ovarian serous adenocarcinoma. The association of Ras and Sos1 protein expression with the tumor-free survival time of the patients awaits further investigation with a larger sample size.

Gene Expression Analysis of Mouse Embryonic Stem Cells Following Levitation in an Ultrasound Standing Wave Trap

PubMed Central

Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

2011-01-01

In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08–0.85 MPa) and times (5–60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. (E-mail: Bazoud@tcd.ie) PMID:21208732

Adipocyte aminopeptidases in obesity and fasting.

PubMed

Alponti, Rafaela Fadoni; Silveira, Paulo Flavio

2015-11-05

This study checked the existence of a diverse array of aminopeptidase (AP) enzymes in high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from adipocytes of control, monosodium glutamate obese and food deprived rats. Gene expression was detected for ArgAP, AspAP, MetAP, and two AlaAP (APM and PSA). APM and PSA had the highest catalytic efficiency, whereas AspAP the highest affinity. Subcellular distribution of AP activities depended on metabolic status. Comparing catalytic levels, AspAP in HDM, LDM and MF was absent in obese and control under food deprivation; PSA in LDM was 3.5-times higher in obese than in normally fed control and control and obese under food deprivation; MetAP in MF was 4.5-times higher in obese than in food deprived obese. Data show new AP enzymes genetically expressed in subcellular compartments of adipocytes, three of them with altered catalytic levels that respond to whole-body energetic demands. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Generative complexity of Gray-Scott model

NASA Astrophysics Data System (ADS)

Adamatzky, Andrew

2018-03-01

In the Gray-Scott reaction-diffusion system one reactant is constantly fed in the system, another reactant is reproduced by consuming the supplied reactant and also converted to an inert product. The rate of feeding one reactant in the system and the rate of removing another reactant from the system determine configurations of concentration profiles: stripes, spots, waves. We calculate the generative complexity-a morphological complexity of concentration profiles grown from a point-wise perturbation of the medium-of the Gray-Scott system for a range of the feeding and removal rates. The morphological complexity is evaluated using Shannon entropy, Simpson diversity, approximation of Lempel-Ziv complexity, and expressivity (Shannon entropy divided by space-filling). We analyse behaviour of the systems with highest values of the generative morphological complexity and show that the Gray-Scott systems expressing highest levels of the complexity are composed of the wave-fragments (similar to wave-fragments in sub-excitable media) and travelling localisations (similar to quasi-dissipative solitons and gliders in Conway's Game of Life).

Effects of exogenous inosine monophosphate on growth performance, flavor compounds, enzyme activity, and gene expression of muscle tissues in chicken.

PubMed

Yan, Junshu; Liu, Peifeng; Xu, Liangmei; Huan, Hailin; Zhou, Weiren; Xu, Xiaoming; Shi, Zhendan

2018-04-01

The goal of this experiment was to examine effects of diets supplemented with exogenous inosine monophosphate (IMP) on the growth performance, flavor compounds, enzyme activity and gene expression of chicken. A total of 1,500 healthy, 1-day-old male 3-yellow chickens were used for a 52-d experimental period. Individuals were randomly divided into 5 groups (group I, II, III, IV, V) with 6 replicates per group, and fed a basal diet supplemented with 0.0, 0.05, 0.1, 0.2, and 0.3% IMP, respectively. There was no significant response to the increasing dietary IMP level in average daily feed intake (ADFI), average daily gain (ADG), and feed:gain ratio (F/G) (P ≥ 0.05). IMP content of the breast and thigh muscle showed an exponential and linear response to the increasing dietary IMP level (P < 0.05), the highest IMP content was obtained when the diet with 0.3% and 0.2% exogenous IMP was fed. There were significant effects of IMP level in diet on free amino acids (FAA) (exponential, linear and quadratic effect, P < 0.05) and delicious amino acids (DAA) (quadratic effect, P < 0.01) content in breast muscle. FAA and DAA content in thigh muscle showed an exponential and linear response (P < 0.05), and quadratic response (P < 0.01) to the increasing dietary IMP level, the highest FAA and DAA content was obtained when the diet with 0.2% exogenous IMP was fed. Dietary IMP supplementation had a quadratic effect on 5΄-NT and the alkaline phosphatase (ALP) enzyme activity in the breast muscle (P < 0.05), and the adenosine triphosphate (ATP) enzyme activity in the thigh muscles increased exponentially and linearly with increasing IMP level in diet (exponential effect, P = 0.061; linear effect, P = 0.059). Cyclohydrolase (ATIC) gene expression in thigh muscle had a quadratic response to the increasing dietary IMP level (P < 0.05), 0.2% exogenous IMP group had the highest (AMPD1) gene expression of the breast muscle and ATIC gene expression of the thigh muscle. These results indicate that dietary IMP did not affect the growth performance of chicken, the diet with 0.2 to 0.3% exogenous IMP is optimal to improve the meat flavor quality in chicken.

Placental expression of EG-VEGF and its receptors PKR1 (prokineticin receptor-1) and PKR2 throughout mouse gestation.

PubMed

Hoffmann, P; Feige, J-J; Alfaidy, N

2007-10-01

Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be disregulated in pre-eclampsia (PE). Recently, we characterised the expression of EG-VEGF (endocrine gland-derived vascular endothelial growth factor), also known as prokineticin 1 (PK1) in human placenta during the first trimester of pregnancy and showed that this factor is likely to play an important role in human placentation. However, because it is impossible to prospectively study placentation in humans, it has been impossible to further characterise EG-VEGF expression throughout complete gestation and especially at critical gestational ages for PE development. In the present study, we used mouse placenta to further characterise EG-VEGF expression throughout gestation. We investigated the pattern of expression of EG-VEGF and its receptors, PKR1 and PKR2 at the mRNA and protein levels. Our results show that EG-VEGF and VEGF exhibit different patterns of expression and different localisations in the mouse placenta. EG-VEGF was mainly localised in the labyrinth whereas VEGF was mainly present in glycogen and giant cells. EG-VEGF mRNA and protein levels were highest before 10.5days post coitus (dpc) whereas those of VEGF showed stable expression throughout gestation. PKR1 protein was localised to the labyrinth layer and showed the same pattern of expression as EG-VEGF whereas PKR2 expression was maintained over 10.5dpc with both trophoblastic and endothelial cell localisations. Altogether these findings suggest that EG-VEGF may have a direct effect on both endothelial and trophoblastic cells and is likely to play an important role in mouse placentation.

Microarray analysis of human milk cells: persistent high expression of osteopontin during the lactation period

PubMed Central

NAGATOMO, T; OHGA, S; TAKADA, H; NOMURA, A; HIKINO, S; IMURA, M; OHSHIMA, K; HARA, T

2004-01-01

To continue the search for immunological roles of breast milk, cDNA microarray analysis on cytokines and growth factors was performed for human milk cells. Among the 240 cytokine-related genes, osteopontin (OPN) gene ranked top of the expression. Real-time PCR revealed that the OPN mRNA levels in colostrum cells were approximately 100 times higher than those in PHA-stimulated peripheral blood mononuclear cells (PBMNCs), and 10 000 times higher than those in PB CD14+ cells. The median levels of OPN mRNA in early milk or mature milk cells were more than three times higher than those in colostrum cells. Western blot analysis of human milk showed appreciable expression of full-length and short form proteins of OPN. The concentrations of full-length OPN in early milk or mature milk whey continued to be higher than those in colostrum whey and plasma as assessed by ELISA. The early milk (3–7 days postpartum) contained the highest concentrations of OPN protein, while the late mature milk cells (1 years postpartum) had the highest expression of OPN mRNA of all the lactating periods. The results of immunohistochemical and immunocytochemical staining indicated that OPN-producing epithelial cells and macrophages are found in actively lactating mammary glands. These results suggest that the persistently and extraordinarily high expression of OPN in human milk cells plays a potential role in the immunological development of breast-fed infants. PMID:15373904

High Expression of CCR7 Predicts Lymph Node Metastasis and Good Prognosis in Triple Negative Breast Cancer.

PubMed

Li, Xuelu; Sun, Siwen; Li, Ning; Gao, Jiyue; Yu, Jing; Zhao, Jinbo; Li, Man; Zhao, Zuowei

2017-01-01

Previous preclinical and clinical studies have reported a positive correlation between the expression of the C-C chemokine receptor 7 (CCR7) and the incidence of lymph node metastasis in breast cancer. However, the prognostic relevance of CCR7 expression in breast cancer remains contradictory till now. The aim of this study is to assess the correlation of the CCR7 expression with other clinicopathological features and prognosis in breast cancer. The CCR7 gene amplification and mRNA expression levels from approximately 3,000 patients were retrieved from human breast cancer databases and analyzed. Furthermore, a total of 188 primary triple negative breast cancer patients were enrolled in this study (diagnosed since January 2009 to January 2013 from the Second Hospital of Dalian Medical University). The protein levels of CCR7 were examined by immunohistochemistry using paraffin-embedded tumor tissues. The analysis of gene amplification and mRNA levels showed the expression of CCR7 in breast cancer correlated with better prognosis. When we compared the CCR7 expressions in different subtypes, the basal-like group showed the highest expression of CCR7 and exhibited a better prognosis. Consistently, Kaplan-Meier analysis of 188 triple negative breast cancer patients showed that the prognosis of patients with positive CCR7 expression was significantly better than those with negative expression (HR=0.642, p=0.0275). Additionally, we also observed a positive correlation between lymph node metastasis and the CCR7 expression (p=0.0096). Our results indicated that elevated CCR7 expression as a marker for increased lymph node metastasis, in addition to serve as an independent prognostic indicator for better overall survival in triple negative breast cancer patients. © 2017 The Author(s). Published by S. Karger AG, Basel.

RNA/DNA ratio and LPL and MyoD mRNA expressions in muscle of Oreochromis niloticus fed with elevated levels of palm oil

NASA Astrophysics Data System (ADS)

Ayisi, Christian Larbi; Zhao, Jinliang

2016-02-01

Palm oil is of great potential as one of the sustainable alternatives to fish oil (FO) in aquafeeds. In this present study, five isonitrogenous diets (32% crude protein) with elevated palm oil levels of 0%, 2%, 4%, 6% and 8% were used during an 8-week feeding trial to evaluate its effects on RNA/DNA ratio and lipoprotein lipase (LPL) and MyoD mRNA expressions in muscle of Oreochromis niloticus. The results showed that RNA, DNA content as well as ratio of RNA to DNA were significantly affected ( P < 0.05), in each case the highest was recorded in fish group subjected to 6% palm oil level. There was a strong positive correlation between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and specific growth rate (SGR), protein efficiency ratio (PER), while a negative correlation existed between nucleic acid concentration (RNA concentration and RNA: DNA ratio) and feed conversion ratio (FCR). The mRNA expressions of LPL and MyoD in muscle were not significantly affected by the different palm oil levels, although the highest expression was observed in fish fed with 6% palm oil level. There also existed a strong positive correlation between the mRNA expression of LPL, MyoD and SGR, PER, while their correlation with FCR was negative. In conclusion, elevated palm oil affected the RNA, DNA concentration as well as RNA/DNA ratio significantly, although the mRNA expression of LPL and MyoD were not affected significantly by elevated palm oil levels.

Effects of different dwarfing interstocks on key enzyme activities and the expression of genes related to malic acid metabolism in Red Fuji apples.

PubMed

Shi, J; Li, F F; Ma, H; Li, Z Y; Xu, J Z

2015-12-22

In this experiment, the test materials were 'Red Fuji' apple trees grafted onto three interstocks (No. 53, No. 111, and No. 236), which were chosen from SH40 seeding interstocks. The content of malic acid, the enzyme activities, and the expression of genes related to malic acid metabolism were determined during fruit development.The results showed that malic acid content in the ripe fruit on interstock No. 53 was higher than that in the interstock No. 111 fruit. The malate dehydrogenase (NAD-MDH) activity in apples on interstock No. 53 was highest on Day 30, Day 100, and Day 160 after bloom, and the malic enzyme (NADP-ME) activity in apples on interstock No. 111 was higher than in the interstock No. 53 fruit from Day 70 to Day 100 after bloom. The relative expression of NAD-MDH genes in interstock No. 53 fruit was higher than in No. 236 fruit on Day 100 after bloom, but the relative expression of NADP-ME in No. 236 interstock fruit was lower than in No. 53 fruit. The relative expression of NAD-MDH genes in No. 53 interstock fruit was highest on Day 160 after bloom. This might have been the main reason for the difference in the accumulation of malic acid in the ripe apples.There was a positive correlation between the relative expression of phosphoenolpyruvate carboxylase (PEPC) and the malic acid content of the fruit, and the content of malic acid in the apples was affected by the PEPC activity during the early developmental stage.

Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi

NASA Astrophysics Data System (ADS)

Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Urbanelli, Sandra; Sassera, Davide; De Marco, Leone; Mereghetti, Valeria; Montagna, Matteo; Ricci, Irene; Favia, Guido; Bandi, Claudio

2014-12-01

In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps.

Functional diversity of arbuscular mycorrhizas extends to the expression of plant genes involved in P nutrition.

PubMed

Burleigh, Stephen H; Cavagnaro, Tim; Jakobsen, Iver

2002-07-01

This study of functional diversity considers symbiotic associations between two plant species, Medicago truncatula and Lycopersicon esculentum, and seven species of arbuscular mycorrhizal fungi (AMF). The objective was to integrate physiological analyses with molecular techniques to test whether functional diversity between AMF species is not only apparent at the level of mycorrhiza formation, plant nutrient uptake and plant growth, but also at the molecular level as observed by variation in the root expression of plant genes involved in the plant's P-starvation response. The seven species of AMF varied widely in their influence on the root expression of MtPT2 and Mt4 from M. truncatula and LePT1 and TPSI1 from L. esculentum. At one extreme was Glomus mosseae, whereby its colonization of M. truncatula resulted in the greatest reduction in MtPT2 and Mt4 gene expression and the highest level of P uptake and growth, while at the other extreme was Gigaspora rosea, whereby colonization resulted in the highest levels of MtPT2 and Mt4 gene expression and the lowest P uptake and growth. The expression of LePT1 and TPSI1 within the roots of L. esculentum was low and relatively uniform across the seven mycorrhizas, reflecting the ability of this cultivar to maintain low and constant shoot P levels despite root colonization by a broad selection of AMF. This study extends current understanding of functional diversity and shows that plants can respond differently to AMF, not only at the level of colonization, nutrient uptake and growth, but also at the level of gene expression.

Cloning HSP70 and HSP90 genes of kaluga (Huso dauricus) and the effects of temperature and salinity stress on their gene expression.

PubMed

Peng, Guogan; Zhao, Wen; Shi, Zhenguang; Chen, Huirong; Liu, Yang; Wei, Jie; Gao, Fengying

2016-03-01

The genes encoding HSP70 and HSP90 proteins were isolated from kaluga by homologous cloning and rapid amplification of complementary DNA (cDNA) ends (RACE). HSP70 (GenBank accession no. KP050541) and HSP90 (GenBank accession no. KP050542) cDNAs were composed of 2275 and 2718 bp and encoded polypeptides of 650 and 725 amino acids, respectively. Basic Local Alignment Search Tool (BLAST) analysis showed that HSP70 and HSP90 of kaluga shared high identities with those of Acipenser ruthenus, Acipenser schrenckii, and Acipenser baerii (98-99 %). Fluorescent real-time RT-PCR under unstressed conditions revealed that HSP70 and HSP90 were expressed in 11 different tissues of kaluga. Messenger RNA (mRNA) expressions of both HSP70 and HSP90 were highest in the intestine and lowest in the muscle. In addition, the patterns of mRNA expression of HSP70 and HSP90 were similar, although the level of expression was more in HSP90 than in HSP70 (P < 0.05).We also analyzed patterns of HSP70 and HSP90 expression in the muscle, gill, and liver of kaluga under different combinations of temperature and salinity stress, including temperatures of 4,10, 25, and 28 °C at 0 ppt salinity, and salinities of 10, 20, 30, and 40 ppt at 16 °C, where 16 °C at 0 ppt (parts per thousand) served as the control. We found that levels of mRNA expression of both HSP70 and HSP90 were highest at 4 °C in the muscle, gill, and liver and changed little with salinity stress. These results increase understanding of the mechanisms of stress response of cold freshwater fish.

Expression of HSPs: an adaptive mechanism during long-term heat stress in goats ( Capra hircus)

NASA Astrophysics Data System (ADS)

Dangi, Satyaveer Singh; Gupta, Mahesh; Dangi, Saroj K.; Chouhan, Vikrant Singh; Maurya, V. P.; Kumar, Puneet; Singh, Gyanendra; Sarkar, Mihir

2015-08-01

Menacing global rise in surface temperature compelled more focus of research over understanding heat stress response mechanism of animals and mitigation of heat stress. Twenty-four goats divided into four groups ( n = 6) such as NHS (non-heat-stressed), HS (heat-stressed), HS + VC (heat-stressed administered with vitamin C), and HS + VE + Se (heat-stressed administered with vitamin E and selenium). Except NHS group, other groups were exposed to repeated heat stress (42 °C) for 6 h on 16 consecutive days. Blood samples were collected at the end of heat exposure on days 1, 6, 11, and 16. When groups compared between days, expression of all heat shock proteins (HSPs) showed a similar pattern as first peak on day 1, reached to basal level on the sixth day, and followed by second peak on day 16. The relative messenger RNA (mRNA) and protein expression of HSP 60, HSP70, and HSP90 was observed highest ( P < 0.05) in HS group, followed by antioxidant-administered group on days 1 and 16, which signifies that antioxidants have dampening effect on HSP expression. HSP105/110 expression was highest ( P < 0.05) on day 16. We conclude that HSP expression pattern is at least two-peak phenomenon, i.e., primary window of HSP protection on the first day followed by second window of protection on day 16. HSP60, HSP70, and HSP90 play an important role during the initial phase of heat stress acclimation whereas HSP105/110 joins this cascade at later phase. Antioxidants may possibly attenuate the HSP expression by reducing the oxidative stress.

pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12.

PubMed

Maurer, Lisa M; Yohannes, Elizabeth; Bondurant, Sandra S; Radmacher, Michael; Slonczewski, Joan L

2005-01-01

Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with an alpha level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.

Extensive nitrification and active ammonia oxidizers in two contrasting coastal systems of the Baltic Sea.

PubMed

Happel, Elisabeth; Bartl, Ines; Voss, Maren; Riemann, Lasse

2018-06-19

Nitrification is important in nitrogen (N) cycling of aquatic environments, but knowledge about its regulation and importance is sparse. Here we examined nitrification and ammonia oxidizers in the Baltic Sea. We investigated two sites with different catchment characteristics (agricultural and forest), the Bay of Gdánsk (south) and the Öre Estuary (north), and measured pelagic nitrification rates and abundance, composition, and expression of ammonia monooxygenase (amoA) genes. Highest nitrification rates were found in the nutrient rich Bay of Gdańsk. Interestingly, abundances of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were orders of magnitude lower than reported from other sites. Although AOA were most abundant at both sites, the highest expression levels were from AOB. Interestingly, few AOA and AOB taxa dominated amoA gene expression, with a Nitrosomarinus related phylotype showing widespread expression. AOA and AOB communities differed between sites and depths, respectively, with the composition in rivers being distinct. A storm event, causing an even depth distribution of nitrification and particles in the Bay of Gdańsk, indicated that the presence of particles stimulate nitrification. The study highlights coastal regions as dynamic sites of extensive pelagic nitrification, which may affect local food web dynamics and loss of N mediated by denitrification. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Genome-wide identification, classification, and analysis of NADP-ME family members from 12 crucifer species.

PubMed

Tao, Peng; Guo, Weiling; Li, Biyuan; Wang, Wuhong; Yue, Zhichen; Lei, Juanli; Zhao, Yanting; Zhong, Xinmin

2016-06-01

NADP-dependent malic enzymes (NADP-MEs) play essential roles in both normal development and stress responses in plants. Here, genome-wide analysis was performed to identify 65 putative NADP-ME genes from 12 crucifer species. These NADP-ME genes were grouped into five categories of syntenic orthologous genes and were divided into three clades of a phylogenic tree. Promoter motif analysis showed that NADP-ME1 genes in Group IV were more conserved with each other than the other NADP-ME genes in Groups I and II. A nucleotide motif involved in ABA responses, desiccation and seed development was found in the promoters of most NADP-ME1 genes. Generally, the NADP-ME genes of Brassica rapa, B. oleracea and B. napus had less introns than their corresponding Arabidopsis orthologs. In these three Brassica species, the NADP-ME genes derived from the least fractionated subgenome have lost less introns than those from the medium fractionated and most fractionated subgenomes. BrNADP-ME1 showed the highest expression in petals and mature embryos. Two paralogous NADP-ME2 genes (BrNADP-ME2a and BrNADP-ME2b) shared similar expression profiles and differential expression levels. BrNADP-ME3 showed down-regulation during embryogenesis and reached its lowest expression in early cotyledonary embryos. BrNADP-ME4 was expressed widely in multiple organs and showed high expression during the whole embryogenesis process. Different NADP-ME genes of B. rapa showed differential gene expression profiles in young leaves after ABA treatment or cold stress. Our genome-wide identification and characterization of NADP-ME genes extend our understanding of the evolution or function of this family in Brassicaceae.

Activation and inhibition of adenylyl cyclase isoforms by forskolin analogs.

PubMed

Pinto, Cibele; Papa, Dan; Hübner, Melanie; Mou, Tung-Chung; Lushington, Gerald H; Seifert, Roland

2008-04-01

Adenylyl cyclase (AC) isoforms 1 to 9 are differentially expressed in tissues and constitute an interesting drug target. ACs 1 to 8 are activated by the diterpene, forskolin (FS). It is unfortunate that there is a paucity of AC isoform-selective activators. To develop such compounds, an understanding of the structure/activity relationships of diterpenes is necessary. Therefore, we examined the effects of FS and nine FS analogs on ACs 1, 2, and 5 expressed in Spodoptera frugiperda insect cells. Diterpenes showed the highest potencies at AC1 and the lowest potencies at AC2. We identified full agonists, partial agonists, antagonists, and inverse agonists, i.e., diterpenes that reduced basal AC activity. Each AC isoform exhibited a distinct pharmacological profile. AC2 showed the highest basal activity of all AC isoforms and highest sensitivity to inverse agonistic effects of 1-deoxy-forskolin, 7-deacetyl-1,9-dideoxy-forskolin, and, particularly, BODIPY-forskolin. In contrast, BODIPY-forskolin acted as partial agonist at the other ACs. 1-Deoxy-forskolin analogs were devoid of agonistic activity at ACs but antagonized the effects of FS in a mixed competitive/noncompetitive manner. At purified catalytic AC subunits, BODIPY-forskolin acted as weak partial agonist/strong partial antagonist. Molecular modeling revealed that the BODIPY group rotates promiscuously outside of the FS-binding site. Collectively, ACs are not uniformly activated and inhibited by FS and FS analogs, demonstrating the feasibility to design isoform-selective FS analogs. The two- and multiple-state models, originally developed to conceptualize ligand effects at G-protein-coupled receptors, can be applied to ACs to explain certain experimental data.

Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish.

PubMed

Laldinsangi, C; Senthilkumaran, B

2018-04-03

C-kit receptor is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analysed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50-75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of C-kit was analysed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression in male compared to females during the pre-spawning phase. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression level of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes involved in germ cell development. Decrease in the levels of serum 11-KT and T were also observed after esiRNA silencing. The findings of this study suggest that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish. Copyright © 2018. Published by Elsevier Inc.

Expression profile and distribution of Efhc1 gene transcript during rodent brain development.

PubMed

Conte, Fábio F; Ribeiro, Patrícia A O; Marchesini, Rafael B; Pascoal, Vinícius D B; Silva, Joelcimar M; Oliveira, Amanda R; Gilioli, Rovílson; Sbragia, Lourenço; Bittencourt, Jackson C; Lopes-Cendes, Iscia

2009-09-01

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

Cultural aspects in social anxiety and social anxiety disorder.

PubMed

Hofmann, Stefan G; Anu Asnaani, M A; Hinton, Devon E

2010-12-01

To examine cultural aspects in social anxiety and social anxiety disorder (SAD), we reviewed the literature on the prevalence rates, expressions, and treatments of social anxiety/SAD as they relate to culture, race, and ethnicity. We further reviewed factors that contribute to the differences in social anxiety/SAD between different cultures, including individualism/collectivism, perception of social norms, self-construal, gender roles, and gender role identification. Our review suggests that the prevalence and expression of social anxiety/SAD depends on the particular culture. Asian cultures typically show the lowest rates, whereas Russian and US samples show the highest rates, of SAD. Taijin kyofusho is discussed as a possible culture-specific expression of social anxiety, although the empirical evidence concerning the validity of this syndrome has been mixed. It is concluded that the individual's social concerns need to be examined in the context of the person's cultural, racial, and ethnic background in order to adequately assess the degree and expression of social anxiety and SAD. This has direct relevance for the upcoming DSM-V. © 2010 Wiley-Liss, Inc.

Cultural Aspects in Social Anxiety and Social Anxiety Disorder

PubMed Central

Hofmann, Stefan G.; Asnaani, Anu; Hinton, Devon E.

2010-01-01

To examine cultural aspects in social anxiety and social anxiety disorder (SAD), we reviewed the literature on the prevalence rates, expressions, and treatments of social anxiety/SAD as they relate to culture, race, and ethnicity. We further reviewed factors that contribute to the differences in social anxiety/SAD between different cultures, including individualism/collectivism, perception of social norms, self-construal, gender roles, and gender role identification. Our review suggests that the prevalence and expression of social anxiety/SAD depends on the particular culture. Asian cultures typically show the lowest rates, whereas Russian and US samples show the highest rates, of SAD. Taijin kyofusho is discussed as a possible culture-specific expression of social anxiety, although the empirical evidence concerning the validity of this syndrome has been mixed. It is concluded that the individual's social concerns need to be examined in the context of the person's cultural, racial, and ethnic background in order to adequately assess the degree and expression of social anxiety and social anxiety disorder. This has direct relevance for the upcoming DSM-V. PMID:21132847

Cloning, characterization, expression and comparative analysis of pig Golgi membrane sphingomyelin synthase 1.

PubMed

Guillén, Natalia; Navarro, María A; Surra, Joaquín C; Arnal, Carmen; Fernández-Juan, Marta; Cebrián-Pérez, Jose Alvaro; Osada, Jesús

2007-02-15

Pig sphingomyelin synthase 1 (SMS1) cDNA was cloned, characterized and compared to the human ortholog. Porcine protein consists of 413 amino acids and displays a 97% sequence identity with human protein. A phylogenic tree of proteins reveals that porcine SMS1 is more closely related to bovine and rodent proteins than to human. Analysis of protein mass was higher than the theoretical prediction based on amino acid sequence suggesting a kind of posttranslational modification. Quantitative representation of tissue distribution obtained by real-time RT-PCR showed that it was widely expressed although important variations in levels were obtained among organs. Thus, the cardiovascular system, especially the heart, showed the highest value of all the tissues studied. Regional differences of expression were observed in the central nervous system and intestinal tract. Analysis of the hepatic mRNA and protein expressions of SMS1 following turpentine treatment revealed a progressive decrease in the former paralleled by a decrease in the protein concentration. These findings indicate the variation in expression in the different tissues might suggest a different requirement of Golgi sphingomyelin for the specific function in each organ and a regulation of the enzyme in response to turpentine-induced hepatic injury.

Identification of differentially expressed genes through RNA sequencing in goats (Capra hircus) at different postnatal stages

PubMed Central

Li, Qian; Lin, Sen

2017-01-01

Intramuscular fat (IMF) content and fatty acid composition of longissimus dorsi muscle (LM) change with growth, which partially determines the flavor and nutritional value of goat (Capra hircus) meat. However, unlike cattle, little information is available on the transcriptome-wide changes during different postnatal stages in small ruminants, especially goats. In this study, the sequencing reads of goat LM tissues collected from kid, youth, and adult period were mapped to the goat genome. Results showed that out of total 24 689 Unigenes, 20 435 Unigenes were annotated. Based on expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), 111 annotated differentially expressed genes (DEGs) were identified among different postnatal stages, which were subsequently assigned to 16 possible expression patterns by series-cluster analysis. Functional classification by Gene Ontology (GO) analysis was used for selecting the genes showing highest expression related to lipid metabolism. Finally, we identified the node genes for lipid metabolism regulation using co-expression analysis. In conclusion, these data may uncover candidate genes having functional roles in regulation of goat muscle development and lipid metabolism during the various growth stages in goats. PMID:28800357

Identification of differentially expressed genes through RNA sequencing in goats (Capra hircus) at different postnatal stages.

PubMed

Lin, Yaqiu; Zhu, Jiangjiang; Wang, Yong; Li, Qian; Lin, Sen

2017-01-01

Intramuscular fat (IMF) content and fatty acid composition of longissimus dorsi muscle (LM) change with growth, which partially determines the flavor and nutritional value of goat (Capra hircus) meat. However, unlike cattle, little information is available on the transcriptome-wide changes during different postnatal stages in small ruminants, especially goats. In this study, the sequencing reads of goat LM tissues collected from kid, youth, and adult period were mapped to the goat genome. Results showed that out of total 24 689 Unigenes, 20 435 Unigenes were annotated. Based on expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), 111 annotated differentially expressed genes (DEGs) were identified among different postnatal stages, which were subsequently assigned to 16 possible expression patterns by series-cluster analysis. Functional classification by Gene Ontology (GO) analysis was used for selecting the genes showing highest expression related to lipid metabolism. Finally, we identified the node genes for lipid metabolism regulation using co-expression analysis. In conclusion, these data may uncover candidate genes having functional roles in regulation of goat muscle development and lipid metabolism during the various growth stages in goats.

Genetic variations in key inflammatory cytokines exacerbates the risk of diabetic nephropathy by influencing the gene expression.

PubMed

Hameed, Iqra; Masoodi, Shariq R; Malik, Perveez A; Mir, Shahnaz A; Ghazanfar, Khalid; Ganai, Bashir A

2018-06-30

Diabetic nephropathy is the single strongest predictor of mortality in patients with diabetes. The development of overt nephropathy involves important inter-individual variations, even after adjusting for potential confounding influences of modifiable and non-modifiable risk factors. Genome-wide transcriptome studies have reported the activation of inflammatory signaling pathways and there is mounting indication of the role of genetic factors. We screened nine genetic variations in three cytokine genes (TNF-α, IL-6 and IL-β) in 1326 unrelated subjects comprising of healthy controls (n = 464), type 2 diabetics with nephropathy (DN, n = 448) and type 2 diabetes without nephropathy (T2D, n = 414) by sequence-specific amplification. Functional implication of SNPs was elucidated by correlation studies and relative gene expression using Realtime-Quantitative PCR (RT-qPCR). Individual SNP analysis showed highest association of IL-1β rs16944-TT genotype (OR = 3.51, 95%CI = 2.36-5.21, P = 0.001) and TNF-α rs1800629-AA genotype (OR = 2.75, 95% CI = 1.64-4.59, P = 0.001) with T2D and DN respectively. The haplotype frequency showed significant risk of seven combinations among T2D and four combinations among DN subjects. The highest risk of T2D and DN was associated with GGTGAGTTT (OR = 4.25, 95%CI = 3.3-14.20, P = 0.0016) and GACGACCTT (OR = 21.3, 95%CI = 15.1-28.33, P = 0.026) haplotypes respectively. Relative expression by RT-qPCR showed increased cytokine expression in cases as compared to controls. TNF-α expression was increased by more than four-folds (n-fold = 4.43 ± 1.11) in DN. TNF-α, IL-6 and IL-1β transcript levels were significantly modulated by promoter region SNPs. The present study implicates a strong association between cytokine TNF-α, IL-6 and IL-1β gene promoter polymorphisms and modulation of transcript levels with susceptibility to nephropathy in diabetes subjects. Copyright © 2018 Elsevier B.V. All rights reserved.

The Potential Role of As-sumo-1 in the Embryonic Diapause Process and Early Embryo Development of Artemia sinica

PubMed Central

Chu, Bing; Yao, Feng; Cheng, Cheng; Wu, Yang; Mei, Yanli; Li, Xuejie; Liu, Yan; Wang, Peisheng; Hou, Lin; Zou, Xiangyang

2014-01-01

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica. PMID:24404204

Effects of salinity on gonadal development, osmoregulation and metabolism of adult male Chinese mitten crab, Eriocheir sinensis

PubMed Central

Zhao, Lei; Ye, Haihui; Cheng, Yongxu; Zeng, Chaoshu

2017-01-01

As a catadromous species, salinity is a key parameter that affects gonadal development of Chinese mitten crab Eriocheir sinensis during reproductive migration. It is however unclear the effects of salinity on the gonadal development of male E. sinensis as well as their physiological responses to salinity during reproductive migration. This study investigated the effects of four salinities (0 ‰, 6 ‰, 12 ‰ and 18 ‰) on gonadal development, osmoregulation and metabolism of adult male E. sinensis over a 40-day period. The results showed that elevating salinity promote gonadal development, increase hemolymph osmolality and K+ and Mg2+ concentrations (P < 0.05). The 12 ‰ salinity resulted in the highest contents of taurine and arginine in the hemolymph while the highest contents of threonine, phenylalanine, lysine, ß-alanine, tryptophan, ornithine and total free amino acids were found for 0 ‰ treatment (P < 0.05). A decreasing trend was detected for the Na+/K+-ATPase activity and its mRNA expression level in the posterior gills with salinity (P < 0.05). Total saturated fatty acids in the anterior gills decreased with increasing salinity (P < 0.05); the 0 ‰ treatment had the highest total polyunsaturated fatty acids in the posterior gills while total n-6 polyunsaturated fatty acids increased with salinity (P < 0.05). The hemolymph glucose and uric acid showed a decreasing trend as salinity while an increasing trend was found for the hemolymph triglyceride and high-density lipoprotein cholesterol (P < 0.05). The 12 ‰ treatment had the highest levels of hemolymph malonaldehyde and hepatopancreatic γ-glutamyltranspeptidase (P < 0.05). In conclusion, these results suggested that the brackish water promote gonadal development of male E. sinensis, and increase osmolality and ionic concentrations in hemolymph while reduced the activity of Na+ /K+- ATPase and its mRNA expression in the posterior gills as well as metabolism. PMID:28628611

Regulation of Banana Phytoene Synthase (MaPSY) Expression, Characterization and Their Modulation under Various Abiotic Stress Conditions

PubMed Central

Kaur, Navneet; Pandey, Ashutosh; Shivani; Kumar, Prateek; Pandey, Pankaj; Kesarwani, Atul K.; Mantri, Shrikant S.; Awasthi, Praveen; Tiwari, Siddharth

2017-01-01

Phytoene synthase (PSY) is a key regulatory enzyme of carotenoid biosynthesis pathway in plants. The present study examines the role of PSY in carotenogenesis and stress management in banana. Germplasm screening of 10 Indian cultivars showed that Nendran (3011.94 μg/100 g dry weight) and Rasthali (105.35 μg/100 g dry weight) contained the highest and lowest amounts of β-carotene, respectively in ripe fruit-pulp. Nendran ripe pulp also showed significantly higher antioxidant activity as compared to Rasthali. Meta-analysis of three banana PSY genes (MaPSY1, MaPSY2, and MaPSY3) was performed to identify their structural features, subcellular, and chromosomal localization in banana genome. The distinct expression patterns of MaPSY1, MaPSY2, and MaPSY3 genes were observed in various tissues, and fruit developmental stages of these two contrasting cultivars, suggesting differential regulation of the banana PSY genes. A positive correlation was observed between the expression of MaPSY1 and β-carotene accumulation in the ripe fruit-peel and pulp of Nendran. The presence of stress responsive cis-regulatory motifs in promoter region of MaPSY genes were correlated with the expression pattern during various stress (abscisic acid, methyl jasmonate, salicylic acid and dark) treatments. The positive modulation of MaPSY1 noticed under abiotic stresses suggested its role in plant physiological functions and defense response. The amino acid sequence analysis of the PSY proteins in contrasting cultivars revealed that all PSY comprises conserved domains related to enzyme activity. Bacterial complementation assay has validated the functional activity of six PSY proteins and among them PSY1 of Nendran (Nen-PSY1) gave the highest activity. These data provide new insights into the regulation of PSY expression in banana by developmental and stress related signals that can be explored in the banana improvement programs. PMID:28421096

Selection and validation of reference genes for gene expression analysis in apomictic and sexual Cenchrus ciliaris

PubMed Central

2013-01-01

Background Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris. Findings Among tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction. Conclusion Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation. PMID:24083672

Gibberellins and stem growth in Arabidopsis thaliana. Effects of photoperiod on expression of the GA4 and GA5 loci.

PubMed

Xu, Y L; Gage, D A; Zeevaart, J A

1997-08-01

Arabidopsis thaliana (L.) Heynh. is a quantitative long-day (LD) rosette plant in which stem growth is mediated by gibberellins (CAs). Application of GAs to plants in short-day (SD) conditions resulted in rapid stem elongation and flower formation, with GA4 and GA9 being equally effective, and GA1 showing lower activity. The effects of photoperiod on the levels of endogenous GAs were measured by combined gas chromatography-mass spectrometry with selected ion monitoring. When plants were transferred from SD to LD conditions there was a slight decrease in the level of GA53 and an increase in the levels of C19-GAs, GA9, GA20, GA1, and GA8, indicating that GA 20-oxidase activity is stimulated in LD conditions. Expression of GA5, which encodes GA 20-oxidase, was highest in elongating stems and was correlated with the rate of stem elongation. By contrast, GA4, which encodes 3 beta-hydroxylase, showed low expression in stems and its expression was not correlated with the rate of stem elongation. We conclude that stem elongation in LD conditions is at least in part due to increased expression of GA5, whereas expression of GA4 is not under photoperiodic control.

DDAH2 mRNA expression is inversely associated with some cardiovascular risk-related features in healthy young adults.

PubMed

Puchau, Blanca; Hermsdorff, Helen Hermana M; Zulet, M Angeles; Martínez, J Alfredo

2009-01-01

The purpose of this study was to evaluate whether the mRNA expression profiles of three genes (PRMT1, DDAH2 and NOS3) are related to ADMA metabolism and signalling, and the potential relationships with anthropometrical, biochemical, lifestyle and inflammatory indicators in healthy young adults. An emphasis on the putative effect of different mRNA expression on cardiovascular risk-related features was paid. Anthropometrical measurements as well as lifestyle features were analyzed in 120 healthy young adults. Fasting blood samples were collected for the measurement of glucose and lipid profiles as well as the concentrations of selected inflammatory markers. Profiles of mRNA expression were assessed for PRMT1, DDAH2 and NOS3 genes from peripheral blood mononuclear cells. Regarding inflammatory biomarkers, DDAH2 was inversely associated with IL-6 and TNF-alpha. Moreover, subjects in the highest quintile of DDAH2 mRNA expression showed a reduced risk to have higher values of waist circumference, and to be more prone to show higher values of HDL-c. Interestingly, DDAH2 gene expression seemed to be related with some anthropometrical, biochemical, lifestyle and inflammatory indicators linked to cardiovascular risk in apparently healthy young adults, emerging as a potential disease marker.

Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response.

PubMed

Inoue, Naoki; Hirouchi, Taisei; Kasai, Atsushi; Higashi, Shintaro; Hiraki, Natsumi; Tanaka, Shota; Nakazawa, Takanobu; Nunomura, Kazuto; Lin, Bangzhong; Omori, Akiko; Hayata-Takano, Atsuko; Kim, Yoon-Jeong; Doi, Takefumi; Baba, Akemichi; Hashimoto, Hitoshi; Shintani, Norihito

2018-01-08

We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart. Copyright © 2017. Published by Elsevier Inc.

Quantitative PCR analysis of CYP1A induction in Atlantic salmon (Salmo salar)

USGS Publications Warehouse

Rees, C.B.; McCormick, S.D.; Vanden, Heuvel J.P.; Li, W.

2003-01-01

Environmental pollutants are hypothesized to be one of the causes of recent declines in wild populations of Atlantic salmon (Salmo salar) across Eastern Canada and the United States. Some of these pollutants, such as polychlorinated biphenyls and dioxins, are known to induce expression of the CYP1A subfamily of genes. We applied a highly sensitive technique, quantitative reverse transcription-polymerase chain reaction (RT-PCR), for measuring the levels of CYP1A induction in Atlantic salmon. This assay was used to detect patterns of CYP1A mRNA levels, a direct measure of CYP1A expression, in Atlantic salmon exposed to pollutants under both laboratory and field conditions. Two groups of salmon were acclimated to 11 and 17??C, respectively. Each subject then received an intraperitoneal injection (50 mg kg-1) of either ??-naphthoflavone (BNF) in corn oil (10 mg BNF ml-1 corn oil) or corn oil alone. After 48 h, salmon gill, kidney, liver, and brain were collected for RNA isolation and analysis. All tissues showed induction of CYP1A by BNF. The highest base level of CYP1A expression (2.56??1010 molecules/??g RNA) was found in gill tissue. Kidney had the highest mean induction at five orders of magnitude while gill tissue showed the lowest mean induction at two orders of magnitude. The quantitative RT-PCR was also applied to salmon sampled from two streams in Massachusetts, USA. Salmon liver and gill tissue sampled from Millers River (South Royalston, Worcester County), known to contain polychlorinated biphenyls (PCBs), showed on average a two orders of magnitude induction over those collected from a stream with no known contamination (Fourmile Brook, Northfield, Franklin County). Overall, the data show CYP1A exists and is inducible in Atlantic salmon gill, brain, kidney, and liver tissue. In addition, the results obtained demonstrate that quantitative PCR analysis of CYP1A expression is useful in studying ecotoxicity in populations of Atlantic salmon in the wild. ?? 2003 Elsevier Science B.V. All rights reserved.

Pheromone Binding Protein EhipPBP1 Is Highly Enriched in the Male Antennae of the Seabuckthorn Carpenterworm and Is Binding to Sex Pheromone Components

PubMed Central

Hu, Ping; Gao, Chenglong; Zong, Shixiang; Luo, Youqing; Tao, Jing

2018-01-01

The seabuckthorn carpenterworm moth Eogystia hippophaecolus is a major threat to seabuckthorn plantations, causing considerable ecological and economic losses in China. Transcriptomic analysis of E. hippophaecolus previously identified 137 olfactory proteins, including three pheromone-binding proteins (PBPs). We investigated the function of E. hippophaecolus PBP1 by studying its mRNA and protein expression profiles and its binding ability with different compounds. The highest levels of expression were in the antennae, particularly in males, with much lower levels of expression in the legs and external genitals. Recombinant PBP1 showed strong binding to sex-pheromone components, suggesting that antennal EhipPBP1 is involved in binding sex-pheromone components during pheromone communication. PMID:29755369

Comparative quantitative analysis of cluster of differentiation 45 antigen expression on lymphocyte subsets.

PubMed

Im, Mijeong; Chae, Hyojin; Kim, Taehoon; Park, Hun-Hee; Lim, Jihyang; Oh, Eun-Jee; Kim, Yonggoo; Park, Yeon-Joon; Han, Kyungja

2011-07-01

Since the recent introduction of radioimmunotherapy (RIT) using antibodies against cluster of differentiation (CD) 45 for the treatment of lymphoma, the clinical significance of the CD45 antigen has been increasing steadily. Here, we analyzed CD45 expression on lymphocyte subsets using flow cytometry in order to predict the susceptibility of normal lymphocytes to RIT. Peripheral blood specimens were collected from 14 healthy individuals aged 25-54 yr. The mean fluorescence intensity (MFI) of the cell surface antigens was measured using a FACSCanto II system (Becton Dickinson Bioscience, USA). MFI values were converted into antibody binding capacity values using a Quantum Simply Cellular microbead kit (Bangs Laboratories, Inc., USA). Among the lymphocyte subsets, the expression of CD45 was the highest (725,368±42,763) on natural killer T (NKT) cells, 674,030±48,187 on cytotoxic/suppressor T cells, 588,750±48,090 on natural killer (NK) cells, 580,211±29,168 on helper T (Th) cells, and 499,436±21,737 on B cells. The Th cells and NK cells expressed a similar level of CD45 (P=0.502). Forward scatter was the highest in NKT cells (P<0.05), whereas side scatter differed significantly between each of the lymphocyte subsets (P<0.05). CD3 expression was highest in the Th and NKT cells. NKT cells express the highest levels of CD45 antigen. Therefore, this lymphocyte subset would be most profoundly affected by RIT or pretargeted RIT. The monitoring of this lymphocyte subset during and after RIT should prove helpful.

Recombinant cell lines expressing shRNA targeting herpes simplex virus 2 VP16 inhibit virus replication.

PubMed

Zhang, Rui; Wang, Yan; Song, Bo; Han, Zhi Qiang; Xu, Yu Ming

2012-01-01

To establish HSV2 VP16 targeting shRNA-expressing cell lines and investigate the antiviral effect of shRNA targeting HSV2 VP16. The cell lines Vero-shRNAs and negative-control Vero-shCON were established. Their inhibition effects on VP16 mRNA expression were tested by real-time fluorescent quantitative polymerase chain reaction (PCR) and their antiviral effects were evaluated by yield reduction assay. The influence of passage numbers on the inhibition ability of cell lines was researched. Vero-shRNA24 targeting the upper stream, Vero-shRNA642 targeting the lower stream and Vero-shCON were established. Vero-shRNA24, Vero-shRNA642 and Vero-shRNA24 + 642 could reduce the VP16 mRNA significantly. Vero-shRNA24 was the most efficient. The HSV2 titers in Vero and Vero-shCON were the highest at 72 h after infection, and started decreasing thereafter. The viral titers of the Vero-shRNA groups reached a peak after 84 h and the highest titers were lower than in the Vero group. The inhibiting effect on VP16 mRNA expression and viral replication of Vero-shRNA24 cell lines of passages 10 and 20 were not significantly different from the primary cell line. Although of no statistical significance, the passage 50 cell line showed decreased inhibiting ability. Recombinant cell lines expressing shRNA targeting HSV2 VP16 were established. They can stably inhibit HSV2 VP16 mRNA expression and viral replication within passage 50. Copyright © 2012 S. Karger AG, Basel.

Improvement in the carcass traits and meat quality of growing-finishing Rongchang pigs by conjugated linoleic acid through altered gene expression of muscle fiber types.

PubMed

Huang, J X; Qi, R L; Chen, X L; You, X Y; Liu, X Q; Yang, F Y; Liu, Z H

2014-03-24

A total of 160 Rongchang pigs (26.76±1.78 kg) were randomly assigned to 5 dietary treatment groups until their body weight (BW) reached 90 kg. The diets were supplemented with 0, 0.5, 1.0, 1.5, and 2.0% conjugated linoleic acid (CLA). Our results showed that the 1.0 to 2.0% CLA-fed pigs had less back fat deposition when their BW reached 90 kg than the pigs that received less than 1% CLA. During the 30 to 60 kg growing period, 1.0, 1.5, and 2.0% CLA treatments improved pork quality by significantly reducing the pork pH (P<0.01) and color value (P<0.05), but they increased marble scaling (P<0.01). Similarly, the 1.5 and 2.0% CLA-fed pigs had more marble than other pigs when their BW reached 90 kg. Furthermore, CLA significantly affected the expression of muscle fiber-type genes. The 1.5% CLA-fed pigs exhibited the highest mRNA expression of MyHC1 and MyHC2a (P<0.05) at 60 kg BW. At 90 kg BW, the highest expression of MyHC1 and MyHC2a (P<0.05) was found in the 2.0% CLA group. However, MyHC2x was downregulated in the CLA-fed pigs at this time. In addition, CLA supplements did not evidently alter mRNA expression of MyHC2b at all times. These results demonstrate that CLA could affect carcass traits and improve the meat quality of growing-finishing pigs by altering the expression of genes related to muscle growth and development; 1-1.5% CLA was the most appropriate CLA dose.

Characterization of the Sucrose Phosphate Phosphatase (SPP) Isoforms from Arabidopsis thaliana and Role of the S6PPc Domain in Dimerization.

PubMed

Albi, Tomás; Ruiz, M Teresa; de Los Reyes, Pedro; Valverde, Federico; Romero, José M

2016-01-01

Sucrose-phosphate phosphatase (SPP) catalyses the final step in the sucrose biosynthesis pathway. Arabidopsis thaliana genome codifies four SPP isoforms. In this study, the four Arabidopsis thaliana genes coding for SPP isoforms have been cloned, expressed in Escherichia coli and the kinetic and regulatory properties of the purified enzymes analysed. SPP2 is the isoform showing the highest activity, with SPP3b and SPP3a showing lower activity levels. No activity was detected for SPP1. We propose that this lack of activity is probably due to the absence of an essential amino acid participating in catalysis and/or in the binding of the substrate, sucrose-6-phosphate (Suc6P). The expression patterns of Arabidopsis SPP genes indicate that SPP2 and SPP3b are the main isoforms expressed in different tissues and organs, although the non-catalytic SPP1 is the main isoform expressed in roots. Thus, SPP1 could have acquired new unknown functions. We also show that the three catalytically active SPPs from Arabidopsis are dimers. By generating a chimeric SPP composed of the monomeric cyanobacterial SPP fused to the higher plant non-catalytic S6PPc domain (from SPP2), we show that the S6PPc domain is responsible for SPP dimerization. This is the first experimental study on the functionality and gene expression pattern of all the SPPs from a single plant species.

Effects of various glutamine concentrations on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

PubMed

Esmaeili, Fatemeh; Ghaheri, Matin; Kahrizi, Danial; Mansouri, Mohsen; Safavi, Seyed Mehdi; Ghorbani, Tayebeh; Muhammadi, Sarre; Rahmanian, Elham; Vaziri, Siavash

2018-02-10

Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

Immune Cell Profiling of IFN-λ Response Shows pDCs Express Highest Level of IFN-λR1 and Are Directly Responsive via the JAK-STAT Pathway.

PubMed

Kelly, Aoife; Robinson, Mark W; Roche, Gerard; Biron, Christine A; O'Farrelly, Cliona; Ryan, Elizabeth J

2016-12-01

The interferon lambda (IFN-λ) cytokines have well-known antiviral properties, yet their contribution to immune regulation is not well understood. Epithelial cells represent the major target cell of IFN-λ; peripheral blood mononuclear cells are generally considered nonresponsive, with the exception of plasmacytoid dendritic cells (pDCs). In this study we aimed to define the potential for discrete subpopulations of cells to directly respond to IFN-λ. Analysis of peripheral blood leukocytes reveals that, while pDCs uniformly express the highest levels of IFN-λ receptor, a small proportion of B cells and monocytes also express the receptor. Nevertheless, B cells and monocytes respond poorly to IFN-λ stimulation in vitro, with minimal STAT phosphorylation and interferon-stimulated gene (ISG) induction observed. We confirm that pDCs respond to IFN-λ in vitro, upregulating their expression of pSTAT1, pSTAT3, and pSTAT5. However, we found that pDCs do not upregulate pSTAT6 in response to IFN-λ treatment. Our results highlight unique aspects of the response to IFN-λ and confirm that while the IFN-λ receptor is expressed by a small proportion of several different circulating immune cell lineages, under normal conditions only pDCs respond to IFN-λ stimulation with robust STAT phosphorylation and ISG induction. The difference in STAT6 responsiveness of pDCs to type I and type III interferons may help explain the divergence in their biological activities.

Relationship between fumonisin production and FUM gene expression in Fusarium verticillioides under different environmental conditions.

PubMed

Fanelli, Francesca; Iversen, Anita; Logrieco, Antonio F; Mulè, Giuseppina

2013-01-01

Fusarium verticillioides is the main source of fumonisins, a group of mycotoxins that can contaminate maize-based food and feed and cause diseases in humans and animals. The study of the effect of different environmental conditions on toxin production should provide information that can be used to develop strategies to minimize the risk. This study analysed the effect of temperature (15°C-35°C), water activity (a(w): 0.999-0.93), salinity (0-125 g l(-1) NaCl) and pH (5-8) on the growth and production of fumonisins B(1) (FB1), B(2) (FB2) and B(3) (FB3) and the expression of FUM1 and FUM21 in F. verticillioides. The highest growth rate was measured at 25°C, a(w) of 0.998-0.99 and 0-25 g l(-1) of NaCl. Optimal conditions for fumonisin production were 30°C, a(w) of 0.99, 25 g l(-1) of NaCl and pH 5; nevertheless, the strain showed a good adaptability and was able to produce moderate levels of fumonisins under a wide range of conditions. Gene expression mirrored fumonisin production profile under all conditions with the exception of temperature: FUM1 and FUM21 expression was highest at 15°C, while maximum fumonisin production was at 30°C. These data indicate that a post-transcriptional regulation mechanism could account for the different optimal temperatures for FUM gene expression and fumonisin production.

Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions.

PubMed

Wan, Qiao; Chen, Shuilian; Shan, Zhihui; Yang, Zhonglu; Chen, Limiao; Zhang, Chanjuan; Yuan, Songli; Hao, Qinnan; Zhang, Xiaojuan; Qiu, Dezhen; Chen, Haifeng; Zhou, Xinan

2017-01-01

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Expression of the ADHD candidate gene Diras2 in the brain.

PubMed

Grünewald, Lena; Becker, Nils; Camphausen, Annika; O'Leary, Aet; Lesch, Klaus-Peter; Freudenberg, Florian; Reif, Andreas

2018-06-01

The distinct subgroup of the Ras family member 2 (DIRAS2) gene has been found to be associated with attention-deficit/hyperactivity disorder (ADHD) in one of our previous studies. This gene is coding for a small Ras GTPase with unknown function. DIRAS2 is highly expressed in the brain. However, the exact neural expression pattern of this gene was unknown so far. Therefore, we investigated the expressional profile of DIRAS2 in the human and murine brain. In the present study, qPCR analyses in the human and in the developing mouse brain, immunocytological double staining on murine hippocampal primary cells and RNA in situ hybridization (ISH) on brain sections of C57BL/6J wild-type mice, have been used to reveal the expression pattern of DIRAS2 in the brain. We could show that DIRAS2 expression in the human brain is the highest in the hippocampus and the cerebral cortex, which is in line with the ISH results in the mouse brain. During mouse brain development, Diras2 levels strongly increase from prenatal to late postnatal stages. Co-expression studies indicate Diras2 expression in glutamatergic and catecholaminergic neurons. Our findings support the idea of DIRAS2 as a candidate gene for ADHD as the timeline of its expression as well as the brain regions and cell types that show Diras2 expression correspond to those assumed to underlie the pathomechanisms of the disease.

Extracellular matrix metalloproteinase inducer expression in the baboon endometrium: menstrual cycle and endometriosis

PubMed Central

Braundmeier, A G; Fazleabas, A T; Nowak, R A

2016-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN; BSG) regulates tissue remodeling through matrix metalloproteinases (MMPs). In human and non-human primates, endometrial remodeling is important for menstruation and the pathogenesis of endometriosis. We hypothesized that as in humans, BSG and MMPs are expressed in the endometrium of cycling baboons, and their expression is hormonally regulated by ovarian hormones, but endometriosis disrupts this regulation. BSG expression was evaluated in the baboon endometrium by q-PCR and immunohistochemistry. In the endometrium of control cycling animals, BSG mRNA levels were highest in late secretory stage tissue. BSG protein localized to glandular epithelial cells during the proliferative phase; whereas, secretory stage tissues expressed BSG in glandular and luminal epithelia with weak stromal staining. Several MMPs were differentially expressed throughout the menstrual cycle with the highest levels found during menstruation. In ovariectomized animals, BSG endometrial mRNA levels were highest with treatment of both estrogen and progesterone than that with only estrogen. Estrogen alone resulted in BSG protein localization primarily in the endometrial glandular epithelia, while estrogen and progesterone treatment displayed BSG protein localization in both the glandular and stromal cells. Exogenous hormone treatment resulted in differential expression patterns of all MMPs compared with the control cycling animals. In the eutopic endometrium of endometriotic animals, BSG mRNA levels and protein were elevated early but decreased later in disease progression. Endometriosis elevated the expression of all MMPs except MMP7 compared with the control animals. In baboons, BSG and MMP endometrial expression is regulated by both ovarian hormones, and their expression patterns are dysregulated in endometriotic animals. PMID:20841363

Expression pattern of zebrafish rxfp2 homologue genes during embryonic development.

PubMed

Donizetti, Aldo; Fiengo, Marcella; Del Gaudio, Rosanna; Iazzetti, Giovanni; Pariante, Paolo; Minucci, Sergio; Aniello, Francesco

2015-11-01

RXFP2 is one of the 4 receptors for relaxin insulin-like peptides, in particular it binds with high affinity the INSL3 peptide. INSL3/RXFP2 pair is essential for testicular descent during placental mammalian development. The evolutionary history of this ligand/receptor pair has received much attention, since its function in vertebrate species lacking testicular descent, such as the fishes, remains elusive. Herein, we analyzed the expression pattern of three rxfp2 homologue genes in zebrafish embryonic development. For all the three rxfp2 genes (rxfp2a, rxfp2b, and rxfp2-like) we showed the presence of maternally derived transcripts. Later in the development, rxfp2a is only expressed at larval stage, whereas rxfp2b is expressed in all the analyzed stage with highest level in the larvae. The rxfp2-like gene is expressed in all the analyzed stage with a transcript level that increased starting at early pharyngula stage. The spatial localization analysis of rxfp2-like gene showed that it is expressed in many cell clusters in the developing brain. In addition, other rxfp2-like-expressing cells were identified in the retina and oral epithelium. This analysis provides new insights to elucidate the evolution of rxfp2 genes in vertebrate lineage and lays the foundations to study their role in vertebrate embryonic development. © 2015 Wiley Periodicals, Inc.

The Expression, Purification, and Characterization of a Ras Oncogene (Bras2) in Silkworm (Bombyx mori).

PubMed

Lv, Zhengbing; Wang, Tao; Zhuang, Wenhua; Wang, Dan; Chen, Jian; Nie, Zuoming; Liu, Lili; Zhang, Wenping; Wang, Lisha; Wang, Deming; Wu, Xiangfu; Li, Jun; Qian, Lian; Zhang, Yaozhou

2013-01-01

The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412 bp. The open reading frame contains 603 bp, which encodes 200 amino acid residues. This recombinant BmBras2 protein was subsequently used as an antigen to raise a rabbit polyclonal antibody. Western blotting and real-time PCR analyses showed that BmBras2 was expressed during four developmental stages. The BmBras2 expression level was the highest in the pupae and was low in other life cycle stages. BmBras2 was expressed in all eight tested tissues, and it was highly expressed in the head, intestine, and epidermis. Subcellular localization studies indicated that BmBras2 was predominantly localized in the nuclei of Bm5 cells, although cytoplasmic staining was also observed to a lesser extent. A cell proliferation assay showed that rBmBras2 could stimulate the proliferation of hepatoma cells. The higher BmBras2 expression levels in the pupal stage, tissue expression patterns, and a cell proliferation assay indicated that BmBras2 promotes cell division and proliferation, most likely by influencing cell signal transduction.

Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multi-gene family

PubMed Central

Dean, Caroline; Elzen, Peter van den; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Of the eight nuclear genes in the plant multi-gene family which encodes the small subunit (rbcS) of Petunia (Mitchell) ribulose bisphosphate carboxylase, one rbcS gene accounts for 47% of the total rbcS gene expression in petunia leaf tissue. Expression of each of five other rbcS genes is detected at levels between 2 and 23% of the total rbcS expression in leaf tissue, while expression of the remaining two rbcS genes is not detected. There is considerable variation (500-fold) in the levels of total rbcS mRNA in six organs of petunia (leaves, sepals, petals, stems, roots and stigmas/anthers). One gene, SSU301, showed the highest levels of steady-state mRNA in each of the organs examined. We discuss the differences in the steady-state mRNA levels of the individual rbcS genes in relation to their gene structure, nucleotide sequence and genomic linkage. ImagesFig. 2.Fig. 3. PMID:16453647

Gene expression analysis of mouse embryonic stem cells following levitation in an ultrasound standing wave trap.

PubMed

Bazou, Despina; Kearney, Roisin; Mansergh, Fiona; Bourdon, Celine; Farrar, Jane; Wride, Michael

2011-02-01

In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) (Bazou et al. 2005a) at variable acoustic pressures (0.08-0.85 MPa) and times (5-60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. Copyright © 2011 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Variation in embryonic mortality and maternal transcript expression among Atlantic cod (Gadus morhua) broodstock: a functional genomics study.

PubMed

Rise, Matthew L; Nash, Gordon W; Hall, Jennifer R; Booman, Marije; Hori, Tiago S; Trippel, Edward A; Gamperl, A Kurt

2014-12-01

Early life stage mortality is an important issue for Atlantic cod aquaculture, yet the impact of the cod maternal (egg) transcriptome on egg quality and mortality during embryonic development is poorly understood. In the present work, we studied embryonic mortality and maternal transcript expression using eggs from 15 females. Total mortality at 7days post-fertilization (7 dpf, segmentation stage) was used as an indice of egg quality. A 20,000 probe (20K) microarray experiment compared the 7hours post-fertilization (7 hpf, ~2-cell stage) egg transcriptome of the two lowest quality females (>90% mortality at 7 dpf) to that of the highest quality female (~16% mortality at 7 dpf). Forty-three microarray probes were consistently differentially expressed in both low versus high quality egg comparisons (25 higher expressed in low quality eggs, and 18 higher expressed in high quality eggs). The microarray experiment also identified many immune-relevant genes [e.g. interferon (IFN) pathway genes ifngr1 and ifrd1)] that were highly expressed in eggs of all 3 females regardless of quality. Twelve of the 43 candidate egg quality-associated genes, and ifngr1, ifrd1 and irf7, were included in a qPCR study with 7 hpf eggs from all 15 females. Then, the genes that were confirmed by qPCR to be greater than 2-fold differentially expressed between 7 hpf eggs from the lowest and highest quality females (dcbld1, ddc, and acy3 more highly expressed in the 2 lowest quality females; kpna7 and hacd1 more highly expressed in the highest quality female), and the 3 IFN pathway genes, were included in a second qPCR study with unfertilized eggs. While some maternal transcripts included in these qPCR studies were associated with extremes in egg quality, there was little correlation between egg quality and gene expression when all females were considered. Both dcbld1 and ddc showed greater than 100-fold differences in transcript expression between females and were potentially influenced by family. The Atlantic cod ddc (dopa decarboxylase) complete cDNA was characterized, and has a 1461bp open reading frame encoding a 486 amino acid protein that contains all eight residues of the conserved pyridoxal 5'-phosphate binding site including the catalytic lysine. This study provides valuable new information and resources related to the Atlantic cod egg transcriptome. Some of these microarray-identified, qPCR-confirmed, Atlantic cod egg transcripts (e.g. ddc, kpna7) play important roles during embryonic development of other vertebrate species, and may have similar functions in Atlantic cod. Copyright © 2014. Published by Elsevier B.V.

Accumulation of kaempferitrin and expression of phenyl-propanoid biosynthetic genes in kenaf (Hibiscus cannabinus).

PubMed

Zhao, Shicheng; Li, Xiaohua; Cho, Dong Ha; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Sang Un

2014-10-23

Kenaf (Hibiscus cannabinus) is cultivated worldwide for its fiber; however, the medicinal properties of this plant are currently attracting increasing attention. In this study, we investigated the expression levels of genes involved in the biosynthesis of kaempferitrin, a compound with many biological functions, in different kenaf organs. We found that phenylalanine ammonia lyase (HcPAL) was more highly expressed in stems than in other organs. Expression levels of cinnamate 4-hydroxylase (HcC4H) and 4-coumarate-CoA ligase (Hc4CL) were highest in mature leaves, followed by stems and young leaves, and lowest in roots and mature flowers. The expression of chalcone synthase (HcCHS), chalcone isomerase (HcCHI), and flavone 3-hydroxylase (HcF3H) was highest in young flowers, whereas that of flavone synthase (HcFLS) was highest in leaves. An analysis of kaempferitrin accumulation in the different organs of kenaf revealed that the accumulation of this compound was considerably higher (>10-fold) in leaves than in other organs. On the basis of a comparison of kaempferitrin contents with the expression levels of different genes in different organs, we speculate that HcFLS plays an important regulatory role in the kaempferitrin biosynthetic pathway in kenaf.

High-level expression of recombinant thermostable β-glucosidase in Escherichia coli by regulating acetic acid.

PubMed

Shi, Xuejia; Xie, Jingcong; Liao, Shiyong; Wu, Tao; Zhao, Lin-Guo; Ding, Gang; Wang, Zhenzhong; Xiao, Wei

2017-10-01

In the fermentation progress, fermentation parameters including the feed rate, induction temperature, and induction pH evidently regulate the accumulation of acetic acid generated by recombinant E. coli in the medium. The production of thermostable β-glucosidase (Tpebgl3) was increased by optimizing the parameters mentioned step by step. The optimal conditions were obtained with the highest enzyme expression (560.4U/mL) and the maximum DCW (65g/L) at the pre-induction specific growth rate of 0.2h -1 followed by a post-induction specific growth rate (0.18h -1 ); induction temperature is 39°C; the pH is 7.2; the concentration of acetic acid was maintained all along below 0.9g/L. Results show it is necessary for the synthesis of Tpebgl3 to regulate the accumulation of acetic acid at the premise of feeding to meet the normal growth of E. coli. The production of Tpebgl3 by recombinant E. coli is the highest reported to date. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cloning and characterization of the Cerasus humilis sucrose phosphate synthase gene (ChSPS1)

PubMed Central

Du, Junjie; Mu, Xiaopeng; Wang, Pengfei

2017-01-01

Sucrose is crucial to the growth and development of plants, and sucrose phosphate synthase (SPS) plays a key role in sucrose synthesis. To understand the genetic and molecular mechanisms of sucrose synthesis in Cerasus humilis, ChSPS1, a homologue of SPS, was cloned using RT-PCR. Sequence analysis showed that the open reading frame (ORF) sequence of ChSPS1 is 3174 bp in length, encoding a predicted protein of 1057 amino acids. The predicted protein showed a high degree of sequence identity with SPS homologues from other species. Real-time RT-PCR analysis showed that ChSPS1 mRNA was detected in all tissues and the transcription level was the highest in mature fruit. There is a significant positive correlation between expression of ChSPS1 and sucrose content. Prokaryotic expression of ChSPS1 indicated that ChSPS1 protein was expressed in E. coli and it had the SPS activity. Overexpression of ChSPS1 in tobacco led to upregulation of enzyme activity and increased sucrose contents in transgenic plants. Real-time RT-PCR analysis showed that the expression of ChSPS1 in transgenic tobacco was significantly higher than in wild type plants. These results suggested that ChSPS1 plays an important role in sucrose synthesis in Cerasus humilis. PMID:29036229

Molecular Characterization of Ferulate 5-Hydroxylase Gene from Kenaf (Hibiscus cannabinus L.)

PubMed Central

Park, Young-Hwan; Lim, Hyoun-Sub; Natarajan, Savithiry; Park, Sang-Un

2013-01-01

The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.). Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84), is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF) encoding 518 amino acids (GenBank Accession number JX524278). The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78%) with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old) of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h), wounding (24 h), cold (24 h), abscisic acid (24 h), and methyl jasmonate (24 h). PMID:24204204

Lin28a is a putative factor in regulating cancer stem cell-like properties in side population cells of oral squamous cell carcinoma

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

Hayashi, S.; Tanaka, J.; Okada, S.

Cancer stem cells (CSCs) are among the target cells of cancer therapy because they are uniquely involved in both cancer progression and sensitivity to chemotherapeutic agents. We identified side population (SP) cells, which are known to be an enriched population of CSC, in five oral squamous cell carcinoma (OSCC) cells (SCC9, SCC25, TOSCC7, TOSCC17, and TOSCC23). The percentages of SP cells ranged from 0% to 3.3%, with TOSCC23 cells showing the highest percentages of SP cells (3.3% of the total cell population). The SP cells isolated from TOSCC23 cells also showed greater cell proliferation and invasion compared to non-SP (MP)more » cells. Therefore, our initial findings suggested that SP cells were enriched for CSC-like cells. Furthermore, DNA microarray analysis revealed that the expression of cell proliferation-related and anti-apoptotic genes was greater in SP cells compared to MP cells. We focused on Lin28a, which showed the highest expression (approximately 22-fold) among the upregulated genes. The overexpression of Lin28a in TOSCC23 cells increased their proliferation, colony formation, and invasion. These findings suggest that Lin28a is an appropriate CSC target molecule for OSCC treatment - Highlights: ► Lin28a is a SP cell-specific factor in oral squamous cell carcinoma (OSCC) cells. ► SP cells in OSCC cells show cancer stem cell-like properties. ► Lin28a regulates OSCC proliferative and invasive activities.« less

Molecular cloning and expression analysis of KIN10 and cold-acclimation related genes in wild banana 'Huanxi' (Musa itinerans).

PubMed

Liu, Weihua; Cheng, Chunzhen; Lai, Gongti; Lin, Yuling; Lai, Zhongxiong

2015-01-01

Banana cultivars may experience chilling or freezing injury in some of their cultivated regions, where wild banana can still grow very well. The clarification of the cold-resistant mechanism of wild banana is vital for cold-resistant banana breeding. In this study, the central stress integrator gene KIN10 and some cold-acclimation related genes (HOS1 and ICE1s) from the cold-resistant wild banana 'Huanxi' (Musa itinerans) were cloned and their expression patterns under different temperature treatments were analyzed. Thirteen full-length cDNA transcripts including 6 KIN10s, 1 HOS1 and 6 ICE1s were successfully cloned. Quantitative real-time PCR (qRT-PCR) results showed that all these genes had the highest expression levels at the critical temperature of banana (13 °C). Under chilling temperature (4 °C), the expression level of KIN10 reduced significantly but the expression of HOS1 was still higher than that at the optimal temperature (28 °C, control). Both KIN10 and HOS1 showed the lowest expression levels at 0 °C, the expression level of ICE1, however, was higher than control. As sucrose plays role in plant cold-acclimation and in regulation of KIN10 and HOS1 bioactivities, the sucrose contents of wild banana under different temperatures were detected. Results showed that the sucrose content increased as temperature lowered. Our result suggested that KIN10 may participate in cold stress response via regulating sucrose biosynthesis, which is helpful in regulating cold acclimation pathway in wild banana.

Distribution and localization of 3β-hydroxysteroid dehydrogenase (3β-HSD) in the brain and its regions of the catfish Heteropneustes fossilis.

PubMed

Mishra, Surabhi; Chaube, Radha

2017-01-15

In vertebrates, steroids are synthesized de novo in the central and peripheral nervous system, independent of peripheral steroidogenic glands, such as the adrenal, gonads and placenta. 3β-Hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD) is a key steroidogenic enzyme in vertebrate gonads, placenta and adrenal. It mediates the oxidation and isomerization reactions of progesterone from pregnenolone, 17-hydroxyprogesterone from 17-hydroxypregnenolone and androstenedione from dehydroepiandrosterone. In the present study, we examined the expression of 3β-HSD cDNA by real time-PCR and localization of the mRNA by in situ hybridization in the brain and its regions during the different phases of the reproductive cycle of the catfish Heteropneustes fossilis. Further, 3β-HSD activity was assayed biochemically to show seasonal variations. We showed significant seasonal and sexual dimorphic changes in the levels of transcript abundance in the whole brain and its regions. In whole brain, level was the highest in post-spawning phase and lowest in spawning phase in males. In females, there was a progressive increase through resting phase to pre-spawning phase, a decline in the spawning phase and increase in the post-spawning phase. In the preparatory phase, the highest transcript level was seen in medulla oblongata and the lowest in pituitary in males. In females, the level was the highest in the hypothalamus and lowest in olfactory bulb and pituitary. However, in the pre-spawning phase, in males it was the highest in telencephalon and hypothalamus and lowest in pituitary. In females, the highest transcript level was in olfactory bulb and lowest in pituitary. 3β-HSD enzyme activity showed significant seasonal variation in the brain, the highest in the resting phase and lowest in the preparatory and spawning phases. In situ hybridization showed the presence of 3β-HSD transcript was especially high in the cerebellum region. The presence of 3β-HSD in the brain may indicate steroidogenesis in the catfish brain. Copyright © 2016 Elsevier Inc. All rights reserved.

Elevated Expression of IRS-1 Associates with p-Akt Expression and Predicts Poor Prognosis of Breast Invasive Ductal Carcinoma.

PubMed

Luo, Jiadi; Feng, Juan; Wen, Qiuyuan; Qoyawayma, Christopher; Wang, Weiyuan; Chen, Lingjiao; Lu, Junmi; Zhan, Yuting; Xu, Lina; Zang, Hongjing; Fan, Songqing; Chu, Shuzhou

2018-03-15

Overexpression of Insulin Receptor Substrate 1 (IRS-1) has been reported to promote cell growth, atypical hyperplasia, and carcinogenesis. And phosphorylated Akt (p-Akt) is certified to be involved in many types of cancers such as breast invasive ductal carcinoma (BIDC). However, the relationship between IRS-1 and Akt, as well as the role of expression of IRS-1 in BIDC, has never been reported. The purpose of this research is to investigate the association between expression of IRS-1 and p-Akt proteins and clinicopathological features of BIDC by immunohistochemistry, as well as the survival status. The results showed that the percentage of either elevated expression of IRS-1 or positive p-Akt expression in BIDC is significantly higher than that in control breast tissue from non-cancer patients (P<.001, P=.001, respectively). Overexpression of IRS-1 was evidently associated with positive expression of p-Akt (r=.337, P<.001). Also, positive percentage of p-Akt expression was statistically different among different molecular subtypes of BIDC (highest in luminal B BIDC, P=.009). Furthermore, significantly worse overall survival was in BIDC patients with high expression of IRS-1 and p-Akt than patients with low expression (P=.006, P=.004, respectively). The multivariate Cox proportional hazard regression analysis showed that high expression of IRS-1 and positive expression of p-Akt protein were independent poor prognostic factors for patients with BIDC (P=.022, P=.046, respectively). In conclusion, we report for the first time that overexpression of IRS-1 protein associates with expression of p-Akt, and overexpression of IRS-1 and positive expression of p-Akt might be independent biomarkers for poor prognosis in BIDC. Copyright © 2018. Published by Elsevier Inc.

Study on differentiation during embryonic development across selective and ancestral breeds.

PubMed

An, Fengli; Wang, Jianlin

2017-06-01

In order to explore the effect of strain on diverging post-hatch muscle properties, muscle regulation during embryo development was investigated in selected and unselected breeds. Four broiler strains were used: JingNing (JN) chicken (a Chinese native chicken), HuangYu (HY) broiler, BaiYu (BY) broiler and Hyline layer (commercial crossbred chickens). Results showed that the four breeds had almost the same characteristic during different incubation periods. BY broilers moved more than JN and Hyline layers from Hamburger & Hamilton stage (HH)24 to HH31 (P < 0.05). HY broilers moved more than JN and Hyline layers from HH27 to HH31 (P < 0.01). All the embryos were heavier daily from HH24 to ED18 (P < 0.05); broilers presented greater body weights than JN and hyline layers (P > 0.05); broilers presented smaller fiber diameter than JN chickens before HH31 (P > 0.05). From then on, JN chicken exhibited smaller fiber diameter compared to the broilers (P > 0.05). Western blotting indicated all the breeds had continuous insulin-like growth factor-I (IGF-I) expression, with the highest expression level in broilers from HH19 to HH24 and highest expression level in JN chicks from HH27 to HH31. The results indicated that the diverging growth among breeds was already shown in embryonic stages; the different expression patterns of IGF-I may be involved in cell proliferation and differentiation. © 2016 Japanese Society of Animal Science.

Molecular characterization and expression profiling of ryanodine receptor gene in the pink stem borer, Sesamia inferens (Walker).

PubMed

Wu, Shun-Fan; Zhao, Dan-Dan; Huang, Jing-Mei; Zhao, Si-Qi; Zhou, Li-Qi; Gao, Cong-Fen

2018-04-01

The susceptibilities of three field populations of pink stem borer (PSB), Sesamia inferens (walker) to diamide insecticides, chlorantraniliprole and flubendiamide, were evaluated in this study. The results showed that these PSB field populations were still sensitive to the two diamide insecticides after many years of exposure. To further understand PSB and diamide insecticide, the full-length ryanodine receptor (RyR) cDNA (named as SiRyR), the molecular target of diamide insecticides was cloned from PSB and characterized. The SiRyR gene contains an open reading frame of 15,420 nucleotides, encoding 5140 amino acid residues, which shares 77% to 98% sequence identity with RyR homologous of other insects. All hallmarks of RyR proteins are conserved in the SiRyR protein, including the conserved C-terminal domain with the consensus calcium-biding EF-hands (calcium-binding motif), the six transmembrane domains, as well as mannosyltransferase, IP3R and RyR (pfam02815) (MIR) domains. Real-time qPCR analysis revealed that the highest mRNA expression levels of SiRyR were observed in pupa and adults, especially in males. SiRyR was expressed at the highest level in thorax, and the lowest level in wing. The full genetic characterization of SiRyR could provide useful information for future functional expression studies and for discovery of new insecticides with selective insecticidal activity. Copyright © 2018 Elsevier Inc. All rights reserved.

Light quality affects flavonoid production and related gene expression in Cyclocarya paliurus.

PubMed

Liu, Yang; Fang, Shengzuo; Yang, Wanxia; Shang, Xulan; Fu, Xiangxiang

2018-02-01

Understanding the responses of plant growth and secondary metabolites to differential light conditions is very important to optimize cultivation conditions of medicinal woody plants. As a highly valued and multiple function tree species, Cyclocarya paliurus is planted and managed for timber production and medical use. In this study, LED-based light including white light (WL), blue light (BL), red light (RL), and green light (GL) were used to affect leaf biomass production, flavonoid accumulation and related gene expression of one-year C. paliurus seedlings in controlled environments. After the treatments of 60 days, the highest leaf biomass appeared in the treatment of WL, while the lowest leaf biomass was found under GL. Compared to WL, the total flavonoid contents of C. paliurus leaves were significantly higher in BL, RL, and GL, but the highest values of selected flavonoids (kaempferol, isoquercitrin and quercetin) were observed under BL. Furthermore, the greatest yields of total and selected flavonoids in C. paliurus leaves per seedling were also achieved under BL, indicating that blue light was effective for inducing the production of flavonoids in C. paliurus leaves. Pearson's correlation analysis showed that there were significantly positive correlations between leaf flavonoid content and relative gene expression of key enzymes (phenylalanine ammonia lyase, PAL; 4-coumaroyl CoA-ligase, 4CL; and chalcone synthase, CHS) in the upstream, which converting phenylalanine into the flavonoid skeleton of tetrahydroxy chalcone. It is concluded that manipulating light quality may be potential mean to achieve the highest yields of flavonoids in C. paliurus cultivation, however this needs to be further verified by more field trials. Copyright © 2018 Elsevier B.V. All rights reserved.

High-fructose corn syrup-55 consumption alters hepatic lipid metabolism and promotes triglyceride accumulation.

PubMed

Mock, Kaitlin; Lateef, Sundus; Benedito, Vagner A; Tou, Janet C

2017-01-01

High-fructose corn syrup-55 (HFCS-55) has been suggested to be more lipogenic than sucrose, which increases the risk for nonalcoholic fatty liver disease (NAFLD) and dyslipidemia. The study objectives were to determine the effects of drinking different sugar-sweetened solutions on hepatic gene expression in relation to liver fatty acid composition and risk of NAFLD. Female rats were randomly assigned (n=7 rats/group) to drink water or water sweetened with 13% (w/v) HFCS-55, sucrose or fructose for 8 weeks. Rats drinking HFCS-55 solution had the highest (P=.03) hepatic total lipid and triglyceride content and histological evidence of fat infiltration. Rats drinking HFCS-55 solution had the highest hepatic de novo lipogenesis indicated by the up-regulation of stearoyl-CoA desaturase-1 and the highest (P<.001) oleic acid (18:1n-9) content. This was accompanied by reduced β-oxidation indicated by down-regulation of hepatic peroxisome proliferator-activated receptor α. Disposal of excess lipids by export of triglyceride-rich lipoprotein from the liver was increased as shown by up-regulation of gene expression of microsomal triglyceride transfer protein in rats drinking sucrose, but not HFCS-55 solution. The observed lipogenic effects were attributed to the slightly higher fructose content of HFCS-55 solution in the absence of differences in macronutrient and total caloric intake between rats drinking HFCS-55 and sucrose solution. Results from gene expression and fatty acid composition analysis showed that, in a hypercaloric state, some types of sugars are more detrimental to the liver. Based on these preclinical study results, excess consumption of caloric sweetened beverage, particularly HFCS-sweetened beverages, should be limited. Published by Elsevier Inc.

The metastasis suppressor gene KISS-1 regulates osteosarcoma apoptosis and autophagy processes.

PubMed

Yin, Yiran; Tang, Lian; Shi, Lei

2017-03-01

The expression of the metastasis suppressor gene KISS-1 in osteosarcoma cells during apoptosis and autophagy was evaluated. MG-63 osteosarcoma cells were transfected with either KISS-1 overexpression or KISS-1 knockdown expression vector in vitro, and compared with cell lines transfected with empty vector. After 12, 24, 48 and 72 h of cell culture, the cell proliferation was examined. The MTT method was used to detect apoptosis by flow cytometry, and the mRNA levels of apoptosis and autophagy markers caspase-3, Bcl-2, Bax, LC3 and Beclin1 were assessed by RT-PCR. Our results showed that cells in the control and low expression group kept proliferating during the cell culture period of 72 h, while the cells in the overexpression group progressively decreased in number. Also, the proliferation rate of the low expression group was significantly higher than that of the control group. The relative mRNA expression levels of caspase-3 and Bax mRNA in the control and low expression group showed no change (the expression was lowest in the low expression group). Moreover, the mRNA level of Bcl-2 increased in both cell groups. The mRNA expression levels of caspase-3 and Bax in the overexpression group were increased, and the level of Bcl-2 was reduced significantly. At the same time, the relative expression level of LC3 and Beclin1 mRNA in the control and low expression groups remained the same, and that of the overexpression group increased. The mRNA levels of LC3 and Beclin1 in the overexpression group were the highest, and that of the low expression group the lowest. The differences were statistically significant (P<0.05). Based on these results, we showed that KISS-1 inhibited the proliferation of osteosarcoma in vitro, probably by accelerating the processes of apoptosis and autophagy in the cells.

Cis-regulation of the amphioxus engrailed gene: insights into evolution of a muscle-specific enhancer.

PubMed

Beaster-Jones, Laura; Schubert, Michael; Holland, Linda Z

2007-08-01

To gain insights into the relation between evolution of cis-regulatory DNA and evolution of gene function, we identified tissue-specific enhancers of the engrailed gene of the basal chordate amphioxus (Branchiostoma floridae) and compared their ability to direct expression in both amphioxus and its nearest chordate relative, the tunicate Ciona intestinalis. In amphioxus embryos, the native engrailed gene is expressed in three domains - the eight most anterior somites, a few cells in the central nervous system (CNS) and a few ectodermal cells. In contrast, in C. intestinalis, in which muscle development is highly divergent, engrailed expression is limited to the CNS. To characterize the tissue-specific enhancers of amphioxus engrailed, we first showed that 7.8kb of upstream DNA of amphioxus engrailed directs expression to all three domains in amphioxus that express the native gene. We then identified the amphioxus engrailed muscle-specific enhancer as the 1.2kb region of upstream DNA with the highest sequence identity to the mouse en-2 jaw muscle enhancer. This amphioxus enhancer directed expression to both the somites in amphioxus and to the larval muscles in C. intestinalis. These results show that even though expression of the native engrailed has apparently been lost in developing C. intestinalis muscles, they express the transcription factors necessary to activate transcription from the amphioxus engrailed enhancer, suggesting that gene networks may not be completely disrupted if an individual component is lost.

Transcriptomic analysis of Salmonella desiccation resistance.

PubMed

Li, Haiping; Bhaskara, Anuhya; Megalis, Christina; Tortorello, Mary Lou

2012-12-01

The survival of Salmonella in low moisture foods and processing environments remains a great challenge for the food industry and public health. To explore the mechanisms of Salmonella desiccation resistance, we studied the transcriptomic responses in Salmonella Tennessee (Tennessee), using Salmonella Typhimurium LT2 (LT2), a strain weakly resistant to desiccation, as a reference strain. In response to 2 h of air-drying at 11% equilibrated relative humidity, approximately one-fourth of the open reading frames (ORFs) in the Tennessee genome and one-fifth in LT2 were differentially expressed (>2-fold). Among all differentially expressed functional groups (>5-fold) in both strains, the expression fold change associated with fatty acid metabolism was the highest, and constituted 51% and 35% of the total expression fold change in Tennessee and LT2, respectively. Tennessee showed greater changes in expression of genes associated with stress response and envelope modification than LT2, while showing lesser changes in protein biosynthesis expression. Expression of flagella genes was significantly more inhibited in stationary phase cells of Tennessee than LT2 both before and after desiccation. The accumulation of the osmolyte trehalose was significantly induced by desiccation in Tennessee, but no increase was detectable in LT2, which is consistent with the expression patterns of the entire trehalose biosynthesis and degradation pathways in both strains. Results from this study present a global view of the dynamic desiccation responses in Salmonella, which will guide future research efforts to control Salmonella in low moisture environments.

Differential gene expression of CYP3A isoforms in equine liver and intestines.

PubMed

Tydén, E; Löfgren, M; Pegolo, S; Capolongo, F; Tjälve, H; Larsson, P

2012-12-01

Recently, seven CYP3A isoforms - CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 - have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin-isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K(m) for the LIPA substrate in the liver and the intestine, while the maximum velocity (V(max)) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses. © 2012 Blackwell Publishing Ltd.

Acute myeloid leukaemia: expression of MYC protein and its association with cytogenetic risk profile and overall survival.

PubMed

Mughal, Muhammad Kashif; Akhter, Ariz; Street, Lesley; Pournazari, Payam; Shabani-Rad, Meer-Taher; Mansoor, Adnan

2017-09-01

Acute myeloid leukaemia (AML) is a clinically aggressive disease with marked genetic heterogeneity. Cytogenetic abnormalities provide the basis for risk stratification into clinically favourable, intermediate, and unfavourable groups. There are additional genetic mutations, which further influence the prognosis of patients with AML. Most of these result in molecular aberrations whose downstream target is MYC. It is therefore logical to study the relationship between MYC protein expression and cytogenetic risk groups. We studied MYC expression by immunohistochemistry in a large cohort (n = 199) of AML patients and correlated these results with cytogenetic risk profile and overall survival (OS). We illustrated differential expression of MYC protein across various cytogenetic risk groups (p = 0.03). Highest expression of MYC was noted in AML patients with favourable cytogenetic risk group. In univariate analysis, MYC expression showed significant negative influence of OS in favourable and intermediate cytogenetic risk group (p = 0.001). Interestingly, MYC expression had a protective effect in the unfavourable cytogenetic risk group. In multivariate analysis, while age and cytogenetic risk group were significant factors influencing survival, MYC expression by immunohistochemistry methods also showed some marginal impact (p = 0.069). In conclusion, we have identified differential expression of MYC protein in relation to cytogenetic risk groups in AML patients and documented its possible impact on OS in favourable and intermediate cytogenetic risk groups. These preliminary observations mandate additional studies to further investigate the routine clinical use of MYC protein expression in AML risk stratification. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

NASA Astrophysics Data System (ADS)

Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi

2013-12-01

First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

Effects of Pectic Polysaccharides Isolated from Leek on the Production of Reactive Oxygen and Nitrogen Species by Phagocytes

PubMed Central

Nikolova, Mariana; Ambrozova, Gabriela; Kratchanova, Maria; Denev, Petko; Kussovski, Veselin; Ciz, Milan

2013-01-01

Abstract The current survey investigates the effect of four polysaccharides isolated from fresh leek or alcohol insoluble substances (AIS) of leek on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) from phagocytes. The ability of the polysaccharides to activate serum complement was also investigated. Despite the lack of antioxidant activity, the pectic polysaccharides significantly decreased the production of ROS by human neutrophils. Polysaccharides isolated from AIS markedly activated RAW 264.7 macrophages for RNS production in a concentration-dependent manner. The Western blot analysis revealed that this effect was due to the stimulation of the inducible nitric oxide synthase protein expression of macrophages. The polysaccharides extracted from AIS with water showed the ability to fix serum complement, especially through the alternative pathway. It was found that the polysaccharide that has the highest complement-fixing effect is characterized by the highest content of uronic acids and the highest molecular weight. PMID:23905651

Expression and regulation of CNTF receptor-alpha in the in situ and in oculo grafted adult rat adrenal medulla.

PubMed

Förander, P; Brené, S; Strömberg, I

2000-02-28

Cultured and transplanted adrenal medullary cells respond to ciliary neurotrophic factor (CNTF) with neurite formation and improved cell survival although the presence of the CNTF receptor-alpha (CNTFRalpha) has been unclear. This study show that CNTFRalpha mRNA was expressed in the postnatal day 1 as well as in the adult rat adrenal medulla. The highest CNTFRalpha mRNA signal was found in the ganglion cells of the adrenal medulla. After transplantation of adrenal medullary tissue the CNTFRalpha mRNA levels were down-regulated in the chromaffin cells. CNTF treatment of grafts did not normalize the receptor levels, but treatment with nerve growth factor (NGF) did. Thus, we demonstrate that CNTFRalpha mRNA is expressed in adrenal medulla, the levels becomes down-regulated after transplantation, but normalized after treatment with NGF.

Expression of growth hormone gene during early development of Siberian sturgeon (Acipenser baerii)

PubMed Central

Abdolahnejad, Zeinab; Pourkazemi, Mohammad; Khoshkholgh, Majid Reza; Yarmohammadi, Mahtab

2015-01-01

The mRNA expression of growth hormone (GH) gene in early development stages of Siberian sturgeon was investigated using RT-PCR method. Samples were collected from unfertilized eggs up to 50 days post hatched (dph) larvae in 11 different times. Ribosomal protein L6 (RPL6) transcripts were used as the internal standard during quantification of GH mRNA expression. The results showed that the GH mRNA could be observed in the eyed eggs and even at unfertilized eggs of Siberian sturgeon. The highest amounts of GH mRNA were found at 25 and 50 dph larvae, while the lowest levels were detected at 1 and 3 dph larvae stage. These findings suggest that, the GH mRNA play a key role during developmental stages of Siberian sturgeon. PMID:27844010

Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions.

PubMed

Guo, Guangyu; Xu, Kun; Zhang, Xiaomei; Zhu, Jinlong; Lu, Mingyang; Chen, Fulu; Liu, Linpo; Xi, Zhang-Ying; Bachmair, Andreas; Chen, Qingshan; Fu, Yong-Fu

2015-01-01

The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth.

Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions

PubMed Central

Zhu, Jinlong; Lu, Mingyang; Chen, Fulu; Liu, Linpo; Xi, Zhang-Ying; Bachmair, Andreas; Chen, Qingshan; Fu, Yong-Fu

2015-01-01

The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth. PMID:26371882

Bioelectrochemical conversion of CO2 to value added product formate using engineered Methylobacterium extorquens.

PubMed

Jang, Jungho; Jeon, Byoung Wook; Kim, Yong Hwan

2018-05-08

The conversion of carbon dioxide to formate is a fundamental step for building C1 chemical platforms. Methylobacterium extorquens AM1 was reported to show remarkable activity converting carbon dioxide into formate. Formate dehydrogenase 1 from M. extorquens AM1 (MeFDH1) was verified as the key responsible enzyme for the conversion of carbon dioxide to formate in this study. Using a 2% methanol concentration for induction, microbial harboring the recombinant MeFDH1 expressing plasmid produced the highest concentration of formate (26.6 mM within 21 hours) in electrochemical reactor. 60 μM of sodium tungstate in the culture medium was optimal for the expression of recombinant MeFDH1 and production of formate (25.7 mM within 21 hours). The recombinant MeFDH1 expressing cells showed maximum formate productivity of 2.53 mM/g-wet cell/hr, which was 2.5 times greater than that of wild type. Thus, M. extorquens AM1 was successfully engineered by expressing MeFDH1 as recombinant enzyme to elevate the production of formate from CO 2 after elucidating key responsible enzyme for the conversion of CO 2 to formate.

Toxic responses of Sox2 gene in the regeneration of the earthworm Eisenia foetida exposed to Retnoic acid.

PubMed

Tao, Jing; Rong, Wei; Diao, Xiaoping; Zhou, Hailong

2018-01-01

Exogenous retinoic acid delays and disturbs the regeneration of Eisenia foetida. The stem cell pluripotency factor, Sox2, can play a crucial role in cell reprogramming and dedifferentiation. In this study, we compared the regeneration of Eisenia foetida in different segments after amputation and the effects of retinoic acid on the regeneration of different segments. The results showed that the regeneration speed of the head and tail was slightly faster than the middle part, and retinoic acid disrupted and delayed the regeneration of the earthworm. The qRT-PCR and Western blot analysis showed that the expression of the Sox2 gene and Sox2 protein was highest on the seventh day in different segments (p<0.05). After treatment with retinoic acid, the expression level of the Sox2 gene and Sox2 protein was significantly reduced (p<0.05). The results indicated that the regeneration of earthworms and the formation of blastema are related to the expression of the Sox2 gene and protein. Retinoic acid delays and interferes with the regeneration of the earthworm by affecting the expression levels of the Sox2 gene and protein. Copyright © 2017 Elsevier Inc. All rights reserved.

A Novel 5-Enolpyruvylshikimate-3-Phosphate Synthase Shows High Glyphosate Tolerance in Escherichia coli and Tobacco Plants

PubMed Central

Zhang, Shengxue; Yang, Xuewen; Chen, Rongrong; Zhang, Yuwen; Lu, Wei; Liu, Yan; Wang, Jianhua; Lin, Min; Wang, Guoying

2012-01-01

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops. PMID:22715408

Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells.

PubMed

Kehl, Debora; Generali, Melanie; Görtz, Sabrina; Geering, Diego; Slamecka, Jaroslav; Hoerstrup, Simon P; Bleul, Ulrich; Weber, Benedikt

2017-10-01

Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

Fundamental research on the action mechanism of the 800 nm semiconductor laser on skin blackheads and coarse pores.

PubMed

Lin, Jie; Jing, Li; Zhu, Hao; Dong, Fu-Sheng

2017-01-01

The aim of the study was to determine the mechanism of action of the 800 nm semiconductor laser on skin blackheads and coarse pores. A total of 24 healthy purebred short-haired male guinea pigs, weighing 350-400 g, were selected and smeared with 0.5 ml coal tar suspension evenly by injector once daily. Treatment was continued for 14 days to form an experimental area of 8×3 cm on the back of the guinea pigs. The animals were divided into the following groups: Normal control group (NC), low-dose laser treatment group (L-LS), high-dose laser treatment group (H-LS), and Q-switched Nd:YAG treatment group (QC). Samples were extracted 1, 7 and 14 days after surgery and hematoxylin and eosin staining was used to identify the following: Epidermis, dermis, sebaceous gland change and hair follicle damage; the expression of proliferating cell nuclear antigen (PCNA) of sebaceous gland cells using immunohistochemistry; sebaceous gland cell apoptosis using TUNEL; and the protein expression of caspase-3, Bax and Bcl-2 using western blot analysis. With the extension of time, we observed inflammatory cell infiltration, an increase in hair follicle distortion and necrosis of the surrounding hair follicles. The expression levels of PCNA of the L-LS, H-LS and QC groups decreased with time. Regarding the respective time points, the NC group was highest, the L-LS and H-LS groups were next highest and the H-LS group was lowest. The difference was statistically significant (P<0.05). The apoptotic rate of the L-LS, H-LS and QC groups increased with time. With regard to the respective time points, the NC group was lowest, the L-LS and QC groups were next lowest and the H-LS group was highest. The difference was statistically significant (P<0.05). The protein expression of caspase-3, Bax and Bcl-2 of the L-LS, H-LS and QC groups increased with time. Regarding the respective time points, caspase-3 and Bax protein expression of the NC group was lowest, the L-LS and QC groups were next lowest and the H-LS group was highest. Bcl-2 protein expression of the NC group was highest, protein expression of the NC group was next highest and the H-LS group was lowest. The difference was statistically significant (P<0.05). In conclusion, the low-dose 800 nm semiconductor laser is an effective treatment on skin blackheads and coarse pores, and promotes hair follicle cell apoptosis without reducing the expression of PCNA.

Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

PubMed

Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

2016-04-07

Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding.

Drivers of the dynamics of diazotrophs and denitrifiers in North Sea bottom waters and sediments

PubMed Central

Fan, Haoxin; Bolhuis, Henk; Stal, Lucas J.

2015-01-01

The fixation of dinitrogen (N2) and denitrification are two opposite processes in the nitrogen cycle. The former transfers atmospheric dinitrogen gas into bound nitrogen in the biosphere, while the latter returns this bound nitrogen back to atmospheric dinitrogen. It is unclear whether or not these processes are intimately connected in any microbial ecosystem or that they are spatially and/or temporally separated. Here, we measured seafloor nitrogen fixation and denitrification as well as pelagic nitrogen fixation by using the stable isotope technique. Alongside, we measured the diversity, abundance, and activity of nitrogen-fixing and denitrifying microorganisms at three stations in the southern North Sea. Nitrogen fixation ranged from undetectable to 2.4 nmol N L−1 d−1 and from undetectable to 8.2 nmol N g−1 d−1 in the water column and seafloor, respectively. The highest rates were measured in August at Doggersbank, both for the water column and for the seafloor. Denitrification ranged from 1.7 to 208.8 μmol m−2 d−1 and the highest rates were measured in May at the Oyster Grounds. DNA sequence analysis showed sequences of nifH, a structural gene for nitrogenase, related to sequences from anaerobic sulfur/iron reducers and sulfate reducers. Sequences of the structural gene for nitrite reductase, nirS, were related to environmental clones from marine sediments. Quantitative polymerase chain reaction (qPCR) data revealed the highest abundance of nifH and nirS genes at the Oyster Grounds. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data revealed the highest nifH expression at Doggersbank and the highest nirS expression at the Oyster Grounds. The distribution of the diazotrophic and denitrifying communities seems to be subject to different selecting factors, leading to spatial and temporal separation of nitrogen fixation and denitrification. These selecting factors include temperature, organic matter availability, and oxygen concentration. PMID:26257718

Retinal dehydrogenase gene expression in stomach and small intestine of rats during postnatal development and in vitamin A deficiency.

PubMed

Bhat, P V

1998-04-17

Retinal dehydrogenase (RALDH) catalyzes the oxidation of retinal to all-trans and 9-cis retinoic acid, which function as ligands controlling RAR and RXR nuclear receptor-signaling pathways. We have recently shown the expression of RALDH transcript in the stomach and small intestine by reverse transcription polymerase chain reaction [Bhat, P.V., Labrecque J., Dumas, F., Lacroix, A. and Yoshida, A. (1995) Gene 166, 303-306]. We have examined RALDH expression in the stomach and small intestine before and during postnatal development and in vitamin A deficiency by assaying for mRNA levels and protein as well as for enzyme activity. In -2 day fetuses, RALDH expression was high in the small intestine, whereas RALDH protein was not detectable in the stomach. However, expression of RALDH was seen in the stomach after birth, and gradually increased with age and reached the highest level at postnatal day 42. In the intestine, RALDH expression decreased postnatally. Vitamin A deficiency up-regulated RALDH expression in the stomach and small intestine, and administration of retinoids down-regulated the RALDH expression in these tissues. These results show the differential expression of RALDH in the stomach and small intestine during postnatal development, and that vitamin A status regulates the expression of RALDH gene in these tissues.

Immunohistochemical expression of heat shock protein27 in the mouse dental pulp after immediate teeth separation

PubMed Central

2011-01-01

Aim After immediate teeth separation, expression of HSP27 in the mouse dental pulp was examined. Immunohistochemistry was performed to examine the incidence of HSP27 expression. Materials and methods A total of 36 8-week-old ddY mice were used as experimental subjects and a wedge was inserted in between maxillary right molars. The wedge was removed 30 min or 3 h after insertion. Animals were immediately sacrificed after the removal of wedge or until 1 week later and serial sections from paraffin-embedded tissues were prepared. Immunohistochemistry was carried out to examine the expression of HSP27. The untreated side served as the control. Results In the control group, the endothelial cells and some pulp fibroblasts weakly expressed HSP27 suggesting that the expression is due to mechanical stress brought about by physiological masticatory force and pressure from the tongue. In both 30 min and 3 h experimental groups, HSP27 expression was highest at 24 h after wedge removal and the expression remained the same or started to decrease thereafter. The expression decreased at the same level as that of the control group 1 week after wedge removal. Conclusion HSP27 may serve as an indicator of stimulus strong enough to show its expression. PMID:22027643

Histological change and heat shock protein 70 expression in different tissues of Japanese flounder Paralichthys olivaceus in response to elevated temperature

NASA Astrophysics Data System (ADS)

Liu, Yifan; Ma, Daoyuan; Xiao, Zhizhong; Xu, Shihong; Wang, Yanfeng; Wang, Yufu; Xiao, Yongshuang; Song, Zongcheng; Teng, Zhaojun; Liu, Qinghua; Li, Jun

2015-01-01

High temperature influences the homeostasis of fish. We investigated the effects of elevated temperature on tissues of Japanese flounder ( Paralichthys olivaceus) by analyzing the histology and heat shock protein 70 ( hsp70) expression of fish reared in warm conditions. In this study, temperature was increased at 1±0.5°C/day starting at 24±0.5°C, and was kept at that temperature for 5 days before the next rise. After raising temperature at the rate up to 32±0.5°C, tissue samples from midgut, spleen, stomach, liver, muscle, gill, heart, trunk kidney and brain were collected for histological analysis and mRNA assay. Almost all the tissues showed changes in morphological structure and hsp70 level at 32±0.5°C. Histological assessment of the tissues indicated that the gill had the most serious damage, including highly severe epithelial lifting and edema, curved tips and hyperemia at the ending of the lamellars, desquamation and necrosis. The next most severe damage was found in liver and kidney. The hsp70 levels in all the tissues first increased and then decreased. The gut, stomach, muscle, heart, and brain had the highest expressions in 6 h, whereas the spleen, liver, gill and kidney had the highest expressions in 2 h. Therefore, tissues with the most significant lesions (especially gill and liver) responded much earlier (2 h) in hsp70 expression than other tissues, and these tissues demonstrated the most marked histological disruption and elevated mRNA levels, making them ideal candidates for further studies on the thermal physiology of this species.

Altered Expression of Porcine Piwi Genes and piRNA during Development

PubMed Central

Kowalczykiewicz, Dorota; Pawlak, Piotr; Lechniak, Dorota; Wrzesinski, Jan

2012-01-01

Three Sus scrofa Piwi genes (Piwil1, Piwil2 and Piwil4) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between Sus scrofa and Homo sapiens. Relative transcript abundance of porcine Piwil1, Piwil2 and Piwil4 genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three Piwi mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the Sus scrofa Piwil1 gene. On the other hand, in testes of neonatal pigs the Piwil1 transcript level was over 2–fold reduced while the level of Piwil2 transcript was higher. As regards the expression of the Piwil4 transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three Piwi genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the Piwil2 gene was characterized by the highest relative transcript abundance among the three Piwi genes analysed. In prepubertal and pubertal oocytes Piwil1 transcript was the most abundant whereas the expression of Piwil4 was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2–3 nucleotides longer than the piRNA from testes. PMID:22952772

Differential expression pattern of heat shock protein 70 gene in tissues and heat stress phenotypes in goats during peak heat stress period.

PubMed

Rout, P K; Kaushik, R; Ramachandran, N

2016-07-01

It has been established that the synthesis of heat shock protein 70 (Hsp70) is temperature-dependent. The Hsp70 response is considered as a cellular thermometer in response to heat stress and other stimuli. The variation in Hsp70 gene expression has been positively correlated with thermotolerance in Drosophila melanogaster, Caenorhabditis elegans, rodents and human. Goats have a wide range of ecological adaptability due to their anatomical and physiological characteristics; however, the productivity of the individual declines during thermal stress. The present study was carried out to analyze the expression of heat shock proteins in different tissues and to contrast heat stress phenotypes in response to chronic heat stress. The investigation has been carried out in Jamunapari, Barbari, Jakhrana and Sirohi goats. These breeds differ in size, coat colour and production performance. The heat stress assessment in goats was carried out at a temperature humidity index (THI) ranging from 85.36-89.80 over the period. Phenotyping for heat stress susceptibility was carried out by combining respiration rate (RR) and heart rate (HR). Based on the distribution of RR and HR over the breeds in the population, individual animals were recognized as heat stress-susceptible (HSS) and heat stress-tolerant (HST). Based on their physiological responses, the selected animals were slaughtered for tissue collection during peak heat stress periods. The tissue samples from different organs such as liver, spleen, heart, testis, brain and lungs were collected and stored at -70 °C for future use. Hsp70 concentrations were analyzed from tissue extract with ELISA. mRNA expression levels were evaluated using the SYBR green method. Kidney, liver and heart had 1.5-2.0-fold higher Hsp70 concentrations as compared to other organs in the tissue extracts. Similarly, the gene expression pattern of Hsp70 in different organs indicated that the liver, spleen, brain and kidney exhibited 5.94, 4.96, 5.29 and 2.63-fold higher expression than control. Liver and brain tissues showed the highest gene expression at mRNA levels as compared to kidney, spleen and heart. HST individuals had higher levels of mRNA level expression than HSS individuals in all breeds. The Sirohi breed showed the highest (6.3-fold) mRNA expression levels as compared to the other three breeds, indicating the better heat stress regulation activity in the breed.

Gene expression of antioxidant enzymes and coffee seed quality during pre- and post-physiological maturity.

PubMed

Santos, F C; Caixeta, F; Clemente, A C S; Pinho, E V; Rosa, S D V F

2014-12-19

Seeds collected at different maturation stages vary in physiological quality and patterns of protective antioxidant systems against deterioration. In this study we investigated the expression of genes that codify catalase (CAT), dismutase superoxide (SOD), and polyphenol oxidase (PPO) during the pre- and post-physiological maturation phases in whole seeds and in endosperms and embryos extracted from the seeds. Coffea arabica L. berries were collected at the green, yellowish-green, cherry, over-ripe, and dry stages, and the seeds were examined physiologically. The transcription levels of the genes were quantified by quantitative real-time polymerase chain reaction using coffee-specific primers. The highest level of SOD expression was observed in the endosperm at the cherry and over-ripe stages; in addition, these seeds presented the greatest physiological quality (assessed via germination test). The highest CAT3 transcript expression was observed at the green stage in whole seeds, and at the green and over-ripe stages in the embryos and endosperms. High expression of the PPO transcript was observed at the green and yellowish-green stages in whole seeds. In embryos and endosperms, peak expression of the PPO transcript was observed at the green stage; subsequently, peaks at the cherry and over-ripe stages were observed. We concluded that the expression patterns of the SOD and CAT3 transcripts were similar at the more advanced maturation stages, which corresponded to enhanced physiological seed quality. High expression of the PPO transcript at the over-ripe stage, also observed in the embryos and endosperms at the cherry stage, coincided with the highest physiological seed quality.

Local oxytocin expression and oxytocin receptor binding in the male rat brain is associated with aggressiveness.

PubMed

Calcagnoli, Federica; de Boer, Sietse F; Beiderbeck, Daniela I; Althaus, Monika; Koolhaas, Jaap M; Neumann, Inga D

2014-03-15

We recently demonstrated in male wild-type Groningen rats that enhancing brain oxytocin (OXT) levels acutely produces marked pro-social explorative and anti-aggressive effects. Moreover, these pharmacologically-induced changes are moderated by the individual's aggressive phenotype, suggesting an inverse relationship between aggressiveness and tonic endogenous OXT signaling properties. Aim of the present study was to verify the hypothesis that variations in OXT expression and/or OXT receptor (OXTR) binding in selected brain regions are associated with different levels or forms of aggression. To this end, male resident wild-type Groningen rats that repeatedly contested and dominated intruder conspecifics were categorized as being low aggressive, highly aggressive or excessively aggressive. Their brains were subsequently collected and quantified for OXT mRNA expression and OXTR binding levels. Our results showed that OXT mRNA expression in the hypothalamic paraventricular nucleus (PVN), but not in the supraoptic nucleus (SON), negatively correlates with the level of offensiveness. In particular, the excessively aggressive group showed a significantly lower OXT mRNA expression in the PVN as compared to both low and highly aggressive groups. Further, the excessively aggressive animals showed the highest OXTR binding in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST). These findings demonstrate that male rats with excessively high levels and abnormal forms of aggressive behavior have diminished OXT transcription and enhanced OXTR binding capacities in specific nodes of the social behavioral brain circuitry. Copyright © 2014 Elsevier B.V. All rights reserved.

Investigation of different concentrations of MS media effects on gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni.

PubMed

Kahrizi, Danial; Ghaheri, Matin; Yari, Zahra; Yari, Khirollah; Bahraminejad, Sohbat

2018-02-10

Stevia rebaudiana Bertoni is one of two species that contains steviol glycosides. Among steviol glycosides that extracted from leaves, stevioside and rebaudioside A are the two major and the sweetest glycosides that are about 200-300 times sweeter than sucrose with zero calories. The best method for stevia propagation is tissue culture. So, for investigation of nutrients in medium, we studied the effect of different concentrations of MS media (MS, 0.5 MS, 0.25 MS, 0 MS) on morphological traits, UGT74G1 and UGT76G1 genes expression and accumulation of steviol glycosides in stevia leaves. The best growth rate (0.472 mm/d) has occurred in plants grown in MS media. Also, the highest gene expression of UGT74G1 gene (1.000 Total lab unit) was seen under MS treatment. However, the highest expression level of UGT76G1 gene (1.701 Total lab unit) was observed at plants grown in 0 MS. The highest amount of both Stevioside and Rebaudioside A (14.23 and 8.12, respectively) were accumulated in plants under MS treatment. Obviously, dilution of MS media associated with decreasing in both expression of the intended genes and accumulation of steviol glycosides.

Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa.

PubMed

Shi, Rui; Wang, Jack P; Lin, Ying-Chung; Li, Quanzi; Sun, Ying-Hsuan; Chen, Hao; Sederoff, Ronald R; Chiang, Vincent L

2017-05-01

Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.

Characterization, expression profiles, intracellular distribution and association analysis of porcine PNAS-4 gene with production traits.

PubMed

Mo, Delin; Zhu, Zhengmao; te Pas, Marinus F W; Li, Xinyun; Yang, Shulin; Wang, Heng; Wang, Huanling; Li, Kui

2008-06-30

In a previous screen to identify differentially expressed genes associated with embryonic development, the porcine PNAS-4 gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and production efficiency, we determined how porcine PNAS-4 gene regulates meat production. Therefore, this gene has been sequenced, expression analyzed and associated with meat production traits. We cloned the full-length cDNA of porcine PNAS-4 gene encoding a protein of 194 amino acids which was expressed in the Golgi complex. This gene was mapped to chromosome 10, q11-16, in a region of conserved synteny with human chromosome 1 where the human homologous gene was localized. Real-time PCR revealed that PNAS-4 mRNA was widely expressed with highest expression levels in skeletal muscle followed by lymph, liver and other tissues, and showed a down-regulated expression pattern during prenatal development while a up-regulated expression pattern after weaning. Association analysis revealed that allele C of SNP A1813C was prevalent in Chinese indigenous breeds whereas A was dominant allele in Landrace and Large White, and the pigs with homozygous CC had a higher fat content than those of the pigs with other genotypes (P < 0.05). Porcine PNAS-4 protein tagged with green fluorescent protein accumulated in the Golgi complex, and its mRNA showed a widespread expression across many tissues and organs in pigs. It may be an important factor affecting the meat production efficiency, because its down-regulated expression pattern during early embryogenesis suggests involvement in increase of muscle fiber number. In addition, the SNP A1813C associated with fat traits might be a genetic marker for molecular-assisted selection in animal breeding.

Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments.

PubMed

Akkiprik, Mustafa; Peker, İrem; Özmen, Tolga; Amuran, Gökçe Güllü; Güllüoğlu, Bahadır M; Kaya, Handan; Özer, Ayşe

2015-11-10

IGFBP5 is an important regulatory protein in breast cancer progression. We tried to identify differentially expressed genes (DEGs) between breast tumor tissues with IGFBP5 overexpression and their adjacent normal tissues. In this study, thirty-eight breast cancer and adjacent normal breast tissue samples were used to determine IGFBP5 expression by qPCR. cDNA microarrays were applied to the highest IGFBP5 overexpressed tumor samples compared to their adjacent normal breast tissue. Microarray analysis revealed that a total of 186 genes were differentially expressed in breast cancer compared with normal breast tissues. Of the 186 genes, 169 genes were downregulated and 17 genes were upregulated in the tumor samples. KEGG pathway analyses showed that protein digestion and absorption, focal adhesion, salivary secretion, drug metabolism-cytochrome P450, and phenylalanine metabolism pathways are involved. Among these DEGs, the prominent top two genes (MMP11 and COL1A1) which potentially correlated with IGFBP5 were selected for validation using real time RT-qPCR. Only COL1A1 expression showed a consistent upregulation with IGFBP5 expression and COL1A1 and MMP11 were significantly positively correlated. We concluded that the discovery of coordinately expressed genes related with IGFBP5 might contribute to understanding of the molecular mechanism of the function of IGFBP5 in breast cancer. Further functional studies on DEGs and association with IGFBP5 may identify novel biomarkers for clinical applications in breast cancer.

Bigger is not always better; How cold-water corals outgrow themselves

NASA Astrophysics Data System (ADS)

Mienis, Furu; Duineveld, Gerard; Lavaleye, Marc; Mohn, Christian; Van Haren, Hans

2015-04-01

Cold-water coral (CWC) framework acts as a sediment trap and as a result kilometres long and up to 360m high mound structures have formed on the SE Rockall Bank. Earlier observations showed that most of the mounds have their summits around 550 m water depth and summits have been reported as being covered with living coral. Pelagia cruises in 2012 and 2013 revealed completely new insights in mound development. Video transects across mounds with different morphology showed that summits of the highest and largest mounds are presently not covered by living coral as opposed to smaller and lower mounds which are covered with a thriving living coral framework. This is also expressed by the vertical mound growth rate measured in sediment cores showing fourfold higher sedimentation rates during the Holocene on small mounds compared to highest mounds. Measurements in the water column with CTD and near-bottom with benthic landers and thermistor string showed that turbulence is likely the most important factor influencing nutrient and food supply and thus coral growth. It seems that the large mounds have outgrown themselves and that their relatively large size and flat summits are limiting turbulence, thereby limiting coral and mound growth.

The Antimicrobial Peptide Lysozyme Is Induced after Multiple Trauma

PubMed Central

Klüter, Tim; Fitschen-Oestern, Stefanie; Lippross, Sebastian; Weuster, Matthias; Pufe, Thomas; Tohidnezhad, Mersedeh; Beyer, Andreas; Seekamp, Andreas; Varoga, Deike

2014-01-01

The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction. PMID:25258475

[Key techniques for precision cultivation of nitrogenous fertilizer of pollution-free ginseng].

PubMed

Guo, Li-Li; Guo, Shuai; Dong, Lin-Lin; Shen, Liang; Li, Xi-Wen; Xu, Jiang; Chen, Shi-Lin

2018-04-01

Planting pollution-free farmland is the main mode of industrialization of ginseng cultivation, fine management of nitrogen fertilizer ginseng pollution-free farmland cultivation technology system is one of the key factors. In order to investigate the effect of nitrogenous fertilizer on the accumulation of ginseng biomass and saponins synthesis in vegetative growth stage, two-years-old ginsengs were used as test materials in this study. The test materials were cultivated by Hoagland medium with different nitrogen concentration (0，10，20，40 mg·L⁻¹) for 40 days. During the cultivation, photosynthetic rate was measured four times. After 40 days cultivation, chlorophyll content, stem diameter and the spatiotemporal expression of saponin synthesis related genes PgHMGR and PgSQE were tested. The results showed that there were significant differences in the photosynthetic rate and chlorophyll content among different nitrogen concentrations. The relative expression level of PgHMGR gene and PgSQE gene in root, stem and leaves of ginseng were different. Ginseng seedlings cultivated by 20 mg·L⁻¹ nitrogen possess the highest photosynthetic rate and chlorophyll content, while PgHMGR and PgSE showed the highest gene expression level. The optimal nitrogen concentration for the growth of 2-years-old ginseng might be 20 mg·L⁻¹ with 57.14 g ammonium nitrate each plant or pure 20.00 mg nitrogen each plant. It is concluded that this concentration is the most suitable concentration for the ginsenoside synthesis. Pollution-free ginseng with fine nitrogen fertilizer cultivation is conducive to the production of high quality and efficient ginseng medicinal materials. It lays a theoretical foundation for the rational fertilization and environment-friendly sustainable ecological ginseng planting industry. Copyright© by the Chinese Pharmaceutical Association.

MyoD and Myf6 gene expression patterns in skeletal muscle during embryonic and posthatch development in the domestic duck (Anas platyrhynchos domestica).

PubMed

Li, H; Zhu, C; Tao, Z; Xu, W; Song, W; Hu, Y; Zhu, W; Song, C

2014-06-01

The MyoD and Myf6 genes, which are muscle regulatory factors (MRFs), play major roles in muscle growth and development and initiate muscle fibre formation via the regulation of muscle-specific gene translation. Therefore, MyoD and Myf6 are potential candidate genes for meat production traits in animals and poultry. The objective of this study was to evaluate MyoD and Myf6 gene expression patterns in the skeletal muscle during early developmental stage of ducks. Gene expression levels were detected using the quantitative RT-PCR method in the breast muscle (BM) and leg muscle (LM) at embryonic days 13, 17, 21, 25, 27, as well as at 1 week posthatching in Gaoyou and Jinding ducks (Anas platyrhynchos domestica). The MyoD and Myf6 gene profiles in the two duck breeds were consistent during early development, and MyoD gene expression showed a 'wave' trend in BM and an approximate 'anti-√' trend in LM. Myf6 gene expression in BM showed the highest level at embryonic day 21, which subsequently decreased, although remained relatively high, while levels at embryonic days 13, 17 and 21 were higher in LM. The results of correlation analysis showed that MyoD and Myf6 gene expression levels were more strongly correlated in LM than in BM in both duck breeds. These results indicated that different expression patterns of the MyoD and Myf6 genes in BM and LM may be related to muscle development and differentiation, suggesting that MyoD and Myf6 are integral to skeletal muscle development. © 2013 Blackwell Verlag GmbH.

Culturing INS-1 cells on CDPGYIGSR-, RGD- and fibronectin surfaces improves insulin secretion and cell proliferation.

PubMed

Kuehn, Carina; Dubiel, Evan A; Sabra, Georges; Vermette, Patrick

2012-02-01

Rat insulinoma cells (INS-1), an immortalized pancreatic beta cell line, were cultured on low-fouling carboxymethyl-dextran (CMD) layers bearing fibronectin, the tripeptide Arg-Gly-Asp (RGD) or CDPGYIGSR, a laminin nonapeptide. INS-1 cells were non-adherent on CMD and RGE but adhered to fibronectin- and peptide-coated CMD surfaces and to tissue culture polystyrene (TCPS). On CMD bearing fibronectin and the peptides, INS-1 cells showed higher glucose-stimulated insulin secretion compared to those on TCPS, bare CMD and RGE. INS-1 cells experienced a net cell growth, with the lowest found after 7 days on CMD and the highest on fibronectin. Similarly, cells on RGD and CDPGYIGSR showed lower net growth rates than those on fibronectin. Expression of E-cadherin and integrins αvβ3 and α5 were similar between the conditions, except for α5 expression on fibronectin, RGD and CDPGYIGSR. Larger numbers of Ki-67-positive cells were found on CDPGYIGSR, TCPS, fibronectin and RGD. Cells in all conditions expressed Pdx1. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

An Assessment of How Facial Mimicry Can Change Facial Morphology: Implications for Identification.

PubMed

Gibelli, Daniele; De Angelis, Danilo; Poppa, Pasquale; Sforza, Chiarella; Cattaneo, Cristina

2017-03-01

The assessment of facial mimicry is important in forensic anthropology; in addition, the application of modern 3D image acquisition systems may help for the analysis of facial surfaces. This study aimed at exposing a novel method for comparing 3D profiles in different facial expressions. Ten male adults, aged between 30 and 40 years, underwent acquisitions by stereophotogrammetry (VECTRA-3D ® ) with different expressions (neutral, happy, sad, angry, surprised). The acquisition of each individual was then superimposed on the neutral one according to nine landmarks, and the root mean square (RMS) value between the two expressions was calculated. The highest difference in comparison with the neutral standard was shown by the happy expression (RMS 4.11 mm), followed by the surprised (RMS 2.74 mm), sad (RMS 1.3 mm), and angry ones (RMS 1.21 mm). This pilot study shows that the 3D-3D superimposition may provide reliable results concerning facial alteration due to mimicry. © 2016 American Academy of Forensic Sciences.

Glucocorticoid receptor expression on circulating leukocytes in healthy and asthmatic adolescents in response to exercise

PubMed Central

Lu, Kim D.; Cooper, Dan; Haddad, Fadia; Zaldivar, Frank; Kraft, Monica; Radom-Aizik, Shlomit

2017-01-01

Background Poor aerobic fitness is associated with worsening of asthma symptoms and fitness training may improve asthma control. The mechanism linking fitness with asthma is not known. We hypothesized that repeated bouts of exercise would lead to a downregulation of glucocorticoid receptor (GR) expression on circulating leukocytes reflecting a reduced responsiveness to stress. Methods In a prospective exercise training intervention of healthy and asthmatic adolescents, GR expression in leukocytes was measured using flow cytometry in response to a brief exercise challenge before and after the training intervention. PBMC gene expression of GR, GRβ, HSP70, and TGFβ1, 2 were determined using RT-PCR. Results Peak V̇O2 increased by 14.6 ± 2.3% indicating an effective training (p<0.01). There was a significant difference in GR expression among leukocyte subtypes, with highest expression in eosinophils. Following the training intervention, there was a significant decrease in baseline GR expression (p<0.05) in leukocyte and monocyte subtypes in both healthy and asthmatic adolescents. Conclusions This is the first study in adolescents to show that exercise training reduces GR expression on circulating leukocytes. We speculate that exercise training downregulates the stress response in general, manifested by decreased GR expression, and may explain why improving fitness improves asthma health. PMID:28796240

Expression of codon-optmized phosphoenolpyruvate carboxylase gene from Glaciecola sp. HTCC2999 in Escherichia coli and its application for C4 chemical production.

PubMed

Park, Soohyun; Pack, Seung Pil; Lee, Jinwon

2012-08-01

We examined the expression of the phosphoenolpyruvate carboxylase (PEPC) gene from marine bacteria in Escherichia coli using codon optimization. The codon-optimized PEPC gene was expressed in the E. coli K-12 strain W3110. SDS-PAGE analysis revealed that the codon-optimized PEPC gene was only expressed in E. coli, and measurement of enzyme activity indicated the highest PEPC activity in the E. coli SGJS112 strain that contained the codon-optimized PEPC gene. In fermentation assays, the E. coli SGJS112 produced the highest yield of oxaloacetate using glucose as the source and produced a 20-times increase in the yield of malate compared to the control. We concluded that the codon optimization enabled E. coli to express the PEPC gene derived from the Glaciecola sp. HTCC2999. Also, the expressed protein exhibited an enzymatic activity similar to that of E. coli PEPC and increased the yield of oxaloacetate and malate in an E. coli system.

Gene expression of apoptosis-related genes, stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress.

PubMed

Guo, Hui; Xian, Jian-An; Li, Bin; Ye, Chao-Xia; Wang, An-Li; Miao, Yu-Tao; Liao, Shao-An

2013-05-01

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress. Copyright © 2013 Elsevier Inc. All rights reserved.

Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

PubMed Central

Liang, Dongchun; Zuo, Aijun; Shao, Hui; Chen, Mingjiazi; Kaplan, Henry J.; Sun, Deming

2014-01-01

The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73+ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses. PMID:25268760

Synergic effects of tactolimus and azole antifungal agents against azole-resistant Candida albican strains.

PubMed

Maesaki, S; Marichal, P; Hossain, M A; Sanglard, D; Vanden Bossche, H; Kohno, S

1998-12-01

We investigated the effects of combining tacrolimus and azole antifungal agents in azole-resistant strains of Candida albicans by comparing the accumulation of [3H]itraconazole. The CDR1-expressing resistant strain C26 accumulated less itraconazole than the CaMDR-expressing resistant strain C40 or the azole-sensitive strain B2630. A CDR1-expressing Saccharomyces cerevisiae mutant, DSY415, showed a marked reduction in the accumulation of both fluconazole and itraconazole. A CaMDR-expressing S. cerevisiae mutant, DSY416, also showed lower accumulation of fluconazole, but not of itraconazole. The addition of sodium azide, an electron-transport chain inhibitor, increased the intracellular accumulation of itraconazole only in the C26 strain, and not in the C40 or B2630 strains. Addition of tacrolimus, an inhibitor of multidrug resistance proteins, resulted in the highest increase in itraconazole accumulation in the C26 strain. The combination of itraconazole and tacrolimus was synergic in azole-resistant C. albicans strains. In the C26 strain, the MIC of itraconazole decreased from >8 to 0.5 mg/L when combined with tacrolimus. Our results showed that two multidrug resistance phenotypes (encoded by the CDR1 and CaMDR genes) in C. albicans have different substrate specificity for azole antifungal agents and that a combination of tacrolimus and azole antifungal agents is effective against azole-resistant strains of C. albicans.

Evaluation of Biosynthesis, Accumulation and Antioxidant Activityof Vitamin E in Sweet Corn (Zea mays L.) during Kernel Development

PubMed Central

Xie, Lihua; Yu, Yongtao; Mao, Jihua; Liu, Haiying; Hu, Jian Guang; Li, Tong; Guo, Xinbo; Liu, Rui Hai

2017-01-01

Sweet corn kernels were used in this research to study the dynamics of vitamin E, by evaluatingthe expression levels of genes involved in vitamin E synthesis, the accumulation of vitamin E, and the antioxidant activity during the different stage of kernel development. Results showed that expression levels of ZmHPT and ZmTC genes increased, whereas ZmTMT gene dramatically decreased during kernel development. The contents of all the types of vitamin E in sweet corn had a significant upward increase during kernel development, and reached the highest level at 30 days after pollination (DAP). Amongst the eight isomers of vitamin E, the content of γ-tocotrienol was the highest, and increased by 14.9 folds, followed by α-tocopherolwith an increase of 22 folds, and thecontents of isomers γ-tocopherol, α-tocotrienol, δ-tocopherol,δ-tocotrienol, and β-tocopherol were also followed during kernel development. The antioxidant activity of sweet corn during kernel development was increased, and was up to 101.8 ± 22.3 μmol of α-tocopherol equivlent/100 g in fresh weight (FW) at 30 DAP. There was a positive correlation between vitamin E contents and antioxidant activity in sweet corn during the kernel development, and a negative correlation between the expressions of ZmTMT gene and vitamin E contents. These results revealed the relations amongst the content of vitamin E isomers and the gene expression, vitamin E accumulation, and antioxidant activity. The study can provide a harvesting strategy for vitamin E bio-fortification in sweet corn. PMID:29261149

Time course of salinity adaptation in a strongly euryhaline estuarine teleost, fundulus heteroclitus: A multivariable approach

USGS Publications Warehouse

Marshall, W.S.; Emberley, T.R.; Singer, T.D.; Bryson, S.E.; McCormick, S.D.

1999-01-01

Freshwater-adapted killifish (Fundulus heteroclitus) were transferred directly from soft fresh water to full-strength sea water for periods of 1h, 3h, 8h and 1, 2, 7, 14 and 30 days. Controls were transferred to fresh water for 24 h. Measured variables included: blood [Na+], osmolality, glucose and cortisol levels, basal and stimulated rates of ion transport and permeability of in vitro opercular epithelium, gill Na+/K+-ATPase and citrate synthase activity and chloride cell ultrastructure. These data were compared with previously published killifish cystic fibrosis transmembrane conductance regulator (kfCFTR) expression in the gills measured over a similar time course. Plasma cortisol levels peaked at 1 h, coincident with a rise in plasma [Na+]. At 8 h after transfer to sea water, a time at which previous work has shown kfCFTR expression to be elevated, blood osmolality and [Na+] were high, and cortisol levels and opercular membrane short-circuit current (I(SC); a measure of Cl- secretion rate) were low. The 24h group, which showed the highest level of kfCFTR expression, had the highest plasma [Na+] and osmolality, elevated plasma cortisol levels, significantly lower opercular membrane resistance, an increased opercular membrane ion secretion rate and collapsed tubule inclusions in mitochondria-rich cells, but no change in gill Na+/K+-ATPase and citrate synthase activity or plasma glucose levels. Apparently, killifish have a rapid (<1h) cortisol response to salinity coupled to subsequent (8-48 h) expression of kfCFTR anion channel proteins in existing mitochondria-rich cells that convert transport from ion uptake to ion secretion.

Evaluation of Biosynthesis, Accumulation and Antioxidant Activityof Vitamin E in Sweet Corn (Zea mays L.) during Kernel Development.

PubMed

Xie, Lihua; Yu, Yongtao; Mao, Jihua; Liu, Haiying; Hu, Jian Guang; Li, Tong; Guo, Xinbo; Liu, Rui Hai

2017-12-20

Sweet corn kernels were used in this research to study the dynamics of vitamin E, by evaluatingthe expression levels of genes involved in vitamin E synthesis, the accumulation of vitamin E, and the antioxidant activity during the different stage of kernel development. Results showed that expression levels of Zm HPT and Zm TC genes increased, whereas Zm TMT gene dramatically decreased during kernel development. The contents of all the types of vitamin E in sweet corn had a significant upward increase during kernel development, and reached the highest level at 30 days after pollination (DAP). Amongst the eight isomers of vitamin E, the content of γ-tocotrienol was the highest, and increased by 14.9 folds, followed by α-tocopherolwith an increase of 22 folds, and thecontents of isomers γ-tocopherol, α-tocotrienol, δ-tocopherol,δ-tocotrienol, and β-tocopherol were also followed during kernel development. The antioxidant activity of sweet corn during kernel development was increased, and was up to 101.8 ± 22.3 μmol of α-tocopherol equivlent/100 g in fresh weight (FW) at 30 DAP. There was a positive correlation between vitamin E contents and antioxidant activity in sweet corn during the kernel development, and a negative correlation between the expressions of Zm TMT gene and vitamin E contents. These results revealed the relations amongst the content of vitamin E isomers and the gene expression, vitamin E accumulation, and antioxidant activity. The study can provide a harvesting strategy for vitamin E bio-fortification in sweet corn.

Potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus

PubMed Central

Ha, Dong-Ho; Pathak, Shiva; Yong, Chul Soon; Kim, Jong Oh; Jeong, Jee-Heon; Park, Jun-Beom

2016-01-01

The aim of the present study is to evaluate the potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus. Tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres were prepared using electrospraying technique. In vitro release study of tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres was performed in phosphate-buffered saline (pH 7.4). Gingiva-derived stem cells were isolated and incubated with tacrolimus or tacrolimus-loaded microspheres. Release study of the microspheres revealed prolonged release profiles of tacrolimus without any significant initial burst release. The microsphere itself did not affect the morphology of the mesenchymal stem cells, and cell morphology was retained after incubation with microspheres loaded with tacrolimus at 1 μg/mL to 10 μg/mL. Cultures grown in the presence of microspheres loaded with tacrolimus at 1 μg/mL showed the highest mineralization. Alkaline phosphatase activity increased with an increase in incubation time. The highest expression of pSmad1/5 was achieved in the group receiving tacrolimus 0.1 μg/mL every third day, and the highest expression of osteocalcin was achieved in the group receiving 1 μg/mL every third day. Biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with tacrolimus promoted mineralization. Microspheres loaded with tacrolimus may be applied for increased osteoblastic differentiation. PMID:27721434

Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats.

PubMed

Adachi, Sachika; Yamada, Shunji; Takatsu, Yoshihiro; Matsui, Hisanori; Kinoshita, Mika; Takase, Kenji; Sugiura, Hitomi; Ohtaki, Tetsuya; Matsumoto, Hirokazu; Uenoyama, Yoshihisa; Tsukamura, Hiroko; Inoue, Kinji; Maeda, Kei-Ichiro

2007-04-01

Metastin/kisspeptin, the KiSS-1 gene product, has been identified as an endogenous ligand of GPR54 that reportedly regulates GnRH/LH surges and estrous cyclicity in female rats. The aim of the present study was to determine if metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges. We demonstrated that preoptic area (POA) infusion of the anti-rat metastin/kisspeptin monoclonal antibody blocked the estrogen-induced LH surge, indicating that endogenous metastin/kisspeptin released around the POA mediates the estrogen positive feedback effect on GnRH/LH release. Metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) may be responsible for mediating the feedback effect because the percentage of c-Fos-expressing KiSS-1 mRNA-positive cells to total KiSS-1 mRNA-positive cells was significantly higher in the afternoon than in the morning in the anteroventral periventricular nucleus (AVPV) of high estradiol (E(2))-treated females. The percentage of c-Fos-expressing metastin/kisspeptin neurons was not different between the afternoon and morning in the arcuate nucleus (ARC). Most of the KiSS-1 mRNA expressing cells contain ERalpha immunoreactivity in the AVPV and ARC. In addition, AVPV KiSS-1 mRNA expressions were highest in the proestrous afternoon and lowest in the diestrus 1 in females and were increased by estrogen treatment in ovariectomized animals. On the other hand, the ARC KiSS-1 mRNA expressions were highest at diestrus 2 and lowest at proestrous afternoon and were increased by ovariectomy and decreased by high estrogen treatment. Males lacking the surge mode of GnRH/LH release showed no obvious cluster of metastin/kisspeptin-immunoreactive neurons in the AVPV when compared with high E(2)-treated females, which showed a much greater density of these neurons. Taken together, the present study demonstrates that the AVPV metastin/kisspeptin neurons are a target of estrogen positive feedback to induce GnRH/LH surges in female rats.

Molecular cloning and sequence analysis of heat shock proteins 70 (HSP70) and 90 (HSP90) and their expression analysis when exposed to benzo(a)pyrene in the clam Ruditapes philippinarum.

PubMed

Liu, Tong; Pan, Luqing; Cai, Yuefeng; Miao, Jingjing

2015-01-25

HSP70 and HSP90 are the most important heat shock proteins (HSPs), which play the key roles in the cell as molecular chaperones and may involve in metabolic detoxification. The present research has obtained full-length cDNAs of genes HSP70 and HSP90 from the clam Ruditapes philippinarum and studied the transcriptional responses of the two genes when exposed to benzo(a)pyrene (BaP). The full-length RpHSP70 cDNA was 2336bp containing a 5' untranslated region (UTR) of 51bp, a 3' UTR of 335bp and an open reading frame (ORF) of 1950bp encoding 650 amino acid residues. The full-length RpHSP90 cDNA was 2839bp containing a 107-bp 5' UTR, a 554-bp 3' UTR and a 2178-bp ORF encoding 726 amino acid residues. The deduced amino acid sequences of RpHSP70 and RpHSP90 shared the highest identity with the sequences of Paphia undulata, and the phylogenetic trees showed that the evolutions of RpHSP70 and RpHSP90 were almost in accord with the evolution of species. The RpHSP70 and RpHSP90 mRNA expressions were detected in all tested tissues in the adult clams (digestive gland, gill, adductor muscle and mantle) and the highest mRNA expression level was observed in the digestive gland compared to other tissues. Quantitative real-time RT-PCR analysis revealed that mRNA expression levels of the clam RpHSP70, RpHSP90 and other xenobiotic metabolizing enzymes (XMEs) (AhR, DD, GST, GPx) in the digestive gland of R. philippinarum were induced by benzo(a)pyrene (BaP) and the absolute expression levels of these genes showed a temporal and dose-dependent response. The results suggested that RpHSP70 and RpHSP90 were involved in the metabolic detoxification of BaP in the clam R. philippinarum. Copyright © 2014 Elsevier B.V. All rights reserved.

Bioinformatics analysis of single and multi-hybrid epitopes of GRA-1, GRA-4, GRA-6 and GRA-7 proteins to improve DNA vaccine design against Toxoplasma gondii.

PubMed

Shaddel, Minoo; Ebrahimi, Mansour; Tabandeh, Mohammad Reza

2018-06-01

Toxoplasma gondii , is a causative agent of morbidity and mortality in immunocompromised and congenitally-infected individuals. Attempts to construct DNA vaccines against T. gondii using surface proteins are increasing. The dense granule antigens are highly expressed in the acute and chronic phases of T. gondii infection and considered as suitable DNA vaccine candidates to control toxoplasmosis. In the present study, bioinformatics tools and online software were used to predict, analyze and compare the structural, physical and chemical characters and immunogenicity of the GRA-1, GRA-4, GRA-6 and GRA-7 proteins. Sequence alignment results indicated that the GRA-1, GRA-4, GRA-6 and GRA-7 proteins had low similarity. The secondary structure prediction demonstrated that among the four proteins, GRA-1 and GRA-6 had similar secondary structure except for a little discrepancy. Hydrophilicity/hydrophobicity analysis showed multiple hydrophilic regions and some classical high hydrophilic domains for each protein sequence. Immunogenic epitope prediction results demonstrated that the GRA-1 and GRA-4 epitopes were stable and GRA-4 showed the highest degree of antigenicity. Although the GRA-7 epitope had the highest score of immunogenicity, this epitope was instable and had the lowest degree of antigenicity and half-time in eukaryotic cell. Also, the results indicated that GRA4-GRA7 epitope and GRA6-GRA7 had the highest degree of antigenicity and immunogenicity among multi-hybrid epitopes, respectively. Totally, in the present study, single epitopes showed the highest degree of antigenicity compared with multi-hybrid epitopes. Given the results, it can be concluded that GRA-4 and GRA-7 can be powerful DNA vaccine candidates against T. gondii .

Effect of the Duck Skin on Quality Characteristics of Duck Hams

PubMed Central

Kim, Dong-Hyun; Kim, Tae-Kyung; Kim, Young-Boong; Sung, Jung-Min; Jang, YoungJin; Shim, Jae-Yun; Han, Sung-Gu; Choi, Yun-Sang

2017-01-01

This study was conducted to investigate the effect of duck skin on cooking loss, emulsion stability, pH, color, protein solubility, texture profile analysis (TPA), apparent viscosity, and sensory characteristics of press type duck ham with different ratio of duck breast meat and duck skin. Five duck ham formulations were produced with the following compositions: T1 (duck breast 70% + duck skin 30%), T2 (duck breast 60% + duck skin 40%), T3 (duck breast 50% + duck skin 50%), T4 (duck breast 40% + duck skin 60%), and T5 (duck breast 30% + duck skin 70%). The cooking loss and fat separation were lower in T1, and the total expressible fluid separations were lower in T1 and T2 than others. The pH ranged from 6.48 to 6.59, with the highest values in T4 and T5. T5 had the highest CIE L*-value, and T1 and T2 had the highest CIE a*-values; however, CIE b*-values did not differ significantly between the duck ham samples. The protein solubility and TPA (hardness, springiness, cohesiveness, gumminess, and chewiness) were the highest in T1. T1 and T2 had higher scores for color, tenderness, and overall acceptability. T1, T2, and T3 showed significantly higher values, but there were no significant differences for flavor and juiciness. Regarding apparent viscosity properties, T1 and T2 had higher viscosity values than the other formulations. In conclusion, the T1 (duck breast 70% + duck skin 30%) and T2 (duck breast 60% + duck skin 40%) duck hams show the highest quality characteristics. PMID:28747821

Metaproteomics reveals abundant transposase expression in mutualistic endosymbionts

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

Kleiner, Manuel; Young, Jacque C; Shah, Manesh B

2013-01-01

Transposases, enzymes that catalyze the movement of mobile genetic elements, are the most abundant genes in nature. While many bacteria encode an abundance of transposases in their genomes, the current paradigm is that transposase gene expression is tightly regulated and generally low due to its severe mutagenic effects. In the current study, we detected the highest number of transposase proteins ever reported in bacteria, in symbionts of the gutless marine worm Olavius algarvensis using metaproteomics. At least 26 different transposases from 12 different families were detected and genomic and proteomic analyses suggest many of these are active. This high expressionmore » of transposases indicates that the mechanisms for their tight regulation have been disabled or destroyed. Based on recent studies on other symbionts and pathogens that showed high transposase transcription, we speculate that abundant transposase expression might be common in symbionts and pathogens.« less

Relationship between Expression of Cellular Receptor-27.8 kDa and Lymphocystis Disease Virus (LCDV) Infection.

PubMed

Wu, Ronghua; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

2015-01-01

The 27.8 kDa membrane protein from flounder (Paralichthys olivaceus) gill (FG) cells was previously identified as a putative cellular receptor involved in lymphocystis disease virus (LCDV) infection. In this paper, the expression of receptor-27.8 kDa (27.8R) and LCDV loads in FG cells and hirame natural embryo (HINAE) cells were investigated upon LCDV infection and anti-27.8R monoclonal antibody (MAb) treatment. The results showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cell lines was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry, providing important new insights into the LCDV replication cycle and the interaction between this virus and the host cells.

Relationship between Expression of Cellular Receptor-27.8kDa and Lymphocystis Disease Virus (LCDV) Infection

PubMed Central

Wu, Ronghua; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

2015-01-01

The 27.8kDa membrane protein from flounder (Paralichthys olivaceus) gill (FG) cells was previously identified as a putative cellular receptor involved in lymphocystis disease virus (LCDV) infection. In this paper, the expression of receptor-27.8kDa (27.8R) and LCDV loads in FG cells and hirame natural embryo (HINAE) cells were investigated upon LCDV infection and anti-27.8R monoclonal antibody (MAb) treatment. The results showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cell lines was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry, providing important new insights into the LCDV replication cycle and the interaction between this virus and the host cells. PMID:26024218

Molecular cloning and characterization of a gene regulating flowering time from Alfalfa (Medicago sativa L.).

PubMed

Zhang, Tiejun; Chao, Yuehui; Kang, Junmei; Ding, Wang; Yang, Qingchuan

2013-07-01

Genes that regulate flowering time play crucial roles in plant development and biomass formation. Based on the cDNA sequence of Medicago truncatula (accession no. AY690425), the LFY gene of alfalfa was cloned. Sequence similarity analysis revealed high homology with FLO/LFY family genes of other plants. When fused to the green fluorescent protein, MsLFY protein was localized in the nucleus of onion (Allium cepa L.) epidermal cells. The RT-qPCR analysis of MsLFY expression patterns showed that the expression of MsLFY gene was at a low level in roots, stems, leaves and pods, and the expression level in floral buds was the highest. The expression of MsLFY was induced by GA3 and long photoperiod. Plant expression vector was constructed and transformed into Arabidopsis by the agrobacterium-mediated methods. PCR amplification with the transgenic Arabidopsis genome DNA indicated that MsLFY gene had integrated in Arabidopsis genome. Overexpression of MsLFY specifically caused early flowering under long day conditions compared with non-transgenic plants. These results indicated MsLFY played roles in promoting flowering time.

Regulation of vascular endothelial genes by dietary flavonoids: structure-expression relationship studies and the role of the transcription factor KLF-2.

PubMed

Martínez-Fernández, Leyre; Pons, Zara; Margalef, Maria; Arola-Arnal, Anna; Muguerza, Begoña

2015-03-01

Physiological concentrations (1 μM) of 15 flavonoids were evaluated in human umbilical vein endothelial cells in the presence of hydrogen peroxide (H₂O₂) for their ability to affect endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) expression in order to establish the structural basis of their bioactivity. Flavonoid effects on eNOS transcription factor Krüpple like factor-2 (KLF-2) expression were also evaluated. All studied flavonoids appeared to be effective compounds for counteracting the oxidative stress-induced effects on vascular gene expression, indicating that flavonoids are an excellent source of functional endothelial regulator products. Notably, the more effective flavonoids for KLF-2 up-regulation resulted in the highest values for eNOS expression, showing that the increment of eNOS expression would take place through KLF-2 induction. Structure-activity relationship studies showed that the combinations of substructures on flavonoid skeleton that regulate eNOS expression are made up of the following elements: glycosylation and hydroxylation of C-ring, double bond C2=C3 at C-ring, methoxylation and hydroxylation of B-ring, ketone group in C4 at C-ring and glycosylation in C7 of A-ring, while flavonoid features involved in the reduction of vasoconstrictor ET-1 expression are as follows: double bond C2=C3 at C-ring glycosylation in C7 of A-ring and ketone group in C4 of C-ring. Copyright © 2015 Elsevier Inc. All rights reserved.

Wood formation from the base to the crown in Pinus radiata: gradients of tracheid wall thickness, wood density, radial growth rate and gene expression

Treesearch

Sheree Cato; Lisa McMillan; Lloyd Donaldson; Thomas Richardson; Craig Echt; Richard Gardner

2006-01-01

Wood formation was investigated at five heights along the bole for two unrelated trees of Pinus radiataBoth trees showed clear gradients in wood properties from the base to the crown. Cambial cells at the base of the tree were dividing 3.3-fold slower than those at the crown, while the average thickness of cell walls in wood was highest at the base....

Chitooligosaccharides suppress the level of protein expression and acetylcholinesterase activity induced by Abeta25-35 in PC12 cells.

PubMed

Lee, Sang-Hoon; Park, Jin-Sook; Kim, Se-Kwon; Ahn, Chang-Bum; Je, Jae-Young

2009-02-01

Clinical applications of acetylcholinesterase (AChE) inhibitors are widespread in Alzheimer's sufferers in order to activate central cholinergic system and alleviate cognitive deficits by inhibiting the hydrolysis of acetylcholine. In this study, six kinds of chitooligosaccharides (COSs) with different molecular weight and degree of deacetylation were examined for their inhibitory effects against AChE. The 90-COSs exhibited potent AChE inhibitory activities compared to 50-COSs, while 90-MMWCOS (1000-5000 Da) in the 90-COSs showed the highest activity. Cell culture experiment revealed that 90-MMWCOS suppressed the level of AChE protein expression and AChE activity induced by Abeta(25-35) in PC12 cell lines.

Molecular genetics of G proteins and atherosclerosis risk.

PubMed

Siffert, W

2001-11-01

Using a classical candidate gene approach, we have described a common C825T polymorphism in the gene GNB3 which encodes the ubiquitously expressed beta3 subunit of heterotrimeric G proteins. The 825T allele is associated with alternative splicing of the gene and the formation of a truncated but functionally active beta3 subunit which is referred to as Gbeta3s. Expression of the splice variant results in an enhanced G protein activation on stimulation of G protein-coupled receptors. Carriers of the 825T allele show an increased risk for hypertension and left ventricular hypertrophy. Homo- and heterozygous 825T allele carriers respond with a stronger decrease in blood pressure to therapy with a thiazide diuretic than homozygous 825C allele carriers. Moreover, 825T allele carriers appear to have an increased risk for obesity which appears sensible given the established role of G protein signaling in adipogenesis. The highest frequencies of the 825T allele are found in ethnicities with the highest lifestyle-dependent risk for obesity, e.g., black Africans and East Asians. This suggests that the 825T allele fulfills the criteria of a thrifty genotype.

Analysis of MUC4 expression in human pancreatic cancer xenografts in immunodeficient mice.

PubMed

Ansari, Daniel; Bauden, Monika P; Sasor, Agata; Gundewar, Chinmay; Andersson, Roland

2014-08-01

Mucin 4 (MUC4) is a cell surface glycoprotein that is overexpressed in most pancreatic tumors. The aim of the present study was to characterize MUC4 expression in experimental pancreatic cancer in order to clarify the correlation between MUC4 and pancreatic cancer histology in vivo. Pancreatic xenograft tumors were generated in immunodeficient mice (n=15) by subcutaneous injection of MUC4(+) human pancreatic cancer cell lines Capan-1, HPAF-II or CD18/HPAF. MUC4 immunoreactivity was compared between the cancer models. Alpha-smooth muscle actin (α-SMA) was used to identify cancer-associated fibroblasts and the amount of collagen fibers was quantified with sirius red. Tumor incidence was 100%. Tumor size showed no difference across groups (p=0.796). The median MUC4 count was highest in Capan-1 tumors (p=0.002). α-SMA and collagen extent were also highest in Capan-1 tumors (p=0.018). The Capan-1 xenograft model could serve as a valuable resource to test new therapeutic strategies targeting MUC4 in pancreatic cancer. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Identification and expression analysis of cobia (Rachycentron canadum) Toll-like receptor 9 gene.

PubMed

Byadgi, Omkar; Puteri, Dinda; Lee, Yan-Horn; Lee, Jai-Wei; Cheng, Ta-Chih

2014-02-01

Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P. damselae subsp. piscicida challenge and CpG ODN stimulation on cobia Toll like receptor 9 (RCTLR9), we used PCR to clone RCTLR9 gene and qRT-PCR to quantify gene expression. The results indicated that RCTLR9 cDNA contains 3141 bp. It encodes 1047 amino acids containing 16 typical structures of leucine-rich repeats (LRRs) including an LRRTYP, LRRCT and a motif involved in PAMP binding was identified at position 240-253 amino acid. Broad expression of RCTLR9 was found in larval, juvenile and adult stages irrespective of the tissues. In larval stage, RCTLR9 mRNA expression decreased at 5 d and then increased at 10 dph. At juvenile stage cobia, the expression was significantly high (p < 0.05) in spleen and intestine compared to gill, kidney, liver and skin. However, at adult stage, the significant high expression was found in gill and intestine. Cobia challenged with P. damselae subsp. piscicida showed significant increase in RCTLR9 expression at 24 h post challenge in intestine, spleen and liver, while in kidney the expression was peak at 12 h and later it decreased at 24 h. The highest expression was 40 fold increase in spleen and the lowest expression was ∼3.6 fold increase in liver. Cobia stimulated with CpG oligonucleotides showed that the induction of these genes was CpG ODN type and time dependent. In spleen and liver, CpG ODNs 1668 and 2006 injected group showed high expression of RCTLR9, IL-1β, chemokine CC compared to other groups. Meanwhile, CpG ODN 2006 has induced high expression of IgM. The CpG ODNs 2395 have induced significant high expression of Mx in spleen and liver. These results demonstrates the potential of using CpG ODN to enhance cobia resistance to P. damselae subsp. piscicida infection and use as an adjuvant in vaccine development. Copyright © 2013 Elsevier Ltd. All rights reserved.

Genomic organization and tissue-specific expression of hepcidin in the pacific mutton hamlet, Alphestes immaculatus (Breder, 1936).

PubMed

Masso-Silva, Jorge; Diamond, Gill; Macias-Rodriguez, Maria; Ascencio, Felipe

2011-12-01

Hepcidin is a cysteine-rich peptide involved in iron metabolism, inflammatory response and as antimicrobial peptide. Despite the fact that hepcidins have been identified in several fish species, only few have been completely characterized. This study, described the identification and complete molecular characterization of the hepcidin antimicrobial peptide 1 (HAMP1) gene of Alphestes immaculatus. Moreover, its specific expression level at both basal and lipopolysaccharide (LPS)-induced conditions in different tissues was also determined by real-time PCR. Results showed that the HAMP1gene consists of three exons and two introns encoding a preprohepcidin composed of 90 aa (24 aa for signal peptide, 40 aa for prodomain and 26 aa for mature peptide). The promoter region analysis revealed a TATA box sequence and several putative transcription factor binding sites. A comparative analysis showed CEBPα, CEBPβ, NF-kB, HNF3, GATA-1 and c-Rel as the most common found in fishes. The mature peptide possesses a pI of 8.34, which is the average among fish hepcidin. In addition, the structural modeling showed a hairpin structure with four putative disulfide bonds. A phylogenetic analysis revealed that this hepcidin gene is a HAMP1 class, and is clustered into the same group with the Serranid fish Epinephelus moara and the Antarctic fish Lycodichthys dearborni. Finally, the relative expression levels showed high basal values in liver and muscle, whereas in LPS-induced fish the relative expression tendency changed, with the highest values in spleen and head kidney tissues. This study describes the completely characterized HAMP1 gene of A. immaculatus and their patterns of expression level at different conditions and in different tissues, showing by first time muscle hepcidin expression could be relevant in the immune response in fish. Copyright © 2011 Elsevier Ltd. All rights reserved.

Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses.

PubMed

Liu, Qiuxu; Qi, Xiao; Yan, Haidong; Huang, Linkai; Nie, Gang; Zhang, Xinquan

2018-01-16

To select the most stable reference genes in annual ryegrass ( Lolium multiflorum ), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different. The expression of the eIF4A gene was the most stable under drought stress. Under saline-alkali stress, Unigene14912 has the highest expression stability. Under acidic aluminum stress, SAMDC expression stability was highest. Under heavy metal stress, Unigene71 expression had the highest stability. According to the software analyses, Unigene14912, HIS3, and eIF4A were the most suitable for analyses of abiotic stress in tissues of annual ryegrass. GAPDH was the least suitable reference gene. In conclusion, selecting appropriate reference genes under abiotic stress not only improves the accuracy of annual ryegrass gene expression analyses, but also provides a theoretical reference for the development of reference genes in plants of the genus Lolium .

Regulated expression and role of c-Myb in the cardiovascular-directed differentiation of mouse embryonic stem cells.

PubMed

Ishida, Masayoshi; El-Mounayri, Omar; Kattman, Steven; Zandstra, Peter; Sakamoto, Hiroshi; Ogawa, Minetaro; Keller, Gordon; Husain, Mansoor

2012-01-20

c-myb null (knockout) embryonic stem cells (ESC) can differentiate into cardiomyocytes but not contractile smooth muscle cells (SMC) in embryoid bodies (EB). To define the role of c-Myb in SMC differentiation from ESC. In wild-type (WT) EB, high c-Myb levels on days 0-2 of differentiation undergo ubiquitin-mediated proteosomal degradation on days 2.5-3, resurging on days 4-6, without changing c-myb mRNA levels. Activin-A and bone morphogenetic protein 4-induced cardiovascular progenitors were isolated by FACS for expression of vascular endothelial growth factor receptor (VEGFR)2 and platelet-derived growth factor receptor (PDGFR)α. By day 3.75, hematopoesis-capable VEGFR2+ cells were fewer, whereas cardiomyocyte-directed VEGFR2+/PDGFRα+ cells did not differ in abundance in knockout versus WT EB. Importantly, highest and lowest levels of c-Myb were observed in VEGFR2+ and VEGFR2+/PDGFRα+ cells, respectively. Proteosome inhibitor MG132 and lentiviruses enabling inducible expression or knockdown of c-myb were used to regulate c-Myb in WT and knockout EB. These experiments showed that c-Myb promotes expression of VEGFR2 over PDGFRα, with chromatin immunopreciptation and promoter-reporter assays defining specific c-Myb-responsive binding sites in the VEGFR2 promoter. Next, FACS-sorted VEGFR2+ cells expressed highest and lowest levels of SMC- and fibroblast-specific markers, respectively, at days 7-14 after retinoic acid (RA) as compared with VEGFR2+/PDGFRα+ cells. By contrast, VEGFR2+/PDGFRα+ cells cultured without RA beat spontaneously, like cardiomyocytes between days 7 and 14, and expressed cardiac troponin. Notably, RA was required to more fully differentiate SMC from VEGFR2+ cells and completely blocked differentiation of cardiomyocytes from VEGFR2+/PDGFRα+ cells. c-Myb is tightly regulated by proteosomal degradation during cardiovascular-directed differentiation of ESC, expanding early-stage VEGFR2+ progenitors capable of RA-responsive SMC formation.

Effects of selenium supplementation on the oxidative state of acute heat stress-exposed quails.

PubMed

Del Vesco, A P; Gasparino, E; Zancanela, V; Grieser, D O; Stanquevis, C E; Pozza, P C; Oliveira Neto, A R

2017-02-01

This study aimed to evaluate the effect of heat stress (HS) and selenium supplementation on markers of stress, meat quality and gene expression. For this, meat quails of 42 days of age were fed a diet that either met [0.33 mg/kg, nutritional demand for selenium (SS)] or did not meet [0.11 mg/kg, selenium deficient (SD)] the nutritional demands for selenium during the 7 days of evaluation. In addition, the animals were kept at either a thermal comfort temperature (25 °C) or exposed to HS (38 °C for 24 h). Glutathione synthetase (GSS), glutathione reductase (GSR) and uncoupling protein (UCP) gene expression were influenced by the interaction between temperature and diet. Animals subjected to HS and fed the SS diet exhibited the highest GSS and GSR gene expression. In terms of UCP gene expression, the lowest values were observed in HS animals on the SD diet. Glutathione peroxidase 7 (GPX7) gene expression, body temperature (BT) and creatine kinase (CK) activity were influenced by both selenium supplementation and HS. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) activity and creatinine content all were influenced by the diet/environment interaction. The highest AST activity, ALT activity and creatinine levels were observed in animals that were both on the SD diet and exposed to HS. HS animals also exhibited an increased heterophil/lymphocyte ratio and lower triiodothyronine (T3) hormone levels than birds that remained at the comfortable temperature. Animals subjected to HS and fed with selenium supplemented diet showed better results regarding gene expression and, thus, better results for the activities of enzymes used as stress markers, which could be due to the higher antioxidant capacity provided by the action of the studied genes. Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.

Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

NASA Technical Reports Server (NTRS)

Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

Teaching Free Expression in Word and Example (Commentary).

ERIC Educational Resources Information Center

Merrill, John

1991-01-01

Suggests that the teaching of free expression may be the highest calling of a communications or journalism professor. Argues that freedom must be tempered by a sense of ethics. Calls upon teachers to encourage students to analyze the questions surrounding free expression. Describes techniques for scrutinizing journalistic myths. (SG)

Differential Expression of Superoxide Dismutase Genes in Aphid-Stressed Maize (Zea mays L.) Seedlings

PubMed Central

Sytykiewicz, Hubert

2014-01-01

The aim of this study was to compare the expression patterns of superoxide dismutase genes (sod2, sod3.4, sod9 and sodB) in seedling leaves of the Zea mays L. Tasty Sweet (susceptible) and Ambrozja (relatively resistant) cultivars infested with one of two hemipteran species, namely monophagous Sitobion avenae F. (grain aphid) or oligophagous Rhopalosiphum padi L. (bird cherry-oat aphid). Secondarily, aphid-elicited alternations in the antioxidative capacity towards DPPH (1,1-diphenyl-2-picrylhydrazyl) radical in insect-stressed plants were evaluated. Comprehensive comparison of expression profiles of the four sod genes showed that both insect species evoked significant upregulation of three genes sod2, sod3.4 and sod9). However, aphid infestation affected non-significant fluctuations in expression of sodB gene in seedlings of both maize genotypes. The highest levels of transcript accumulation occurred at 8 h (sod2 and sod3.4) or 24 h (sod9) post-infestation, and aphid-induced changes in the expression of sod genes were more dramatic in the Ambrozja cultivar than in the Tasty Sweet variety. Furthermore, bird cherry-oat aphid colonization had a more substantial impact on levels of DPPH radical scavenging activity in infested host seedlings than grain aphid colonization. Additionally, Ambrozja plants infested by either hemipteran species showed markedly lower antioxidative capacity compared with attacked Tasty Sweet plants. PMID:24722734

Differential expression of superoxide dismutase genes in aphid-stressed maize (Zea mays L.) seedlings.

PubMed

Sytykiewicz, Hubert

2014-01-01

The aim of this study was to compare the expression patterns of superoxide dismutase genes (sod2, sod3.4, sod9 and sodB) in seedling leaves of the Zea mays L. Tasty Sweet (susceptible) and Ambrozja (relatively resistant) cultivars infested with one of two hemipteran species, namely monophagous Sitobion avenae F. (grain aphid) or oligophagous Rhopalosiphum padi L. (bird cherry-oat aphid). Secondarily, aphid-elicited alternations in the antioxidative capacity towards DPPH (1,1-diphenyl-2-picrylhydrazyl) radical in insect-stressed plants were evaluated. Comprehensive comparison of expression profiles of the four sod genes showed that both insect species evoked significant upregulation of three genes sod2, sod3.4 and sod9). However, aphid infestation affected non-significant fluctuations in expression of sodB gene in seedlings of both maize genotypes. The highest levels of transcript accumulation occurred at 8 h (sod2 and sod3.4) or 24 h (sod9) post-infestation, and aphid-induced changes in the expression of sod genes were more dramatic in the Ambrozja cultivar than in the Tasty Sweet variety. Furthermore, bird cherry-oat aphid colonization had a more substantial impact on levels of DPPH radical scavenging activity in infested host seedlings than grain aphid colonization. Additionally, Ambrozja plants infested by either hemipteran species showed markedly lower antioxidative capacity compared with attacked Tasty Sweet plants.

Identification and characterisation of multiple glutathione S-transferase genes from the diamondback moth, Plutella xylostella.

PubMed

Chen, Xi'en; Zhang, Ya-lin

2015-04-01

The diamondback moth (DBM), Plutella xylostella, is one of the most harmful insect pests on crucifer crops worldwide. In this study, 19 cDNAs encoding glutathione S-transferases (GSTs) were identified from the genomic and transcriptomic database for DBM (KONAGAbase) and further characterized. Phylogenetic analysis showed that the 19 GSTs were classified into six different cytosolic classes, including four in delta, six in epsilon, three in omega, two in sigma, one in theta and one in zeta. Two GSTs were unclassified. RT-PCR analysis revealed that most GST genes were expressed in all developmental stages, with higher expression in the larval stages. Six DBM GSTs were expressed at the highest levels in the midgut tissue. Twelve purified recombinant GSTs showed varied enzymatic properties towards 1-chloro-2,4-dinitrobenzene and glutathione, whereas rPxGSTo2, rPxGSTz1 and rPxGSTu2 had no activity. Real-time quantitative PCR revealed that expression levels of the 19 DBM GST genes were varied and changed after exposure to acephate, indoxacarb, beta-cypermethrin and spinosad. PxGSTd3 was significantly overexpressed, while PxGSTe3 and PxGSTs2 were significantly downregulated by all four insecticide exposures. The changes in DBM GST gene expression levels exposed to different insecticides indicate that they may play individual roles in tolerance to insecticides and xenobiotics. © 2014 Society of Chemical Industry.

Molecular cloning and gene expression analysis of Ercc6l in Sika deer (Cervus nippon hortulorum).

PubMed

Yin, Yupeng; Tang, Lina; Zhang, Jiabao; Tang, Bo; Li, Ziyi

2011-01-01

One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42-82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals.

Molecular Cloning and Gene Expression Analysis of Ercc6l in Sika Deer (Cervus nippon hortulorum)

PubMed Central

Zhang, Jiabao; Tang, Bo; Li, Ziyi

2011-01-01

Background One important protein family that functions in nucleotide excision repair (NER) factors is the SNF2 family. A newly identified mouse ERCC6-like gene, Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like), has been shown to be another developmentally related member of the SNF2 family. Methodology/Principal Findings In this study, Sika deer Ercc6l cDNA was first cloned and then sequenced. The full-length cDNA of the Sika deer Ercc6l gene is 4197 bp and contains a 3732 bp open reading frame that encodes a putative protein of 1243 amino acids. The similarity of Sika deer Ercc6l to Bos taurus Ercc6l is 94.05% at the amino acid sequence level. The similarity, however, is reduced to 68.42–82.21% when compared to Ercc6l orthologs in other mammals and to less than 50% compared to orthologs in Gallus gallus and Xenopus. Additionally, the expression of Ercc6l mRNA was investigated in the organs of fetal and adult Sika deer (FSD and ASD, respectively) by quantitative RT-PCR. The common expression level of Ercc6l mRNA in the heart, liver, spleen, lung, kidney, and stomach from six different developmental stages of 18 Sika deer were examined, though the expression levels in each organ varied among individual Sika deer. During development, there was a slight trend toward decreased Ercc61 mRNA expression. The highest Ercc6l expression levels were seen at 3 months old in every organ and showed the highest level of detection in the spleen of FSD. The lowest Ercc6l expression levels were seen at 3 years old. Conclusions/Significance We are the first to successfully clone Sika deer Ercc6l mRNA. Ercc6l transcript is present in almost every organ. During Sika deer development, there is a slight trend toward decreased Ercc61 mRNA expression. It is possible that Ercc6l has other roles in embryonic development and in maintaining the growth of animals. PMID:21695076

Temperature effects on gene expression and morphological development of European eel, Anguilla anguilla larvae

PubMed Central

Mazurais, David; Servili, Arianna; Zambonino-Infante, Jose-Luis; Miest, Joanna J.; Sørensen, Sune R.; Tomkiewicz, Jonna; Butts, Ian A. E.

2017-01-01

Temperature is important for optimization of rearing conditions in aquaculture, especially during the critical early life history stages of fish. Here, we experimentally investigated the impact of temperature (16, 18, 20, 22 and 24°C) on thermally induced phenotypic variability, from larval hatch to first-feeding, and the linked expression of targeted genes [heat shock proteins (hsp), growth hormone (gh) and insulin-like growth factors (igf)] associated to larval performance of European eel, Anguilla anguilla. Temperature effects on larval morphology and gene expression were investigated throughout early larval development (in real time from 0 to 18 days post hatch) and at specific developmental stages (hatch, jaw/teeth formation, and first-feeding). Results showed that hatch success, yolk utilization efficiency, survival, deformities, yolk utilization, and growth rates were all significantly affected by temperature. In real time, increasing temperature from 16 to 22°C accelerated larval development, while larval gene expression patterns (hsp70, hsp90, gh and igf-1) were delayed at cold temperatures (16°C) or accelerated at warm temperatures (20–22°C). All targeted genes (hsp70, hsp90, gh, igf-1, igf-2a, igf-2b) were differentially expressed during larval development. Moreover, expression of gh was highest at 16°C during the jaw/teeth formation, and the first-feeding developmental stages, while expression of hsp90 was highest at 22°C, suggesting thermal stress. Furthermore, 24°C was shown to be deleterious (resulting in 100% mortality), while 16°C and 22°C (~50 and 90% deformities respectively) represent the lower and upper thermal tolerance limits. In conclusion, the high survival, lowest incidence of deformities at hatch, high yolk utilization efficiency, high gh and low hsp expression, suggest 18°C as the optimal temperature for offspring of European eel. Furthermore, our results suggest that the still enigmatic early life history stages of European eel may inhabit the deeper layer of the Sargasso Sea and indicate vulnerability of this critically endangered species to increasing ocean temperature. PMID:28806748

Characterization of a RacGTPase up-regulated in the large yellow croaker Pseudosciaena crocea immunity.

PubMed

Han, Fang; Wang, Xiaoqing; Yang, Qilian; Cai, Mingyi; Wang, Zhi Yong

2011-02-01

The Rac proteins are members of the Rho family of small G proteins and are implicated in the regulation of several pathways, including those leading to cytoskeleton reorganization, gene expression, cell proliferation, cell adhesion and cell migration and survival. In this investigation, a Rac gene (named as LycRac gene) was obtained from the large yellow croaker and it was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LycRac). Moreover, the GTP-binding assay showed that the LycRac protein had GTP-binding activity. The LycRac gene was ubiquitously transcribed and expressed in 9 tissues. Quantitative real-time RT-PCR and Western blot analysis revealed the highest expression in gill and the weakest expression in spleen. Time-course analysis revealed that LycRac expression was obviously up-regulated in blood, spleen and liver after immunization with polyinosinic polycytidynic acid (poly I:C), formalin-inactive Gram-negative bacterium Vibrio parahemolyticus and bacterial lipopolysaccharides (LPS). These results suggested that LycRac protein might play an important role in the immune response against microorganisms in large yellow croaker. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.

PubMed

Nomura, S; Ogami, K; Kawamura, K; Tsukamoto, I; Kudo, Y; Kanakura, Y; Kitamura, Y; Miyazaki, H; Kato, T

1997-07-01

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

Methylation of microRNA genes regulates gene expression in bisexual flower development in andromonoecious poplar.

PubMed

Song, Yuepeng; Tian, Min; Ci, Dong; Zhang, Deqiang

2015-04-01

Previous studies showed sex-specific DNA methylation and expression of candidate genes in bisexual flowers of andromonoecious poplar, but the regulatory relationship between methylation and microRNAs (miRNAs) remains unclear. To investigate whether the methylation of miRNA genes regulates gene expression in bisexual flower development, the methylome, microRNA, and transcriptome were examined in female and male flowers of andromonoecious poplar. 27 636 methylated coding genes and 113 methylated miRNA genes were identified. In the coding genes, 64.5% of the methylated reads mapped to the gene body region; by contrast, 60.7% of methylated reads in miRNA genes mainly mapped in the 5' and 3' flanking regions. CHH methylation showed the highest methylation levels and CHG showed the lowest methylation levels. Correlation analysis showed a significant, negative, strand-specific correlation of methylation and miRNA gene expression (r=0.79, P <0.05). The methylated miRNA genes included eight long miRNAs (lmiRNAs) of 24 nucleotides and 11 miRNAs related to flower development. miRNA172b might play an important role in the regulation of bisexual flower development-related gene expression in andromonoecious poplar, via modification of methylation. Gynomonoecious, female, and male poplars were used to validate the methylation patterns of the miRNA172b gene, implying that hyper-methylation in andromonoecious and gynomonoecious poplar might function as an important regulator in bisexual flower development. Our data provide a useful resource for the study of flower development in poplar and improve our understanding of the effect of epigenetic regulation on genes other than protein-coding genes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Methylation of microRNA genes regulates gene expression in bisexual flower development in andromonoecious poplar

PubMed Central

Song, Yuepeng; Tian, Min; Ci, Dong; Zhang, Deqiang

2015-01-01

Previous studies showed sex-specific DNA methylation and expression of candidate genes in bisexual flowers of andromonoecious poplar, but the regulatory relationship between methylation and microRNAs (miRNAs) remains unclear. To investigate whether the methylation of miRNA genes regulates gene expression in bisexual flower development, the methylome, microRNA, and transcriptome were examined in female and male flowers of andromonoecious poplar. 27 636 methylated coding genes and 113 methylated miRNA genes were identified. In the coding genes, 64.5% of the methylated reads mapped to the gene body region; by contrast, 60.7% of methylated reads in miRNA genes mainly mapped in the 5′ and 3′ flanking regions. CHH methylation showed the highest methylation levels and CHG showed the lowest methylation levels. Correlation analysis showed a significant, negative, strand-specific correlation of methylation and miRNA gene expression (r=0.79, P <0.05). The methylated miRNA genes included eight long miRNAs (lmiRNAs) of 24 nucleotides and 11 miRNAs related to flower development. miRNA172b might play an important role in the regulation of bisexual flower development-related gene expression in andromonoecious poplar, via modification of methylation. Gynomonoecious, female, and male poplars were used to validate the methylation patterns of the miRNA172b gene, implying that hyper-methylation in andromonoecious and gynomonoecious poplar might function as an important regulator in bisexual flower development. Our data provide a useful resource for the study of flower development in poplar and improve our understanding of the effect of epigenetic regulation on genes other than protein-coding genes. PMID:25617468

Pharmacological characterization of a β-adrenergic-like octopamine receptor in Plutella xylostella.

PubMed

Huang, Qing-Ting; Ma, Hai-Hao; Deng, Xi-Le; Zhu, Hang; Liu, Jia; Zhou, Yong; Zhou, Xiao-Mao

2018-04-25

The β-adrenergic-like octopamine receptor (OA2B2) belongs to the class of G-protein coupled receptors. It regulates important physiological functions in insects, thus is potentially a good target for insecticides. In this study, the putative open reading frame sequence of the Pxoa2b2 gene in Plutella xylostella was cloned. Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. PxOA2B2 was transiently expressed in HEK-293 cells. It was found that PxOA2B2 could be activated by both octopamine and tyramine, resulting in increased intracellular cyclic AMP (cAMP) levels, whereas dopamine and serotonin were not effective in eliciting cAMP production. Further studies with series of PxOA2B2 agonists and antagonists showed that all four tested agonists (e.g., naphazoline, clonidine, 2-phenylethylamine, and amitraz) could activate the PxOA2B2 receptor, and two of tested antagonists (e.g., phentolamine and mianserin) had significant antagonistic effects. However, antagonist of yohimbine had no effects. Quantitative real-time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P. xylostella and that the highest expression occurred in male adults. Further analysis with fourth-instar P. xylostella larvae showed that the Pxoa2b2 gene was mainly expressed in Malpighian tubule, epidermal, and head tissues. This study provides both a pharmacological characterization and the gene expression patterns of the OA2B2 in P. xylostella, facilitating further research for insecticides using PxOA2B2 as a target. © 2018 Wiley Periodicals, Inc.

Improvement of isolated caprine islet survival and functionality in vitro by enhancing of PDX1 gene expression.

PubMed

Hani, Homayoun; Allaudin, Zeenathul Nazariah; Mohd-Lila, Mohd-Azmi; Sarsaifi, Kazhal; Rasouli, Mina; Tam, Yew Joon; Tengku-Ibrahim, Tengku-Azmi; Othman, Abas Mazni

2017-05-01

Dead islets replaced with viable islets are a promising offer to restore normal insulin production to a person with diabetes. The main reason for establishing a new islet source for transplantation is the insufficiency of human donor pancreas while using xenogeneic islets perhaps assists this problem. The expression of PDX1 is essential for the pancreas expansion. In mature β-cells, PDX1 has several critical roles such as glucose sensing, insulin synthesis, and insulin secretion. In this study, we aimed to evaluate the expression of pancreatic duodenal homeobox-1 (PDX1) in treated caprine islets in culture and to assess the protective effects of antioxidant factors on the PDX1 gene in cultured caprine islets. Purified islets were treated with serum-free, serum, IBMX, tocopherol, or IBMX and tocopherol media. Quantitative polymerase chain reaction and Western blotting were carried out to compare the expression levels of PDX1 in treated purified islets cultured with different media. Islets treated with IBMX/tocopherol exhibited the highest fold change in the relative expression of PDX1 on day 5 post-treatment (relative expression: 6.80±2.08), whereas serum-treated islets showed the lowest fold changes in PDX1 expression on day 5 post-treatment (0.67±0.36), as compared with the expression on day 1 post-treatment. Insulin production and viability tests of purified islets showed superiority of islet at supplemented serum-free media with IBMX/tocopherol compared to other cultures (53.875%±1.59%). Our results indicated that supplemented serum-free medium with tocopherol and IBMX enhances viability and PDX1 gene expression compared to serum-added and serum-free media. © 2017 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.

Functional Characterization of a Strong Bi-directional Constitutive Plant Promoter Isolated from Cotton Leaf Curl Burewala Virus

PubMed Central

Khan, Zainul A.; Abdin, Malik Z.; Khan, Jawaid A.

2015-01-01

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells. PMID:25799504

Characterization, expression and function analysis of DAX1 gene of scallop ( Chlamys farreri jones and preston 1904) during its gametogenesis

NASA Astrophysics Data System (ADS)

Li, Hailong; Liu, Jianguo; Huang, Xiaoting; Wang, Dan; Zhang, Zhifeng

2014-08-01

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop ( Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at mRNA and protein levels. The cDNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame (ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher ( P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.

Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

PubMed

Khan, Zainul A; Abdin, Malik Z; Khan, Jawaid A

2015-01-01

Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.

Tissue Distribution of the 27.8 kDa Receptor and its Dynamic Expression in Response to Lymphocystis Disease Virus Infection in Flounder (Paralichthys olivaceus).

PubMed

Wu, R-H; Tang, X-Q; Sheng, X-Z; Zhan, W-B

2015-11-01

Lymphocystis disease virus (LCDV) enters and infects the gill cells of flounder (Paralichthys olivaceus) via a 27.8 kDa membrane protein receptor. In the present study, immunohistochemistry was performed to locate the tissue distribution of this molecule in healthy flounder and showed that it was widely distributed in the tissues tested. Indirect enzyme-linked immunosorbent assay (ELISA) showed that the expression of the receptor in healthy flounder was highest in the gills and stomach, then in the skin, intestine and liver, followed by the spleen, head kidney, heart, ovary and brain and finally the kidney. On LCDV infection, ELISA indicated that the expression of the receptor, as determined by ELISA, was significantly upregulated in all tissues of LCDV-infected flounder compared with controls, but this expression decreased over the 4 weeks post infection. In contrast, real-time quantitative polymerase chain reaction demonstrated that the copy number of the LCDV gene in the tissues increased with time post infection, and that viral loads were higher in the tissues with higher expressions of the receptor. These results point to a correlation between high expression of the 27.8 kDa receptor and efficient LCDV propagation. The wide tissue distribution of the receptor might be one reason why LCDV can infect various tissues leading to systemic infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cloning, functional expression, and characterization of a chalcone 3-hydroxylase from Cosmos sulphureus.

PubMed

Schlangen, Karin; Miosic, Silvija; Thill, Jana; Halbwirth, Heidi

2010-07-01

A chalcone 3-hydroxylase (CH3H) cDNA clone was isolated and characterized from Cosmos sulphureus petals accumulating butein (2',3,4,4'-tetrahydroxychalcone) derivatives as yellow flower pigments. The recombinant protein catalyses the introduction of an additional hydroxyl group in the B-ring of chalcones, a reaction with high similarity to the hydroxylation of flavonoids catalysed by the well-studied flavonoid 3'-hydroxylase (F3'H). CH3H shows high specificity for chalcones, but a low F3'H activity was also detected. By contrast, the common F3'H from C. sulphureus does not accept chalcones as substrates and is therefore unlikely to be involved in the creation of the B-ring hydroxylation pattern of the yellow flower pigments. CH3H was primarily expressed in young buds, the main tissue for chalcone pigment formation. Expression levels in open flowers and 3-d-old seedlings were lower and almost no CH3H expression was observed in leaves. F3'H, in contrast, showed the highest expression also in buds, but comparable expression rates in all other tissues tested. Recombinant hybrid proteins constructed from CH3H and F3'H fragments demonstrated that amino acid residues at a substrate recognition site and an insertion of four amino acid residues in a putative loop region have an impact on chalcone acceptance. This is the first identification of a CH3H cDNA from any plant species.

Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding.

PubMed

Petznick, Andrea; Madigan, Michele C; Garrett, Qian; Sweeney, Deborah F; Evans, Margaret D M

2013-01-01

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding. Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs. The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining. Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.

The Differential Expression of Immune Genes between Water Buffalo and Yellow Cattle Determines Species-Specific Susceptibility to Schistosoma japonicum Infection

PubMed Central

Yang, Jianmei; Fu, Zhiqiang; Hong, Yang; Wu, Haiwei; Jin, Yamei; Zhu, Chuangang; Li, Hao; Lu, Ke; Shi, Yaojun; Yuan, Chunxiu; Cheng, Guofeng; Feng, Xingang; Liu, Jinming; Lin, Jiaojiao

2015-01-01

Water buffalo are less susceptible to Schistosoma japonicum infection than yellow cattle. The factors that affect such differences in susceptibility remain unknown. A Bos taurus genome-wide gene chip was used to analyze gene expression profiles in the peripheral blood of water buffalo and yellow cattle pre- and post-infection with S. japonicum. This study showed that most of the identified differentially expressed genes(DEGs) between water buffalo and yellow cattle pre- and post-infection were involved in immune-related processes, and the expression level of immune genes was lower in water buffalo. The unique DEGs (390) in yellow cattle were mainly associated with inflammation pathways, while the unique DEGs (2,114) in water buffalo were mainly associated with immune-related factors. The 83 common DEGs may be the essential response genes during S. japonicum infection, the highest two gene ontology (GO) functions were associated with the regulation of fibrinolysis. The pathway enrichment analysis showed that the DEGs constituted similar immune-related pathways pre- and post-infection between the two hosts. This first analysis of the transcriptional profiles of natural hosts has enabled us to gain new insights into the mechanisms that govern their susceptibility or resistance to S. japonicum infections. PMID:26125181

Head and Neck Squamous Cell Carcinomas Do Not Express EGFRvIII

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

Melchers, Lieuwe J., E-mail: l.j.melchers@umcg.nl; Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen; Clausen, Martijn J.A.M.

2014-10-01

Purpose: To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription–polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. Results: None of the 531 casesmore » showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription–polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. Conclusions: The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker.« less

Growth hormone and Prolactin-1 gene transcription in natural populations of the black-chinned tilapia Sarotherodon melanotheron acclimatised to different salinities.

PubMed

Tine, M; de Lorgeril, J; Panfili, J; Diop, K; Bonhomme, F; Durand, J-D

2007-07-01

The effects of salinity on the expression of genes coding for growth hormone (GH) and prolactin-1 (PRL1) were studied in various natural populations of the black-chinned tilapia Sarotherodon melanotheron from West Africa. Individuals were sampled in June 2005 in six locations in Senegal and the Gambia, at various salinities between 0 and 101. The poorest condition factors were recorded in the most saline sampling site and the best growth in the fish from a marine environment. The pituitary GH mRNA levels were significantly higher in fish adapted to seawater, whereas the PRL1 mRNA levels were highest in fish adapted to fresh- and brackish water. These results show that the PRL1 mRNA levels seem to reflect relatively well the differences in environmental salinity, in contrast to those of GH, which would tend instead to reflect the individual growth in each environment. However, no relation could be found between growth in the hypersaline areas and the expression profile of GH. Although the fish analysed were morphologically identical, the expression of genes coding for GH and PRL1 showed large differences between individuals. This inter-individual variation in gene expression remains poorly understood.

A systems-based approach to analyse the host response in murine lung macrophages challenged with respiratory syncytial virus

PubMed Central

2013-01-01

Background Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infection in young children. The degree of disease severity is determined by the host response to infection. Lung macrophages play an important early role in the host response to infection and we have used a systems-based approach to examine the host response in RSV-infected lung-derived macrophage cells. Results Lung macrophage cells could be efficiently infected (>95%) with RSV in vitro, and the expression of several virus structural proteins could be detected. Although we failed to detect significant levels of virus particle production, virus antigen could be detected up until 96 hours post-infection (hpi). Microarray analysis indicated that 20,086 annotated genes were expressed in the macrophage cells, and RSV infection induced an 8.9% and 11.3% change in the global gene transcriptome at 4 hpi and 24 hpi respectively. Genes showing up-regulated expression were more numerous and exhibited higher changes in expression compared to genes showing down-regulated expression. Based on gene ontology, genes with cytokine, antiviral, cell death, and signal transduction functions showed the highest increases in expression, while signalling transduction, RNA binding and protein kinase genes showed the greatest reduction in expression levels. Analysis of the global gene expression profile using pathway enrichment analysis confirmed that up-regulated expression of pathways related to pathogen recognition, interferon signalling and antigen presentation occurred in the lung macrophage cells challenged with RSV. Conclusion Our data provided a comprehensive analysis of RSV-induced gene expression changes in lung macrophages. Although virus gene expression was detected, our data was consistent with an abortive infection and this correlated with the activation of several antivirus signalling pathways such as interferon type I signalling and cell death signalling. RSV infection induced a relatively large increase in pro-inflammatory cytokine expression, however the maintenance of this pro-inflammatory response was not dependent on the production of infectious virus particles. The sustained pro-inflammatory response even in the absence of a productive infection suggests that drugs that control the pro-inflammatory response may be useful in the treatment of patients with severe RSV infection. PMID:23506210

IRF8 Governs Expression of Genes Involved in Innate and Adaptive Immunity in Human and Mouse Germinal Center B Cells

PubMed Central

Morse, Herbert C.

2011-01-01

IRF8 (Interferon Regulatory Factor 8) is a transcription factor expressed throughout B cell differentiation except for mature plasma cells. Previous studies showed it is part of the transcriptional network governing B cell specification and commitment in the bone marrow, regulates the distribution of mature B cells into the splenic follicular and marginal zone compartments, and is expressed at highest levels in germinal center (GC) B cells. Here, we investigated the transcriptional programs and signaling pathways affected by IRF8 in human and mouse GC B cells as defined by ChIP-chip analyses and transcriptional profiling. We show that IRF8 binds a large number of genes by targeting two distinct motifs, half of which are also targeted by PU.1. Over 70% of the binding sites localized to proximal and distal promoter regions with ∼25% being intragenic. There was significant enrichment among targeted genes for those involved in innate and adaptive immunity with over 30% previously defined as interferon stimulated genes. We also showed that IRF8 target genes contributes to multiple aspects of the biology of mature B cells including critical components of the molecular crosstalk among GC B cells, T follicular helper cells, and follicular dendritic cells. PMID:22096565

Molecular identification and characterization of haptoglobin in teleosts revealed an important role on fish viral infections.

PubMed

Cordero, Héctor; Li, Chang Hong; Chaves-Pozo, Elena; Esteban, María Ángeles; Cuesta, Alberto

2017-11-01

Haptoglobin (Hp) molecule has been cloned and characterized in two marine teleosts (gilthead seabream and European sea bass), obtaining putative proteins of 319 residues encoded by an ORF of 960 bp in both species. However, the matrix of similarity revealed low identities among bony fish species 78.9% (seabream-sea bass), 43% (seabream/seabass-zebrafish) and lower than 20% with sharks and human. The protein sequences showed a signal peptide from the position 1 to 23, a trypsin domain from 47 to 297, and several predicted disulfide bridges and glycosylation sites. The expression of hp transcript levels during ontogeny showed a progressive increase of expression in seabream whilst remained almost unaltered in sea bass. By tissues, this gene was found constitutively expressed with the highest levels on liver in both species. The main results on hp transcript levels showed the up-regulation in gilthead seabream suffering from naturally occurring lymphocystis disease; and the down-regulation and up-regulation after nodavirus infection in the resistant gilthead seabream and the susceptible European sea bass, respectively. These findings demonstrate for the first time an important role of haptoglobin against viral infections, operating differently in two of the most important marine farmed fish species. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transformation of Beauveria bassiana to produce EGFP in Tenebrio molitor for use as animal feed additives.

PubMed

Kim, Jae Su; Choi, Jae Young; Lee, Se Jin; Lee, Ju Hyun; Fu, Zhenli; Skinner, Margaret; Parker, Bruce L; Je, Yeon Ho

2013-07-01

Efforts are underway to develop more effective and safer animal feed additives. Entomopathogenic fungi can be considered practical expression platforms of functional genes within insects which have been used as animal feed additives. In this work, as a model, the enhanced green fluorescent protein (egfp) gene was expressed in yellow mealworms, Tenebrio molitor by highly infective Beauveria bassiana ERL1170. Among seven test isolates, ERL1170 treatment showed 57.1% and 98.3% mortality of mealworms 2 and 5 days after infection, respectively. The fungal transformation vector, pABeG containing the egfp gene, was inserted into the genomic DNA of ERL1170 using the restriction enzyme-mediated integration method. This resulted in the generation of the transformant, Bb-egfp#3, which showed the highest level of fluorescence. Bb-egfp#3-treated mealworms gradually turned dark brown, and in 7-days mealworm sections showed a strong fluorescence. This did not occur in the wild-type strain. This work suggests that further valuable proteins can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Role of EG-VEGF in human placentation: Physiological and pathological implications.

PubMed

Hoffmann, Pascale; Saoudi, Yasmina; Benharouga, Mohamed; Graham, Charles H; Schaal, Jean-Patrick; Mazouni, Chafika; Feige, Jean-Jacques; Alfaidy, Nadia

2009-08-01

Pre-eclampsia (PE), the major cause of maternal morbidity and mortality, is thought to be caused by shallow invasion of the maternal decidua by extravillous trophoblasts (EVT). Data suggest that a fine balance between the expressions of pro- and anti-invasive factors might regulate EVT invasiveness. Recently, we showed that the expression of the new growth factor endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is high in early pregnancy but falls after 11 weeks, suggesting an essential role for this factor in early pregnancy. Using human villous explants and HTR-8/SVneo, a first trimester extravillous trophoblast cell line, we showed differential expression of EG-VEGF receptors, PKR1 and PKR2, in the placenta and demonstrated that EG-VEGF inhibits EVT migration, invasion and tube-like organisation. EG-VEGF inhibitory effect on invasion was supported by a decrease in matrix metalloproteinase (MMP)-2 and MMP-9 production. Interference with PKR2 expression, using specific siRNAs, reversed the EG-VEGF-induced inhibitory effects. Furthermore, we determined EG-VEGF circulating levels in normal and PE patients. Our results showed that EG-VEGF levels were highest during the first trimester of pregnancy and decreased thereafter to non-pregnant levels. More important, EG-VEGF levels were significantly elevated in PE patients compared with age-matched controls. These findings identify EG-VEGF as a novel paracrine regulator of trophoblast invasion. We speculate that a failure to correctly down-regulate placental expression of EG-VEGF at the end of the first trimester of pregnancy might lead to PE.

Effect of heat shock on the fatty acid and protein profiles of Cronobacter sakazakii BCRC 13988 as well as its growth and survival in the presence of various carbon, nitrogen sources and disinfectants.

PubMed

Li, Po-Ting; Hsiao, Wan-Ling; Yu, Roch-Chui; Chou, Cheng-Chun

2013-12-01

In the present study, Cronobacter sakazakii, a foodborne pathogen, was first subjected to heat shock at 47 °C for 15 min. Effect of heat shock on the fatty acid and protein profiles, carbon and nitrogen source requirements as well as the susceptibilities of C. sakazakii to Clidox-S, a chlorine-containing disinfectant and Quatricide, a quaternary ammonium compound were investigated. Results revealed that heat shock increased the proportion of myristic acid (14:0), palmitic acid (16:0) and the ratio of saturated fatty acid to unsaturated fatty acid, while reducing the proportion of palmitoleic acid (16:1) and cis-vacceric acid (18:1). In addition, eleven proteins showed enhanced expression, while one protein showed decreased expression in the heat-shocked compared to the non-heat-shocked cells. Non-heat-shocked cells in the medium supplemented with beef extract exhibited the highest maximum population. On the contrary, the highest maximum population of heat-shocked C. sakazakii was noted in the medium having either tryptone or yeast extract as the nitrogen source. Among the various carbon sources examined, the growth of the test organism, regardless of heat shock, was greatest in the medium having glucose as the carbon source. Furthermore, heat shock enhanced the resistance of C. sakazakii to Clidox-S or Quatricide. Copyright © 2013 Elsevier Ltd. All rights reserved.

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

PubMed

Schrevel, Marlies; Osse, E Michelle; Prins, Frans A; Trimbos, J Baptist M Z; Fleuren, Gert Jan; Gorter, Arko; Jordanova, Ekaterina S

2017-06-01

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB‑EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB‑EGF had the highest mRNA expression and HB‑EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB‑EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB‑EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB‑EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB‑EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB‑EGF. Therefore, we propose an autocrine EGFR stimulation model in cervical carcinomas.

Quantitative Mapping of Cocaine-Induced ΔFosB Expression in the Striatum of Male and Female Rats

PubMed Central

Sato, Satoru M.; Wissman, Anne Marie; McCollum, Andrew F.; Woolley, Catherine S.

2011-01-01

ΔFosB plays a critical role in drug-induced long-term changes in the brain. In the current study, we evaluated locomotor activity in male and female rats treated with saline or cocaine for 2 weeks and quantitatively mapped ΔFosB expression in the dorsal striatum and nucleus accumbens of each animal by using an anti-FosB antibody that recognizes ΔFosB isoforms preferentially. Behavioral analysis showed that while there was little difference between males and females that sensitized to cocaine, nonsensitizing rats showed a large sex difference. Nonsensitizing males showed low behavioral activation in response to cocaine on the first day of treatment, and their activity remained low. In contrast, nonsensitizing females showed high activation on the first day of treatment and their activity remained high. Western blot and immunohistochemical analyses indicated that basal levels of ΔFosB were higher in the nucleus accumbens than the dorsal striatum, but that the effect of cocaine on ΔFosB was greater in the dorsal striatum. Immunostaining showed that the effect of cocaine in both the dorsal striatum and nucleus accumbens was primarily to increase the intensity of ΔFosB immunoreactivity in individual neurons, rather than to increase the number of cells that express ΔFosB. Detailed mapping of ΔFosB-labeled nuclei showed that basal ΔFosB levels were highest in the medial portion of the dorsal striatum and dorsomedial accumbens, particularly adjacent to the lateral ventricle, whereas the cocaine-induced increase in ΔFosB was most pronounced in the lateral dorsal striatum, where basal ΔFosB expression was lowest. Sex differences in ΔFosB expression were small and independent of cocaine treatment. We discuss implications of the sex difference in locomotor activation and regionally-specific ΔFosB induction by cocaine. PMID:21747956

Antioxidant and Anti-Inflammatory Activities of Hydrolysates and Peptide Fractions Obtained by Enzymatic Hydrolysis of Selected Heat-Treated Edible Insects.

PubMed

Zielińska, Ewelina; Baraniak, Barbara; Karaś, Monika

2017-09-02

This study investigated the effect of heat treatment of edible insects on antioxidant and anti-inflammatory activities of peptides obtained by in vitro gastrointestinal digestion and absorption process thereof. The antioxidant potential of edible insect hydrolysates was determined as free radical-scavenging activity, ion chelating activity, and reducing power, whereas the anti-inflammatory activity was expressed as lipoxygenase and cyclooxygenase-2 inhibitory activity. The highest antiradical activity against DPPH • (2,2-diphenyl-1-picrylhydrazyl radical) was noted for a peptide fraction from baked cricket Gryllodes sigillatus hydrolysate (IC 50 value 10.9 µg/mL) and that against ABTS •+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical) was the highest for raw mealworm Tenebrio molitor hydrolysate (inhibitory concentration (IC 50 value) 5.3 µg/mL). The peptides obtained from boiled locust Schistocerca gregaria hydrolysate showed the highest Fe 2+ chelation ability (IC 50 value 2.57 µg/mL); furthermore, the highest reducing power was observed for raw G. sigillatus hydrolysate (0.771). The peptide fraction from a protein preparation from the locust S. gregaria exhibited the most significant lipoxygenase and cyclooxygenase-2 inhibitory activity (IC 50 value 3.13 µg/mL and 5.05 µg/mL, respectively).

Antioxidant and Anti-Inflammatory Activities of Hydrolysates and Peptide Fractions Obtained by Enzymatic Hydrolysis of Selected Heat-Treated Edible Insects

PubMed Central

Zielińska, Ewelina; Baraniak, Barbara; Karaś, Monika

2017-01-01

This study investigated the effect of heat treatment of edible insects on antioxidant and anti-inflammatory activities of peptides obtained by in vitro gastrointestinal digestion and absorption process thereof. The antioxidant potential of edible insect hydrolysates was determined as free radical-scavenging activity, ion chelating activity, and reducing power, whereas the anti-inflammatory activity was expressed as lipoxygenase and cyclooxygenase-2 inhibitory activity. The highest antiradical activity against DPPH• (2,2-diphenyl-1-picrylhydrazyl radical) was noted for a peptide fraction from baked cricket Gryllodes sigillatus hydrolysate (IC50 value 10.9 µg/mL) and that against ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical) was the highest for raw mealworm Tenebrio molitor hydrolysate (inhibitory concentration (IC50 value) 5.3 µg/mL). The peptides obtained from boiled locust Schistocerca gregaria hydrolysate showed the highest Fe2+ chelation ability (IC50 value 2.57 µg/mL); furthermore, the highest reducing power was observed for raw G. sigillatus hydrolysate (0.771). The peptide fraction from a protein preparation from the locust S. gregaria exhibited the most significant lipoxygenase and cyclooxygenase-2 inhibitory activity (IC50 value 3.13 µg/mL and 5.05 µg/mL, respectively). PMID:28869499

Analysis of micromineral contents of school meals.

PubMed

Shin, Dongsoon

2014-08-01

Korean ordinary diets are referred to be good for human health in worldwide. However it is uncertain whether they provide microminerals enough for growth and health of teenagers. A main purpose of this study was to identify micromineral contents in school meals. The fifty cuisines were collected from elementary schools and middle schools in Gyeongnam area. The contents of Fe, Zn, Cu and Mn among microminerals were analyzed by using ICP-OES method. Data were expressed as mean, standard deviation and range value and linear regression analysis performed. Fe level of Pangibuseotpaprika-salad was the highest among side-dishes (average 346.6 µg) and Zn level of Sullung-tang was highest among soups (average 229.1 µg). Cu level of Buchu-kimchi was the highest among kimchies (average 217.5 µg) and Mn level of Gumeunkongyangnyum-gui was highest among side-dishes (average 198.4 µg). Generally cooked-rices as main dish had relative smaller amounts of microminerals than the other cuisines. The results showed that the ratio of Cu : Fe : Zn was approximately 12 : 4 : 1 and the relationship between Fe versus Zn or Fe versus Cu was significantly positive. Comparing to Korean Dietary Recommended Intakes (KDRI) level, school meals provided not sufficient amount (<25% DRI) of Fe, Zn or Mn, while they did excessive amount (>125% DRI) of Cu.

CD4+ T cell-mediated cytotoxicity is associated with MHC class II expression on malignant CD19+ B cells in diffuse large B cell lymphoma.

PubMed

Zhou, Yong; Zha, Jie; Lin, Zhijuan; Fang, Zhihong; Zeng, Hanyan; Zhao, Jintao; Luo, Yiming; Li, Zhifeng; Xu, Bing

2018-01-15

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy with approximately 30% of patients present relapsed or refractory disease after first-line therapy. Research of further treatment options is needed. Cytotoxic CD4 + T cells express cytolytic molecules and have potential antitumor function. Here, we showed that the CD19 + cells from DLBCL patients presented significantly reduced expression of MHC II molecules than those from healthy controls. Three years after the first-line treatment, patients that presented relapsed disease had significantly lower MHC II expression on their CD19 + cells than patients who did not show recurrence. Examining cytotoxic CD4 + T cells show that DLBCL patients presented significantly elevated frequencies of granzyme A-, granzyme B-, and/or perforin-expressing cytotoxic CD4 + T cells. Also, frequency of cytotoxic CD4 + T cells in DLBCL patients was positively correlated with the MHC II expression level. Subsequently, the cytotoxic potential of CD4 + T cells against autologous CD19 + cells was investigated. We found that the cytotoxic potential of CD4 + T cells was highest in MHC II-high, intermediate in MHC II-mid, and lowest in MHC II-low patients. The percentage of MHC II-expressing viable CD19 + cells presented a significant reduction after longer incubation with cytotoxic CD4 + T cells, suggesting that cytotoxic CD4 + T cells preferentially eliminated MHC II-expressing CD19 + cells. Blocking MHC II on CD19 + cells significantly reduced the cytolytic capacity of CD4 + T cells. Despite these discoveries, the frequency of cytotoxic CD4 + T cells did not predict the clinical outcome of DLBCL patients. Together, these results demonstrated that cytotoxic CD4 + T cells presented an MHC II-dependent cytotoxic potential against autologous CD19 + cells and could potentially represent a future treatment option for DLBCL. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of anisodamine on the expressions of vascular endothelial growth factor and intercellular adhesion molecule 1 in experimental infusion phlebitis.

PubMed

Zhang, Zhen-Xiang; Wang, Peng; Zhang, Qiu-Shi; Pan, Xue; Zhao, Qing-Xia; Wang, Xiao-Kai

2012-01-01

Infusion phlebitis is the most common side effect of clinical intravenous drug therapy and several clinical studies have demonstrated that anisodamine can effectively prevent the occurrence of infusion phlebitis. This study was designed to investigate effects of anisodamine on the expressions of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) in a rabbit model of infusion phlebitis and to analyze the mechanisms of anisodamine effect on the prevention and treatment of experimental infusion phlebitis. Twenty-four specific pathogen-free male Japanese white rabbits were randomly assigned to the control group, the model group, the magnesium sulfate group and the anisodamine group. The rabbit model of infusion phlebitis, induced by intravenous administration, was established and expressions of VEGF and ICAM-1 were determined and contrasted with the control group treated with normal saline. We evaluated expression by histopathology, immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blotting assay. Pathohistological changes of the model group were observed, such as loss of venous endothelial cells, inflammatory cell infiltration, edema and thrombus. The magnesium sulfate group and the anisodamine group showed significant protective effects on vascular congestion, inflammatory cell infiltration, proliferation, swelling of endothelium and perivascular hemorrhage. The model group showed the highest expressions of VEGF and ICAM-1 of the four groups (P < 0.01). On the contrary, anisodamine alleviated the inflammatory damage by significantly reducing the expressions of VEGF and ICAM-1 compared with the model group (P < 0.01). There was no significant difference in the expressions of VEGF and ICAM-1 between the magnesium sulfate group and the anisodamine group (P > 0.05). Anisodamine alleviates inflammatory damage by significantly reducing the expressions of VEGF and ICAM-1, and shows significant protective effects in an animal model of infusion phlebitis.

[Epidemiology of type 1 diabetes in children and adolescents aged less than 15 years in the provinces of Castilla y León].

PubMed

Bahíllo Curieses, Maria P; Hermoso López, F; García Fernández, J A; Ochoa Sangrador, C; Rodrigo Palacios, J; de la Torre Santos, S I; Marugán de Miguelsanz, J M; Manzano Recio, F; García Velázquez, J; Lema Garret, T J

2006-07-01

The incidence of type 1 diabetes shows wide geographical variability and heterogeneity. The aim of this study was to determine the incidence and prevalence of type 1 diabetes in children and adolescents ages less than 15 years in the different provinces of Castilla-León. To determine incidence, all new cases of type 1 diabetes with onset under 15 years of age in 2003-2004 were obtained. Incidence was expressed as the crude value with the corresponding confidence interval and as standardized incidence. The capture-recapture method was used to calculate the completeness of ascertainment. To determine prevalence, all cases of type 1 diabetes in persons ages less than 15 years at 31 December 2004 were obtained. Incidence showed wide variability among the different provinces of Castilla-León. The highest values were found in Segovia (38.77/100,000/year), Valladolid (32.07/100,000/ year) and Avila (23.21/100,000/year) and the lowest in Zamora (8.14/100,000/year). Incidences were highest in the 5-9 years age group in all provinces except Burgos. Prevalence was highest in Segovia (1.54/1,000), Valladolid (1.41/1,000), Avila (1.38/1,000) and Zamora (1.32/1,000) and lowest in Burgos (0.91/1,000). Castilla-León seems to have one of the highest incidences of type 1 diabetes in Spain; several of its provinces have values similar to those in Northern Europe.

Evaluation of the potential application of three different biomaterials combined with bone morphological proteins for enhancing tendon-bone integration.

PubMed

Pan, Weimin; Cao, Zheng; Li, Dan; Zhang, Mingjun

2013-04-01

Secure tendon-bone integration is crucial for successful anterior cruciate ligament (ACL) reconstruction. Previous studies have applied different types of biomaterial or biomaterial combined with bone-growth factors to enhance tendon-bone integration. However, which approach is better remains controversial. This comparison evaluation could help identify a suitable composite biomaterial for osteointegration of grafted tendon. Three different composite biomaterials mixed with bone morphological proteins (BMPs) were fabricated. The in vitro study investigates cell metabolism, osteogenic gene expression and the growth behaviour of bone marrow stromal cells (BMSCs) on fibrin glue-BMPs (FGB), calcium phosphate cement-BMPs (CPCB) and recombined bone xenograft (RBX), which are commercially, clinically available biomaterials. Meanwhile, the changes in the physical, morphological and mechanical properties between the three composites and the original biomaterials were also observed. The in vivo study mainly examined the osteogenic ability of the three composites through rat ectopic testing. The porosity structure of three biomaterials was improved after being combined with BMPs powder for SEM observation, and the setting times of the injectable composites were not significantly delayed. More importantly, there were no significant decreases in compressive strength between the three composite biomaterials and the original biomaterials. The highest proliferation rate of BMSCs was found in the RBX group, followed by the CPCB and FGB groups. BMSCs seeded onto an RBX showed the highest alkaline phosphatase (ALPase) activity and gene expression of collagen I (P < 0.05). Histological examination showed endochondral new bone formation in the specimens of all groups, but the ALPase activity of newly formed tissue in the RBX group showed the highest level (P < 0.01). Our results indicate that RBX seems to be a very good choice for accelerating tendon-bone integration, and CPCB also has a large potential ability to be used. However, these two composites still need to be modified, and we postulate that a combination of them would be more favourable for tendon osteointegration after ACL reconstruction than either composite used alone. Copyright © 2012 Elsevier Ltd. All rights reserved.

Improved Sp1 and Betaine Homocysteine-S-Methyltransferase Expression and Homocysteine Clearance Are Involved in the Effects of Zinc on Oxidative Stress in High-Fat-Diet-Pretreated Mice.

PubMed

Wu, Li; Zhou, Xihong; Li, Tiejun; He, Juyun; Huang, Linli; Ouyang, Zicheng; He, Liuqin; Wei, Tao; He, Qinghua

2017-12-04

Zinc plays a role in alleviating oxidative stress. However, the related mechanisms remain to be further elucidated. The present study was conducted to investigate whether the recovery of oxidative stress in high-fat-diet (HFD)-pretreated mice was affected by zinc. Male mice received either an HFD or a low-fat-diet (LFD) for 8 weeks. Then, the mice fed with HFD and LFD were both assigned to either a control diet (30 mg zinc, ZD) or a no-added zinc diet (NZD) for an additional 4 weeks. The results showed that after feeding with NZD for 4 weeks, the HFD-pretreated mice had the highest plasma glucose and insulin concentrations, while had the lowest CuZn-SOD and glutathione concentrations. Moreover, after feeding with NZD for 4 weeks, the HFD-pretreated mice had the highest hepatic ROS and homocysteine concentrations, while had the lowest glutathione and methionine concentrations. Furthermore, the HFD-pretreated mice fed with NZD for 4 weeks had the lowest gene and protein expression of betaine homocysteine-S-methyltransferase (BHMT), cystathionine β-synthase, and Sp1. The results suggested that zinc was critical for oxidative stress alleviation and homocysteine clearance in HFD-pretreated mice. It was further elucidated that improved Sp1 and BHMT expression are involved in the effects of zinc on oxidative stress.

Regulatory heterochronies and loose temporal scaling between sea star and sea urchin regulatory circuits.

PubMed

Gildor, Tsvia; Hinman, Veronica; Ben-Tabou-De-Leon, Smadar

2017-01-01

It has long been argued that heterochrony, a change in relative timing of a developmental process, is a major source of evolutionary innovation. Heterochronic changes of regulatory gene activation could be the underlying molecular mechanism driving heterochronic changes through evolution. Here, we compare the temporal expression profiles of key regulatory circuits between sea urchin and sea star, representative of two classes of Echinoderms that shared a common ancestor about 500 million years ago. The morphologies of the sea urchin and sea star embryos are largely comparable, yet, differences in certain mesodermal cell types and ectodermal patterning result in distinct larval body plans. We generated high resolution temporal profiles of 17 mesodermally-, endodermally- and ectodermally-expressed regulatory genes in the sea star, Patiria miniata, and compared these to their orthologs in the Mediterranean sea urchin, Paracentrotus lividus. We found that the maternal to zygotic transition is delayed in the sea star compared to the sea urchin, in agreement with the longer cleavage stage in the sea star. Interestingly, the order of gene activation shows the highest variation in the relatively diverged mesodermal circuit, while the correlations of expression dynamics are the highest in the strongly conserved endodermal circuit. We detected loose scaling of the developmental rates of these species and observed interspecies heterochronies within all studied regulatory circuits. Thus, after 500 million years of parallel evolution, mild heterochronies between the species are frequently observed and the tight temporal scaling observed for closely related species no longer holds.

Determination of optimized oxygen partial pressure to maximize the liver regenerative potential of the secretome obtained from adipose-derived stem cells.

PubMed

Lee, Sang Chul; Kim, Kee-Hwan; Kim, Ok-Hee; Lee, Sang Kuon; Hong, Ha-Eun; Won, Seong Su; Jeon, Sang-Jin; Choi, Byung Jo; Jeong, Wonjun; Kim, Say-June

2017-08-03

A hypoxic-preconditioned secretome from stem cells reportedly promotes the functional and regenerative capacity of the liver more effectively than a control secretome. However, the optimum oxygen partial pressure (pO 2 ) in the cell culture system that maximizes the therapeutic potential of the secretome has not yet been determined. We first determined the cellular alterations in adipose tissue-derived stem cells (ASCs) cultured under different pO 2 (21%, 10%, 5%, and 1%). Subsequently, partially hepatectomized mice were injected with the secretome of ASCs cultured under different pO 2 , and then sera and liver specimens were obtained for analyses. Of all AML12 cells cultured under different pO 2 , the AML12 cells cultured under 1% pO 2 showed the highest mRNA expression of proliferation-associated markers (IL-6, HGF, and VEGF). In the cell proliferation assay, the AML12 cells cultured with the secretome of 1% pO 2 showed the highest cell proliferation, followed by the cells cultured with the secretome of 21%, 10%, and 5% pO 2 , in that order. When injected into the partially hepatectomized mice, the 1% pO 2 secretome most significantly increased the number of Ki67-positive cells, reduced serum levels of proinflammatory mediators (IL-6 and TNF-α), and reduced serum levels of liver transaminases. In addition, analysis of the liver specimens indicated that injection with the 1% pO 2 secretome maximized the expression of the intermediate molecules of the PIP3/Akt and IL-6/STAT3 signaling pathways, all of which are known to promote liver regeneration. The data of this study suggest that the secretome of ASCs cultured under 1% pO 2 has the highest liver reparative and regenerative potential of all the secretomes tested here.

Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms.

PubMed

Syrjänen, Leo; Luukkaala, Tiina; Leppilampi, Mari; Kallioinen, Matti; Pastorekova, Silvia; Pastorek, Jaromir; Waheed, Abdul; Sly, William S; Parkkila, Seppo; Karttunen, Tuomo

2014-09-01

Purpose of the study was to evaluate the presence of hypoxia-inducible, tumour-associated carbonic anhydrases IX and XII in normal skin and a series of cutaneous tumours. Human tumour samples were taken during surgical operations performed on 245 patients and were immunohistochemically stained. A histological score value was calculated for statistical analyses which were performed using SPSS for Windows, versions 17.0 and 20.0. In normal skin, the highest expression of CA IX was detected in hair follicles, sebaceous glands, and basal parts of epidermis. CA XII was detected in all epithelial components of skin. Both CA IX and CA XII expression levels were significantly different in epidermal, appendigeal, and melanocytic tumour categories. Both CA IX and XII showed the most intense immunostaining in epidermal tumours, whereas virtually all melanocytic tumours were devoid of CA IX and XII immunostaining. In premalignant lesions, CA IX expression significantly increased when the tumours progressed to more severe dysplasia forms. Both CA IX and XII are highly expressed in different epithelial components of skin. They are also highly expressed in epidermal tumours, in which CA IX expression levels also correlate with the dysplasia grade. Interestingly, both isozymes are absent in melanocytic tumours. © 2014 APMIS. Published by John Wiley & Sons Ltd.

Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio)

PubMed Central

Tierbach, Alena; Groh, Ksenia J; Schönenberger, René; Schirmer, Kristin

2018-01-01

Abstract Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations. PMID:29361160

Expression of hygromycin B resistance in oyster culinary-medicinal mushroom, Pleurotus ostreatus (Jacq.:Fr.)P. Kumm. (higher Basidiomycetes) using three gene expression systems.

PubMed

Dong, Xiaoya; Zhang, Ke; Gao, Yuqian; Qi, Yuancheng; Shen, Jinwen; Qiu, Liyou

2012-01-01

Three hygromycin B phosphotransferase (hph) gene expression systems for culinary-medicinal Oyster mushroom, Pleurotus ostreatus, plasmid pSHC, pAN7-1, and pBHt1 were evaluated through PEG/CaCl(2)-mediated protoplast transformation. Plasmid pSHC is a newly constructed hph gene expression system, composed of Escherichia coli hph gene, the P. ostreatus sdi promoter, and the CaMV35S terminator. The vector pAN7-1 was commonly used for integrative transformation in filamentous fungi. Plasmid pBHtl is a T-DNA binary vector, usually introduced into fungi by Agrobacterium-mediated transformation. The results showed that plasmids pSHC, pAN7-1, and pBHt1 were all integrated into the host chromosomes and expressed hygromycin B resistance in P. ostreatus. pAN7-1 had the highest transformation efficiency and hph gene expression level, pSHC the second, and pBHt1 the lowest. Growth rates of the transformants on plates containing hygromycin B were in correspondence with their hph gene expression levels. To our knowledge, this is the first report on integrated transformation of plasmid pAN7-1 and pBHt1 in P. ostreatus.

GPNMB promotes proliferation of developing eosinophils.

PubMed

Hwang, Sae Mi; Kang, Jin Hyun; Kim, Bo Kyum; Uhm, Tae Gi; Kim, Hye Jeong; Lee, Hyune-Hwan; Binas, Bert; Chung, Il Yup

2017-08-01

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane protein that is expressed in a wide variety of cell types, including haematopoietic lineages. We previously demonstrated that GPNMB is one of the most highly expressed genes at an early and intermediate stage of eosinophil development. We herein examined GPNMB expression and its possible functional effect using cord blood (CB) CD34+ haematopoietic stem cells differentiating toward eosinophils during a 24-day culture period. Western blot and confocal microscopy analyses showed that GPNMB reached its highest levels at day 12 with most GPNMB-positive cells also expressing major basic protein 1 (MBP1), an eosinophil granule protein. GPNMB declined thereafter, but was still present at an appreciable level at day 24, the time when CB eosinophils most abundantly expressed MBP1 and were thus considered fully differentiated. When the developing CB cells were cultured in the presence of a blocking anti-GPNMB antibody, cell proliferation was significantly reduced. In agreement, ectopic expression of GPNMB in heterologous cells resulted in a significant increase in cell proliferation, while small interfering RNA of GPNMB inhibited the GPNMB-mediated proliferation. Thus, GPNMB is expressed in a temporal manner during eosinophil development and delivers a proliferative signal upon activation. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

The expression characteristics of mt-ND2 gene in chicken.

PubMed

Zhang, Wenwen; Hou, Lingling; Wang, Ting; Lu, Weiwei; Tao, Yafei; Chen, Wen; Du, Xiaohui; Huang, Yanqun

2016-09-01

Subunit 2 of NADH dehydrogenase (ND2) is encoded by the mt-ND2 gene and plays a critical role in controlling the production of the mitochondrial reactive oxygen species. Our study focused on exploring the mt-ND2 tissue expression patterns and the effects of energy restriction and dietary fat (linseed oil, corn oil, sesame oil or lard) level (2.5% and 5%) on its expression in chicken. The results showed that mt-ND2 gene was expressed in the 15 tissues of hybrid chickens with the highest level in heart and lowest level in pancreas tissue; 30% energy restriction did not significantly affect mt-ND2 mRNA level in chicken liver tissue. Both the mt-ND2 mRNA levels in chicken pectoralis (p < 0.05) and hepatic tissues (p < 0.05) at 42 d-old were affected by the type of dietary fats in 5% level, while not in abdominal fat tissues. The expression of mt-ND2 in hepatic tissues was down-regulated with chicken age (p < 0.01). The interactive effect of dietary fat types with chicken age (p < 0.05) was significant on mt-ND2 mRNA level. The study demonstrated that mt-ND2 gene was extensively expressed in tissues, and the expression was affected by dietary fat types and chicken age.

Monotreme Lactation Protein Is Highly Expressed in Monotreme Milk and Provides Antimicrobial Protection

PubMed Central

Enjapoori, Ashwantha Kumar; Grant, Tom R.; Nicol, Stewart C.; Lefèvre, Christophe M.; Nicholas, Kevin R.; Sharp, Julie A.

2014-01-01

Monotremes (platypus and echidna) are the descendants of the oldest ancestor of all extant mammals distinguished from other mammals by mode of reproduction. Monotremes lay eggs following a short gestation period and after an even briefer incubation period, altricial hatchlings are nourished over a long lactation period with milk secreted by nipple-less mammary patches located on the female’s abdomen. Milk is the sole source of nutrition and immune protection for the developing young until weaning. Using transcriptome and mass spectrometry analysis of milk cells and milk proteins, respectively, a novel Monotreme Lactation Protein (MLP) was identified as a major secreted protein in milk. We show that platypus and short-beaked echidna MLP genes show significant homology and are unique to monotremes. The MLP transcript was shown to be expressed in a variety of tissues; however, highest expression was observed in milk cells and was expressed constitutively from early to late lactation. Analysis of recombinant MLP showed that it is an N-linked glycosylated protein and biophysical studies predicted that MLP is an amphipathic, α-helical protein, a typical feature of antimicrobial proteins. Functional analysis revealed MLP antibacterial activity against both opportunistic pathogenic Staphylococcus aureus and commensal Enterococcus faecalis bacteria but showed no effect on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Salmonella enterica. Our data suggest that MLP is an evolutionarily ancient component of milk-mediated innate immunity absent in other mammals. We propose that MLP evolved specifically in the monotreme lineage supporting the evolution of lactation in these species to provide bacterial protection, at a time when mammals lacked nipples. PMID:25245409

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

PubMed

Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

1998-12-01

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

Cytokine mRNA expression in normal skin of various age populations before and after engraftment onto nude mice.

PubMed

Gilhar, A; Ullmann, Y; Shalagino, R; Weisinger, G

1998-01-01

Whether the impact of skin biological age on cytokine expression is a result of this tissue's proliferation potential or not is an important issue in dermatology. We investigated these questions by monitoring cytokine marker mRNA expression from human skin samples from healthy groups of individuals. The skin samples studied represented three age groups: fetal (17-21 weeks), young (18-35 years) and aged (76-88 years). Furthermore, upon skin transplantation of tissue from different age groups onto nude mice, we investigated whether cytokine marker RNA levels would change or normalize. Interestingly, both TNF-alpha and P53 mRNA showed a similar pattern of expression. Both were significantly higher in fetal skin (p < 0.0001 and p < 0.05, respectively), and no difference was noted between aged versus young skin. In contrast to this, IL1-alpha mRNA was expressed at its lowest and highest levels in fetal and young skin, respectively. Following skin transplantation, cytokines and P53 mRNA expression were normalized to similar levels in all age groups. This study implies that when cytokine expression was determined directly at the mRNA level, post-natal expression was not significantly different at either age group. Furthermore, it seems that the environmental conditions surrounding the grafted human skin found on nude mice encouraged normalization of donor cytokine expression.

Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant.

PubMed Central

Rentsch, D; Hirner, B; Schmelzer, E; Frommer, W B

1996-01-01

A yeast mutant lacking SHR3, a protein specifically required for correct targeting of plasma membrane amino acid permeases, was used to study the targeting of plant transporters and as a tool to isolate new SHR3-independent amino acid transporters. For this purpose, an shr3 mutant was transformed with an Arabidopsis cDNA library. Thirty-four clones were capable of growth under selective conditions, but none showed homology with SHR3. However, genes encoding eight different amino acid transporters belonging to three different transporter families were isolated. Five of these are members of the general amino acid permease (AAP) gene family, one is a member of the NTR family, encoding an oligopeptide transporter, and two belong to a new class of transporter genes. A functional analysis of the latter two genes revealed that they encode specific proline transporters (ProT) that are distantly related to the AAP gene family. ProT1 was found to be expressed in all organs, but highest levels were found in roots, stems, and flowers. Expression in flowers was highest in the floral stalk phloem that enters the carpels and was downregulated after fertilization, indicating a specific role in supplying the ovules with proline. ProT2 transcripts were found ubiquitously throughout the plant, but expression was strongly induced under water or salt stress, implying that ProT2 plays an important role in nitrogen distribution during water stress, unlike members of the AAP gene family whose expression was repressed under the same conditions. These results corroborate the general finding that under water stress, amino acid export is impaired whereas proline export is increased. PMID:8776904

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

PubMed

Norzagaray-Valenzuela, Claudia D; Germán-Báez, Lourdes J; Valdez-Flores, Marco A; Hernández-Verdugo, Sergio; Shelton, Luke M; Valdez-Ortiz, Angel

2018-05-16

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD 600  = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Endothelial pro-atherosclerotic response to extracellular diabetic-like environment: possible role of thioredoxin-interacting protein.

PubMed

Zitman-Gal, Tali; Green, Janice; Pasmanik-Chor, Metsada; Oron-Karni, Varda; Bernheim, Jacques

2010-07-01

BACKGROUND. High blood and tissue concentrations of glucose and advanced glycation end-products (AGEs) are thought to play an important role in the development of vascular diabetic complications. Therefore, the impact of extracellular AGEs and different glucose concentrations was evaluated by studying the gene expressions and the underlying cellular pathways involved in the development of inflammatory pro-atherosclerotic processes observed in cultured endothelial cells. METHODS. Fresh human umbilical vein cord endothelial cells (HUVEC) were treated in the presence of elevated extracellular glucose concentrations (5.5-28 mmol/l) with and without AGE-human serum albumin (HSA). Affymetrix GeneChip(R) Human Gene 1.0 ST arrays were used for gene expression analysis (total 20 chips). Genes of interest were further validated using real-time PCR and western blot techniques. RESULTS. Microarray analysis revealed significant changes in some gene expressions in the presence of the different stimuli, suggesting that different pathways are involved. Six genes were selected for validation as follows: thioredoxin-interacting protein (TXNIP), thioredoxin (TXN), nuclear factor of kappa B (NF-kappaB), interleukin 6 (IL6), interleukin 8 (IL8) and receptor of advanced glycation end-products (RAGE). Interestingly, it was found that the association of AGEs together with the highest pathophysiological concentration of glucose (28 mmol/l) diminished the expression of these specific genes, excluding TXN. CONCLUSIONS. In the present model that mimics a diabetic environment, the relatively short-term experimental conditions used showed an unexpected blunting action of AGEs in the presence of the highest glucose concentration (28 mmol/l). The interactive cellular pathways involved in these processes should be further investigated.

Genome-wide identification and characterization of aquaporin gene family in common bean (Phaseolus vulgaris L.).

PubMed

Ariani, Andrea; Gepts, Paul

2015-10-01

Plant aquaporins are a large and diverse family of water channel proteins that are essential for several physiological processes in living organisms. Numerous studies have linked plant aquaporins with a plethora of processes, such as nutrient acquisition, CO2 transport, plant growth and development, and response to abiotic stresses. However, little is known about this protein family in common bean. Here, we present a genome-wide identification of the aquaporin gene family in common bean (Phaseolus vulgaris L.), a legume crop essential for human nutrition. We identified 41 full-length coding aquaporin sequences in the common bean genome, divided by phylogenetic analysis into five sub-families (PIPs, TIPs, NIPs, SIPs and XIPs). Residues determining substrate specificity of aquaporins (i.e., NPA motifs and ar/R selectivity filter) seem conserved between common bean and other plant species, allowing inference of substrate specificity for these proteins. Thanks to the availability of RNA-sequencing datasets, expression levels in different organs and in leaves of wild and domesticated bean accessions were evaluated. Three aquaporins (PvTIP1;1, PvPIP2;4 and PvPIP1;2) have the overall highest mean expressions, with PvTIP1;1 having the highest expression among all aquaporins. We performed an EST database mining to identify drought-responsive aquaporins in common bean. This analysis showed a significant increase in expression for PvTIP1;1 in drought stress conditions compared to well-watered environments. The pivotal role suggested for PvTIP1;1 in regulating water homeostasis and drought stress response in the common bean should be verified by further field experimentation under drought stress.

The GmFAD7 gene family from soybean: identification of novel genes and tissue-specific conformations of the FAD7 enzyme involved in desaturase activity.

PubMed

Andreu, Vanesa; Lagunas, Beatriz; Collados, Raquel; Picorel, Rafael; Alfonso, Miguel

2010-07-01

The FAD7 gene encodes a omega3 fatty acid desaturase which catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts. A novel GmFAD7 gene (named GmFAD7-2) has been identified in soybean, with high homology to the previously annotated GmFAD7 gene. Genomic sequencing analysis together with searches at the soybean genome database further confirmed that both GmFAD7 genes were located in two different loci within the soybean genome, suggesting that the soybean omega3 plastidial desaturase FAD7 is encoded by two different paralogous genes. Both GmFAD7-1 and GmFAD7-2 genes were expressed in all soybean tissues examined, displaying their highest mRNA accumulation in leaves. This expression profile contrasted with GmFAD3A and GmFAD3B mRNA accumulation, which was very low in this tissue. These results suggested a concerted control of plastidial and reticular omega3 desaturase gene expression in soybean mature leaves. Analysis of GmFAD7 protein distribution in different soybean tissues showed that, in mature leaves, two bands were detected, coincident with the higher expression level of both GmFAD7 genes and the highest 18:3 fatty acid accumulation. By contrast, in seeds, where FAD7 activity is low, specific GmFAD7 protein conformations were observed. These GmFAD7 protein conformations were affected in vitro by changes in the redox conditions of thiol groups and iron availability. These results suggest the existence of tissue-specific post-translational regulatory mechanisms affecting the distribution and conformation of the FAD7 enzymes related with the control of its activity.

Preclinical validation of 111In-girentuximab-F(ab')2 as a tracer to image hypoxia related marker CAIX expression in head and neck cancer xenografts.

PubMed

Huizing, Fokko J; Hoeben, Bianca A W; Franssen, Gerben; Lok, Jasper; Heskamp, Sandra; Oosterwijk, Egbert; Boerman, Otto C; Bussink, Johan

2017-09-01

Hypoxia is a major cause of radio- and chemoresistance. Carbonic anhydrase IX (CAIX) is an endogenous hypoxia-related marker and an important prognostic marker. Assessment of CAIX expression may allow patient selection for hypoxia or CAIX-targeted treatment. The radioactive tracer 111 In-girentuximab-F(ab') 2 targets CAIX and can be used for SPECT imaging. Aim of this study was to validate and optimize 111 In-girentuximab-F(ab') 2 for imaging of CAIX expression in head and neck tumor xenografts. Affinity and internalization kinetics of 111 In-girentuximab-F(ab') 2 were determined in vitro using CAIX-expressing SK-RC-52 cells. Tumor targeting characteristics were determined in athymic mice with six different head and neck squamous cell carcinoma (SCCNij) xenografts. Tracer uptake was measured by ex vivo radioactivity counting. Intratumoral distribution of tracer uptake was measured using autoradiography and CAIX expression was determined immunohistochemically. 26% of the tracer was internalized into the SK-RC-52 cells within 24h. The half maximal inhibitory concentration (IC 50 ) was 0.69±0.08nM. In biodistribution studies SCCNij153 tumors showed the highest tracer uptake: 4.1±0.8ID/g at 24h p.i. Immunohistochemical and autoradiographic analyses of the xenografts showed a distinct spatial correlation between localization of the tracer and CAIX expression. 111 In-girentuximab-F(ab') 2 has a high affinity for CAIX. In vivo tumor uptake correlated strongly with CAIX expression in different head and neck xenografts. These results suggest that 111 In-girentuximab-F(ab') 2 is a promising tracer for imaging of hypoxia-related CAIX expression. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of fourteen novel obesity-related genes in Zucker diabetic fatty rats.

PubMed

Schmid, Peter M; Heid, Iris; Buechler, Christa; Steege, Andreas; Resch, Markus; Birner, Christoph; Endemann, Dierk H; Riegger, Guenter A; Luchner, Andreas

2012-07-13

Genome-wide association studies (GWAS) are useful to reveal an association between single nucleotide polymorphisms and different measures of obesity. A multitude of new loci has recently been reported, but the exact function of most of the according genes is not known. The aim of our study was to start elucidating the function of some of these genes. We performed an expression analysis of fourteen genes, namely BDNF, ETV5, FAIM2, FTO, GNPDA2, KCTD15, LYPLAL1, MCR4, MTCH2, NEGR1, NRXN3, TMEM18, SEC16B and TFAP2B, via real-time RT-PCR in adipose tissue of the kidney capsule, the mesenterium and subcutaneum as well as the hypothalamus of obese Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats at an age of 22 weeks. All of our target genes except for SEC16B showed the highest expression in the hypothalamus. This suggests a critical role of these obesity-related genes in the central regulation of energy balance. Interestingly, the expression pattern in the hypothalamus showed no differences between obese ZDF and lean ZL rats. However, LYPLAL1, TFAP2B, SEC16B and FAIM2 were significantly lower expressed in the kidney fat of ZDF than ZL rats. NEGR1 was even lower expressed in subcutaneous and mesenterial fat, while MTCH2 was higher expressed in the subcutaneous and mesenterial fat of ZDF rats. The expression pattern of the investigated obesity genes implies for most of them a role in the central regulation of energy balance, but for some also a role in the adipose tissue itself. For the development of the ZDF phenotype peripheral rather than central mechanisms of the investigated genes seem to be relevant.

Genetic divergence in the transcriptional engram of chronic alcohol abuse: A laser-capture RNA-seq study of the mouse mesocorticolimbic system.

PubMed

Mulligan, Megan K; Mozhui, Khyobeni; Pandey, Ashutosh K; Smith, Maren L; Gong, Suzhen; Ingels, Jesse; Miles, Michael F; Lopez, Marcelo F; Lu, Lu; Williams, Robert W

2017-02-01

Genetic factors that influence the transition from initial drinking to dependence remain enigmatic. Recent studies have leveraged chronic intermittent ethanol (CIE) paradigms to measure changes in brain gene expression in a single strain at 0, 8, 72 h, and even 7 days following CIE. We extend these findings using LCM RNA-seq to profile expression in 11 brain regions in two inbred strains - C57BL/6J (B6) and DBA/2J (D2) - 72 h following multiple cycles of ethanol self-administration and CIE. Linear models identified differential expression based on treatment, region, strain, or interactions with treatment. Nearly 40% of genes showed a robust effect (FDR < 0.01) of region, and hippocampus CA1, cortex, bed nucleus stria terminalis, and nucleus accumbens core had the highest number of differentially expressed genes after treatment. Another 8% of differentially expressed genes demonstrated a robust effect of strain. As expected, based on similar studies in B6, treatment had a much smaller impact on expression; only 72 genes (p < 0.01) are modulated by treatment (independent of region or strain). Strikingly, many more genes (415) show a strain-specific and largely opposite response to treatment and are enriched in processes related to RNA metabolism, transcription factor activity, and mitochondrial function. Over 3 times as many changes in gene expression were detected in D2 compared to B6, and weighted gene co-expression network analysis (WGCNA) module comparison identified more modules enriched for treatment effects in D2. Substantial strain differences exist in the temporal pattern of transcriptional neuroadaptation to CIE, and these may drive individual differences in risk of addiction following excessive alcohol consumption. Copyright © 2016 Elsevier Inc. All rights reserved.

Insights into TREM2 biology by network analysis of human brain gene expression data

PubMed Central

Forabosco, Paola; Ramasamy, Adaikalavan; Trabzuni, Daniah; Walker, Robert; Smith, Colin; Bras, Jose; Levine, Adam P.; Hardy, John; Pocock, Jennifer M.; Guerreiro, Rita; Weale, Michael E.; Ryten, Mina

2013-01-01

Rare variants in TREM2 cause susceptibility to late-onset Alzheimer's disease. Here we use microarray-based expression data generated from 101 neuropathologically normal individuals and covering 10 brain regions, including the hippocampus, to understand TREM2 biology in human brain. Using network analysis, we detect a highly preserved TREM2-containing module in human brain, show that it relates to microglia, and demonstrate that TREM2 is a hub gene in 5 brain regions, including the hippocampus, suggesting that it can drive module function. Using enrichment analysis we show significant overrepresentation of genes implicated in the adaptive and innate immune system. Inspection of genes with the highest connectivity to TREM2 suggests that it plays a key role in mediating changes in the microglial cytoskeleton necessary not only for phagocytosis, but also migration. Most importantly, we show that the TREM2-containing module is significantly enriched for genes genetically implicated in Alzheimer's disease, multiple sclerosis, and motor neuron disease, implying that these diseases share common pathways centered on microglia and that among the genes identified are possible new disease-relevant genes. PMID:23855984

Morphological, physicochemical, and antioxidant profile of noncommercial banana cultivars

PubMed Central

Anyasi, Tonna A; Jideani, Afam IO; Mchau, Godwin A

2015-01-01

Banana cultivars––Luvhele (MusaABB), Mabonde (MusaAAA), and Muomva-red (Musa balbisiana) ––were characterized for morphological, physicochemical, and antioxidant properties. All three cultivars varied significantly (P < 0.05) in their morphology, pH, titratable acidity and total soluble solids with no significant difference in their ash content. Individual cultivars showed variations in flour starch granule when observed using a scanning electron microscope. Characterization of cultivars for total polyphenols (TPs) and antioxidant activity upon pretreatment with ascorbic, citric, and lactic acid shows that the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay of samples varied significantly as Muomva-red cultivar (1.02 ± 0.01 mg GA/g) expressed the highest DPPH activity at lactic acid concentration of 20 g/L. Total polyphenol content was also highest for Muomva-red [1091.76 ± 122.81 mg GAE/100 g (d.w.)]. The high amount of TPs present in these cultivars make them suitable source of bio-nutrients with great medicinal and health functions. PMID:25987997

Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment.

PubMed

Abd Wahid, Muhamad Azhar; Megat Mohd Noor, Megat Johari; Goto, Masafumi; Sugiura, Norio; Othman, Nor'azizi; Zakaria, Zuriati; Ahmad Mohammed, Thamer; Jusoh, Ahmad; Hara, Hirofumi

2017-08-01

The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.

Induction of TNF-alfa and CXCL-2 mRNAs in different organs of mice infected with pathogenic Leptospira.

PubMed

da Silva, Josefa B; Carvalho, Enéas; Covarrubias, Ambart E; Ching, Ana Tung C; Mattaraia, Vania G M; Paiva, Delhi; de Franco, Marcelo; Fávaro, Regiane Degan; Pereira, Martha M; Vasconcellos, Silvio; Zorn, Telma T M; Ho, Paulo Lee; Martins, Elizabeth A L

2012-04-01

The role of innate immune response in protection against leptospirosis is poorly understood. We examined the expression of the chemokine CXCL2/MIP-2 and the cytokine TNF-α in experimental resistant and susceptible mice models, C3H/HeJ, C3H/HePas and BALB/c strains, using a virulent strain of Leptospira interrogans serovar Copenhageni. Animals were infected intraperitoneally with 10(7) cells and the development of the disease was followed. Mortality of C3H/HeJ mice was observed whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. The infection was confirmed by the presence of leptospiral DNA in the organs of the animals, demonstrated by PCR. Sections of the organs were analyzed, after H&E stain. The relative expression of mRNA of chemokine CXCL2/MIP-2 and cytokine TNF-α was measured in lung, kidney and liver of the mice by qPCR. The concentrations of these proteins were measured in extracts of tissues and in serum of the animals, by ELISA. Increasing levels of transcripts and protein CXCL2/MIP-2 were detected since the first day of infection. The highest expression was observed at third day of infection in kidney, liver and lung of BALB/c mice. In C3H/HeJ the expression of CXCL2/MIP-2 was delayed, showing highest protein concentration in lung and kidney at the 5th day. Increasing in TNF-α transcripts were detected after infection, in kidney and liver of animals from the three mice strains. The expression of TNF-α protein in C3H/HeJ was also delayed, being detected in kidney and lung. Our data demonstrated that Leptospira infection stimulates early expression of CXCL2/MIP-2 and TNF-α in the resistant strain of mice. Histological analysis suggests that the expression of those molecules may be related to the influx of distinct immune cells and plays a role in the naturally acquired protective immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

PubMed

Cui, Yulin; Zhao, Jialin; Hou, Shichang; Qin, Song

2016-05-01

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

A Tomato Peroxidase Involved in the Synthesis of Lignin and Suberin1

PubMed Central

Quiroga, Mónica; Guerrero, Consuelo; Botella, Miguel A.; Barceló, Araceli; Amaya, Iraida; Medina, María I.; Alonso, Francisco J.; de Forchetti, Silvia Milrad; Tigier, Horacio; Valpuesta, Victoriano

2000-01-01

The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin. PMID:10759507

Effects of Methionine Supplementation on the Expression of Protein Deposition-Related Genes in Acute Heat Stress-Exposed Broilers

PubMed Central

Grieser, Daiane Oliveira; Zancanela, Vittor; Voltolini, Débora Marques; Khatlab, Angélica Souza; Guimarães, Simone Eliza Facioni; Soares, Maria Amélia Menck; Neto, Adhemar Rodrigues Oliveira

2015-01-01

The objective of this study was to evaluate the effect of heat stress and methionine supplementation on the gene expression of insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), phosphatidylinositol 3-kinase, and regulatory 1 (PI3KR1) in the liver, as well as the expression of the atrogin 1 and cathepsin L2 (CTSL2) genes in the breast muscle of broilers. Broilers from 1–21 and 22–42 days of age were divided into three treatments related to methionine supplementation as follows: without methionine supplementation (MD), recommended level of methionine (DL1), and excess supplementation of methionine (DL2). The animals were either maintained at a thermal comfort temperature or exposed to heat stress (HS) (38°C for 24 hours, starting on day 20 or day 41 for experiments 1 and 2, respectively). The heat stress increased the body temperature at both ages. Starter period: The HS animals presented increased plasma creatinine content (P<0.0001) and the highest CTSL2 gene expression (P<0.0001). The methionine supplementation increased the IGF-I (P = 0.0144) and GHR (P = 0.0011) gene expression and decreased the CTSL2 (P = 0.0004) and atrogin 1 (P = 0.0012) gene expression. Grower period: Significant effects for the interaction between supplementation and environment were observed for GHR (P = 0.0252) and CTSL2 (P = 0.0011) gene expression. The highest GHR expression was observed in animals that remained in thermal comfort on the DL2 diet, and the lowest expression occurred in the HS animals fed the MD diet. For CTSL2, the HS animals fed the MD diet presented the highest CTSL2 gene expression, and the lowest expression was observed in the animals maintained at thermal comfort on DL1 and DL2 diets. Only methionine supplementation had effect on atrogin-1 gene expression (P<0.0001), with higher methionine content in the diet lower atrogin-1 gene expression was observed. Our results suggest that heat stress induces greater protein degradation and that methionine supplementation could induce protein deposition because methionine increased the expression of genes related to protein synthesis and decreased the expression of genes related to protein breakdown. PMID:25714089

Calmodulin Gene Family in Potato: Developmental and Touch-Induced Expression of the mRNA Encoding a Novel Isoform

NASA Technical Reports Server (NTRS)

Takezawa, D.; Liu, Z. H.; An, G.; Poovaiah, B. W.

1995-01-01

Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots

PubMed Central

2011-01-01

Background Tropane alkaloids (TA) including anisodamine, anisodine, hyoscyamine and scopolamine are a group of important anticholinergic drugs with rapidly increasing market demand, so it is significant to improve TA production by biotechnological approaches. Putrescine N-methyltransferase (PMT) was considered as the first rate-limiting upstream enzyme while tropinone reductase I (TRI) was an important branch-controlling enzyme involved in TA biosynthesis. However, there is no report on simultaneous introduction of PMT and TRI genes into any TA-producing plant including Anisodus acutangulus (A. acutangulus), which is a Solanaceous perennial plant that is endemic to China and is an attractive resource plant for production of TA. Results In this study, 21 AaPMT and AaTRI double gene transformed lines (PT lines), 9 AaPMT single gene transformed lines (P lines) and 5 AaTRI single gene transformed lines (T lines) were generated. RT-PCR and real-time fluorescence quantitative analysis results revealed that total AaPMT (AaPMT T) and total AaTRI (AaTRI T) gene transcripts in transgenic PT, P and T lines showed higher expression levels than native AaPMT (AaPMT E) and AaTRI (AaTRI E) gene transcripts. As compared to the control and single gene transformed lines (P or T lines), PT transgenic hairy root lines produced significantly higher levels of TA. The highest yield of TA was detected as 8.104 mg/g dw in line PT18, which was 8.66, 4.04, and 3.11-times higher than those of the control (0.935 mg/g dw), P3 (highest in P lines, 2.004 mg/g dw) and T12 (highest in T lines, 2.604 mg/g dw), respectively. All the tested samples were found to possess strong radical scavenging capacity, which were similar to control. Conclusion In the present study, the co-expression of AaPMT and AaTRI genes in A. acutangulus hairy roots significantly improved the yields of TA and showed higher antioxidant activity than control because of higher total TA content, which is the first report on simultaneous introduction of PMT and TRI genes into TA-producing plant by biotechnological approaches. PMID:21526999

The ability to regulate emotion is associated with greater well-being, income, and socioeconomic status.

PubMed

Côté, Stéphane; Gyurak, Anett; Levenson, Robert W

2010-12-01

Are people who are best able to implement strategies to regulate their emotional expressive behavior happier and more successful than their counterparts? Although past research has examined individual variation in knowledge of the most effective emotion regulation strategies, little is known about how individual differences in the ability to actually implement these strategies, as assessed objectively in the laboratory, are associated with external criteria. In two studies, we examined how individual variation in the ability to modify emotional expressive behavior in response to evocative stimuli is related to well-being and financial success. Study 1 showed that individuals who can best suppress their emotional reaction to an acoustic startle are happiest with their lives. Study 2 showed that individuals who can best amplify their emotional reaction to a disgust-eliciting movie are happiest with their lives and have the highest disposable income and socioeconomic status. Thus, being able to implement emotion regulation strategies in the laboratory is closely linked to well-being and financial success.

Designing appropriate blended courses: a students' perspective.

PubMed

Tsai, Chia-Wen

2010-10-01

The computing education in Taiwan's vocational schools usually focuses on how to help students enhance their professional skills and pass certified examinations. In addition, due to national education policy and universities' regulations, pure online courses are not permitted in Taiwan. In order to design appropriate blended learning (BL) courses, the author explored the effects of web-mediated self-regulated learning (SRL) with variations in online class frequency on enhancing students' computing skills and their perspective of the blended courses. A total of 172 students, divided into four groups, participated in the experiment. The results showed that students in the SRL and BL group with five online classes had the highest scores for using a database management system (DBMS), and the highest pass rate on certified examinations. Students in this group also expressed their positive perspective on the arrangement of their blended course with the intervention of web-mediated SRL.

Extracellular invertase is involved in the regulation of clubroot disease in Arabidopsis thaliana.

PubMed

Siemens, Johannes; González, Maria-Cruz; Wolf, Sebastian; Hofmann, Christina; Greiner, Steffen; DU, Yejie; Rausch, Thomas; Roitsch, Thomas; Ludwig-Müller, Jutta

2011-04-01

Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms. © 2010 The Authors. Molecular Plant Pathology © 2010 BSPP and Blackwell Publishing Ltd.

Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.).

PubMed

Lin, L S; Varner, J E

1991-05-01

The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall "loosening."

Transcript profiling of pattern recognition receptors in a semi domesticated breed of buffalo, Toda, of India.

PubMed

Vignesh, A R; Dhanasekaran, S; Raj, G Dhinakar; Balachandran, C; Pazhanivel, N; Sreekumar, C; Tirumurugaan, K G; Raja, A; Kumanan, K

2012-06-15

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of the ABA Receptor VlPYL1 That Regulates Anthocyanin Accumulation in Grape Berry Skin

PubMed Central

Gao, Zhen; Li, Qin; Li, Jing; Chen, Yujin; Luo, Meng; Li, Hui; Wang, Jiyuan; Wu, Yusen; Duan, Shuyan; Wang, Lei; Song, Shiren; Xu, Wenping; Zhang, Caixi; Wang, Shiping; Ma, Chao

2018-01-01

ABA plays a crucial role in controlling several ripening-associated processes in grape berries. The soluble proteins named as PYR (pyrabactin resistant)/PYL (PYR-like)/RCAR (regulatory component of ABA receptor) family have been characterized as ABA receptors. Here, the function of a grape PYL1 encoding gene involved in the response to ABA was verified through heterologous expression. The expression level of VlPYL1 was highest in grape leaf and fruit tissues of the cultivar Kyoho, and the expression of VlPYL1 was increased during fruit development and showed a reduction in ripe berries. Over-expression of VlPYL1 enhances ABA sensitivity in Arabidopsis. Using the transient overexpression technique, the VlPYL1 gene was over-expressed in grape berries. Up-regulation of the VlPYL1 gene not only promoted anthocyanin accumulation but also induced a set of ABA-responsive gene transcripts, including ABF2 and BG3. Although tobacco rattle virus (TRV)-induced gene silencing (VIGS) was not successfully applied in the “Kyoho” grape, the application of the transient overexpression technique in grape fruit could be used as a novel tool for studying grape fruit development. PMID:29868057

On angry leaders and agreeable followers. How leaders' emotions and followers' personalities shape motivation and team performance.

PubMed

Van Kleef, Gerben A; Homan, Astrid C; Beersma, Bianca; van Knippenberg, Daan

2010-12-01

Do followers perform better when their leader expresses anger or when their leader expresses happiness? We propose that this depends on the follower's level of agreeableness. Anger is associated with hostility and conflict-states that are at odds with agreeable individuals' goals. Happiness facilitates affiliation and positive relations-states that are in line with agreeable individuals' goals. Accordingly, the two studies we conducted showed that agreeableness moderates the effects of a leader's emotional displays. In a scenario study, participants with lower levels of agreeableness responded more favorably to an angry leader, whereas participants with higher levels of agreeableness responded more favorably to a neutral leader. In an experiment involving four-person teams, teams composed of participants with lower average levels of agreeableness performed better when their leader expressed anger, whereas teams composed of participants with higher average levels of agreeableness performed better when their leader expressed happiness. Team performance was mediated by experienced workload, which was highest among agreeable followers with an angry leader. Besides having important practical implications, the findings shed new light on the fundamental question of how emotional expressions regulate social behavior.

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

PubMed

Masoomi Dezfooli, Seyedehsara; Tan, Wen Siang; Tey, Beng Ti; Ooi, Chien Wei; Hussain, Siti Aslina

2016-01-01

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers.

Unicellular cyanobacteria Synechocystis accommodate heterotrophic bacteria with varied enzymatic and metal resistance properties.

PubMed

Abdulaziz, Anas; Sageer, Saliha; Chekidhenkuzhiyil, Jasmin; Vijayan, Vijitha; Pavanan, Pratheesh; Athiyanathil, Sujith; Nair, Shanta

2016-08-01

The interactions between heterotrophic bacteria and primary producers have a profound impact on the functioning of marine ecosystem. We characterized the enzymatic and metal resistance properties of fourteen heterotrophic bacteria isolated from a unicellular cyanobacterium Synechocystis sp. that came from a heavy metal contaminated region of Cochin estuary, southwest coast of India. Based on 16S rRNA gene sequence similarities, the heterotrophic bacteria were grouped into three phyla: namely Actinobacteria, Firmicute, and Proteobacteria. Overall Proteobacteria showed a higher level of enzyme expression while Actinobacteria and Firmicutes showed higher tolerance to heavy metals. Among Proteobacteria, an isolate of Marinobacter hydrocarbonoclasticus (MMRF-584) showed highest activities of β-glucosidase (1.58 ± 0.2 μMml(-1)  min(-1) ) and laminarinase (1170.17 ± 95.4 μgml(-1)  min(-1) ), while other two isolates of M. hydrocarbonoclasticus, MMRF-578 and 581, showed highest phosphatase (44.71 ± 0.2 μMml(-1)  min(-1) ) and aminopeptidase (33.22 ± 0 μMml(-1)  min(-1) ) activities respectively. Among Firmicutes, the Virgibacillus sp. MMRF-571 showed exceptional resistance against the toxic heavy metals Cd (180 mM), Pb (150 mM), and Hg (0.5 mM). Bacillus cereus, MMRF-575, showed resistance to the highest concentrations of Co (250 mM), Cd (150 mM), Pb (180 mM), Hg (0.5 mM), Ni (280 mM), and Zn (250 mM) tested. Our results show that heterotrophic bacteria with varied enzymatic and metal resistance properties are associated with Synechocystis sp. Further studies to delineate the role of these heterotrophic bacteria in protecting primary producers from toxic effects of heavy metals and their potential application in bioremediation will be appreciated. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulated expression of a calmodulin isoform alters growth and development in potato.

PubMed

Poovaiah, B W; Takezawa, D; An, G; Han, T J

1996-01-01

A transgene approach was taken to study the consequences of altered expression of a calmodulin isoform on plant growth and development. Eight genomic clones of potato calmodulin (PCM1 to 8) have been isolated and characterized (Takezawa et al., 1995). Among the potato calmodulin isoforms studied, PCM1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM1 fused to the CaMV 35S promoter. Transgenic plants showing a moderate increase in PCM1 mRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM1 mRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM1 mRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM1 protein in transgenic plants, indicating that the expression of both mRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM1 alters growth and development in potato plants.

Quantitative comparison of the expression of myogenic regulatory factors in flounder ( Paralichthys olivaceus) embryos and adult tissues

NASA Astrophysics Data System (ADS)

Zhang, Yuqing; Tan, Xungang; Xu, Peng; Sun, Wei; Xu, Yongli; Zhang, Peijun

2010-03-01

MyoD, Myf5, and myogenin are myogenic regulatory factors that play important roles during myogenesis. It is thought that MyoD and Myf5 are required for myogenic determination, while myogenin is important for terminal differentiation and lineage maintenance. To better understand the function of myogenic regulatory factors in muscle development of flounder, an important economic fish in Asia, real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD, Myf5, and myogenin at early stages of embryo development, and in different tissues of the adult flounder. The results show that, Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin. The expressions of Myf5, yoD, and myogenin at the early stages have a common characteristic: expression gradually increased to a peak level, and then gradually decreased to an extremely low level. In the adult flounder, the expression of the three genes in muscle is much higher than that in other tissues, indicating that they are important for muscle growth and maintenance of grown fish. During embryonic stages, the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation. When the MyoD expression is over 30% of its highest level, the muscle cells enter the differentiation stage.

Gene expression of amino acid transporter in pigeon (Columbia livia) intestine during post-hatch development and its correlation with amino acid in pigeon milk.

PubMed

Zhang, X Y; Zhang, N N; Wan, X P; Li, L L; Zou, X T

2017-05-01

This study was conducted to evaluate gene expression of the amino acid transporter in post-hatch pigeon small intestine and the association of pigeon milk amino acid with the above transporter's gene expression. A total of 48 pigeon breeding families were randomly allocated to 8 groups of 6 replicates of one parental pigeon pair and 2 squabs. Samples of pigeon milk and duodenum, jejunum, and ileum were collected on d 1, 2, 3, 4, 6, 8, 10, and 14 post hatch. The results showed that levels of crude protein (8.93 to 15.56%) were highest in pigeon milk on an air-dry basis. Amino acid content in pigeon milk remained constant in the first 4 d, declined abruptly at d 6, then increased dramatically from d 8 to 14. There was a significant effect of interaction between age and intestinal segments on those amino acid transporters gene expression. mRNA abundance of ATB0'+, SNAT-2, LAT-4, rBAT, b0'+AT, EAAT-3 and PAT-1 was highest in the ileum; B0AT1, asc-1, and IMINO were predominate in the jejunum; and CAT-1 and y+LAT2 were greatest in the duodenum. Age-related changes of amino acid transporter mRNA was inconsistent. mRNA levels of SNAT-2, rBAT, y+LAT2, b0'+AT, and EAAT-3 ascended with age, whereas that of asc-1, CAT-1, and IMINO diminished significantly. Levels of B0AT1 and PAT-1 mRNA abundance were minimized at d 6. However, few correlations were found between pigeon milk amino acid and the amino acid transporter gene expressions in squab small intestine. Our findings provide a comprehensive elaboration on ontogeny of the amino acid transporter in post-hatch pigeon intestine. © 2016 Poultry Science Association Inc.

Quantitative analysis of the expression and distribution of calcium channel alpha 1 subunit mRNA in the atria and ventricles of the rat heart.

PubMed

Larsen, Janice K; Mitchell, Jennifer W; Best, Philip M

2002-05-01

Two distinct calcium currents are present in mammalian cardiac myocytes. Utilizing quantitative RT-PCR methods, we have analysed the expression patterns and abundance of four calcium channel alpha 1 subunit mRNAs in different regions of the rat heart and compared them to the known density of calcium currents recorded from rat atria. Our results show that Ca(V)1.2 is the most abundant of the four alpha 1 subunit transcripts in the rat heart. The Ca(V)1.2 message is more abundant in ventricle than in atria and does not vary in expression as a function of developmental age. Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 mRNAs are 10-100 times less abundant than Ca(V)1.2. Interestingly, Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 are expressed in both atria and ventricle. The abundance of atrial Ca(V)3.1 mRNA does not change significantly during development and remains high in older animals. In contrast, levels of atrial Ca(V)3.2 mRNA are high in embryonic tissue and at 3- and 4-weeks postnatal but become undetectable at 5 weeks. Expression of atrial Ca(V)2.3 mRNA is highest at 4-weeks postnatal and then declines gradually. We have previously documented that the LVA calcium current density is highest within 4-5 weeks after birth and then declines gradually reaching less than 30% of its maximal value at 12-14 weeks. The complex relationship between atrial LVA current density and the abundance of Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 mRNA suggests that their contribution to the cardiac LVA current may vary as a function of postnatal age. Copyright 2002 Academic Press.

Association Study between an SNP in CD147 and Its Expression With Acute Coronary Syndrome in a Jiangsu Chinese Population.

PubMed

Yan, Jinchuan; Mao, Yu; Wang, Cuiping; Wang, Zhongqun

2015-10-01

CD147 is an important molecule in the inflammation and proteolysis process. This molecule crucially contributes to the initial and progression of atherosclerotic lesions. A single nucleotide polymorphism in CD147 gene, the rs8259 T/A in the 3'-untranslated region, is responsible for its expression in various cells. This study assessed whether the genetic variation rs8259 is associated with acute coronary syndrome (ACS) and CD147. A total of 943 ACS subjects and 439 stable angina subjects, and 851 controls were genotyped for rs8259 polymorphism by polymerase chain reaction restriction fragment length polymorphism and DNA-sequencing method. Plasma soluble CD147 (sCD147) level was measured by enzyme-linked immunosorbent assay. CD147 mRNA and protein expression in peripheral blood mononuclear cells were tested by real-time quantitative polymerase chain reaction and western blot, respectively. We found that TT genotype and T-allele frequency of CD147 rs8259 in ACS patients were much lower than the other patient groups. Significant difference was not observed between stable angina and controls. CD147 T allele was negatively related to ACS. ACS patients exhibited the highest CD147 expression in peripheral blood mononuclear cells and plasma sCD147 level. The plasma sCD147 levels in the culprit vessel were higher than those in the radial artery. In ACS patients, AA gene carriers had the highest CD147 levels, whereas TT gene carriers had the lowest CD147 levels. Linear regression analysis showed that genotypes and disease conditions contributed 49% to the change of the plasma CD147 level. These results suggested that the single nucleotide polymorphism of CD147 gene rs8259 T/A was associated with ACS susceptibility. Allele T gene may decrease the relative risk of suffering from ACS through downregulation of CD147 expression.

Targeted Imaging of the Atypical Chemokine Receptor 3 (ACKR3/CXCR7) in Human Cancer Xenografts.

PubMed

Behnam Azad, Babak; Lisok, Ala; Chatterjee, Samit; Poirier, John T; Pullambhatla, Mrudula; Luker, Gary D; Pomper, Martin G; Nimmagadda, Sridhar

2016-06-01

The atypical chemokine receptor ACKR3 (formerly CXCR7), overexpressed in various cancers compared with normal tissues, plays a pivotal role in adhesion, angiogenesis, tumorigenesis, metastasis, and tumor cell survival. ACKR3 modulates the tumor microenvironment and regulates tumor growth. The therapeutic potential of ACKR3 has also been demonstrated in various murine models of human cancer. Literature findings underscore the importance of ACKR3 in disease progression and suggest it as an important diagnostic marker for noninvasive imaging of ACKR3-overexpressing malignancies. There are currently no reports on direct receptor-specific detection of ACKR3 expression. Here we report the evaluation of a radiolabeled ACKR3-targeted monoclonal antibody (ACKR3-mAb) for the noninvasive in vivo nuclear imaging of ACKR3 expression in human breast, lung, and esophageal squamous cell carcinoma cancer xenografts. ACKR3 expression data were extracted from Cancer Cell Line Encyclopedia, The Cancer Genome Atlas, and the Clinical Lung Cancer Genome Project. (89)Zr-ACKR3-mAb was evaluated in vitro and subsequently in vivo by PET and ex vivo biodistribution studies in mice xenografted with breast (MDA-MB-231-ACKR3 [231-ACKR3], MDA-MB-231 [231], MCF7), lung (HCC95), or esophageal (KYSE520) cancer cells. In addition, ACKR3-mAb was radiolabeled with (125)I and evaluated by SPECT imaging and ex vivo biodistribution studies. ACKR3 transcript levels were highest in lung squamous cell carcinoma among the 21 cancer type data extracted from The Cancer Genome Atlas. Also, Clinical Lung Cancer Genome Project data showed that lung squamous cell carcinoma had the highest CXCR7 transcript levels compared with other lung cancer subtypes. The (89)Zr-ACKR3-mAb was produced in 80% ± 5% radiochemical yields with greater than 98% radiochemical purity. In vitro cell uptake of (89)Zr-ACKR3-mAb correlated with gradient levels of cell surface ACKR3 expression observed by flow cytometry. In vivo PET imaging and ex vivo biodistribution studies in mice with breast, lung, and esophageal cancer xenografts consistently showed enhanced (89)Zr-ACKR3-mAb uptake in high-ACKR3-expressing tumors. SPECT imaging of (125)I-ACKR3-mAb showed the versatility of ACKR3-mAb for in vivo monitoring of ACKR3 expression. Data from this study suggest ACKR3 to be a viable diagnostic marker and demonstrate the utility of radiolabeled ACKR3-mAb for in vivo visualization of ACKR3-overexpressing malignancies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-09-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

PubMed Central

Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

1990-01-01

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility. Images PMID:1697032

Altered expression of Matrix Remodeling Associated 7 (MXRA7) in psoriatic epidermis: evidence for a protective role in the psoriasis imiquimod mouse model.

PubMed

Ning, Jinling; Shen, Ying; Wang, Ting; Wang, Mengru; Liu, Wei; Sun, Yonghu; Zhang, Furen; Chen, Lingling; Wang, Yiqiang

2018-05-21

Preliminary datamining performed with Gene Expression Omnibus datasets implied that psoriasis may involve the matrix remodeling associated 7 (MXRA7), a gene with little function information yet. To test that hypothesis, studies were performed in human samples and murine models. Immunohistochemistry in normal human skin showed that MXRA7 proteins were present across the full epidermal layer, with highest expression level detected in the basal layer. In psoriatic samples, MXRA7 proteins were absent in the basal stem cells layer while suprabasal keratinocytes stained at a higher level than in normal tissues. In an imiquimod-induced psoriasis-like disease model in mice, diseased skins manifested similar MXRA7 expression pattern and change as in human samples, and MXRA7-deficient mice developed severer psoriasis-like diseases than wild-type mice did. While levels of pro-psoriatic genes (e.g. IL17, IL22, IL23, etc) in imiquimod-stimulated MXRA7-deficient mice were higher than in wild-type mice, keratinocytes isolated from MXRA7-deficient mice showed increased proliferation upon differentiation induction in culture. These data demonstrated that MXRA7 gene might function as a negative modulator in psoriasis development when pro-psoriatic factors attack, presumably via expression alteration or redistribution of MXRA7 proteins in keratinocytes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Expression of Gas1 in Mouse Brain: Release and Role in Neuronal Differentiation.

PubMed

Bautista, Elizabeth; Zarco, Natanael; Aguirre-Pineda, Nicolás; Lara-Lozano, Manuel; Vergara, Paula; González-Barrios, Juan Antonio; Aguilar-Roblero, Raúl; Segovia, José

2018-05-01

Growth arrest-specific 1 (Gas1) is a pleiotropic protein that induces apoptosis of tumor cells and has important roles during development. Recently, the presence of two forms of Gas1 was reported: one attached to the cell membrane by a GPI anchor; and a soluble extracellular form shed by cells. Previously, we showed that Gas1 is expressed in different areas of the adult mouse CNS. Here, we report the levels of Gas1 mRNA protein in different regions and analyzed its expressions in glutamatergic, GABAergic, and dopaminergic neurons. We found that Gas1 is expressed in GABAergic and glutamatergic neurons in the Purkinje-molecular layer of the cerebellum, hippocampus, thalamus, and fastigial nucleus, as well as in dopaminergic neurons of the substantia nigra. In all cases, Gas1 was found in the cell bodies, but not in the neuropil. The Purkinje and the molecular layers show the highest levels of Gas1, whereas the granule cell layer has low levels. Moreover, we detected the expression and release of Gas1 from primary cultures of Purkinje cells and from hippocampal neurons as well as from neuronal cell lines, but not from cerebellar granular cells. In addition, using SH-SY5Y cells differentiated with retinoic acid as a neuronal model, we found that extracellular Gas1 promotes neurite outgrowth, increases the levels of tyrosine hydroxylase, and stimulates the inhibition of GSK3β. These findings demonstrate that Gas1 is expressed and released by neurons and promotes differentiation, suggesting an important role for Gas1 in cellular signaling in the CNS.

Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver

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

Al-Dosari, Mohammed; Zhang Guisheng; Knapp, Joseph E.

2006-01-13

Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), {alpha}-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken {beta} actin (ACT), nuclear factor {kappa} B (NF{kappa}B), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promotermore » exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within First 5-7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein-Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.« less

In Vivo and In Vitro Effects of ATM/ATR Signaling Pathway on Proliferation, Apoptosis, and Radiosensitivity of Nasopharyngeal Carcinoma Cells.

PubMed

Wang, Ming; Liu, Gang; Shan, Guo-Ping; Wang, Bing-Bing

2017-08-01

The study investigated the ability of ataxia-telangiectasia mutated (ATM)/Rad3-related (ATR) signaling pathway to influence the proliferation, apoptosis, and radiosensitivity of nasopharyngeal carcinoma (NPC) cells. NPC tissues and corresponding adjacent normal tissues were collected from 143 NPC patients. The NPC CNE2 cells were assigned into a control group, X-ray group, CGK-733 group, and X-ray+CGK-733 group. The mRNA levels of ATM and ATR were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) and the protein levels of ATM and ATR using western blotting. The positive expression of ATM and ATR in tissues and nude mouse tumor tissues was determined by immunohistochemistry. Cell proliferation, migration, invasion, and apoptosis rates were analyzed by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay, scratch test, transwell assay, and flow cytometry, respectively. A nude mouse model of NPC was established to observe tumor volume and growth. The mRNA levels of ATR and ATM and the expression of ATR and ATM protein in NPC tissues were significantly higher than those in adjacent normal tissues. The colony formation assay showed that the colony-forming rate decreased, showing radiation dose-dependent and CGK-733 concentration-dependent manners. Expression of ATM, ATR, Chk1, and Chk2 was evidently increased in the X-ray, CGK-733, and X-ray+CGK-733groups compared with the control group, and the aforementioned expression was highest in the X-ray+CGK-733 group among the four groups. The cell proliferation, invasion, and migration were decreased, tumor volume decreased and cell apoptosis increased in the X-ray, CGK-733, and X-ray+CGK-733 groups compared with the control group; the X-ray+CGK-733 group exhibited lowest cell proliferation, invasion and migration, smallest tumor volume, and highest cell apoptosis among the four groups. Inhibition of ATM/ATR signaling pathway reduces proliferation and enhances apoptosis and radiosensitivity of NPC cells.

Flow cytometric single cell analysis reveals heterogeneity between adipose depots

PubMed Central

Boumelhem, Badwi B.; Assinder, Stephen J.; Bell-Anderson, Kim S.; Fraser, Stuart T.

2017-01-01

ABSTRACT Understanding adipose tissue heterogeneity is hindered by the paucity of methods to analyze mature adipocytes at the single cell level. Here, we report a system for analyzing live adipocytes from different adipose depots in the adult mouse. Single cell suspensions of buoyant adipocytes were separated from the stromal vascular fraction and analyzed by flow cytometry. Compared to other lipophilic dyes, Nile Red uptake effectively distinguished adipocyte populations. Nile Red fluorescence increased with adipocyte size and granularity and could be combined with MitoTracker® Deep Red or fluorescent antibody labeling to further dissect adipose populations. Epicardial adipocytes exhibited the least mitochondrial membrane depolarization and highest fatty-acid translocase CD36 surface expression. In contrast, brown adipocytes showed low surface CD36 expression. Pregnancy resulted in reduced mitochondrial membrane depolarisation and increased CD36 surface expression in brown and epicardial adipocyte populations respectively. Our protocol revealed unreported heterogeneity between adipose depots and highlights the utility of flow cytometry for screening adipocytes at the single cell level. PMID:28453382

Canonical Genetic Signatures of the Adult Human Brain

PubMed Central

Hawrylycz, Michael; Miller, Jeremy A.; Menon, Vilas; Feng, David; Dolbeare, Tim; Guillozet-Bongaarts, Angela L.; Jegga, Anil G.; Aronow, Bruce J.; Lee, Chang-Kyu; Bernard, Amy; Glasser, Matthew F.; Dierker, Donna L.; Menche, Jörge; Szafer, Aaron; Collman, Forrest; Grange, Pascal; Berman, Kenneth A.; Mihalas, Stefan; Yao, Zizhen; Stewart, Lance; Barabási, Albert-László; Schulkin, Jay; Phillips, John; Ng, Lydia; Dang, Chinh; Haynor, David R.; Jones, Allan; Van Essen, David C.; Koch, Christof; Lein, Ed

2015-01-01

The structure and function of the human brain are highly stereotyped, implying a conserved molecular program responsible for its development, cellular structure, and function. We applied a correlation-based metric of “differential stability” (DS) to assess reproducibility of gene expression patterning across 132 structures in six individual brains, revealing meso-scale genetic organization. The highest DS genes are highly biologically relevant, with enrichment for brain-related biological annotations, disease associations, drug targets, and literature citations. Using high DS genes we identified 32 anatomically diverse and reproducible gene expression signatures, which represent distinct cell types, intracellular components, and/or associations with neurodevelopmental and neurodegenerative disorders. Genes in neuron-associated compared to non-neuronal networks showed higher preservation between human and mouse; however, many diversely-patterned genes displayed dramatic shifts in regulation between species. Finally, highly consistent transcriptional architecture in neocortex is correlated with resting state functional connectivity, suggesting a link between conserved gene expression and functionally relevant circuitry. PMID:26571460

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets – “Sand out and Gold Stays”

PubMed Central

Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y.; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T.; Wang, Hong; Yang, Xiao-feng

2016-01-01

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: 1) Histone enzymes are differentially expressed in cardiovascular, immune and other tissues; 2) Our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, histone methylation/demethylation are in the highest varieties; and 3) Histone enzymes are more downregulated than upregulated in metabolic diseases and Treg polarization/differentiation, but not in tumors. These results have demonstrated a new working model of “sand out and gold stays,” where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity. PMID:26746407

Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets--"Sand Out and Gold Stays".

PubMed

Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T; Wang, Hong; Yang, Xiao-feng

2016-02-01

To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: (1) Histone enzymes are differentially expressed in cardiovascular, immune, and other tissues; (2) our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, and histone methylation/demethylation are in the highest varieties; and (3) histone enzymes are more downregulated than upregulated in metabolic diseases and regulatory T cell (Treg) polarization/ differentiation, but not in tumors. These results have demonstrated a new working model of "Sand out and Gold stays," where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity.

Identification of a p-cresol degradation pathway by a GFP-based transposon in Pseudomonas and its dominant expression in colonies.

PubMed

Cho, Ah Ra; Lim, Eun Jin; Veeranagouda, Yaligara; Lee, Kyoung

2011-11-01

In this study, the chromosome-encoded pcuRCAXB genes that are required for p-cresol degradation have been identified by using a newly constructed green fluorescent protein (GFP)-based promoter probe transposon in the long-chain alkylphenol degrader Pseudomonas alkylphenolia. The deduced amino acid sequences of the genes showed the highest identities at the levels of 65-93% compared with those in the databases. The transposon was identified to be inserted in the pcuA gene, with the promoterless gfp gene being under the control of the pcu catabolic gene promoter. The expression of GFP was positively induced by p-cresol and was about 10 times higher by cells grown on agar than those in liquid culture. In addition, phydroxybenzoic acid was detected during p-cresol degradation. These results indicate that P. alkylphenolia additionally possesses a protocatechuate ortho-cleavage route for pcresol degradation that is dominantly expressed in colonies.

Transient Gene Expression in Maize, Rice, and Wheat Cells Using an Airgun Apparatus 1

PubMed Central

Oard, James H.; Paige, David F.; Simmonds, John A.; Gradziel, Thomas M.

1990-01-01

An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The β-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 × 10−3. In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals. Images Figure 7 Figure 8 Figure 9 PMID:16667278

Gelam honey potentiates ex vivo corneal keratocytes proliferation with desirable phenotype expression.

PubMed

Yusof, Alia Md; Abd Ghafar, Norzana; Kamarudin, Taty Anna; Hui, Chua Kien; Yusof, Yasmin Anum Mohd

2016-02-24

This study aimed to evaluate the effects of Gelam honey on corneal keratocytes proliferative capacity and phenotypic characterization via MTT assay, gene expression and immunocytochemistry. Corneal keratocytes from New Zealand white rabbits were cultured in basal medium (BM) and serum enriched medium (BMS). Serial dilutions of Gelam honey (GH) were added to both media and cells were cultured until passage 1. MTT assay was performed on corneal keratocytes in both media to ascertain the optimal dose of GH that produced maximum proliferation. Gelam honey at the concentration of 0.0015% in both media showed the highest proliferative capacity with no morphological changes compared to their respective controls. The gene expression of aldehyde dehydrogenase (ALDH), a marker for quiescent keratocytes and vimentin, a marker for fibroblast, were higher in the GH enriched groups. The alpha smooth muscle actin (α-SMA) expression, marker for myofibroblast, was lower in GH treated groups compared to the controls. Immunocytochemistry results were in accordance to the gene expression analyses. Gelam honey at a concentration of 0.0015% promotes ex vivo corneal keratocytes proliferation while retaining desirable phenotype expression. The results serve as a basis for the development of Gelam honey as a potential natural product in promoting corneal wound healing.

The Omics Dashboard for interactive exploration of gene-expression data.

PubMed

Paley, Suzanne; Parker, Karen; Spaulding, Aaron; Tomb, Jean-Francois; O'Maille, Paul; Karp, Peter D

2017-12-01

The Omics Dashboard is a software tool for interactive exploration and analysis of gene-expression datasets. The Omics Dashboard is organized as a hierarchy of cellular systems. At the highest level of the hierarchy the Dashboard contains graphical panels depicting systems such as biosynthesis, energy metabolism, regulation and central dogma. Each of those panels contains a series of X-Y plots depicting expression levels of subsystems of that panel, e.g. subsystems within the central dogma panel include transcription, translation and protein maturation and folding. The Dashboard presents a visual read-out of the expression status of cellular systems to facilitate a rapid top-down user survey of how all cellular systems are responding to a given stimulus, and to enable the user to quickly view the responses of genes within specific systems of interest. Although the Dashboard is complementary to traditional statistical methods for analysis of gene-expression data, we show how it can detect changes in gene expression that statistical techniques may overlook. We present the capabilities of the Dashboard using two case studies: the analysis of lipid production for the marine alga Thalassiosira pseudonana, and an investigation of a shift from anaerobic to aerobic growth for the bacterium Escherichia coli. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Omics Dashboard for interactive exploration of gene-expression data

PubMed Central

Paley, Suzanne; Parker, Karen; Spaulding, Aaron; Tomb, Jean-Francois; O’Maille, Paul

2017-01-01

Abstract The Omics Dashboard is a software tool for interactive exploration and analysis of gene-expression datasets. The Omics Dashboard is organized as a hierarchy of cellular systems. At the highest level of the hierarchy the Dashboard contains graphical panels depicting systems such as biosynthesis, energy metabolism, regulation and central dogma. Each of those panels contains a series of X–Y plots depicting expression levels of subsystems of that panel, e.g. subsystems within the central dogma panel include transcription, translation and protein maturation and folding. The Dashboard presents a visual read-out of the expression status of cellular systems to facilitate a rapid top-down user survey of how all cellular systems are responding to a given stimulus, and to enable the user to quickly view the responses of genes within specific systems of interest. Although the Dashboard is complementary to traditional statistical methods for analysis of gene-expression data, we show how it can detect changes in gene expression that statistical techniques may overlook. We present the capabilities of the Dashboard using two case studies: the analysis of lipid production for the marine alga Thalassiosira pseudonana, and an investigation of a shift from anaerobic to aerobic growth for the bacterium Escherichia coli. PMID:29040755

High MACC1 expression in combination with mutated KRAS G13 indicates poor survival of colorectal cancer patients.

PubMed

Ilm, Katharina; Kemmner, Wolfgang; Osterland, Marc; Burock, Susen; Koch, Gudrun; Herrmann, Pia; Schlag, Peter M; Stein, Ulrike

2015-02-14

The metastasis-associated in colon cancer 1 (MACC1) gene has been identified as prognostic biomarker for colorectal cancer (CRC). Here, we aimed at the refinement of risk assessment by separate and combined survival analyses of MACC1 expression with any of the markers KRAS mutated in codon 12 (KRAS G12) or codon 13 (KRAS G13), BRAF V600 mutation and MSI status in a retrospective study of 99 CRC patients with tumors UICC staged I, II and III. We showed that only high MACC1 expression (HR: 6.09, 95% CI: 2.50-14.85, P < 0.001) and KRAS G13 mutation (HR: 5.19, 95% CI: 1.06-25.45, P = 0.042) were independent prognostic markers for shorter metastasis-free survival (MFS). Accordingly, Cox regression analysis revealed that patients with high MACC1 expression and KRAS G13 mutation exhibited the worst prognosis (HR: 14.48, 95% CI: 3.37-62.18, P < 0.001). Patients were classified based on their molecular characteristics into four clusters with significant differences in MFS (P = 0.003) by using the SPSS 2-step cluster function and Kaplan-Meier survival analysis. According to our results, patients with high MACC1 expression and mutated KRAS G13 exhibited the highest risk for metachronous metastases formation. Moreover, we demonstrated that the "Traditional pathway" with an intermediate risk for metastasis formation can be further subdivided by assessing MACC1 expression into a low and high risk group with regard to MFS prognosis. This is the first report showing that identification of CRC patients at high risk for metastasis is possible by assessing MACC1 expression in combination with KRAS G13 mutation.

Global Genetic Variations Predict Brain Response to Faces

PubMed Central

Dickie, Erin W.; Tahmasebi, Amir; French, Leon; Kovacevic, Natasa; Banaschewski, Tobias; Barker, Gareth J.; Bokde, Arun; Büchel, Christian; Conrod, Patricia; Flor, Herta; Garavan, Hugh; Gallinat, Juergen; Gowland, Penny; Heinz, Andreas; Ittermann, Bernd; Lawrence, Claire; Mann, Karl; Martinot, Jean-Luc; Nees, Frauke; Nichols, Thomas; Lathrop, Mark; Loth, Eva; Pausova, Zdenka; Rietschel, Marcela; Smolka, Michal N.; Ströhle, Andreas; Toro, Roberto; Schumann, Gunter; Paus, Tomáš

2014-01-01

Face expressions are a rich source of social signals. Here we estimated the proportion of phenotypic variance in the brain response to facial expressions explained by common genetic variance captured by ∼500,000 single nucleotide polymorphisms. Using genomic-relationship-matrix restricted maximum likelihood (GREML), we related this global genetic variance to that in the brain response to facial expressions, as assessed with functional magnetic resonance imaging (fMRI) in a community-based sample of adolescents (n = 1,620). Brain response to facial expressions was measured in 25 regions constituting a face network, as defined previously. In 9 out of these 25 regions, common genetic variance explained a significant proportion of phenotypic variance (40–50%) in their response to ambiguous facial expressions; this was not the case for angry facial expressions. Across the network, the strength of the genotype-phenotype relationship varied as a function of the inter-individual variability in the number of functional connections possessed by a given region (R2 = 0.38, p<0.001). Furthermore, this variability showed an inverted U relationship with both the number of observed connections (R2 = 0.48, p<0.001) and the magnitude of brain response (R2 = 0.32, p<0.001). Thus, a significant proportion of the brain response to facial expressions is predicted by common genetic variance in a subset of regions constituting the face network. These regions show the highest inter-individual variability in the number of connections with other network nodes, suggesting that the genetic model captures variations across the adolescent brains in co-opting these regions into the face network. PMID:25122193

Identification of genes involved in reproduction and lipid pathway metabolism in wild and domesticated shrimps.

PubMed

Rotllant, Guiomar; Wade, Nicholas M; Arnold, Stuart J; Coman, Gregory J; Preston, Nigel P; Glencross, Brett D

2015-08-01

The aims of this study were to identify genes involved in reproduction and lipid pathway metabolism in Penaeus monodon and correlate their expression with reproductive performance. Samples of the hepatopancreas and ovaries were obtained from a previous study of the reproductive performance of wild and domesticated P. monodon broodstock. Total mRNA from the domesticated broodstock was used to create two next generation sequencing cDNA libraries enabling the identification of 11 orthologs of key genes in reproductive and nutritional metabolic pathways in P. monodon. These were identified from the library of de novo assembled contigs, including the description of 6 newly identified genes. Quantitative RT-PCR of these genes in the hepatopancreas prior to spawning showed that the domesticated mature females significantly showed higher expression of the Pm Elovl4, Pm COX and Pm SUMO genes. The ovaries of domesticated females had a significantly decreased expression of the Pm Elovl4 genes. In the ovaries of newly spawned females, a significant correlation was observed between hepatosomatic index and the expression of Pm FABP and also between total lipid content and the expression of Pm CYP4. Although not significant, the highest levels of correlation were found between relative fecundity and Pm CRP and Pm CYP4 expression, and between hatching rate and Pm Nvd and Pm RXR expression. This study reports the discovery of genes involved in lipid synthesis, steroid biosynthesis and reproduction in P. monodon. These results indicate that genes encoding enzymes involved in lipid metabolism pathways might be potential biomarkers to assess reproductive performance. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression profile of IGF-I-calcineurin-NFATc3-dependent pathway genes in skeletal muscle during early development between duck breeds differing in growth rates.

PubMed

Shu, Jingting; Li, Huifang; Shan, Yanju; Xu, Wenjuan; Chen, Wenfeng; Song, Chi; Song, Weitao

2015-06-01

The insulin-like growth factor I (IGF-I)-calcineurin (CaN)-NFATc signaling pathways have been implicated in the regulation of myocyte hypertrophy and fiber-type specificity. In the present study, the expression of the CnAα, NFATc3, and IGF-I genes was quantified by RT-PCR for the first time in the breast muscle (BM) and leg muscle (LM) on days 13, 17, 21, 25, and 27 of embryonic development, as well as at 7 days posthatching (PH), in Gaoyou and Jinding ducks, which differ in their muscle growth rates. Consistent expression patterns of CnAα, NFATc3, and IGF-I were found in the same anatomical location at different development stages in both duck breeds, showing significant differences in an age-specific fashion. However, the three genes were differentially expressed in the two different anatomical locations (BM and LM). CnAα, NFATc3, and IGF-I messenger RNA (mRNA) could be detected as early as embryonic day 13 (ED13), and the highest level appeared at this stage in both BM and LM. Significant positive relationships were observed in the expression of the studied genes in the BM and LM of both duck breeds. Also, the expression of these three genes showed a positive relationship with the percentage of type IIb fibers and a negative relationship with the percentage of type I fibers and type IIa fibers. Our data indicate differential expression and coordinated developmental regulation of the selected genes involved in the IGF-I-calcineurin-NFATc3 pathway in duck skeletal muscle during embryonic and early PH growth and development; these data also indicate that this signaling pathway might play a role in the regulation of myofiber type transition.

BAC Recombineering of the Agouti Loci from Spotted Gar and Zebrafish Reveals the Evolutionary Ancestry of Dorsal-Ventral Pigment Asymmetry in Fish.

PubMed

Cal, Laura; MegÍas, Manuel; Cerdá-Reverter, José Miguel; Postlethwait, John H; Braasch, Ingo; Rotllant, Josep

2017-11-01

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts. © 2017 Wiley Periodicals, Inc.

Peripheral blood mononuclear cells: a potential cellular system to understand differential heat shock response across native cattle (Bos indicus), exotic cattle (Bos taurus), and riverine buffaloes (Bubalus bubalis) of India.

PubMed

Kishore, Amit; Sodhi, Monika; Kumari, Parvesh; Mohanty, A K; Sadana, D K; Kapila, Neha; Khate, K; Shandilya, Umesh; Kataria, R S; Mukesh, M

2014-09-01

Circulating leukocytes can be used as an effective model to understand the heat stress response of different cattle types and buffaloes. This investigation aimed to determine the temporal profile of HSPs (HSP40, HSP60, HSP70, and HSP90) expression in circulating peripheral blood mononuclear cells (PBMCs) of Murrah buffaloes, Holstein-Friesian (HF), and Sahiwal cows in response to sublethal heat shock at 42 °C. The viability data indicated HF PBMCs to be the most affected to the heat shock, whereas Sahiwal PBMCs were least affected, indicating its better survivability during the heat stress condition. The qRT-PCR expression data showed significant increase in mRNA expression of the analyzed HSPs genes after heat stimuli to the PBMCs under in vitro condition. In each case, the HSPs were most upregulated at 2 h after the heat stress. Among the HSPs, HSP70 was relatively more expressed followed by HSP60 indicating the action of molecular chaperones to stabilize the native conformation of proteins. However, PBMCs from different cattle types and buffaloes showed difference in the extent of transcriptional response. The level of expression of HSPs throughout the time period of heat stress was highest in buffaloes, followed by HF and Sahiwal cows. The higher abundance of HSP70 mRNA at each time point after heat stress showed prolonged effect of heat stress in HF PBMCs. The data presented here provided initial evidence of transcriptional differences in PBMCs of different cattle types and buffaloes and warrant further research.

Role of HIF-1α and CASPASE-3 in cystogenesis of odontogenic cysts and tumors.

PubMed

da Costa, Natacha M M; de Siqueira, Adriane S; Ribeiro, André L R; da Silva Kataoka, Maria S; Jaeger, Ruy G; de Alves-Júnior, Sérgio M; Smith, Andrew M; de Jesus Viana Pinheiro, João

2018-01-01

Odontogenic cysts and tumors are the most relevant lesions that affect the gnathic bones. These lesions have in common the formation of cystic areas and this common feature may suggest involvement of similar mechanisms. The hypoxia inducible factor 1 alpha (HIF-1α), a responsive protein to hypoxia and caspase-3, an irreversible apoptosis marker, may contribute to cyst formation. Thus, this study aimed to investigate the immunoexpression of these proteins in odontogenic cysts and tumors. Twenty cases of ameloblastoma, keratocystic odontogenic tumor (KOT) (n = 20), radicular cyst (RC) (n = 18), dentigerous cyst (DC) (n = 11), calcifying cystic odontogenic tumor (n = 8), and dental follicle (DF) (n = 10) were used to investigate HIF-1α and caspase-3 expression in sequential serial cuts by immunohistochemistry. HIF-1α was overexpressed in RC, DC, and ameloblastoma when compared with DF. The basal and sometimes the lower suprabasal layer showed no or very low expression in DC, KOT, and ameloblastoma, the last also showing strong expression in solid epithelial areas and initial cystic formation regions. Caspase-3 was found to be overexpressed in all lesions, with the highest expression in odontogenic cysts compared to tumors. HIF-1α and caspase-3 were localized in similar areas of the same lesions, especially in the epithelium surrounding cystic formations. This study showed distinct immunoexpression of HIF-1α and caspase-3 in odontogenic cyst and tumors, with higher expression observed in odontogenic cysts. These findings suggest a possible correlation between hypoxia, apoptosis, and cystogenesis, leading to understand the mechanisms responsible to cystic formation in odontogenic lesions.

Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus.

PubMed

Sourial, Mary; Doering, Laurie C

2017-07-01

The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST + CD15 + CD133 + ) and an increased proportion of neuroblasts (PSA-NCAM + CD15 + ) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G 2 /M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles following Peripheral Nerve Injury

PubMed Central

Wiberg, Rebecca; Jonsson, Samuel; Novikova, Liudmila N.; Kingham, Paul J.

2015-01-01

Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres) and soleus (slow type) muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides) at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury. PMID:26691660

Molecular cloning, expression, and stress response of the estrogen-related receptor gene (AccERR) from Apis cerana cerana

NASA Astrophysics Data System (ADS)

Zhang, Weixing; Zhu, Ming; Zhang, Ge; Liu, Feng; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

2016-04-01

Estrogen-related receptor (ERR), which belongs to the nuclear receptor superfamily, has been implicated in diverse physiological processes involving the estrogen signaling pathway. However, little information is available on ERR in Apis cerana cerana. In this report, we isolated the ERR gene and investigated its involvement in antioxidant defense. Quantitative real-time polymerase chain reaction (qPCR) revealed that the highest mRNA expression occurred in eggs during different developmental stages. The expression levels of AccERR were highest in the muscle, followed by the rectum. The predicted transcription factor binding sites in the promoter of AccERR suggested that AccERR potentially functions in early development and in environmental stress responses. The expression of AccERR was induced by cold (4 °C), heat (42 °C), ultraviolet light (UV), HgCl2, and various types of pesticides (phoxim, deltamethrin, triadimefon, and cyhalothrin). Western blot was used to measure the expression levels of AccERR protein. These data suggested that AccERR might play a vital role in abiotic stress responses.

Identification and expression analysis of glucosinolate biosynthetic genes and estimation of glucosinolate contents in edible organs of Brassica oleracea subspecies.

PubMed

Yi, Go-Eun; Robin, Arif Hasan Khan; Yang, Kiwoung; Park, Jong-In; Kang, Jong-Goo; Yang, Tae-Jin; Nou, Ill-Sup

2015-07-20

Glucosinolates are anti-carcinogenic, anti-oxidative biochemical compounds that defend plants from insect and microbial attack. Glucosinolates are abundant in all cruciferous crops, including all vegetable and oilseed Brassica species. Here, we studied the expression of glucosinolate biosynthesis genes and determined glucosinolate contents in the edible organs of a total of 12 genotypes of Brassica oleracea: three genotypes each from cabbage, kale, kohlrabi and cauliflower subspecies. Among the 81 genes analyzed by RT-PCR, 19 are transcription factor-related, two different sets of 25 genes are involved in aliphatic and indolic biosynthesis pathways and the rest are breakdown-related. The expression of glucosinolate-related genes in the stems of kohlrabi was remarkably different compared to leaves of cabbage and kale and florets of cauliflower as only eight genes out of 81 were expressed in the stem tissues of kohlrabi. In the stem tissue of kohlrabi, only one aliphatic transcription factor-related gene, Bol036286 (MYB28) and one indolic transcription factor-related gene, Bol030761 (MYB51), were expressed. The results indicated the expression of all genes is not essential for glucosinolate biosynthesis. Using HPLC analysis, a total of 16 different types of glucosinolates were identified in four subspecies, nine of them were aliphatic, four of them were indolic and one was aromatic. Cauliflower florets measured the highest number of 14 glucosinolates. Among the aliphatic glucosinolates, only gluconapin was found in the florets of cauliflower. Glucoiberverin and glucobrassicanapin contents were the highest in the stems of kohlrabi. The indolic methoxyglucobrassicin and aromatic gluconasturtiin accounted for the highest content in the florets of cauliflower. A further detailed investigation and analyses is required to discern the precise roles of each of the genes for aliphatic and indolic glucosinolate biosynthesis in the edible organs.

Hepatic Oval Cells Have the Side Population Phenotype Defined by Expression of ATP-Binding Cassette Transporter ABCG2/BCRP1

PubMed Central

Shimano, Koichi; Satake, Makoto; Okaya, Atsuhito; Kitanaka, Junichi; Kitanaka, Nobue; Takemura, Motohiko; Sakagami, Masafumi; Terada, Nobuyuki; Tsujimura, Tohru

2003-01-01

Organ-specific stem cells can be identified by the side population (SP) phenotype, which is defined by the property to effectively exclude the Hoechst 33342 dye. The ATP-binding cassette transporter ABCG2/BCRP1 mediates the SP phenotype. Because hepatic oval cells possess several characteristics of stem cells, we examined whether they have the SP phenotype using the 2-acetylaminofluorene/partial hepatectomy (PH) model. Fluorescence-activated cell sorting analysis showed that a population of non-parenchymal cells containing oval cells, prepared on day 7 after PH, carried a significant number of SP cells, whereas that of non-parenchymal cells without oval cells, prepared on day 0 after PH, did not. Northern blot analysis using total liver RNA obtained on various days after PH showed that the expression of ABCG2/BCRP1 mRNA increased after PH, reaching the highest level on day 7, and then gradually decreased. This pattern of changes in the ABCG2/BCRP1 mRNA level was well correlated to that in the number of oval cells. Furthermore, in situ hybridization revealed that oval cells were the sites of expression of ABCG2/BCRP1 mRNA. These results indicate that oval cells have the SP phenotype defined by expression of ABCG2/BCRP1, suggesting that oval cells may represent stem cells in the liver. PMID:12819005

Phenotypic and Functional Heterogeneity of Bovine Blood Monocytes

PubMed Central

Hussen, Jamal; Düvel, Anna; Sandra, Olivier; Smith, David; Sheldon, Iain Martin; Zieger, Peter; Schuberth, Hans-Joachim

2013-01-01

Murine and human peripheral blood monocytes are heterogeneous in size, granularity, nuclear morphology, phenotype and function. Whether and how bovine blood monocytes follow this pattern was analyzed in this study. Flow cytometrically, classical monocytes (cM) CD14+ CD16−, intermediate monocytes (intM) CD14+ CD16+ and nonclassical monocytes (ncM) CD14+ CD16+ were identified, with cM being the predominant subset (89%). cM showed a significant lower expression of CD172a, intM expressed the highest level of MHC class II molecules, and ncM were low positive for CD163. Compared to cM and intM, ncM showed a significantly reduced phagocytosis capacity, a significantly reduced generation of reactive oxygen species, and reduced mRNA expression of CXCL8, CXCL1 and IL-1β after LPS stimulation. Based on IL-1β secretion after LPS/ATP stimulation, the inflammasome could be activated in cM and intM, but not in ncM. IFNγ increased the expression of CD16 selectively on cM and induced a shift from cM into intM in vitro. In summary, bovine CD172a-positive mononuclear cells define three monocyte subsets with distinct phenotypic and functional differences. Bovine cM and intM share homologies with their human counterparts, whereas bovine ncM are not inflammatory monocytes. PMID:23967219

Thyme and Savory Essential Oil Efficacy and Induction of Resistance against Botrytis cinerea through Priming of Defense Responses in Apple

PubMed Central

Banani, Houda; Olivieri, Leone; Santoro, Karin; Garibaldi, Angelo; Gullino, Maria Lodovica

2018-01-01

The efficacy of thyme and savory essential oils were investigated against Botrytis cinerea on apple fruit. Apples treated with thyme and savory essential oils showed significantly lower gray mold severity and incidence. Thyme essential oil at 1% concentration showed the highest efficacy, with lower disease incidence and smaller lesion diameter. The expression of specific pathogenesis-related (PR) genes PR-8 and PR-5 was characterized in apple tissues in response to thyme oil application and B. cinerea inoculation. After 6 h of pathogen inoculation, thyme essential oil induced a 2.5-fold increase of PR-8 gene expression compared to inoculated fruits. After 24 h of inoculation, PR-8 was highly induced (7-fold) in both thyme oil-treated and untreated apples inoculated with B. cinerea. After 48 h of inoculation, PR-8 expression in thyme-treated and inoculated apples was 4- and 6-fold higher than in inoculated and water-treated apples. Neither thyme oil application nor B. cinerea inoculation markedly affected PR-5 expression. These results suggest that thyme oil induces resistance against B. cinerea through the priming of defense responses in apple fruit, and the PR-8 gene of apple may play a key role in the mechanism by which thyme essential oil effectively inhibits gray mold in apple fruit. PMID:29360731

[Mechanism of apoptosis of nasopharyngeal carcinoma cells induced by polysaccharides extracts from Hedyotic diffusa].

PubMed

Jing Yan; Kang Min; Liu Jin; Li Jingyu; Tang Anzhou

2015-04-01

To explore the proliferation inhibition and apoptosis of polysaccharides extracts from polysaccharides extracts from Hedyotic diffusa (PEHD) on Human Nasopharyngeal Carcinoma (NPC)cell line CNE2 cells in vitro. CNE2 cells treated with various concentrations of PEHD were detected by MTT assay at 24 h, 48 h, and 72 h. The apoptotic cells were analyzed by flow cytometry with Annexin V/PI staining. The expression levels of Bax, Bcl-2 and caspase-3 protein were examined by Western blotting method. The growth of CNE2 cells were suppressed after treatment with PEHD (P < 0.05), MTT assay showed that the highest cell inhibition rate reached to 76.5%, the inhibition in the doses from 2 to 6 mg/ml showed dose-and-time-dependent. The percent of apoptosis in 4 and 6 mg/ml PEHD treatment groups for 48 h were 31.32%, 46.28%, respectively, and significantly higher than that in control groups, 4.86% (P < 0.01). After the cells being treated with PEHD for 48 h, the expression of Bax and caspase-3 protein increased, and the expression of Bcl-2 protein decreased gradually. PEHD could inhibited the growth of CNE2 cells and was dose-and-time-dependent, the mechanism may involve induction of cell apoptosis, which was associated with the activation of Bax and caspase-3 protein and the down-regulation of Bcl-2 protein expression.

Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis.

PubMed

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J; Considine, Robert V; Sethi, Jaswinder K; Vidal-Puig, Antonio; O'Rahilly, Stephen

2004-03-19

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2-4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.

Thyme and Savory Essential Oil Efficacy and Induction of Resistance against Botrytis cinerea through Priming of Defense Responses in Apple.

PubMed

Banani, Houda; Olivieri, Leone; Santoro, Karin; Garibaldi, Angelo; Gullino, Maria Lodovica; Spadaro, Davide

2018-01-23

The efficacy of thyme and savory essential oils were investigated against Botrytis cinerea on apple fruit. Apples treated with thyme and savory essential oils showed significantly lower gray mold severity and incidence. Thyme essential oil at 1% concentration showed the highest efficacy, with lower disease incidence and smaller lesion diameter. The expression of specific pathogenesis-related (PR) genes PR-8 and PR-5 was characterized in apple tissues in response to thyme oil application and B. cinerea inoculation. After 6 h of pathogen inoculation, thyme essential oil induced a 2.5-fold increase of PR-8 gene expression compared to inoculated fruits. After 24 h of inoculation, PR-8 was highly induced (7-fold) in both thyme oil-treated and untreated apples inoculated with B. cinerea . After 48 h of inoculation, PR-8 expression in thyme-treated and inoculated apples was 4- and 6-fold higher than in inoculated and water-treated apples. Neither thyme oil application nor B. cinerea inoculation markedly affected PR-5 expression. These results suggest that thyme oil induces resistance against B. cinerea through the priming of defense responses in apple fruit, and the PR-8 gene of apple may play a key role in the mechanism by which thyme essential oil effectively inhibits gray mold in apple fruit.

Isolation and Characterization of a Novel Pathogenesis-Related Protein Gene (GmPRP) with Induced Expression in Soybean (Glycine max) during Infection with Phytophthora sojae

PubMed Central

Jiang, Liangyu; Wu, Junjiang; Fan, Sujie; Li, Wenbin; Dong, Lidong; Cheng, Qun; Xu, Pengfei; Zhang, Shuzhen

2015-01-01

Pathogenesis-related proteins (PR proteins) play crucial roles in the plant defense system. A novel PRP gene was isolated from highly resistant soybean infected with Phytophthora sojae (P. sojae) and was named GmPRP (GenBank accession number: KM506762). The amino acid sequences of GmPRP showed identities of 74%, 73%, 72% and 69% with PRP proteins from Vitis vinifera, Populus trichocarpa, Citrus sinensis and Theobroma cacao, respectively. Quantitative real-time reverse transcription PCR (qRT-PCR) data showed that the expression of GmPRP was highest in roots, followed by the stems and leaves. GmPRP expression was upregulated in soybean leaves infected with P. sojae. Similarly, GmPRP expression also responded to defense/stress signaling molecules, including salicylic acid (SA), ethylene (ET), abscisic acid (ABA) and jasmonic acid (JA). GmPRP was localized in the cell plasma membrane and cytoplasm. Recombinant GmPRP protein exhibited ribonuclease activity and significant inhibition of hyphal growth of P. sojae 1 in vitro. Overexpression of the GmPRP gene in T2 transgenic tobacco and T2 soybean plants resulted in enhanced resistance to Phytophthora nicotianae (P. nicotianae) and P. sojae race 1, respectively. These results indicated that the GmPRP protein played an important role in the defense of soybean against P. sojae infection. PMID:26114301

Molecular Cloning, Characterization, and Differential Expression of a Glucoamylase Gene from the Basidiomycetous Fungus Lentinula edodes

PubMed Central

Zhao, J.; Chen, Y. H.; Kwan, H. S.

2000-01-01

The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus. PMID:10831434

Aldehyde dehydrogenase expression in Metaphire posthuma as a bioindicator to monitor heavy metal pollution in soil.

PubMed

Panday, Raju; Bhatt, Padam Shekhar; Bhattarai, Tribikram; Shakya, Kumudini; Sreerama, Lakshmaiah

2016-11-21

Soil contamination and associated pollution plays a detrimental role in soil flora and fauna. Soil is processed and remodeled by subterranean earthworms, accordingly are referred to as soil chemical engineers. These worms, besides processing carbon and nitrogen, serve as minors for processing metals. In heavy metal contaminated soils, they accumulate heavy metals, which in turn cause altered gene expression, including aldehyde dehydrogenase (ALDH) enzymes. This study explores the possibility of ALDH expression in earthworms as a novel biomarker for the heavy metal contamination of soil. Earthworms cultured in contaminated soils accumulated significantly higher levels of Pb and Cd. Similarly, significantly higher levels of ALDH enzyme activities were observed in earthworms cultured in soils contaminated with Pb and Cd. The ALDH activity was found to be highest in worms cultured in 5 ppm heavy metal contaminated soils. Although, ALDH activities decreased as the heavy metal concentration in soil increased, they were significantly higher when compared to control worms cultured in uncontaminated soils. The accumulation of heavy metal in earthworms measured after 28 days decreased as the heavy metal concentration in soil increased. Levels of ALDH expression correlated with total Pb and Cd concentration in the earthworm tissue. This study showed that the ALDH activity in earthworms could potentially be used as a biomarker to show heavy metal pollution in soil.

Alteration of runt-related transcription factor 3 gene expression and biologic behavior of esophageal carcinoma TE-1 cells after 5-azacytidine intervention.

PubMed

Wang, Shuai; Liu, Hong; Akhtar, Javed; Chen, Hua-Xia; Wang, Zhou

2013-01-01

5-Azacytidine (5-azaC) was originally identified as an anticancer drug (NSC102876) which can cause hypomethylation of tumor suppressor genes. To assess its effects on runt-related transcription factor 3 (RUNX3), expression levels and the promoter methylation status of the RUNX3 gene were assessed. We also investigated alteration of biologic behavior of esophageal carcinoma TE-1 cells. MTT assays showed 5-azaC inhibited the proliferation of TE-1 cells in a time and dose-dependent way. Although other genes could be demethylated after 5-azaC intervention, we focused on RUNX3 gene in this study. The expression level of RUNX3 mRNA increased significantly in TE-1 cells after treatment with 5-azaC at hypotoxic levels. RT-PCR showed 5-azaC at 50 μM had the highest RUNX3-induction activity. Methylation-specific PCR indicated that 5-azaC induced RUNX3 expression through demethylation. Migration and invasion of TE-1 cells were inhibited by 5-azaC, along with growth of Eca109 xenografts in nude mice. In conclusion, we demonstrate that the RUNX3 gene can be reactivated by the demethylation reagent 5-azaC, which inhibits the proliferation, migration and invasion of esophageal carcinoma TE-1 cells.

Expression of modulators of extracellular matrix structure after anterior cruciate ligament injury.

PubMed

Haslauer, Carla M; Proffen, Benedikt L; Johnson, Victor M; Murray, Martha M

2014-01-01

The ability of the anterior cruciate ligament (ACL) to heal after injury declines within the first 2 weeks after ACL rupture. To begin to explore the mechanism behind this finding, we quantified the expression of genes for collagen I and III, decorin, tenascin-C, and alpha smooth muscle actin, as well as matrix metalloproteinase (MMP)-1 and -13 gene expression within multiple tissues of the knee joint after ACL injury in a large animal model over a 2-week postinjury period. Gene expression of collagen I and III, decorin, and MMP-1 was highest in the synovium, whereas the highest MMP-13 gene expression levels were found in the ACL. The gene expression for collagen and decorin increased over the 2 weeks to levels approaching that in the ligament and synovium; however, no significant increase in either of the MMPs was found in the provisional scaffold. This suggests that although the ACL and synovium up-regulate both anabolic and catabolic factors, the provisional scaffold is primarily anabolic in function. The relative lack of provisional scaffold formation within the joint environment may thus be one of the key reasons for ACL degradation after injury. © 2014 by the Wound Healing Society.

Electrospun nanofibrous SF/P(LLA-CL) membrane: a potential substratum for endothelial keratoplasty.

PubMed

Chen, Junzhao; Yan, Chenxi; Zhu, Mengyu; Yao, Qinke; Shao, Chunyi; Lu, Wenjuan; Wang, Jing; Mo, Xiumei; Gu, Ping; Fu, Yao; Fan, Xianqun

2015-01-01

Cornea transplant technology has progressed markedly in recent decades, allowing surgeons to replace diseased corneal endothelium by a thin lamellar structure. A thin, transparent, biocompatible, tissue-engineered substratum with corneal endothelial cells for endothelial keratoplasty is currently of interest. Electrospinning a nanofibrous structure can simulate the extracellular matrix and have beneficial effects for cell culture. Silk fibroin (SF) has good biocompatibility but poor mechanical properties, while poly(L-lactic acid-co-ε-caprolactone) (P(LLA-CL)) has good mechanical properties but poor biocompatibility. Blending SF with P(LLA-CL) can maintain the advantages of both these materials and overcome their disadvantages. Blended electrospun nanofibrous membranes may be suitable for regeneration of the corneal endothelium. The aim of this study was to produce a tissue-engineered construct suitable for endothelial keratoplasty. Five scaffolds containing different SF:P(LLA-CL) blended ratios (100:0, 75:25, 50:50, 25:75, 0:100) were manufactured. A human corneal endothelial (B4G12) cell line was cultured on the membranes. Light transmission, speed of cell adherence, cell viability (live-dead test), cell proliferation (Ki-67, BrdU staining), and cell monolayer formation were detected on membranes with the different blended ratios, and expression of some functional genes was also detected by real-time polymerase chain reaction. Different blended ratios of scaffolds had different light transmittance properties. The 25:75 blended ratio membrane had the best transmittance among these scaffolds. All electrospun nanofibrous membranes showed improved speed of cell adherence when compared with the control group, especially when the P(LLA-CL) ratio increased. The 25:75 blended ratio membranes also had the highest cell proliferation. B4G12 cells could form a monolayer on all scaffolds, and most functional genes were also stably expressed on all scaffolds. Only two genes showed changes in expression. All blended ratios of SF:P(LLA-CL) scaffolds were evaluated and showed good biocompatibility for cell adherence and monolayer formation. Among them, the 25:75 blended ratio SF:P(LLA-CL) scaffold had the best transmittance and the highest cell proliferation. These attributes further the potential application of the SF:P(LLA-CL) scaffold for corneal endothelial transplantation.

Methallothionein expression on the gills and stomach of Chinese pond mussels exposed to lead (Pb)

NASA Astrophysics Data System (ADS)

Kartikaningsih, H.; Suryanto, A. M.; Arfiati, D.

2018-04-01

In freshwaters area, Pb originates from rocks (naturally), industries, and pesticides. The ability of Chinese pond mussels as biofilters to absorb heavy metal (Pb) was demonstrated in water circulation system using ten 8 cm mussels. PbNO3 (0, 10, 20, and 30 ppm) was administered into water containing mussels. Carp culture was done for 30 days, and Pb accumulation in carps was measured every week (week 0, 1, 2, and 3). The results showed that the highest Pb ion accumulation was found in the gills of mussels. The examination using hematoxylin-eosin showed that tissues were damage due to haemorrhage, cell ruptures, and cell deaths. The results of the measurement of metallothionein (MT) showed that MT molecular weight was 12.84 kDa.

Influence of micellar calcium and phosphorus on rennet coagulation properties of cows milk.

PubMed

Malacarne, Massimo; Franceschi, Piero; Formaggioni, Paolo; Sandri, Sandro; Mariani, Primo; Summer, Andrea

2014-05-01

The main requirement for milk processed in most cheese typologies is its rennet coagulation ability. Despite the increasing number of studies, the causes for abnormal coagulation of milk are not fully understood. The aim of this study was to ascertain relationships between milk characteristics and its rennet coagulation ability, focusing on the influence of calcium (Ca) and phosphorus (P). Ca and P are essential constituents of the micelles. Micellar P can be present as part of colloidal calcium phosphate (inorganic-P) or covalently bound to caseins as phosphate groups (casein-P). Eighty one herd milk samples (SCC<400 000 cell/ml) were classified as Optimal (8), Suboptimal (39) Poor (29) and Non-coagulating milk (5), according to their rennet coagulation parameters as assessed by lactodynamographic test. Samples were analysed for their chemical composition (basic composition, protein fractions, minerals and salt equilibria), physicochemical parameters (pH and titratable acidity) and rheological properties. Optimal milk was characterised by the highest contents of major constituents, protein fractions and minerals, lowest content of chloride and highest values of titratable acidity. Non-coagulating milk was characterised by the highest values of pH and the lowest of titratable acidity. At micellar level, Optimal milk showed the highest values of colloidal Ca, casein-P and colloidal Mg (g/100 g casein), while Non-coagulating milk showed the lowest values. Interestingly, there was no statistical difference regarding the content of colloidal inorganic-P (g/100 g casein) between Optimal and Non-coagulating milks. Overall, high mineralisation of the micelle (expressed as g inorganic-P/100 g casein) positively affect its rennetability. However, excessive mineralisation could lead to a reduction of the phosphate groups (g casein-P/100 g casein) available for curd formation.

Molecular characterization and expression analysis of the myostatin gene and its association with growth traits in Noble scallop (Chlamys nobilis).

PubMed

Fan, Sigang; Xu, Youhou; Liu, Baosuo; He, Wenyao; Zhang, Bo; Su, Jiaqi; Yu, Dahui

2017-10-01

Myostatin (MSTN), also called growth and differentiation factor-8 (GDF-8), is a member of the transforming growth factor-β (TGF-β) superfamily and an inhibitor of muscle differentiation and growth. In this report, we identified and characterized a MSTN gene (CnMSTN) from the scallop Chlamys nobilis. The open reading frame of CnMSTN was 1374bp in length, encoding 457 amino acids. The structure of CnMSTN included a putative signal peptide, a TGF-β propeptide domain, and a conserved TGF-β domain. Phylogenetic analysis showed that the CnMSTN gene was clustered in the same subgroup with the MSTN gene found in Mollusca. Quantitative real-time PCR showed that the CnMSTN gene was widely expressed in all tissues tested, with the highest expression level observed in the adductor muscle. Six single nucleotide polymorphisms (SNPs) were identified in the promoter region, but no SNP was detected in the exon regions. Association analysis showed that SNP g.-579A/C had significant effects on body mass, soft-tissue mass, and adductor muscle mass. The CC and AC genotypes of g.-579A/C had significantly higher growth trait values than that of genotype AA (P<0.05). These results suggest that CnMSTN could be used as a candidate gene for the selective breeding of C. nobilis. Copyright © 2017 Elsevier Inc. All rights reserved.

CD63 tetraspanin is a negative driver of epithelial-to-mesenchymal transition in human melanoma cells.

PubMed

Lupia, Antonella; Peppicelli, Silvia; Witort, Ewa; Bianchini, Francesca; Carloni, Vinicio; Pimpinelli, Nicola; Urso, Carmelo; Borgognoni, Lorenzo; Capaccioli, Sergio; Calorini, Lido; Lulli, Matteo

2014-12-01

The CD63 tetraspanin is highly expressed in the early stages of melanoma and decreases in advanced lesions, suggesting it as a possible suppressor of tumor progression. We employed loss- and gain-of-gene-function approaches to investigate the role of CD63 in melanoma progression and acquisition of the epithelial-to-mesenchymal transition (EMT) program. We used two human melanoma cell lines derived from primary tumors and one primary human melanoma cell line isolated from a cutaneous metastasis, differing by levels of CD63 expression. CD63-silenced melanoma cells showed enhanced motility and invasiveness with downregulation of E-cadherin and upregulation of N-cadherin and Snail. In parallel experiments, transient and stable ectopic expression of CD63 resulted in a robust reduction of cell motility, invasiveness, and protease activities, which was proportional to the increase in CD63 protein level. Transfected cells overexpressing the highest level of CD63 when transplanted into immunodeficient mice showed a reduced incidence and rate of tumor growth. Moreover, these cells showed a reduction of N-cadherin, Vimentin, Zeb1, and a-SMA, and a significant resistance to undergo an EMT program both in basal condition and in the following stimulation with TGFβ. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

An Oleuropein β-Glucosidase from Olive Fruit Is Involved in Determining the Phenolic Composition of Virgin Olive Oil

PubMed Central

Velázquez-Palmero, David; Romero-Segura, Carmen; García-Rodríguez, Rosa; Hernández, María L.; Vaistij, Fabián E.; Graham, Ian A.; Pérez, Ana G.; Martínez-Rivas, José M.

2017-01-01

Phenolic composition of virgin olive oil is determined by the enzymatic and/or chemical reactions that take place during olive fruit processing. Of these enzymes, β-glucosidase activity plays a relevant role in the transformation of the phenolic glycosides present in the olive fruit, generating different secoiridoid derivatives. The main goal of the present study was to characterize olive fruit β-glucosidase genes and enzymes responsible for the phenolic composition of virgin olive oil. To achieve that, we have isolated an olive β-glucosidase gene from cultivar Picual (OepGLU), expressed in Nicotiana benthamiana leaves and purified its corresponding recombinant enzyme. Western blot analysis showed that recombinant OepGLU protein is detected by an antibody raised against the purified native olive mesocarp β-glucosidase enzyme, and exhibits a deduced molecular mass of 65.0 kDa. The recombinant OepGLU enzyme showed activity on the major olive phenolic glycosides, with the highest levels with respect to oleuropein, followed by ligstroside and demethyloleuropein. In addition, expression analysis showed that olive GLU transcript level in olive fruit is spatially and temporally regulated in a cultivar-dependent manner. Furthermore, temperature, light and water regime regulate olive GLU gene expression in olive fruit mesocarp. All these data are consistent with the involvement of OepGLU enzyme in the formation of the major phenolic compounds present in virgin olive oil. PMID:29163620

The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower

PubMed Central

Tan, Jiafu; Wang, Maojun; Tu, Lili; Nie, Yichun; Lin, Yongjun; Zhang, Xianlong

2013-01-01

Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development. PMID:23951318

Determination of the Michaelis-Menten kinetics and the genes expression involved in phyto-degradation of cyanide and ferri-cyanide.

PubMed

Yu, Xiao-Zhang; Zhang, Xue-Hong

2016-07-01

Hydroponic experiments were conducted with different species of plants (rice, maize, soybean and willow) exposed to ferri-cyanide to investigate the half-saturation constant (K M ) and the maximal metabolic capacity (v max ) involved in phyto-assimilation. Three varieties for each testing species were collected from different origins. Measured concentrations show that the uptake rates responded biphasically to ferri-cyanide treatments by showing increases linearly at low and almost constant at high concentrations from all treatments, indicating that phyto-assimilation of ferri-cyanide followed the Michaelis-Menten kinetics. Using non-linear regression, the highest v max was by rice, followed by willows. The lowest v max was found for soybean. All plants, except maize (DY26) and rice (XJ12), had a similar K M value, suggesting the same enzyme was active in phyto-assimilation of ferri-cyanide. Transcript level, by real-time quantitative PCR, of enzymes involved in degradation of cyanides showed that the analyzed genes were differently expressed during different cyanides exposure. The expression of CAS and ST genes responded positively to KCN exposure, suggesting that β-CAS and ST pathways were two possible pathways for cyanide detoxification in rice. The transcript level of NIT and ASPNASE genes also showed a remarkable up-regulation to KCN, implying the contribution to the pool of amino acid aspartate, which is an end product of CN metabolism. Up-regulation of GS genes suggests that acquisition of ammonium released from cyanide degradation may be an additional nitrogen source for plant nutrition. Results also revealed that the expressions of these genes, except for GS, were relatively constant during iron cyanide exposure, suggesting that they are likely metabolized by plants through a non-defined pathway rather than the β-CAS pathway.

Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize.

PubMed

Johnson, Eric T; Berhow, Mark A; Dowd, Patrick F

2007-04-18

Hi II maize (Zea mays) plants were engineered to express maize p1 cDNA, a Myb transcription factor, controlled by a putative silk specific promoter, for secondary metabolite production and corn earworm resistance. Transgene expression did not enhance silk color, but about half of the transformed plant silks displayed browning when cut, which indicated the presence of p1-produced secondary metabolites. Levels of maysin, a secondary metabolite with insect toxicity, were highest in newly emerged browning silks. The insect resistance of transgenic silks was also highest at emergence, regardless of maysin levels, which suggests that other unidentified p1-induced molecules likely contributed to larval mortality. Mean survivor weights of corn earworm larvae fed mature browning transgenic silks were significantly lower than weights of those fed mature nonbrowning transgenic silks. Some transgenic pericarps browned with drying and contained similar molecules found in pericarps expressing a dominant p1 allele, suggesting that the promoter may not be silk-specific.

Aerobic glycolysis in the human brain is associated with development and neotenous gene expression

PubMed Central

Goyal, Manu S.; Hawrylycz, Michael; Miller, Jeremy A.; Snyder, Abraham Z.; Raichle, Marcus E.

2015-01-01

SUMMARY Aerobic glycolysis (AG), i.e., non-oxidative metabolism of glucose despite the presence of abundant oxygen, accounts for 10–12% of glucose used by the adult human brain. AG varies regionally in the resting state. Brain AG may support synaptic growth and remodeling; however, data supporting this hypothesis are sparse. Here, we report on investigations on the role of AG in the human brain. Meta-analysis of prior brain glucose and oxygen metabolism studies demonstrates that AG increases during childhood, precisely when synaptic growth rates are highest. In resting adult humans, AG correlates with persistence of gene expression typical of infancy (transcriptional neoteny). In brain regions with the highest AG, we find increased gene expression related to synapse formation and growth. In contrast, regions high in oxidative glucose metabolism express genes related to mitochondria and synaptic transmission. Our results suggest that brain AG supports developmental processes, particularly those required for synapse formation and growth. PMID:24411938

ABCC3 as a marker for multidrug resistance in non-small cell lung cancer

PubMed Central

Zhao, Yanbin; Lu, Hailing; Yan, An; Yang, Yanmei; Meng, Qingwei; Sun, Lichun; Pang, Hui; Li, Chunhong; Dong, Xiaoqun; Cai, Li

2013-01-01

Multidrug resistance (MDR) contributes to the failure of chemotherapy and high mortality in non-small cell lung cancer (NSCLC). We aim to identify MDR genes that predict tumor response to chemotherapy. 199 NSCLC fresh tissue samples were tested for chemosensitivity by MTT assay. cDNA microarray was done with 5 samples with highest resistance and 6 samples with highest sensitivity. Expression of ABCC3 mRNA and protein was detected by real-time PCR and immunohistochemisty, respectively. The association between gene expression and overall survival (OS) was examined using Cox proportional hazard regression. 44 genes were upregulated and 168 downregulated in the chemotherapy-resistant group. ABCC3 was one of the most up-regulated genes in the resistant group. ABCC3-positive expression correlated with lymph node involvement, advanced TNM stage, more malignant histological type, multiple-resistance to anti-cancer drugs, and reduced OS. ABCC3 expression may serve as a marker for MDR and predictor for poor clinical outcome of NSCLC. PMID:24176985

CEACAM1 is overexpressed in oral tumors and related to tumorigenesis.

PubMed

Wang, Fu-Fang; Guan, Bing-Xin; Yang, Jing-Yan; Wang, Hai-Tao; Zhou, Cheng-Jun

2017-03-01

Carcinoembryonic antigen-related adhesion molecule 1 (CEACAM1) is a type 1 transmembrane glycoprotein belonging to the CEA family, which has been known to exist as either soluble forms in body fluids or membrane-bound forms on the cell surface. Aberrant CEACAM1 expression is associated with tumorigenesis and has been reported in a variety of human tumors, especially malignancies. The aim of this study is to determine the expression of CEACAM1 in oral tumors, trying to study CEACAM1 different expressions as a function of histotype. CEACAM1 expression was observed by immunohistochemistry (IHC) with mouse anti-human antibody for CEACAM1. IHC was performed using avidin-biotin-diaminobenzidine staining. The results were expressed as average score ± SD (0 = negative/8 = highest) for each histotype. Oral tumors expressed more CEACAM1 than normal tissues including squamous and salivary epithelia (P < 0.05). In malignancies, the squamous cell carcinoma overexpressed CEACAM1, compared to well-differentiated squamous cell with more membranous expression; the intermediately and poorly differentiated squamous cell carcinoma showed more cytoplasmic expression (P < 0.05). In addition, the salivary tumors significantly expressed more CEACAM1 than squamous cell carcinoma (P < 0.05). So, we thought oral tumors overexpressed CEACAM1 and the cytoplasmic CEACAM1 might be involved in tumorigenesis, and also CEACAM1 might be regarded as a marker of salivary glandular tumors.

Characterization of Pseudomonas putida Genes Responsive to Nutrient Limitation

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

Syn, Chris K.; Magnuson, Jon K.; Kingsley, Mark T.

2004-06-01

The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation/biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high nutrient- and oxygen-availability. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 was examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate, or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7, and NRM17, were selected for identification of the tagged genes. In themore » mutant strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased between 4.9- to 26.4-fold under various low nutrient conditions. In NRM7, expression of the novel NADPH:quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low nutrient and anoxic conditions. The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low nutrient conditions but its absolute expression levels was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that in E. coli.« less

A family of tissue-specific resistin-like molecules

PubMed Central

Steppan, Claire M.; Brown, Elizabeth J.; Wright, Christopher M.; Bhat, Savitha; Banerjee, Ronadip R.; Dai, Charlotte Y.; Enders, Gregory H.; Silberg, Debra G.; Wen, Xiaoming; Wu, Gary D.; Lazar, Mitchell A.

2001-01-01

We have identified a family of resistin-like molecules (RELMs) in rodents and humans. Resistin is a hormone produced by fat cells. RELMα is a secreted protein that has a restricted tissue distribution with highest levels in adipose tissue. Another family member, RELMβ, is a secreted protein expressed only in the gastrointestinal tract, particularly the colon, in both mouse and human. RELMβ gene expression is highest in proliferative epithelial cells and is markedly increased in tumors, suggesting a role in intestinal proliferation. Resistin and the RELMs share a cysteine composition and other signature features. Thus, the RELMs together with resistin comprise a class of tissue-specific signaling molecules. PMID:11209052

A family of tissue-specific resistin-like molecules.

PubMed

Steppan, C M; Brown, E J; Wright, C M; Bhat, S; Banerjee, R R; Dai, C Y; Enders, G H; Silberg, D G; Wen, X; Wu, G D; Lazar, M A

2001-01-16

We have identified a family of resistin-like molecules (RELMs) in rodents and humans. Resistin is a hormone produced by fat cells. RELMalpha is a secreted protein that has a restricted tissue distribution with highest levels in adipose tissue. Another family member, RELMbeta, is a secreted protein expressed only in the gastrointestinal tract, particularly the colon, in both mouse and human. RELMbeta gene expression is highest in proliferative epithelial cells and is markedly increased in tumors, suggesting a role in intestinal proliferation. Resistin and the RELMs share a cysteine composition and other signature features. Thus, the RELMs together with resistin comprise a class of tissue-specific signaling molecules.

Faster embryonic segmentation through elevated Delta-Notch signalling

PubMed Central

Liao, Bo-Kai; Jörg, David J.; Oates, Andrew C.

2016-01-01

An important step in understanding biological rhythms is the control of period. A multicellular, rhythmic patterning system termed the segmentation clock is thought to govern the sequential production of the vertebrate embryo's body segments, the somites. Several genetic loss-of-function conditions, including the Delta-Notch intercellular signalling mutants, result in slower segmentation. Here, we generate DeltaD transgenic zebrafish lines with a range of copy numbers and correspondingly increased signalling levels, and observe faster segmentation. The highest-expressing line shows an altered oscillating gene expression wave pattern and shortened segmentation period, producing embryos with more, shorter body segments. Our results reveal surprising differences in how Notch signalling strength is quantitatively interpreted in different organ systems, and suggest a role for intercellular communication in regulating the output period of the segmentation clock by altering its spatial pattern. PMID:27302627

A comparison of Helicobacter pylori and non-Helicobacter pylori Helicobacter spp. Binding to canine gastric mucosa with defined gastric glycophenotype.

PubMed

Amorim, Irina; Freitas, Daniela P; Magalhães, Ana; Faria, Fátima; Lopes, Célia; Faustino, Augusto M; Smet, Annemieke; Haesebrouck, Freddy; Reis, Celso A; Gärtner, Fátima

2014-08-01

The gastric mucosa of dogs is often colonized by non-Helicobacter pylori helicobacters (NHPH), while H. pylori is the predominant gastric Helicobacter species in humans. The colonization of the human gastric mucosa by H. pylori is highly dependent on the recognition of host glycan receptors. Our goal was to define the canine gastric mucosa glycophenotype and to evaluate the capacity of different gastric Helicobacter species to adhere to the canine gastric mucosa. The glycosylation profile in body and antral compartments of the canine gastric mucosa, with focus on the expression of histo-blood group antigens was evaluated. The in vitro binding capacity of FITC-labeled H. pylori and NHPH to the canine gastric mucosa was assessed in cases representative of the canine glycosylation pattern. The canine gastric mucosa lacks expression of type 1 Lewis antigens and presents a broad expression of type 2 structures and A antigen, both in the surface and glandular epithelium. Regarding the canine antral mucosa, H. heilmannii s.s. presented the highest adhesion score whereas in the body region the SabA-positive H. pylori strain was the strain that adhered more. The canine gastric mucosa showed a glycosylation profile different from the human gastric mucosa suggesting that alternative glycan receptors may be involved in Helicobacter spp. binding. Helicobacter pylori and NHPH strains differ in their ability to adhere to canine gastric mucosa. Among the NHPH, H. heilmannii s.s. presented the highest adhesion capacity in agreement with its reported colonization of the canine stomach. © 2014 John Wiley & Sons Ltd.

Short-term effects of T-2 toxin or deoxynivalenol on lipid peroxidation and the glutathione system in common carp.

PubMed

Pelyhe, Csilla; Kövesi, Benjámin; Zándoki, Erika; Kovács, Balázs; Szabó-Fodor, Judit; Mézes, Miklós; Balogh, Krisztián

2016-12-01

The purpose of this study was to investigate the short-term effects of a single oral dose of T-2 and HT-2 toxin at 0.15, 0.33 and 1.82 mg kg -1 body weight, or deoxynivalenol (DON) and 15-acetyl-DON at 0.13, 0.31 and 1.75 mg kg -1 body weight in common carp. Conjugated dienes and trienes (the early markers of lipid peroxidation) were elevated in all DON-treated groups at the 16th hour, while thiobarbituric acid reactive substances (TBARS; termination marker) were increased at the highest dose of DON at the 16th and 24th hours. T-2 toxin did not cause changes in these parameters. Glutathione content and glutathione peroxidase activity showed higher levels at the 16th hour as the effect of both mycotoxins. The expression of glutathione peroxidase (GPx4) genes (gpx4a and gpx4b) revealed a dual response. Downregulation was observed at the 8th hour, followed by an induction at the 16th hour, at the lowest dose of both mycotoxins. Higher doses revealed long-drawn emergence and an elevation was observed only at the 24th hour. However, at the lowest and highest doses of DON or T-2 toxin the changes in gene expression were delayed, which may be related to the low oxidative stress response, as suggested by the expression profiles of the nrf2, keap1, gpx4a and gpx4b genes.

Stimulation of growth by proteorhodopsin phototrophy involves regulation of central metabolic pathways in marine planktonic bacteria.

PubMed

Palovaara, Joakim; Akram, Neelam; Baltar, Federico; Bunse, Carina; Forsberg, Jeremy; Pedrós-Alió, Carlos; González, José M; Pinhassi, Jarone

2014-09-02

Proteorhodopsin (PR) is present in half of surface ocean bacterioplankton, where its light-driven proton pumping provides energy to cells. Indeed, PR promotes growth or survival in different bacteria. However, the metabolic pathways mediating the light responses remain unknown. We analyzed growth of the PR-containing Dokdonia sp. MED134 (where light-stimulated growth had been found) in seawater with low concentrations of mixed [yeast extract and peptone (YEP)] or single (alanine, Ala) carbon compounds as models for rich and poor environments. We discovered changes in gene expression revealing a tightly regulated shift in central metabolic pathways between light and dark conditions. Bacteria showed relatively stronger light responses in Ala compared with YEP. Notably, carbon acquisition pathways shifted toward anaplerotic CO2 fixation in the light, contributing 31 ± 8% and 24 ± 6% of the carbon incorporated into biomass in Ala and YEP, respectively. Thus, MED134 was a facultative double mixotroph, i.e., photo- and chemotrophic for its energy source and using both bicarbonate and organic matter as carbon sources. Unexpectedly, relative expression of the glyoxylate shunt genes (isocitrate lyase and malate synthase) was >300-fold higher in the light--but only in Ala--contributing a more efficient use of carbon from organic compounds. We explored these findings in metagenomes and metatranscriptomes and observed similar prevalence of the glyoxylate shunt compared with PR genes and highest expression of the isocitrate lyase gene coinciding with highest solar irradiance. Thus, regulatory interactions between dissolved organic carbon quality and central metabolic pathways critically determine the fitness of surface ocean bacteria engaging in PR phototrophy.

Physical and chemical quality characteristics and antioxidant properties of strawberry cultivars (Fragaria × ananassa Duch.) in Greece: assessment of their sensory impact.

PubMed

Zeliou, Konstantina; Papasotiropoulos, Vassilis; Manoussopoulos, Yiannis; Lamari, Fotini N

2018-02-01

There are many factors determining the strawberry organoleptic profile and they are difficult to define. In this study, the sensory, physical, and chemical quality characteristics, the antioxidant properties as examined using ferric reducing antioxidant power (FRAP) and 1-diphenyl-2-picrylhydrazyl (DPPH) assays, the lactone concentration, and the FaFAD1 expression of ripe strawberries (cv. Camarosa, Florida Fortuna, and Sabrina) from Greece were evaluated and their interrelationships were investigated. 'Camarosa' had the highest antioxidant capacity and polyphenol content, although significant intra-cultivar variations of sugars, solid soluble content/titratable acidity (SSC/TA), red color intensity, sweetness, and hardness were recorded. In 'Sabrina' there was a constant lactone presence and FaFAD1 expression; it also had the lowest ascorbic acid content, the highest pH, SSC/TA index, firmness, and sweetness. 'Fortuna' showed the lowest sweetness and aroma indices, whereas 'Camarosa' had intermediate ones. Overall, firmness was correlated with hardness, while pH and SSC/TA index correlated with juiciness and sweetness. Both γ-decalactone and γ-dodecalactone concentrations were correlated with FaFAD1 expression and pH, but they did not solely determine the aroma sensory perception. In total, FRAP values were positively correlated with ascorbic acid and polyphenol content, and negatively with pH. Significant inter- and intra-cultivar variation was recorded, revealing the impact of the genotype and underlining the effect of microenvironmental and cultivation conditions on quality and sensory perception. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Enhanced cell surface expression, immunogenicity and genetic stability resulting from a spontaneous truncation of HIV Env expressed by a recombinant MVA

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

Wyatt, Linda S.; Belyakov, Igor M.; Earl, Patricia L.

2008-03-15

During propagation of modified vaccinia virus Ankara (MVA) encoding HIV 89.6 Env, a few viral foci stained very prominently. Virus cloned from such foci replicated to higher titers than the parent and displayed enhanced genetic stability on passage. Sequence analysis showed a single nucleotide deletion in the 89.6 env gene of the mutant that caused a frame shift and truncation of 115 amino acids from the cytoplasmic domain. The truncated Env was more highly expressed on the cell surface, induced higher antibody responses than the full-length Env, reacted with HIV neutralizing monoclonal antibodies and mediated CD4/co-receptor-dependent fusion. Intramuscular (IM), intradermalmore » (ID) needleless, and intrarectal (IR) catheter inoculations gave comparable serum IgG responses. However, intraoral (IO) needleless injector route gave the highest IgA in lung washings and IR gave the highest IgA and IgG responses in fecal extracts. Induction of CTL responses in the spleens of individual mice as assayed by intracellular cytokine staining was similar with both the full-length and truncated Env constructs. Induction of acute and memory CTL in the spleens of mice immunized with the truncated Env construct by ID, IO, and IR routes was comparable and higher than by the IM route, but only the IR route induced CTL in the gut-associated lymphoid tissue. Thus, truncation of Env enhanced genetic stability as well as serum and mucosal antibody responses, suggesting the desirability of a similar modification in MVA-based candidate HIV vaccines.« less

Engineering transcription factors to improve tolerance against alkane biofuels in Saccharomyces cerevisiae.

PubMed

Ling, Hua; Pratomo Juwono, Nina Kurniasih; Teo, Wei Suong; Liu, Ruirui; Leong, Susanna Su Jan; Chang, Matthew Wook

2015-01-01

Biologically produced alkanes can be used as 'drop in' to existing transportation infrastructure as alkanes are important components of gasoline and jet fuels. Despite the reported microbial production of alkanes, the toxicity of alkanes to microbial hosts could pose a bottleneck for high productivity. In this study, we aimed to improve the tolerance of Saccharomyces cerevisiae, a model eukaryotic host of industrial significance, to alkane biofuels. To increase alkane tolerance in S. cerevisiae, we sought to exploit the pleiotropic drug resistance (Pdr) transcription factors Pdr1p and Pdr3p, which are master regulators of genes with pleiotropic drug resistance elements (PDREs)-containing upstream sequences. Wild-type and site-mutated Pdr1p and Pdr3p were expressed in S. cerevisiae BY4741 pdr1Δ pdr3Δ (BYL13). The point mutations of PDR1 (F815S) and PDR3 (Y276H) in BYL13 resulted in the highest tolerance to C10 alkane, and the expression of wild-type PDR3 in BYL13 led to the highest tolerance to C11 alkane. To identify and verify the correlation between the Pdr transcription factors and tolerance improvement, we analyzed the expression patterns of genes regulated by the Pdr transcription factors in the most tolerant strains against C10 and C11 alkanes. Quantitative PCR results showed that the Pdr transcription factors differentially regulated genes associated with multi-drug resistance, stress responses, and membrane modifications, suggesting different extents of intracellular alkane levels, reactive oxygen species (ROS) production and membrane integrity. We further showed that (i) the expression of Pdr1mt1 + Pdr3mt reduced intracellular C10 alkane by 67 % and ROS by 53 %, and significantly alleviated membrane damage; and (ii) the expression of the Pdr3wt reduced intracellular C11 alkane by 72 % and ROS by 21 %. Alkane transport assays also revealed that the reduction of alkane accumulation was due to higher export (C10 and C11 alkanes) and lower import (C11 alkane). We improved yeast's tolerance to alkane biofuels by modulating the expression of the wild-type and site-mutated Pdr1p and Pdr3p, and extensively identified the correlation between Pdr transcription factors and tolerance improvement by analyzing gene patterns, alkane transport, ROS, and membrane integrity. These findings provide valuable insights into manipulating transcription factors in yeast for improved alkane tolerance and productivity.

Identification and expression profiling of novel plant cell wall degrading enzymes from a destructive pest of palm trees, Rhynchophorus ferrugineus.

PubMed

Antony, B; Johny, J; Aldosari, S A; Abdelazim, M M

2017-08-01

Plant cell wall degrading enzymes (PCWDEs) from insects were recently identified as a multigene family of proteins that consist primarily of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) and play essential roles in the degradation of the cellulose/hemicellulose/pectin network in the invaded host plant. Here we applied transcriptomic and degenerate PCR approaches to identify the PCWDEs from a destructive pest of palm trees, Rhynchophorus ferrugineus, followed by a gut-specific and stage-specific differential expression analysis. We identified a total of 27 transcripts encoding GH family members and three transcripts of the CE family with cellulase, hemicellulase and pectinase activities. We also identified two GH9 candidates, which have not previously been reported from Curculionidae. The gut-specific quantitative expression analysis identified key cellulases, hemicellulases and pectinases from R. ferrugineus. The expression analysis revealed a pectin methylesterase, RferCE8u02, and a cellulase, GH45c34485, which showed the highest gut enriched expression. Comparison of PCWDE expression patterns revealed that cellulases and pectinases are significantly upregulated in the adult stages, and we observed specific high expression of the hemicellulase RferGH16c4170. Overall, our study revealed the potential of PCWDEs from R. ferrugineus, which may be useful in biotechnological applications and may represent new tools in R. ferrugineus pest management strategies. © 2017 The Royal Entomological Society.

Piwil1 mediates meiosis during spermatogenesis in chicken.

PubMed

Xu, Lu; Chang, Guobin; Ma, Teng; Wang, Hongzhi; Chen, Jing; Li, Zhiteng; Guo, Xiaomin; Wan, Fang; Ren, Lichen; Lu, Wei; Chen, Guohong

2016-03-01

Piwil1 mediates spermatogenesis and ensures stable cell division rates in germline cells in mammals. However, the involvement of Piwil1 in poultry spermatogenesis and meiosis is poorly understood. In the present study, we used TaqMan RT-qPCR to characterize Piwil1 mRNA expression in different types of spermatogenic cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia cells (Sa), tetraploid cells (Tp), round sperm cells (Rs), mature sperm, and in PGCs treated with retinoic acid. Our results revealed that Piwil1 is differentially expressed during spermatogenesis in chicken. Compared to PGCs, SSCs, Tp, and Sa, Rs cells presented the highest Piwil1 mRNA expression levels. Retinoic acid significantly upregulated Piwil1 and Stra8 mRNA expression as well as Piwil1 levels in chicken PGCs. In addition, retinoic acid induced PGCs to progress through all the meiotic stages, eventually leading to haploid cell formation, which was determined using flow cytometry and western blot analysis. Taken together, our results showed that during spermatogenesis, Piwil1 was first expressed at low levels in germ stem cells, PGCs, and SSCs. Its expression levels increased during later meiosis stages. Finally, no expression was detected in mature sperm after meiosis. Treatment of PGCs with retinoic acid further demonstrated that Piwil1 plays a key role in meiosis during chicken spermatogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of classical components of the renin-angiotensin system in the human eye.

PubMed

White, Andrew J R; Cheruvu, Sarat C; Sarris, Maria; Liyanage, Surabhi S; Lumbers, Eugenie; Chui, Jeanie; Wakefield, Denis; McCluskey, Peter J

2015-03-01

The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions. © The Author(s) 2014.

Prostate-specific membrane antigen in breast cancer: a comprehensive evaluation of expression and a case report of radionuclide therapy.

PubMed

Tolkach, Yuri; Gevensleben, Heidrun; Bundschuh, Ralph; Koyun, Aydan; Huber, Daniela; Kehrer, Christina; Hecking, Thomas; Keyver-Paik, Mignon-Denise; Kaiser, Christina; Ahmadzadehfar, Hojjat; Essler, Markus; Kuhn, Walther; Kristiansen, Glen

2018-06-01

Prostate-specific membrane antigen (PSMA), a protein product of the folate hydrolase 1 (FOLH1) gene, is gaining increasing acceptance as a target for positron emission tomography/computer tomography (PET/CT) imaging in patients with several cancer types, including breast cancer. So far, PSMA expression in breast cancer endothelia has not been sufficiently characterized. This study comprised 315 cases of invasive carcinoma of no special type (NST) and lobular breast cancer (median follow-up time 9.0 years). PSMA expression on tumor endothelia was detected by immunohistochemistry. Further, vascular mRNA expression of the FOLH1 gene (PSMA) was investigated in a cohort of patients with invasive breast cancer provided by The Cancer Genome Atlas (TCGA). Sixty percent of breast cancer cases exhibited PSMA-positive endothelia with higher expression rates in tumors of higher grade, NST subtype with Her2-positivity, and lack of hormone receptors. These findings were confirmed on mRNA expression levels. The highest PSMA rates were observed in triple-negative carcinomas (4.5 × higher than in other tumors). Further, a case of a patient with metastatic breast cancer showing PSMA expression in PET/CT imaging and undergoing PSMA radionuclide therapy is discussed in detail. This study provides a rationale for the further development of PSMA-targeted imaging in breast cancer, especially in triple-negative tumors.

Specialized Cortex Glial Cells Accumulate Lipid Droplets in Drosophila melanogaster.

PubMed

Kis, Viktor; Barti, Benjámin; Lippai, Mónika; Sass, Miklós

2015-01-01

Lipid droplets (LDs) are common organelles of the majority of eukaryotic cell types. Their biological significance has been extensively studied in mammalian liver cells and white adipose tissue. Although the central nervous system contains the highest relative amount and the largest number of different lipid species, neither the spatial nor the temporal distribution of LDs has been described. In this study, we used the brain of the fruitfly, Drosophila melanogaster, to investigate the neuroanatomy of LDs. We demonstrated that LDs are exclusively localised in glial cells but not in neurons in the larval nervous system. We showed that the brain's LD pool, rather than being constant, changes dynamically during development and reaches its highest value at the beginning of metamorphosis. LDs are particularly enriched in cortex glial cells located close to the brain surface. These specialized superficial cortex glial cells contain the highest amount of LDs among glial cell types and encapsulate neuroblasts and their daughter cells. Superficial cortex glial cells, combined with subperineurial glial cells, express the Drosophila fatty acid binding protein (Dfabp), as we have demonstrated through light- and electron microscopic immunocytochemistry. To the best of our best knowledge this is the first study that describes LD neuroanatomy in the Drosophila larval brain.

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

USGS Publications Warehouse

Alexander, Courtney R.; Hooper, Michael J.; Cacela, Dave; Smelker, Kim D.; Calvin, Caleshia S.; Dean, Karen M.; Bursian, Steve J.; Cunningham, Fred L.; Hanson-Dorr, Katie C.; Horak, Katherine E.; Isanhart, John P.; Link, Jane E.; Shriner, Susan A.; Godard-Codding, Céline A.J.

2017-01-01

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20 ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5 ml/kg bw oil or 10 ml/kg bw oil, 21 day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10 ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Transcription factor AtTCP14 regulates embryonic growth potential during seed germination in Arabidopsis thaliana.

PubMed

Tatematsu, Kiyoshi; Nakabayashi, Kazumi; Kamiya, Yuji; Nambara, Eiji

2008-01-01

To understand the molecular mechanisms underlying regulation of seed germination, we searched enriched cis elements in the upstream regions of Arabidopsis genes whose transcript levels increased during seed germination. Using available published microarray data, we found that two cis elements, Up1 or Up2, which regulate outgrowth of Arabidopsis axillary shoots, were significantly over-represented. Classification of Up1- and Up2-containing genes by gene ontology revealed that protein synthesis-related genes, especially ribosomal protein genes, were highly over-represented. Expression analysis using a reporter gene driven by a synthetic promoter regulated by these elements showed that the Up1 is necessary and sufficient for germination-associated gene induction, whereas Up2 acts as an enhancer of Up1. Up1-mediated gene expression was suppressed by treatments that blocked germination. Up1 is almost identical to the site II motif, which is the predicted target of TCP transcription factors. Of 24 AtTCP genes, AtTCP14, which showed the highest transcript level just prior to germination, was functionally characterized to test its involvement in the regulation of seed germination. Transposon-tagged lines for AtTCP14 showed delayed germination. In addition, germination of attcp14 mutants exhibited hypersensitivity to exogenously applied abscisic acid and paclobutrazol, an inhibitor of gibberellin biosynthesis. AtTCP14 was predominantly expressed in the vascular tissues of the embryo, and affected gene expression in radicles in a non-cell-autonomous manner. Taken together, these results indicate that AtTCP14 regulates the activation of embryonic growth potential in Arabidopsis seeds.

Dendritic sodium channels promote active decorrelation and reduce phase locking to parkinsonian input oscillations in model globus pallidus neurons

PubMed Central

Edgerton, Jeremy R.; Jaeger, Dieter

2011-01-01

Correlated firing among populations of neurons is present throughout the brain and is often rhythmic in nature, observable as an oscillatory fluctuation in the local field potential. Although rhythmic population activity is believed to be critical for normal function in many brain areas, synchronized neural oscillations are associated with disease states in other cases. In the globus pallidus (GP in rodents, homolog of the primate GPe), pairs of neurons generally have uncorrelated firing in normal animals despite an anatomical organization suggesting that they should receive substantial common input. By contrast, correlated and rhythmic GP firing is observed in animal models of Parkinson's disease (PD). Based in part on these findings it has been proposed that an important part of basal ganglia function is active decorrelation, whereby redundant information is compressed. Mechanisms that implement active decorrelation, and changes that cause it to fail in PD, are subjects of great interest. Rat GP neurons express fast, transient voltage-dependent sodium channels (NaF channels) in their dendrites, with the expression level being highest near asymmetric synapses. We recently showed that the dendritic NaF density strongly influences the responsiveness of model GP neurons to synchronous excitatory inputs. In the present study we use rat GP neuron models to show that dendritic NaF channel expression is a potential cellular mechanism of active decorrelation. We further show that model neurons with lower dendritic NaF channel expression have a greater tendency to phase lock with oscillatory synaptic input patterns like those observed in PD. PMID:21795543

Purification, biochemical characterization, and genetic cloning of the phytase produced by Burkholderia sp. strain a13.

PubMed

Graminho, Eduardo Rezende; Takaya, Naoki; Nakamura, Akira; Hoshino, Takayuki

2015-01-01

A phytase-producing bacterium, Burkholderia sp. a13 (JCM 30421), was isolated from Lake Kasumigaura by enrichment cultivation using minimum medium containing phytic acid as the sole phosphorus source. The phytase production by strain a13 was induced by the presence of phytic acid and repressed by the addition of glucose. The purified enzyme had a molecular weight of 44 kDa and a phytase activity of 174 μmol min(-1) mg(-1). The enzyme showed broad substrate specificity, but the highest activity was observed with phytic acid. The enzyme activity was strongly inhibited by Cu(2+), Zn(2+), Hg(2+), and iodoacetic acid, indicating the requirement of a thiol group for the activity. Genetic cloning reveals that the mature portion of this enzyme consists of 428 amino acids with a calculated molecular weight of 46 kDa. The amino acid sequence showed the highest similarity to the phytase produced by Hafnia alvei with 48% identity; it also contained histidine acid phosphatase (HAP) motifs (RHGXRXP and HD), indicating the classification of this enzyme in the HAP phytase family. We have successfully expressed the cloned gene in Escherichia coli from its putative initiation codon, showing that the gene actually encodes the phytase.

High lactic acid and fructose production via Mn2+-mediated conversion of inulin by Lactobacillus paracasei.

PubMed

Petrov, Kaloyan; Popova, Luiza; Petrova, Penka

2017-06-01

Lactobacillus paracasei DSM 23505 is able to produce high amounts of lactic acid (LA) by simultaneous saccharification and fermentation (SSF) of inulin. Aiming to obtain the highest possible amounts of LA and fructose, the present study is devoted to evaluate the impact of bivalent metal ions on the process of inulin conversion. It was shown that Mn 2+ strongly increases the activity of the purified key enzyme β-fructosidase. In vivo, batch fermentation kinetics revealed that the high Mn 2+ concentrations accelerated inulin hydrolysis by raise of the inulinase activity, and increased sugars conversion to LA through enhancement of the whole glycolytic flux. The highest LA concentration and yield were reached by addition of 15 mM Mn 2+ -151 g/L (corresponding to 40% increase) and 0.83 g/g, respectively. However, the relative quantification by real-time reverse transcription assay showed that the presence of Mn 2+ decreases the expression levels of fosE gene encoding β-fructosidase. Contrariwise, the full exclusion of metal ions resulted in fosE gene expression enhancement, blocked fructose transport, and hindered fructose conversion thus leading to huge fructose accumulation. During fed-batch with optimized medium and fermentation parameters, the fructose content reached 35.9% (w/v), achieving yield of 467 g fructose from 675 g inulin containing chicory flour powder (0.69 g/g). LA received in course of the batch fermentation and fructose gained by the fed-batch are the highest amounts ever obtained from inulin, thus disclosing the key role of Mn 2+ as a powerful tool to guide inulin conversion to targeted bio-chemicals.

Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

PubMed Central

Park, Jong-Won; Beyene, Getu; Buenrostro-Nava, Marco T.; Molina, Joe; Wang, Xiaofeng; Ciomperlik, Jessica J.; Manabayeva, Shuga A.; Alvarado, Veria Y.; Rathore, Keerti S.; Scholthof, Herman B.; Mirkov, T. Erik

2013-01-01

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48–96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane. PMID:23799071

CB1 Cannabinoid Receptor Expression in the Striatum: Association with Corticostriatal Circuits and Developmental Regulation

PubMed Central

Van Waes, Vincent; Beverley, Joel A.; Siman, Homayoun; Tseng, Kuei Y.; Steiner, Heinz

2012-01-01

Corticostriatal circuits mediate various aspects of goal-directed behavior and are critically important for basal ganglia-related disorders. Activity in these circuits is regulated by the endocannabinoid system via stimulation of CB1 cannabinoid receptors. CB1 receptors are highly expressed in projection neurons and select interneurons of the striatum, but expression levels vary considerably between different striatal regions (functional domains). We investigated CB1 receptor expression within specific corticostriatal circuits by mapping CB1 mRNA levels in striatal sectors defined by their cortical inputs in rats. We also assessed changes in CB1 expression in the striatum during development. Our results show that CB1 expression is highest in juveniles (P25) and then progressively decreases toward adolescent (P40) and adult (P70) levels. At every age, CB1 receptors are predominantly expressed in sensorimotor striatal sectors, with considerably lower expression in associative and limbic sectors. Moreover, for most corticostriatal circuits there is an inverse relationship between cortical and striatal expression levels. Thus, striatal sectors with high CB1 expression (sensorimotor sectors) tend to receive inputs from cortical areas with low expression, while striatal sectors with low expression (associative/limbic sectors) receive inputs from cortical regions with higher expression (medial prefrontal cortex). In so far as CB1 mRNA levels reflect receptor function, our findings suggest differential CB1 signaling between different developmental stages and between sensorimotor and associative/limbic circuits. The regional distribution of CB1 receptor expression in the striatum further suggests that, in sensorimotor sectors, CB1 receptors mostly regulate GABA inputs from local axon collaterals of projection neurons, whereas in associative/limbic sectors, CB1 regulation of GABA inputs from interneurons and glutamate inputs may be more important. PMID:22416230

Identification of a locus control region for quadruplicated green-sensitive opsin genes in zebrafish

PubMed Central

Tsujimura, Taro; Chinen, Akito; Kawamura, Shoji

2007-01-01

Duplication of opsin genes has a crucial role in the evolution of visual system. Zebrafish have four green-sensitive (RH2) opsin genes (RH2–1, RH2–2, RH2–3, and RH2–4) arrayed in tandem. They are expressed in the short member of the double cones (SDC) but differ in expression areas in the retina and absorption spectra of their encoding photopigments. The shortest and the second shortest wavelength subtypes, RH2–1 and RH2–2, are expressed in the central-to-dorsal retina. The longer wavelength subtype, RH2–3, is expressed circumscribing the RH2–1/RH2–2 area, and the longest subtype, RH2–4, is expressed further circumscribing the RH2–3 area and mainly occupying the ventral retina. The present report shows that a 0.5-kb region located 15 kb upstream of the RH2 gene array is an essential regulator for their expression. When the 0.5-kb region was deleted from a P1-artificial chromosome (PAC) clone encompassing the four RH2 genes and when one of these genes was replaced with a reporter GFP gene, the GFP expression in SDCs was abolished in the zebrafish to which a series of the modified PAC clones were introduced. Transgenic studies also showed that the 0.5-kb region conferred the SDC-specific expression for promoters of a non-SDC (UV opsin) and a nonretinal (keratin 8) gene. Changing the location of the 0.5-kb region in the PAC clone conferred the highest expression for its proximal gene. The 0.5-kb region was thus designated as RH2-LCR analogous to the locus control region of the L-M opsin genes of primates. PMID:17646658

Age-related alterations in basal expression and in vitro, tumour necrosis factor alpha mediated, upregulation of CD11b.

PubMed

Armstrong, M E; Alexander, H D; Ritchie, J L; McMillan, S A; Rea, I M

2001-01-01

The beta(2-)integrin CD11b (Mac-1) plays a crucial role in the firm attachment of leucocytes to the endothelium during the inflammatory response. This study aimed to determine whether the increased incidence of infections witnessed in elderly individuals compared to their younger counterparts was associated with deficiencies in basal expression and/or upregulation of CD11b. Flow cytometry was used to measure CD11b expression, before and after in vitro tumour necrosis factor alpha (TNF-alpha) stimulation, on neutrophils, monocytes and lymphocytes from healthy volunteers aged less than 36 years and Senieur-approximated 70-85 and over 85 year olds. The TNF-alpha levels in serum were measured using a commercially available enzyme-linked immunoassay technique. The basal expression of CD11b on monocytes and lymphocytes was highest in the 70-85-year-olds and lowest in the > 85-year-olds. Following in vitro stimulation using low (10 IU) and high (100 IU) TNF-alpha concentrations, subjects > 85 years consistently showed significantly lower increases in CD11b expression on each of the three cell types. The maximal increase in CD11b expression was in the 70-85-year age group for neutrophils and monocytes and in < 36-year-olds for lymphocytes. Serum TNF-alpha was significantly higher in the elderly groups. Regression analysis showed a significant association between TNF-alpha and expression of CD11b on lymphocytes before and after TNF-alpha stimulation and for neutrophils before stimulation. The results of this study suggest that CD11b expression on leucocytes may not be consistent throughout life. Such age-related changes could compromise the inflammatory response, rendering individuals > 85 years old more susceptible to infections. Alternatively, the lower levels of CD11b expression in this group may represent downregulation and protection against excess leucocyte activation within the vascular system and may, therefore, provide a mechanism for successful ageing. Copyright 2001 S. Karger AG, Basel

Drug transporter gene expression in human colorectal tissue and cell lines: modulation with antiretrovirals for microbicide optimization.

PubMed

Mukhopadhya, Indrani; Murray, Graeme I; Berry, Susan; Thomson, John; Frank, Bruce; Gwozdz, Garry; Ekeruche-Makinde, Julia; Shattock, Robin; Kelly, Charles; Iannelli, Francesco; Pozzi, Gianni; El-Omar, Emad M; Hold, Georgina L; Hijazi, Karolin

2016-02-01

The objectives of this study were to comprehensively assess mRNA expression of 84 drug transporters in human colorectal biopsies and six representative cell lines, and to investigate the alteration of drug transporter gene expression after exposure to three candidate microbicidal antiretroviral (ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium. The outcome of the objectives informs development of optimal ARV-based microbicidal formulations for prevention of HIV-1 infection. Drug transporter mRNA expression was quantified from colorectal biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression was quantified in Caco-2 cells and colorectal explants after induction with ARVs. Data were analysed using Pearson's product moment correlation (r), hierarchical clustering and principal component analysis (PCA). Expression of 58 of the 84 transporters was documented in colorectal biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the highest expression. No difference was noted between individual subjects when analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r = 0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by immunohistochemical staining. Similarity between colorectal tissue and cell-line drug transporter gene expression was variable (r = 0.64-0.84). PCA showed distinct clustering of human colorectal biopsy samples, with the Caco-2 cells defined as the best surrogate system. Induction of Caco-2 cell lines with ARV drugs suggests that darunavir-based microbicides incorporating tenofovir may result in drug-drug interactions likely to affect distribution of individual drugs to sub-epithelial target cells. These findings will help optimize complex formulations of rectal microbicides to realize their full potential as an effective approach for pre-exposure prophylaxis against HIV-1 infection. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Overexpression of PvPin1, a Bamboo Homolog of PIN1-Type Parvulin 1, Delays Flowering Time in Transgenic Arabidopsis and Rice

PubMed Central

Zheng, Zhigang; Yang, Xiaoming; Fu, Yaping; Zhu, Longfei; Wei, Hantian; Lin, Xinchun

2017-01-01

Because of the long and unpredictable flowering period in bamboo, the molecular mechanism of bamboo flowering is unclear. Recent study showed that Arabidopsis PIN1-type parvulin 1 (Pin1At) is an important floral activator and regulates floral transition by facilitating the cis/trans isomerization of the phosphorylated Ser/Thr residues preceding proline motifs in suppressor of overexpression of CO 1 (SOC1) and agamous-like 24 (AGL24). Whether bamboo has a Pin1 homolog and whether it works in bamboo flowering are still unknown. In this study, we cloned PvPin1, a homolog of Pin1At, from Phyllostachys violascens (Bambusoideae). Bioinformatics analysis showed that PvPin1 is closely related to Pin1-like proteins in monocots. PvPin1 was widely expressed in all tested bamboo tissues, with the highest expression in young leaf and lowest in floral bud. Moreover, PvPin1 expression was high in leaves before bamboo flowering then declined during flower development. Overexpression of PvPin1 significantly delayed flowering time by downregulating SOC1 and AGL24 expression in Arabidopsis under greenhouse conditions and conferred a significantly late flowering phenotype by upregulating OsMADS56 in rice under field conditions. PvPin1 showed subcellular localization in both the nucleus and cytolemma. The 1500-bp sequence of the PvPin1 promoter was cloned, and cis-acting element prediction showed that ABRE and TGACG-motif elements, which responded to abscisic acid (ABA) and methyl jasmonate (MeJA), respectively, were characteristic of P. violascens in comparison with Arabidopsis. On promoter activity analysis, exogenous ABA and MeJA could significantly inhibit PvPin1 expression. These findings suggested that PvPin1 may be a repressor in flowering, and its delay of flowering time could be regulated by ABA and MeJA in bamboo. PMID:28951734

A novel class I Chitinase from Hippophae rhamnoides: Indications for participating in ICE-CBF cold stress signaling pathway.

PubMed

Kashyap, Prakriti; Deswal, Renu

2017-06-01

Plant chitinases are the members of PR (Pathogenesis related) proteins family and protect plants from biotic and abiotic stress. A novel chitinase HrCHI1 (Accession number JQ289153) of 954bp ORF encoding 317 amino acids protein was cloned, expressed and characterized from seabuckthorn, a cold/freeze tolerant shrub. The 3D structure (predicted with I-TASSER server) showed highest homology with Oryza sativa class I chitinase (PDB 2dkvA). Putative promoter region (obtained by genome walking) showed GCC box, E-boxes, the binding site for bHLH proteins and DRE elements, the CBF (C-repeat binding factor) binding site besides TATA and CAAT boxes. The gel shift assay with the nuclear extract indicated that the HrCHI1 might be participating in CBF/ERF dependent cold stress signaling pathway. The quantitative transcript profiling supported this observation as cold induced expression of HrCBF peaked earlier (at 1h) while HrCHI1 peaked latter (after 3h) indicating HrCHI1 expression might be induced by HrCBF. Further, HrCHI1 expression was methyl jasmonate (MeJa) dependent and salicylic acid (SA) independent. HrCHI1 was expressed in E. coli and purified using chitin affinity chromatography. It showed 512U/mg chitinase hydrolytic activity and resolved as a 34kDa spot with a slightly basic pI (8.5) on a 2-D gel. The E. coli cells containing recombinant chitinase showed higher rate of growth in cold in comparison with the cells containing the empty vector. In conclusion, we have isolated and characterized a cold responsive basic class I chitinase which is regulated by MeJa and seems to be functioning via CBF/ERF dependent cold stress signaling pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Overexpression of PvPin1, a Bamboo Homolog of PIN1-Type Parvulin 1, Delays Flowering Time in Transgenic Arabidopsis and Rice.

PubMed

Zheng, Zhigang; Yang, Xiaoming; Fu, Yaping; Zhu, Longfei; Wei, Hantian; Lin, Xinchun

2017-01-01

Because of the long and unpredictable flowering period in bamboo, the molecular mechanism of bamboo flowering is unclear. Recent study showed that Arabidopsis PIN1-type parvulin 1 (Pin1At) is an important floral activator and regulates floral transition by facilitating the cis/trans isomerization of the phosphorylated Ser/Thr residues preceding proline motifs in suppressor of overexpression of CO 1 (SOC1) and agamous-like 24 (AGL24). Whether bamboo has a Pin1 homolog and whether it works in bamboo flowering are still unknown. In this study, we cloned PvPin1 , a homolog of Pin1At , from Phyllostachys violascens (Bambusoideae). Bioinformatics analysis showed that PvPin1 is closely related to Pin1-like proteins in monocots. PvPin1 was widely expressed in all tested bamboo tissues, with the highest expression in young leaf and lowest in floral bud. Moreover, PvPin1 expression was high in leaves before bamboo flowering then declined during flower development. Overexpression of PvPin1 significantly delayed flowering time by downregulating SOC1 and AGL24 expression in Arabidopsis under greenhouse conditions and conferred a significantly late flowering phenotype by upregulating OsMADS56 in rice under field conditions. PvPin1 showed subcellular localization in both the nucleus and cytolemma. The 1500-bp sequence of the PvPin1 promoter was cloned, and cis -acting element prediction showed that ABRE and TGACG-motif elements, which responded to abscisic acid (ABA) and methyl jasmonate (MeJA), respectively, were characteristic of P. violascens in comparison with Arabidopsis . On promoter activity analysis, exogenous ABA and MeJA could significantly inhibit PvPin1 expression. These findings suggested that PvPin1 may be a repressor in flowering, and its delay of flowering time could be regulated by ABA and MeJA in bamboo.

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

PubMed Central

Mayer-Jaekel, R E; Baumgartner, S; Bilbe, G; Ohkura, H; Glover, D M; Hemmings, B A

1992-01-01

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner. Images PMID:1320961

The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human renal cell carcinoma cell growth.

PubMed

Su, Ying; Zhao, An; Cheng, Guoping; Xu, Jingjing; Ji, Enming; Sun, Wenyong

2017-07-04

Renal cell carcinoma (RCC) is the highest mortality rate of the genitourinary cancers, and the treatment options are very limited. Thus, identification of molecular mechanisms underlying RCC tumorigenesis, is critical for identifying biomarkers for RCC diagnosis and prognosis. To validate whether the IGF-I/JAK2-STAT3/miR-21 signaling pathway is associated with human RCC cell growth. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels, respectively. The MTT assay was performed to determine cell survival rate. The Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. We employed RCC tissues and cell lines (A498; ACHN; Caki-1; Caki-2 and 786-O) in the study. IGF-I, and its inhibitor (NT-157) were administrated to detect the effects of IGF-I on the expression of miR-21 and p-JAK2. JAK2 inhibitor (AG490), and si-STAT3 were used to detect the effects of JAK2/STAT3 signaling pathway on the expression of miR-21. In our study, we firstly showed that the expression levels of IGF-I and miR-21 were up-regulated in RCC tissues and cell lines. After exogenous IGF-I treatment, the expression levels of miR-21, p-IGF-IR and p-JAK2 were significantly increased, whereas NT-157 treatment showed the reversed results. Further study indicated that JAK2 inhibitor or si-STAT3 significantly reversed the IGF-I-induced miR-21 expression level. Finally, we found that IGF-I treatment significantly prompted human RCC cell survival and inhibited cell apoptosis, and NT-157 treatment showed the reversed results. The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human RCC cell growth.

Heterogeneity of glutamatergic and GABAergic release machinery in cerebral cortex: analysis of synaptogyrin, vesicle-associated membrane protein, and syntaxin.

PubMed

Bragina, L; Giovedì, S; Barbaresi, P; Benfenati, F; Conti, F

2010-02-03

To define whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, we studied the degree of co-localization of synaptogyrin (SGYR) 1 and 3, vesicle-associated membrane protein (VAMP) 1 and 2, syntaxin (STX) 1A and 1B in vesicular glutamate transporter (VGLUT)1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta and synaptic vesicles in the rat cerebral cortex. Co-localization studies showed that SGYR1 and 3 were expressed in about 90% of VGLUT1+, 70% of VGLUT2+ and 80% of VGAT+ puncta; VAMP1 was expressed in approximately 45% of VGLUT1+, 55% of VGLUT2+, and 80% of VGAT+ puncta; VAMP2 in about 95% of VGLUT1+, 75% of VGLUT2+, and 80% of VGAT+ puncta; STX1A in about 65% of VGLUT1+, 30% of VGLUT2+, and 3% of VGAT+ puncta, and STX1B in approximately 45% of VGLUT1+, 35% of VGLUT2+, and 70% of VGAT+ puncta. Immunoisolation studies showed that while STX1A was completely segregated and virtually absent from VGAT synaptic vesicles, STX1B, VAMP1/VAMP2, SGYR1/SGYR3 showed a similar pattern with the highest expression in VGLUT1 immunoisolated vesicles and the lowest in VGAT immunoisolated vesicles. Moreover, we studied the localization of STX1B at the electron microscope and found that a population of axon terminals forming symmetric synapses were STX1B-positive.These results extend our previous observations on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of glutamatergic and GABAergic release machinery can be contributed by both the presence or absence of a given protein in a nerve terminal and the amount of protein expressed by synaptic vesicles. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

High Heregulin Expression Is Associated with Activated HER3 and May Define an Actionable Biomarker in Patients with Squamous Cell Carcinomas of the Head and Neck

PubMed Central

Shames, David S.; Carbon, Juliet; Walter, Kim; Jubb, Adrian M.; Kozlowski, Cleopatra; Januario, Tom; An, Do; Fu, Ling; Xiao, Yuanyuan; Raja, Rajiv; Jiang, Brittany; Malekafzali, Ashi; Stern, Howard; Settleman, Jeff; Wilson, Timothy R.; Hampton, Garret M.; Yauch, Robert L.; Pirzkall, Andrea; Amler, Lukas C.

2013-01-01

Purpose Tumors with oncogenic dependencies on the HER family of receptor tyrosine kinases (RTKs) often respond well to targeted inhibition. Our previous work suggested that many cell lines derived from squamous cell carcinomas of the head and neck (SCCHNs) depend on autocrine signaling driven by HER2/3 dimerization and high-level co-expression of HRG. Additionally, results from a Phase I trial of MEHD7495A, a dual-action antibody that blocks ligand binding to EGFR and HER3, suggest that high-level HRG expression was associated with clinical response in SCCHN patients. Here we explore the hypothesis that high-level HRG expression defines a subpopulation of SCCHNs with activated HER3. Experimental Design qRT-PCR expression profiling was performed on >750 tumors of diverse origin, including >150 therapy-naïve, primary, and recurrent SCCHNs. Activated HER3, defined by immunoprecipitation of phospho-HER3, was compared to HRG expression in SCCHN samples. Paracrine versus autocrine expression was evaluated using RNA-in situ hybridization. Results SCCHN tumors express the highest levels of HRG compared to a diverse collection of other tumor types. We show that high HRG expression is associated with activated HER3, whereas low HRG expression is associated with low HER3 activation in SCCHN tumors. Furthermore, HRG expression is higher in recurrent SCCHN compared to patient-matched therapy naïve specimens. Conclusions HRG expression levels define a biologically distinct subset of SCCHN patients. We propose that high-level expression of HRG is associated with constitutive activation of HER3 in SCCHN and thus defines an actionable biomarker for interventions targeting HER3. PMID:23468880

Deregulation of Genes Associated with Alternate Drug Resistance Mechanisms in Mycobacterium tuberculosis.

PubMed

Sriraman, Kalpana; Nilgiriwala, Kayzad; Saranath, Dhananjaya; Chatterjee, Anirvan; Mistry, Nerges

2018-04-01

Alternate mechanisms of drug resistance involving intrinsic defense pathways play an important role in development of drug resistance. Deregulation of drug efflux, cellular metabolism, and DNA repair have been indicated to have effect on drug tolerance and persistence. Here we chose eight genes from these pathways to investigate their association with development of multidrug resistance (MDR). We generated mono drug resistant and MDR strains of rifampicin and isoniazid and examined the differential expression of genes belonging to efflux, DNA repair and cell wall lipid synthesis pathways. Rv1687c, recB, ppsD and embC genes showed significant (P <0.05) upregulation in mono-resistant (both rifampicin and isoniazid) as well as MDR strains. mmr showed significant upregulation with rifampicin resistance while Rv1457c showed significant upregulation only with mono-resistant strains. Highest expression change was observed with Rv1687c and ppsD. The study identified potential key genes that are significantly associated with development of drug resistance in vitro. These genes may help identify clinical strains predisposed to acquiring drug resistance in patients during the course of treatment or help in management of MDR forms of tuberculosis.

The Role of Emotional Reactivity, Self-regulation, and Puberty in Adolescents' Prosocial Behaviors

PubMed Central

Carlo, Gustavo; Crockett, Lisa J.; Wolff, Jennifer M.; Beal, Sarah J.

2017-01-01

This study was designed to examine the roles of emotional reactivity, self-regulation, and pubertal timing in prosocial behaviors during adolescence. Participants were 850 sixth graders (50% female, Mean age = 11.03, SD = .17) who were followed up at age 15. In hierarchical regression models, measures of emotional reactivity, self-regulation, pubertal timing and their interactions were used to predict (concurrently and over time) adolescents’ prosocial behaviors in the home and with peers. Overall, the findings provide evidence for pubertal and temperament based predictors of prosocial behaviors expressed in different contexts. Self-regulation was positively related to both forms of prosocial behavior, concurrently and longitudinally. Emotional reactivity showed moderately consistent effects, showing negative concurrent relations to prosocial behavior with peers and negative longitudinal relations (four years later) to prosocial behavior at home. Some curvilinear effects of temperament on prosocial behaviors were also found. Effects of pubertal timing were found to interact with gender, such that boys who were early maturers showed the highest levels of prosocial behavior at home concurrently. Discussion focuses on the role of temperament-based mechanisms in the expression of prosocial behaviors in different contexts in adolescence. PMID:28316370

Social issues in high school biology textbooks: 1963- 1983

NASA Astrophysics Data System (ADS)

Rosenthal, Dorothy B.

Twenty-two high school biology textbooks published between 1963 and 1983 were analyzed for their treatment of social issues. Textbooks were selected from among those used most frequently by teachers and/or having the highest sales. The textbooks were read in random order and the amount of space, to the nearest tenth of a page, devoted to each social issue was expressed as a percentage of total length of text. The results showed that attention to social issues decreased between 1963 and 1983 in the textbooks studied. The implications of these results for biology education in the 1980s are discussed.

The complexity of gene expression dynamics revealed by permutation entropy

PubMed Central

2010-01-01

Background High complexity is considered a hallmark of living systems. Here we investigate the complexity of temporal gene expression patterns using the concept of Permutation Entropy (PE) first introduced in dynamical systems theory. The analysis of gene expression data has so far focused primarily on the identification of differentially expressed genes, or on the elucidation of pathway and regulatory relationships. We aim to study gene expression time series data from the viewpoint of complexity. Results Applying the PE complexity metric to abiotic stress response time series data in Arabidopsis thaliana, genes involved in stress response and signaling were found to be associated with the highest complexity not only under stress, but surprisingly, also under reference, non-stress conditions. Genes with house-keeping functions exhibited lower PE complexity. Compared to reference conditions, the PE of temporal gene expression patterns generally increased upon stress exposure. High-complexity genes were found to have longer upstream intergenic regions and more cis-regulatory motifs in their promoter regions indicative of a more complex regulatory apparatus needed to orchestrate their expression, and to be associated with higher correlation network connectivity degree. Arabidopsis genes also present in other plant species were observed to exhibit decreased PE complexity compared to Arabidopsis specific genes. Conclusions We show that Permutation Entropy is a simple yet robust and powerful approach to identify temporal gene expression profiles of varying complexity that is equally applicable to other types of molecular profile data. PMID:21176199

Suppression of GHS-R in AgRP neurons mitigates diet-induced obesity by activating thermogenesis

USDA-ARS?s Scientific Manuscript database

Ghrelin, an orexigenic hormone released primarily from the gut, signals the hypothalamus to stimulate growth hormone release, enhance appetite and promote weight gain. The ghrelin receptor, aka Growth Hormone Secretagogue Receptor (GHS-R), is highly expressed in the brain, with highest expression in...

Comparative gene expression profiling of ADAMs, MMPs, TIMPs, EMMPRIN, EGF-R and VEGFA in low grade meningioma.

PubMed

Rooprai, Harcharan K; Martin, Andrew J; King, Andrew; Appadu, Usha D; Jones, Huw; Selway, Richard P; Gullan, Richard W; Pilkington, Geoffrey J

2016-12-01

MMPs (matrix metalloproteinases), ADAMs (a disintegrin and metalloproteinase) and TIMPs (tissue inhibitors of metalloproteinases) are implicated in invasion and angiogenesis: both are tissue remodeling processes involving regulated proteolysis of the extracellular matrix, growth factors and their receptors. The expression of these three groups and their correlations with clinical behaviour has been reported in gliomas but a similar comprehensive study in meningiomas is lacking. In this study, we aimed to evaluate the patterns of expression of 23 MMPs, 4 TIMPs, 8 ADAMs, selective growth factors and their receptors in 17 benign meningiomas using a quantitative real-time polymerase chain reaction (qPCR). Results indicated very high gene expression of 13 proteases, inhibitors and growth factors studied: MMP2 and MMP14, TIMP-1, -2 and -3, ADAM9, 10, 12, 15 and 17, EGF-R, EMMPRIN and VEGF-A, in almost every meningioma. Expression pattern analysis showed several positive correlations between MMPs, ADAMs, TIMPs and growth factors. Furthermore, our findings suggest that expression of MMP14, ADAM9, 10, 12, 15 and 17, TIMP-2, EGF-R and EMMPRIN reflects histological subtype of meningioma such that fibroblastic subtype had the highest mRNA expression, transitional subtype was intermediate and meningothelial type had the lowest expression. In conclusion, this is the first comprehensive study characterizing gene expression of 8 ADAMs in meningiomas. These neoplasms, although by histological definition benign, have invasive potential. Taken together, the selected elevated gene expression pattern may serve to identify targets for therapeutic intervention or indicators of biological progression and recurrence.

Characterization of the Expression of the Petunia Glycine-Rich Protein-1 Gene Product 1

PubMed Central

Condit, Carol M.; McLean, B. Gail; Meagher, Richard B.

1990-01-01

We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178-181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:16667509

Intermittent hydrostatic pressure enhances growth factor-induced chondroinduction of human adipose-derived mesenchymal stem cells.

PubMed

Safshekan, Farzaneh; Tafazzoli-Shadpour, Mohammad; Shokrgozar, Mohammad Ali; Haghighipour, Nooshin; Mahdian, Reza; Hemmati, Alireza

2012-12-01

Hydrostatic pressure (HP) plays an essential role in regulating function of chondrocytes and chondrogenic differentiation. The objective of this study was to examine effects of intermittent HP on chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs) in the presence or absence of chemical chondrogenic medium. Cells were isolated from abdominal fat tissue and confirmed for expression of ASC surface proteins and differentiation potential. Passage 3 pellets were treated with chemical (growth factor), mechanical (HP of 5 MPa and 0.5 Hz with duration of 4 h/day for 7 consecutive days), and combined chemical-mechanical stimuli. Using real-time polymerase chain reaction, the expression of Sox9, collagen II, and aggrecan as three major chondrogenic markers were quantified among three experimental groups and compared to those of stem cells and human cartilage tissue. In comparison to the chemical and mechanical groups, the chemical-mechanical group showed the highest expression for all three chondrogenic genes close to that of cartilage tissue. Results show the beneficial role of intermittent HP on chondrogenic differentiation of hASCs, and that this loading regime in combination with chondrogenic medium can be used in cartilage tissue engineering. © 2012, Copyright the Authors. Artificial Organs © 2012, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Characterization and expression of genes encoding three small heat shock proteins in Sesamia inferens (Lepidoptera: Noctuidae).

PubMed

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2014-12-12

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae)

PubMed Central

Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

2014-01-01

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens. PMID:25514417

Specific regions of the brain are capable of fructose metabolism.

PubMed

Oppelt, Sarah A; Zhang, Wanming; Tolan, Dean R

2017-02-15

High fructose consumption in the Western diet correlates with disease states such as obesity and metabolic syndrome complications, including type II diabetes, chronic kidney disease, and non-alcoholic fatty acid liver disease. Liver and kidneys are responsible for metabolism of 40-60% of ingested fructose, while the physiological fate of the remaining fructose remains poorly understood. The primary metabolic pathway for fructose includes the fructose-transporting solute-like carrier transport proteins 2a (SLC2a or GLUT), including GLUT5 and GLUT9, ketohexokinase (KHK), and aldolase. Bioinformatic analysis of gene expression encoding these proteins (glut5, glut9, khk, and aldoC, respectively) identifies other organs capable of this fructose metabolism. This analysis predicts brain, lymphoreticular tissue, placenta, and reproductive tissues as possible additional organs for fructose metabolism. While expression of these genes is highest in liver, the brain is predicted to have expression levels of these genes similar to kidney. RNA in situ hybridization of coronal slices of adult mouse brains validate the in silico expression of glut5, glut9, khk, and aldoC, and show expression across many regions of the brain, with the most notable expression in the cerebellum, hippocampus, cortex, and olfactory bulb. Dissected samples of these brain regions show KHK and aldolase enzyme activity 5-10 times the concentration of that in liver. Furthermore, rates of fructose oxidation in these brain regions are 15-150 times that of liver slices, confirming the bioinformatics prediction and in situ hybridization data. This suggests that previously unappreciated regions across the brain can use fructose, in addition to glucose, for energy production. Copyright © 2016 Elsevier B.V. All rights reserved.

Specific regions of the brain are capable of fructose metabolism

PubMed Central

Oppelt, Sarah A.; Zhang, Wanming; Tolan, Dean R.

2017-01-01

High fructose consumption in the Western diet correlates with disease states such as obesity and metabolic syndrome complications, including type II diabetes, chronic kidney disease, and nonalcoholic fatty acid liver disease. Liver and kidneys are responsible for metabolism of 40–60% of ingested fructose, while the physiological fate of the remaining fructose remains poorly understood. The primary metabolic pathway for fructose includes the fructose-transporting solute-like carrier transport proteins 2a (SLC2a or GLUT), including GLUT5 and GLUT9, ketohexokinase (KHK), and aldolase. Bioinformatic analysis of gene expression encoding these proteins (glut5, glut9, khk, and aldoC, respectively) identifies other organs capable of this fructose metabolism. This analysis predicts brain, lymphoreticular tissue, placenta, and reproductive tissues as possible additional organs for fructose metabolism. While expression of these genes is highest in liver, the brain is predicted to have expression levels of these genes similar to kidney. RNA in situ hybridization of coronal slices of adult mouse brains validate the in silico expression of glut5, glut9, khk, and aldoC, and show expression across many regions of the brain, with the most notable expression in the cerebellum, hippocampus, cortex, and olfactory bulb. Dissected samples of these brain regions show KHK and aldolase enzyme activity 5–10 times the concentration of that in liver. Furthermore, rates of fructose oxidation in these brain regions are 15–150 times that of liver slices, confirming the bioinformatics prediction and in situ hybridization data. This suggests that previously unappreciated regions across the brain can use fructose, in addition to glucose, for energy production. PMID:28034722

Glucocorticoid receptor gene expression and promoter CpG modifications throughout the human brain.

PubMed

Cao-Lei, Lei; Suwansirikul, Songkiet; Jutavijittum, Prapan; Mériaux, Sophie B; Turner, Jonathan D; Muller, Claude P

2013-11-01

Glucocorticoids and the glucocorticoid (GR) and mineralocorticoid (MR) receptors have been implicated in many processes, particularly in negative feedback regulation of the hypothalamic-pituitary-adrenal axis. Epigenetically programmed GR alternative promoter usage underlies transcriptional control of GR levels, generation of GR 3' splice variants, and the overall GC response in the brain. No detailed analysis of GR first exons or GR transcript variants throughout the human brain has been reported. Therefore we investigated post mortem tissues from 28 brain regions of 5 individuals. GR first exons were expressed throughout the healthy human brain with no region-specific usage patterns. First exon levels were highly inter-correlated suggesting that they are co-regulated. GR 3' splice variants (GRα and GR-P) were equally distributed in all regions, and GRβ expression was always low. GR/MR ratios showed significant differences between the 28 tissues with the highest ratio in the pituitary gland. Modification levels of individual CpG dinucleotides, including 5-mC and 5-hmC, in promoters 1D, 1E, 1F, and 1H were low, and diffusely clustered; despite significant heterogeneity between the donors. In agreement with this clustering, sum modification levels rather than individual CpG modifications correlated with GR expression. Two-way ANOVA showed that this sum modification was both promoter and brain region specific, but that there was however no promoter*tissue interaction. The heterogeneity between donors may however hide such an interaction. In both promoters 1F and 1H modification levels correlated with GRα expression suggesting that 5-mC and 5-hmC play an important role in fine tuning GR expression levels throughout the brain. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

PubMed

Tuan, Pham Anh; Park, Sang Un

2013-01-01

Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress. Copyright © 2012 Elsevier GmbH. All rights reserved.

Expression analysis of metallothioneins and mineral contents in tomato (Lycopersicon esculentum) under heavy metal stress.

PubMed

Kısa, Dursun; Öztürk, Lokman; Doker, Serhat; Gökçe, İsa

2017-04-01

Heavy metals are considered to be the most important pollutants in the contamination of soils; they adversely affect plant growth and development and cause some physiological and molecular changes. The contamination of agricultural soils by heavy metals has changed the mineral element content of vegetables. Plant metallothioneins (MTs) are thought to have the functional role in heavy metal homeostasis, and they are used as the biomarkers for evaluating environmental pollution. We aimed to evaluate the expression of MT isoforms (MT1, 2, 3 and 4) and some mineral element composition of tomato roots, leaves and fruits exposed to copper and lead. Heavy metal applications increased MT1 and MT2 gene expressions compared to the control in the tissues of tomato. The highest level of MT1 and MT2 transcripts was found in roots and leaves, respectively. The expression of MT3 is induced in roots, leaves and fruits except for Pb treatment in roots. MT4 expression increased in fruits; however, other tissues did not show a clear change. Our results indicated that Cu content was higher than Pb in all tissues of tomato. The lower doses of Cu (10 ppm) increased the content of Mg, Fe, Ca and Mn in roots. Pb generally increased the level of minerals in leaves and fruits, but it decreased Mg, Mn and Fe contents in roots. Both heavy metals not only moved to aerial parts but also caused alterations to mineral element levels. These results show that MT transcripts are regulated by Cu and Pb, and expression pattern changes to MT isoforms and tissue types. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Exploring valid internal-control genes in Porphyra yezoensis (Bangiaceae) during stress response conditions

NASA Astrophysics Data System (ADS)

Wang, Wenlei; Wu, Xiaojie; Wang, Chao; Jia, Zhaojun; He, Linwen; Wei, Yifan; Niu, Jianfeng; Wang, Guangce

2014-07-01

To screen the stable expression genes related to the stress (strong light, dehydration and temperature shock) we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test genes in P orphyra yezoensis, which has been regarded as a potential model species responding the stress conditions in the intertidal. Absolute real-time PCR technology was applied to determine the transcription numbers of the selected test genes in P orphyra yezoensis, which has been regarded as a potential model species in stress responding. According to the results of photosynthesis parameters, we observed that Y(II) and F v/ F m were significantly affected when stress was imposed on the thalli of P orphyra yezoensis, but underwent almost completely recovered under normal conditions, which were collected for the following experiments. Then three samples, which were treated with different grade stresses combined with salinity, irradiation and temperature, were collected. The transcription numbers of seven constitutive expression genes in above samples were determined after RNA extraction and cDNA synthesis. Finally, a general insight into the selection of internal control genes during stress response was obtained. We found that there were no obvious effects in terms of salinity stress (at salinity 90) on transcription of most genes used in the study. The 18S ribosomal RNA gene had the highest expression level, varying remarkably among different tested groups. RPS8 expression showed a high irregular variance between samples. GAPDH presented comparatively stable expression and could thus be selected as the internal control. EF-1α showed stable expression during the series of multiple-stress tests. Our research provided available references for the selection of internal control genes for transcripts determination of P. yezoensis.

Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum

PubMed Central

2011-01-01

Background Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported. Results De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes. Conclusion This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, DsIV genes expressed in the host and also improves our current understanding of this host-parasitoid interaction. PMID:21906285

Molecular cloning and characterization of amh, dax1 and cyp19a1a genes and their response to 17α-methyltestosterone in Pengze crucian carp.

PubMed

Li, Meng; Wang, Lihong; Wang, Houpeng; Liang, Hongwei; Zheng, Yao; Qin, Fang; Liu, Shaozhen; Zhang, Yingying; Wang, Zaizhao

2013-05-01

The proteins encoded by amh, dax1 and cyp19a1a play important roles in gonad differentiation. Their functions have been far less studied in teleosts. In this study, the full-length cDNAs of amh, dax1 and cyp19a1a were cloned and characterized in a triploid gynogenic fish, the Pengze crucian carp. Their expression profilings in juvenile development, adult tissues and juveniles exposed to 100 ng/L 17α-methyltestosterone (MT) were investigated. Results showed that their putative proteins shared high identities to their counterparts in cyprinid fish species, respectively. The tissue distribution results indicated that amh and cyp19a1a were predominantly expressed in the ovary and dax1 was dominantly expressed in the liver. Gene profiling in the developmental stages showed that all the three target genes had a consistent highest expression at 48 days post hatching (dph). The period of 48 dph appeared to be a key time during the process of the gonad development of Pengze crucian carp. 100 ng/L MT significantly increased the mRNA expression of amh at 2- and 4-week exposures and enhanced dax1 and cyp19a1a at 6-week exposure. The present study indicated that MT could influence the gonad development in Pengze crucian carp by disturbing sex-differentiation associated gene expression. Furthermore, the present study will be of great significance to broaden the understanding of molecular mechanisms of the physiological processes of reproduction in fish. Copyright © 2013 Elsevier Inc. All rights reserved.

CD47 is an adverse prognostic factor and a therapeutic target in gastric cancer

PubMed Central

Yoshida, Kazumichi; Tsujimoto, Hironori; Matsumura, Kouji; Kinoshita, Manabu; Takahata, Risa; Matsumoto, Yusuke; Hiraki, Shuichi; Ono, Satoshi; Seki, Shuhji; Yamamoto, Junji; Hase, Kazuo

2015-01-01

CD47 is an antiphagocytic molecule that acts via ligation to signal regulatory protein alpha on phagocytes; its enhanced expression and therapeutic targeting have recently been reported for several malignancies. However, CD47 expression in gastric cancer is not well documented. Immunohistochemical expression of CD47 in surgical specimens was investigated. Expression of CD47 and CD44, a known gastric cancer stem cell marker, were investigated in gastric cancer cell lines by flow cytometry. MKN45 and MKN74 gastric cancer cells were sorted by fluorescence-activated cell sorting according to CD44 and CD47 expression levels, and their in vitro proliferation, spheroid-forming capacity, and in vivo tumorigenicity were studied. In vitro phagocytosis of cancer cells by human macrophages in the presence of a CD47 blocking monoclonal antibody (B6H12) and the survival of immunodeficient mice intraperitoneally engrafted with MKN45 cells and B6H12 were compared to experiments using control antibodies. Immunohistochemistry of the clinical specimens indicated that CD47 was positive in 57 out of 115 cases, and its positivity was an independent adverse prognostic factor. Approximately 90% of the MKN45 and MKN74 cells expressed CD47 and CD44. CD47hi gastric cancer cells showed significantly higher proliferation and spheroid colony formation than CD47lo, and CD44hiCD47hi cells showed the highest proliferation in vitro and tumorigenicity in vivo. B6H12 significantly enhanced in vitro phagocytosis of cancer cells by human macrophages and prolonged the survival of intraperitoneal cancer dissemination in mice compared to control antibodies. In conclusion, CD47 is an adverse prognostic factor and promising therapeutic target in gastric cancer. PMID:26077800

Progressive increase of glucose transporter-3 (GLUT-3) expression in estrogen-induced breast carcinogenesis.

PubMed

Kocdor, M A; Kocdor, H; Pereira, J S; Vanegas, J E; Russo, I H; Russo, J

2013-01-01

Increased glucose uptake and glycolysis are main metabolic characteristics of malignant cells. A family of glucose transporters (GLUTs) facilitates glucose movement across the plasma membranes in a tumor-specific manner. Glucose transporter-1 (GLUT-1), GLUT-3 and recently GLUT-12, have been previously shown in breast cancer cells and are found to be associated with poor prognosis. In addition, it has been shown that estrogen plays critical roles in GLUT regulation, however, the stage-specific GLUT regulation of mammary carcinogenesis is unclear. GLUT expression patterns were investigated in an in vitro-in vivo progressive, estrogen-induced, mammary carcinogenesis model which consisted of four cell lines, with same genetic background. In this model, different stages of tumor initiation and progression are represented, MCF-10F being the normal stage, E2 cells the transformed stage by estrogen, C5 cells, the invasive stage, and T4 cells the tumorigenic stage. In addition, loss of ductulogenesis and solid mass formation in collagen matrix and invasiveness of the cells were counted. Real time PCR showed that GLUT1 expression was downregulated in MCF10F after treatment with 17β-estradiol (E2), and in the invasive cell type (C5), but not in the tumor cells (T4), which had no changes compared to MCF10F. C5 and T4 cells showed the highest rate of GLUT-3 expression. These cells were also found to be associated with loss of ductulogenesis, solid mass formation and higher invasive capacity, whereas, GLUT-12 was downregulated in C5 and T4 cells. Estrogen-induced malignant transformation is associated with remarkable and progressive GLUT-3 expression, GLUT-1 re-expression at further stages, as well as GLUT-12 downregulation.

Human Peripheral Blood Mononuclear Cells Exhibit Heterogeneous CD52 Expression Levels and Show Differential Sensitivity to Alemtuzumab Mediated Cytolysis

PubMed Central

Rao, Sambasiva P.; Sancho, Jose; Campos-Rivera, Juanita; Boutin, Paula M.; Severy, Peter B.; Weeden, Timothy; Shankara, Srinivas; Roberts, Bruce L.; Kaplan, Johanne M.

2012-01-01

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact. PMID:22761788

Cloning and Expression Analysis of a PISTILLATA Homologous Gene from Pineapple (Ananas comosus L. Merr)

PubMed Central

Lv, Ling-Ling; Duan, Jun; Xie, Jiang-Hui; Liu, Yu-Ge; Wei, Chang-Bin; Liu, Sheng-Hui; Zhang, Jian-Xia; Sun, Guang-Ming

2012-01-01

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants. PMID:22312303

Cloning and expression analysis of a PISTILLATA homologous gene from pineapple (Ananas comosus L. Merr).

PubMed

Lv, Ling-Ling; Duan, Jun; Xie, Jiang-Hui; Liu, Yu-Ge; Wei, Chang-Bin; Liu, Sheng-Hui; Zhang, Jian-Xia; Sun, Guang-Ming

2012-01-01

PISTILLATA (PI)-like genes are crucial regulators of flowering in angiosperms. A homologue of PI, designated as AcPI (Genbank accession number HQ717796), was isolated from pineapple cultivar Comte de Paris by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of AcPI is 907 bp in length and contains an open reading frame of 594 bp, which encodes a protein of 197 amino acids. The molecular weight was 2.29 kDa and the isoelectric point was 9.28. The alignment showed that AcPI had a high identity with CsPIC2 (78.6%), AoPI (77.4%), OrcPI (75.7%) and HPI2 (72.4%). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses in different tissues showed that the expression pattern of AcPI was different from the B-class genes in eudicots. AcPI was expressed in all the tissues investigated. The expression level was very low in fruit stems, bracts, leaves and sepals, high in petals and carpels, and moderate in apical meristems, flesh and stamens. The qRT-PCR analyses in different stages indicated that the expression of AcPI reached the highest level at 40 days after flower inducement, when the multiple fruit and floral organs were forming. It proved the important role of AcPI in floral organs and fruit development. The 35S::AcPI transgenic Arabidopsis plants flowered earlier and had more inflorescences or branches than wild type plants.

Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

PubMed Central

Deplus, Rachel; Lampe, Xavier; Krusy, Nathalie; Calonne, Emilie; Delbecque, Katty; Kridelka, Frederic; Fuks, François; Ennaji, My Mustapha; Delvenne, Philippe

2012-01-01

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. PMID:22880087

Larvicidal Activity against Aedes aegypti and Chemical Characterization of the Inflorescences of Tagetes patula

PubMed Central

Antonelli-Ushirobira, Tânia Mara; Panizzon, Gean; Sereia, Ana Luiza; de Souza, José Roberto Pinto; Zequi, João Antonio Cyrino; Novello, Cláudio Roberto; Lopes, Gisely Cristiny; de Medeiros, Daniela Cristina; Silva, Denise Brentan; Leite-Mello, Eneri Vieira de Souza

2017-01-01

The crude acetone extract (CAE) of defatted inflorescences of Tagetes patula was partitioned into five semipurified fractions: n-hexane (HF), dichloromethane (DF), ethyl acetate (EAF), n-butanol (BF), and aqueous (AQF). BF was fractionated by reversed-phase polyamide column chromatography, obtaining 34 subfractions, which were subjected to HSCCC, where patuletin and patulitrin were isolated. CAE and the fractions BF, EAF, DF, and AQF were analyzed by LC-DAD-MS, and patuletin and patulitrin were determined as the major substances in EAF and BF, respectively. BF was also analyzed by HPLC and capillary electrophoresis (CE), and patulitrin was again determined to be the main substance in this fraction. CAE and the semipurified fractions (750, 500, 300, 100, and 50 mg/L) were assayed for larvicidal activity against Aedes aegypti, with mortality rate expressed as percentage. All fractions except AQF showed insecticidal activity after 24 h exposure of larvae to the highest concentration. However, EAF showed the highest activity with more than 50% reduction in larval population at 50 mg/L. The insecticidal activity observed with EAF might have been due to the higher concentration of patuletin present in this fraction. PMID:29362590

Using heterologous expression systems to characterize potassium and sodium transport activities.

PubMed

Rodríguez, Alonso; Benito, Begoña; Cagnac, Olivier

2012-01-01

The expression of plant transporters in simple well-characterized cell systems is an irreplaceable technique for gaining insights into the kinetic and energetic features of plant transporters. Among all the available expression systems, yeast cells offer the highest simplicity and have the capacity to mimic the in vivo properties of plant transporters. Here, we describe the use of yeast mutants to express K(+) and Na(+) plant transporters and discuss some experimental problems that can produce misleading results.

Iron Biofortification and Homeostasis in Transgenic Cassava Roots Expressing the Algal Iron Assimilatory Gene, FEA1

PubMed Central

Ihemere, Uzoma E.; Narayanan, Narayanan N.; Sayre, Richard T.

2012-01-01

We have engineered the tropical root crop cassava (Manihot esculenta) to express the Chlamydomonas reinhardtii iron assimilatory gene, FEA1, in its storage roots with the objective of enhancing the root nutritional qualities. Iron levels in mature cassava storage roots were increased from 10 to 36 ppm in the highest iron accumulating transgenic lines. These iron levels are sufficient to meet the minimum daily requirement for iron in a 500 g meal. Significantly, the expression of the FEA1 gene in storage roots did not alter iron levels in leaves. Transgenic plants also had normal levels of zinc in leaves and roots consistent with the specific uptake of ferrous iron mediated by the FEA1 protein. Relative to wild-type plants, fibrous roots of FEA1 expressing plants had reduced Fe (III) chelate reductase activity consistent with the more efficient uptake of iron in the transgenic plants. We also show that multiple cassava genes involved in iron homeostasis have altered tissue-specific patterns of expression in leaves, stems, and roots of transgenic plants consistent with increased iron sink strength in transgenic roots. These results are discussed in terms of strategies for the iron biofortification of plants. PMID:22993514

Molecular Cloning and Characterization of a P-Glycoprotein from the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae)

PubMed Central

Tian, Lixia; Yang, Jiaqiang; Hou, Wenjie; Xu, Baoyun; Xie, Wen; Wang, Shaoli; Zhang, Youjun; Zhou, Xuguo; Wu, Qingjun

2013-01-01

Macrocyclic lactones such as abamectin and ivermectin constitute an important class of broad-spectrum insecticides. Widespread resistance to synthetic insecticides, including abamectin and ivermectin, poses a serious threat to the management of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), a major pest of cruciferous plants worldwide. P-glycoprotein (Pgp), a member of the ABC transporter superfamily, plays a crucial role in the removal of amphiphilic xenobiotics, suggesting a mechanism for drug resistance in target organisms. In this study, PxPgp1, a putative Pgp gene from P. xylostella, was cloned and characterized. The open reading frame (ORF) of PxPgp1 consists of 3774 nucleotides, which encodes a 1257-amino acid peptide. The deduced PxPgp1 protein possesses structural characteristics of a typical Pgp, and clusters within the insect ABCB1. PxPgp1 was expressed throughout all developmental stages, and showed the highest expression level in adult males. PxPgp1 was highly expressed in midgut, malpighian tubules and testes. Elevated expression of PxPgp1 was observed in P. xylostella strains after they were exposed to the abamectin treatment. In addition, the constitutive expressions of PxPgp1 were significantly higher in laboratory-selected and field-collected resistant strains in comparison to their susceptible counterpart. PMID:24264038

Job satisfaction among academic coordinators of clinical education in physical therapy.

PubMed

Harris, M J; Fogel, M; Blacconiere, M

1987-06-01

The Academic Coordinator of Clinical Education is the physical therapy faculty member who is responsible for the clinical component of the curriculum. The responsibilities involved in the ACCE's job are such that ACCEs seem to be at risk for job dissatisfaction and burnout. The purpose of this descriptive study was to investigate the levels and patterns of job satisfaction among ACCEs in physical therapy. A questionnaire, including a 32-item job satisfaction inventory, was sent to the ACCE at each accredited entry-level education program for physical therapists and physical therapist assistants (N = 169). One hundred twelve (66.3%) responses were received and analyzed. Demographic characteristics of the respondents are reported. The results of the study showed that ACCEs, in general, expressed low levels of occupational dissatisfaction and burnout. Satisfaction with the aspects of the job involving self-esteem, achievement, and creativity seems to outweight dissatisfaction with the time available, the work load, and organizational efficiency. Those ACCEs with doctoral degrees expressed the highest levels of dissatisfaction and burnout. Those ACCEs working in entry-level master's degree programs expressed the lowest level of dissatisfaction; those in tenure-track positions expressed the lowest level of burnout. Factors contributing to job satisfaction and dissatisfaction are discussed.

Neuropilin-2 promotes extravasation and metastasis by interacting with endothelial α5 integrin.

PubMed

Cao, Ying; Hoeppner, Luke H; Bach, Steven; E, Guangqi; Guo, Yan; Wang, Enfeng; Wu, Jianmin; Cowley, Mark J; Chang, David K; Waddell, Nicola; Grimmond, Sean M; Biankin, Andrew V; Daly, Roger J; Zhang, Xiaohui; Mukhopadhyay, Debabrata

2013-07-15

Metastasis, the leading cause of cancer death, requires tumor cell intravasation, migration through the bloodstream, arrest within capillaries, and extravasation to invade distant tissues. Few mechanistic details have been reported thus far regarding the extravasation process or re-entry of circulating tumor cells at metastatic sites. Here, we show that neuropilin-2 (NRP-2), a multifunctional nonkinase receptor for semaphorins, vascular endothelial growth factor (VEGF), and other growth factors, expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular extravasation and metastasis in zebrafish and murine xenograft models of clear cell renal cell carcinoma (RCC) and pancreatic adenocarcinoma. In tissue from patients with RCC, NRP-2 expression is positively correlated with tumor grade and is highest in metastatic tumors. In a prospectively acquired cohort of patients with pancreatic cancer, high NRP-2 expression cosegregated with poor prognosis. Through biochemical approaches as well as Atomic Force Microscopy (AFM), we describe a unique mechanism through which NRP-2 expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular adhesion and extravasation. Taken together, our studies reveal a clinically significant role of NRP-2 in cancer cell extravasation and promotion of metastasis. ©2013 AACR.

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio).

PubMed

Wang, Yeda; Li, Zeming; Lu, Yuanan; Hu, Guangfu; Lin, Li; Zeng, Lingbing; Zhou, Yong; Liu, Xueqin

2016-10-09

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp ( Cyprinus carpio ) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro.

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio)

PubMed Central

Wang, Yeda; Li, Zeming; Lu, Yuanan; Hu, Guangfu; Lin, Li; Zeng, Lingbing; Zhou, Yong; Liu, Xueqin

2016-01-01

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp (Cyprinus carpio) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro. PMID:27735853

HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer.

PubMed

Foley, Kendra C; Spear, Timothy T; Murray, David C; Nagato, Kaoru; Garrett-Mayer, Elizabeth; Nishimura, Michael I

2017-06-16

T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and β TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cβ2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

[Effect of different oxygen tension on the cytoskeleton remodeling of goat temporomandibular joint disc cells].

PubMed

Xiaolan, He; Guangjie, Bao; Linglu, Sun; Xue, Zhang; Shanying, Bao; Hong, Kang

2017-08-01

Objective The effect of different oxygen tensions on the cytoskeleton remodeling of goat temporomandibular joint (TMJ) disc cells were investigated. Methods Goat TMJ disc cells were cultured under normoxia (21% O₂) and hypoxia (2%, 4%, and 8% O₂). Toluidine blue, picrosirius red, and type Ⅰ collagen immunocytochemical staining were performed to observe the changes in cell phenotype under different oxygen levels. Immunofluorescent staining and real-time reverse transcription-polymerase chain reaction analysis were then performed to identify actin, tubulin, and vimentin in the cultured disc cells. Results TMJ disc cells still displayed fibroblast characteristics under different oxygen levels and their cytoskeletons had regular arrangement. The fluorescence intensities of actin and vimentin were lowest at 4% O₂(P<0.05), whereas that of tubulin was highest at 2% O₂ (P<0.05). No significant difference among the other groups was observed (P>0.05). Actin mRNA levels were considerably decreased at 2% O₂ and 4% O₂ in hypoxic conditions, while actin mRNA expression was highest in 21% O₂. Tubulin mRNA levels considerably increased at 2% O₂, while tubulin mRNA expression was lowest in 8% O₂ (P<0.05). Vimentin mRNA expression was lowest at 4% O₂ and highest at 21% O₂, and significant differences were observed between vimentin mRNA expression levels among these oxygen levels (P<0.05). Conclusion Cytoskeletons were reconstructed in different oxygen tensions, and 2% O₂ may be the optimal oxygen level required to proliferate TMJ disc cells.

HDAC1 and HDAC2 are Differentially Expressed in Endometriosis

PubMed Central

Colón-Díaz, Maricarmen; Báez-Vega, Perla; García, Miosotis; Ruiz, Abigail; Monteiro, Janice B.; Fourquet, Jessica; Bayona, Manuel; Alvarez-Garriga, Carolina; Achille, Alexandra; Seto, Edward; Flores, Idhaliz

2012-01-01

Epigenetic mechanisms have been ascribed important roles in endometriosis. Covalent histone modifications at lysine residues have been shown to regulate gene expression and thus contribute to pathological states in many diseases. In endometriosis, histone deacetylase inhibition (HDACi) resulted in reactivation of E-cadherin, attenuation of invasion, decreased proliferation of endometriotic cells, and caused lesion regression in an animal model. This study was conducted to assess basal and hormone-regulated gene expression levels of HDAC1 and HDAC2 (HDAC1/2) in cell lines and protein expression levels in tissues. Basal and steroid hormone-regulated HDAC1/2 gene expression levels were determined by quantitative polymerase chain reaction in cell lines and tissues. Protein levels were measured by immunohistochemistry (IHC) in tissues on an endometriosis tissue microarray (TMA). Basal HDAC1/2 gene expression levels were significantly higher in endometriotic versus endometrial stromal cells, which was confirmed by Western blot analysis. Estradiol (E2) and progesterone (P4) significantly downregulated HDAC1 expression in endometrial epithelial cells. Levels of HDAC2 were upregulated by E2 and downregulated by E2 + P4 in endometrial stromal cells. Hormone modulation of HDAC1/2 gene expression was lost in the endometriotic cell line. Immunohistochemistry showed that HDAC1/2 proteins were expressed in a substantial proportion of lesions and endometrium from patients, and their expression levels varied according to lesion localization. The highest proportion of strong HDAC1 immunostaining was seen in ovarian, skin, and gastrointestinal lesions, and of HDAC2 in skin lesions and endometrium from patients with endometriosis. These studies suggest that endometriosis etiology may be partially explained by epigenetic regulation of gene expression due to dysregulations in the expression of HDACs. PMID:22344732

Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90β) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C.

PubMed

Yan, Jie; Liang, Xiao; Zhang, Yin; Li, Yang; Cao, Xiaojuan; Gao, Jian

2017-07-01

Heat shock protein 70 (HSP70) and 90 (HSP90) are the most broadly studied proteins in HSP families. They play key roles in cells as molecular chaperones, in response to stress conditions such as thermal stress. In this study, full-length cDNA sequences of HSP70, HSP90α and HSP90β from loach Misgurnus anguillicaudatus were cloned. The full-length cDNA of HSP70 in loach was 2332bp encoding 644 amino acids, while HSP90α and HSP90β were 2586bp and 2678bp in length, encoding 729 and 727 amino acids, respectively. The deduced amino acid sequences of HSP70 in loach shared the highest identity with those of Megalobrama amblycephala and Cyprinus carpio. The deduced amino acid sequences of HSP90α and HSP90β in loach both shared the highest identity with those of M. amblycephala. Their mRNA tissue expression results showed that the maximum expressions of HSP70, HSP90α and HSP90β were respectively present in the intestine, brain and kidney of loach. Quantitative real-time PCR was employed to analyze the temporal expressions of HSP70, HSP90α and HSP90β in livers of loaches fed with different levels of vitamin C under thermal stress. Expression levels of the three HSP genes in loach fed the diet without vitamin C supplemented at 0 h of thermal stress were significantly lower than those at 2 h, 6 h, 12 h and 24 h of thermal stress. It indicated that expressions of the three HSP genes were sensitive to thermal stress in loach. The three HSP genes in loaches fed with 1000 mg/kg vitamin C expressed significantly lower than other vitamin C groups at many time points of thermal stress, suggesting 1000 mg/kg dietary vitamin C might decrease the body damages caused by the thermal stress. This study will be of value for further studies into thermal stress tolerance in loach. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gene expression of Vibrio parahaemolyticus growing in laboratory isolation conditions compared to those common in its natural ocean environment.

PubMed

García, Katherine; Yáñez, Cristian; Plaza, Nicolás; Peña, Francisca; Sepúlveda, Pedro; Pérez-Reytor, Diliana; Espejo, Romilio T

2017-05-19

Vibrio parahaemolyticus is an autochthonous marine bacterial species comprising strains able to grow in broth containing bile salts at 37 °C, a condition seldom found in the ocean. However, this condition is used for isolation in the laboratory because it is considered a necessary property for pathogenesis. In this context, revealing how gene expression enables V. parahaemolyticus to adapt to this particular condition -common to almost all V. parahaemolyticus isolates- will improve our understanding of the biology of this important pathogen. To determine the genes of V. parahaemolyticus differentially expressed when growing in isolation condition (37 °C, 0.9% NaCl, and 0.04% bile salts) referred to those at the temperature and salt concentration prevailing in ocean south of Chile (marine-like condition; 12 °C, 3% NaCl, and absence of bile salts) we used high-throughput sequencing of RNA. Our results showed that in the isolation condition, among the 5034 genes annotated in the V. parahaemolyticus RIMD2210633 genome, 344 were upregulated and 433 downregulated referred to the marine-like condition, managing an adjusted P-value (Padj) < E -5 . Between the 50 more highly expressed genes, among the small RNAs (sRNA), the three carbon storage regulators B (CsrB) were up four to six times, while RyhB, related to iron metabolism besides motility control, was down about eight times. Among proteins, BfdA, a hemolysin-co-regulated protein (Hcp1) secreted by T6SS1, one of the most highly expressed genes, was about 140 times downregulated in isolation condition. The highest changes in relative expression were found among neighboring genes coding for proteins related to respiration, which were about 40 times upregulated. When V. parahaemolyticus is grown in conditions used for laboratory isolation 777 genes are up- or downregulated referred to conditions prevailing in the marine-like condition; the most significantly overrepresented categories among upregulated processes were those related to transport and localization, while secretion and pathogenesis were overrepresented among downregulated genes. Genes with the highest differential expression included the sRNAs CsrB and RhyB and the mRNAs related with secretion, nutritional upshift, respiration and rapid growing.

Primary study on the lesions and specific proteins in BEAS-2B cells induced with the 2009 A (H1N1) influenza virus.

PubMed

Fang, Shisong; Zhang, Kaining; Wang, Ting; Wang, Xin; Lu, Xing; Peng, Bo; Wu, Weihua; Zhang, Ran; Chen, Shiju; Zhang, Renli; Xue, Hong; Yu, Muhua; Cheng, Jinquan

2014-12-01

In order to investigate the lesions and proteins with differential expression in cells infected with the 2009 A (H1N1) virus and to determine the specific proteins involved in cell damage, the present study has been performed. BEAS-2B cells were infected with the 2009 A (H1N1) influenza virus or the seasonal H1N1 influenza virus for 12, 24, 48, and 72 h, and cell cycle and apoptosis were analyzed with flow cytometry. Total cellular proteins were extracted and underwent two-dimensional gel electrophoresis. The differentially expressed proteins underwent mass spectrometry for identification. The results showed that after 12 h, cells infected with the virus strain sourced from severe cases had the highest apoptosis rate (P < 0.05). After 48 h, cells infected with the virus strain sourced from fatal cases and severe cases had the highest apoptosis rate (P < 0.05), and after 72 h, cells infected with virus strains from fatal cases and ordinary cases had the highest apoptosis rate (P < 0.05). All the four influenza virus strains induced cell cycle arrest mainly at the G0/G1 phase. Eighteen differentially expressed proteins were identified, including galectin-1, cofilin-1, protein DJ-1, proteasome subunit α type-5, macrophage migration inhibitory factor, translationally controlled tumor protein, profilin 1, and interferon α-2. Galectin-1 was specifically observed in BEAS-2B infected with 2009 A (H1N1) influenza viruses, and cofilin-1 was specifically observed in BEAS-2B cells in the late stage of 2009 A (H1N1) influenza virus infection. In conclusion, differential effects of the 2009 A (H1N1) influenza virus and seasonal H1N1 influenza virus were identified on the cell cycle and apoptosis, and galectin-1 may play a role in cell apoptosis induced by 2009 A (H1N1) influenza virus.

An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

PubMed

Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

2015-05-01

An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular cloning and expression analysis of the canine chemokine receptor CCR9.

PubMed

Maeda, Shingo; Ohno, Koichi; Tsukamoto, Atsushi; Nakashima, Ko; Fukushima, Kenjiro; Goto-Koshino, Yuko; Fujino, Yasuhito; Tsujimoto, Hajime

2012-01-15

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD. Copyright © 2011 Elsevier B.V. All rights reserved.

High matrix metalloproteinase activity is a hallmark of periapical granulomas.

PubMed

de Paula-Silva, Francisco Wanderley Garcia; D'Silva, Nisha J; da Silva, Léa Assed Bezerra; Kapila, Yvonne Lorraine

2009-09-01

The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tukey test. We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. Our findings indicate that high enzymatic MMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts.

High Matrix Metalloproteinase Activity is a Hallmark of Periapical Granulomas

PubMed Central

de Paula e Silva, Francisco Wanderley Garcia; D'Silva, Nisha J.; da Silva, Léa Assed Bezerra; Kapila, Yvonne Lorraine

2009-01-01

Introduction Inability to distinguish periapical cysts from granulomas prior to performing root canal treatment leads to uncertainty in treatment outcomes, because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Methods Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Since matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed using one-way ANOVA followed by Tukey test. Results We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the matrix metalloproteinase (MMP) family. Compared to cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs), in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared to cysts. Conclusion Our findings indicate that high enzymatic MMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas, unlike periapical cysts. PMID:19720222

Chitinase mRNA Levels Determined by QPCR in Crab-Eating Monkey (Macaca fascicularis) Tissues: Species-Specific Expression of Acidic Mammalian Chitinase and Chitotriosidase.

PubMed

Uehara, Maiko; Tabata, Eri; Ishii, Kazuhiro; Sawa, Akira; Ohno, Misa; Sakaguchi, Masayoshi; Matoska, Vaclav; Bauer, Peter O; Oyama, Fumitaka

2018-05-09

Mice and humans express two active chitinases: acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Both chitinases are thought to play important roles in specific pathophysiological conditions. The crab-eating monkey ( Macaca fascicularis ) is one of the most frequently used nonhuman primate models in basic and applied biomedical research. Here, we performed gene expression analysis of two chitinases in normal crab-eating monkey tissues by way of quantitative real-time polymerase chain reaction (qPCR) using a single standard DNA molecule. Levels of AMCase and CHIT1 messenger RNAs (mRNAs) were highest in the stomach and the lung, respectively, when compared to other tissues. Comparative gene expression analysis of mouse, monkey, and human using monkey⁻mouse⁻human hybrid standard DNA showed that the AMCase mRNA levels were exceptionally high in mouse and monkey stomachs while very low in the human stomach. As for the CHIT1 mRNA, we detected higher levels in the monkey lung when compared with those of mouse and human. The differences of mRNA expression between the species in the stomach tissues were basically reflecting the levels of the chitinolytic activities. These results indicate that gene expression of AMCase and CHIT1 differs between mammalian species and requiring special attention in handling data in chitinase-related studies in particular organisms.

Cloning and expression of two 9-cis-epoxycarotenoid dioxygenase genes during fruit development and under stress conditions from Malus.

PubMed

Xia, Hui; Wu, Shan; Ma, Fengwang

2014-10-01

There is now biochemical and genetic evidence that oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. To understand the expression characteristics of NCED during ABA biosynthesis in apple (Malus), two NCED genes cDNA sequence were cloned from Malus prunifolia using RT-PCR techniques, named MpNCED1 and MpNCED2. The two cDNA sequences have full-length open reading frame, encoding a polypeptide of 607 and 614 amino acids, respectively. Sequences analysis showed that the deduced two apple NCED proteins were highly homologous to other NCED proteins from different plant species. Real-time PCR analysis revealed MpNCED2 were expressed continuously during the whole period of apple fruit development with the pattern of "higher-low-highest", while the expression of MpNCED1 clearly declined to a steady low level in the mid-later period of fruit development. Expression of the MpNCED2 increased under the drought stress, high temperature and low temperature strongly and rapidly, whereas expression of the MpNCED1 was detected in response to temperature stress, but did not detected under drought stress. These results revealed that MpNCED1 and MpNCED2 may play different roles in regulation of the ABA biosynthesis in fruit development and various stresses response.

Molecular Cloning, Bioinformatic Analysis, and Expression of Bombyx mori Lebocin 5 Gene Related to Beauveria bassiana Infection.

PubMed

Lü, Dingding; Hou, Chengxiang; Qin, Guangxing; Gao, Kun; Chen, Tian; Guo, Xijie

2017-01-01

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori , by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana , the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.

Effect of systemic administration of lipopolysaccharides derived from Porphyromonas gingivalis on gene expression in mice kidney.

PubMed

Harada, Fumiya; Uehara, Osamu; Morikawa, Tetsuro; Hiraki, Daichi; Onishi, Aya; Toraya, Seiko; Adhikari, Bhoj Raj; Takai, Rie; Yoshida, Koki; Sato, Jun; Nishimura, Michiko; Chiba, Itsuo; Wu, Ching Zong; Abiko, Yoshihiro

2018-01-31

Although an association between periodontitis and chronic kidney disease (CKD) has been suggested, the mechanism involved remains unclear. Herein, we examined the global gene expression profile in a mouse model that showed no acute inflammation in the kidney following stimulation with lipopolysaccharides (LPS) derived from Porphyromonas gingivalis (PG-LPS). The mice were injected with PG-LPS at a concentration of 5 mg/kg intraperitoneally, every 3 days, for 1 month. Microarray analysis was used to identify 10 genes with the highest expression levels in the kidney stimulated with PG-LPS. Among them, the functions of five genes (Saa3, Ticam2, Reg3b, Ocxt2a, and Xcr1) were known. The upregulation of these genes was confirmed by quantitative polymerase chain reaction assay. Furthermore, we examined whether the expression of these upregulated genes were altered in endothelial cells derived from the kidney, in vitro. The mRNA expression levels of all five genes were significantly higher in the experimental group than in the controls (no LPS stimulation; *p < 0.05). In conclusion, the responses noted in the kidney may have arisen mainly from the endothelial cells. Moreover, upregulation of the expression levels of Saa3, Ticam2, Reg3b, Ocxt2a, and Xcr1 may be associated with the pathogenesis of CKD.

Correlation of EGFR expression, gene copy number and clinicopathological status in NSCLC.

PubMed

Gaber, Rania; Watermann, Iris; Kugler, Christian; Reinmuth, Nils; Huber, Rudolf M; Schnabel, Philipp A; Vollmer, Ekkehard; Reck, Martin; Goldmann, Torsten

2014-09-17

Epidermal Growth Factor Receptor (EGFR) targeting therapies are currently of great relevance for the treatment of lung cancer. For this reason, in addition to mutational analysis immunohistochemistry (IHC) of EGFR in lung cancer has been discussed for the decision making of according therapeutic strategies. The aim of this study was to obtain standardization of EGFR-expression methods for the selection of patients who might benefit of EGFR targeting therapies. As a starting point of a broad investigation, aimed at elucidating the expression of EGFR on different biological levels, four EGFR specific antibodies were analyzed concerning potential differences in expression levels by Immunohistochemistry (IHC) and correlated with fluorescence in situ hybridization (FISH) analysis and clinicopathological data. 206 tumor tissues were analyzed in a tissue microarray format employing immunohistochemistry with four different antibodies including Dako PharmDx kit (clone 2-18C9), clone 31G7, clone 2.1E1 and clone SP84 using three different scoring methods. Protein expression was compared to FISH utilizing two different probes. EGFR protein expression determined by IHC with Dako PharmDx kit, clone 31G7 and clone 2.1E1 (p ≤ 0.05) correlated significantly with both FISH probes independently of the three scoring methods; best correlation is shown for 31G7 using the scoring method that defined EGFR positivity when ≥ 10% of the tumor cells show membranous staining of moderate and severe intensity (p=0.001). Overall, our data show differences in EGFR expression determined by IHC, due to the applied antibody. Highest concordance with FISH is shown for antibody clone 31G7, evaluated with score B (p=0.001). On this account, this antibody clone might by utilized for standard evaluation of EGFR expression by IHC. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_165.

Human Milk and Matched Serum Demonstrate Concentration of Select miRNAs.

PubMed

Qin, Wenyi; Dasgupta, Santanu; Corradi, John; Sauter, Edward R

Pregnancy-associated breast cancers (PABCs), especially those diagnosed after childbirth, are often aggressive, with a poor prognosis. Factors influencing PABC are largely unknown. Micro(mi)RNAs are present in many human body fluids and shown to influence cancer development and/or growth. In six nursing mothers, we determined if breast cancer-associated miRNAs were (1) detectable in human breast milk and (2) if detectable, their relative expression in milk fractions compared to matched serum. We evaluated by quantitative PCR the expression of 11 cancer-associated miRNAs (10a-5p, 16, 21, 100, 140, 145, 155, 181, 199, 205, 212) in breast milk cells, fat and supernatant (skim milk), and matched serum. miRNA expression was detectable in all samples. For 10/11 miRNAs, mean relative expression compared to control (ΔCt) values was lowest in milk cells, the exception being miR205. Relative concentration was highest in the skim fraction of milk for all miRNAs. Expression was higher in skim milk than matched serum for 7/11 miRNAs and in serum for 4/11 miRNAs. miR205 expression was higher in all milk fractions than in matched serum. In conclusion, the expression of breast cancer-associated miRNAs is detectable in human breast milk and serum samples. The concentration is highest in skim milk, but is also detectable in milk fat and milk cells.

Characterization of Three Novel Fatty Acid- and Retinoid-Binding Protein Genes (Ha-far-1, Ha-far-2 and Hf-far-1) from the Cereal Cyst Nematodes Heterodera avenae and H. filipjevi.

PubMed

Qiao, Fen; Luo, Lilian; Peng, Huan; Luo, Shujie; Huang, Wenkun; Cui, Jiangkuan; Li, Xin; Kong, Lingan; Jiang, Daohong; Chitwood, David J; Peng, Deliang

2016-01-01

Heterodera avenae and H. filipjevi are major parasites of wheat, reducing production worldwide. Both are sedentary endoparasitic nematodes, and their development and parasitism depend strongly on nutrients obtained from hosts. Secreted fatty acid- and retinol-binding (FAR) proteins are nematode-specific lipid carrier proteins used for nutrient acquisition as well as suppression of plant defenses. In this study, we obtained three novel FAR genes Ha-far-1 (KU877266), Ha-far-2 (KU877267), Hf-far-1 (KU877268). Ha-far-1 and Ha-far-2 were cloned from H. avenae, encoding proteins of 191 and 280 amino acids with molecular masses about 17 and 30 kDa, respectively and sequence identity of 28%. Protein Blast in NCBI revealed that Ha-FAR-1 sequence is 78% similar to the Gp-FAR-1 protein from Globodera pallida, while Ha-FAR-2 is 30% similar to Rs-FAR-1 from Radopholus similis. Only one FAR protein Hf-FAR-1was identified in H. filipjevi; it had 96% sequence identity to Ha-FAR-1. The three proteins are alpha-helix-rich and contain the conserved domain of Gp-FAR-1, but Ha-FAR-2 had a remarkable peptide at the C-terminus which was random-coil-rich. Both Ha-FAR-1 and Hf-FAR-1 had casein kinase II phosphorylation sites, while Ha-FAR-2 had predicted N-glycosylation sites. Phylogenetic analysis showed that the three proteins clustered together, though Ha-FAR-1 and Hf-FAR-1 adjoined each other in a plant-parasitic nematode branch, but Ha-FAR-2 was distinct from the other proteins in the group. Fluorescence-based ligand binding analysis showed the three FAR proteins bound to a fluorescent fatty acid derivative and retinol and with dissociation constants similar to FARs from other species, though Ha-FAR-2 binding ability was weaker than that of the two others. In situ hybridization detected mRNAs of Ha-far-1 and Ha-far-2 in the hypodermis. The qRT-PCR results showed that the Ha-far-1and Ha-far-2 were expressed in all developmental stages; Ha-far-1 expressed 70 times more than Ha-far-2 in all stages. The highest expression level of Ha-far-1 was observed in fourth-stage juvenile (J4), whereas the highest expression level of Ha-far-2 occurred in second-stage juvenile (J2). In conclusion, we have identified two novel far genes from H. avenae and one from H. filipjevi and have provided further indication that nematode far genes are present in a variety of nematode species, where the FAR proteins share similar basic structure, expression pattern and biochemical activities.

Gene family size conservation is a good indicator of evolutionary rates.

PubMed

Chen, Feng-Chi; Chen, Chiuan-Jung; Li, Wen-Hsiung; Chuang, Trees-Juen

2010-08-01

The evolution of duplicate genes has been a topic of broad interest. Here, we propose that the conservation of gene family size is a good indicator of the rate of sequence evolution and some other biological properties. By comparing the human-chimpanzee-macaque orthologous gene families with and without family size conservation, we demonstrate that genes with family size conservation evolve more slowly than those without family size conservation. Our results further demonstrate that both family expansion and contraction events may accelerate gene evolution, resulting in elevated evolutionary rates in the genes without family size conservation. In addition, we show that the duplicate genes with family size conservation evolve significantly more slowly than those without family size conservation. Interestingly, the median evolutionary rate of singletons falls in between those of the above two types of duplicate gene families. Our results thus suggest that the controversy on whether duplicate genes evolve more slowly than singletons can be resolved when family size conservation is taken into consideration. Furthermore, we also observe that duplicate genes with family size conservation have the highest level of gene expression/expression breadth, the highest proportion of essential genes, and the lowest gene compactness, followed by singletons and then by duplicate genes without family size conservation. Such a trend accords well with our observations of evolutionary rates. Our results thus point to the importance of family size conservation in the evolution of duplicate genes.

The effects of exogenous CCK-8 on the acquisition and expression of morphine-induced CPP.

PubMed

Wen, Di; Cong, Bin; Ma, Chunling; Yang, Shengchang; Yu, Hailei; Ni, Zhiyu; Li, Shujin

2012-02-21

Cholecystokinin octapeptide (CCK-8) is the most potent endogenous anti-opioid peptide and regulates a variety of physiological processes. In our previous study, we found that exogenous CCK-8 attenuated naloxone-induced withdrawal symptoms, but the possible regulative effects of CCK-8 on the rewarding effects of morphine were not examined. In the present study, we aimed to determine the exact effects of exogenous CCK-8 at various doses on the rewarding action of morphine by utilizing the unbiased conditioned place preference (CPP) paradigm. We therefore examined the effects of CCK-8 on the acquisition, expression and extinction of morphine-induced CPP and on locomotor activity. The results showed that CCK-8 (0.01-1μg, i.c.v.), administered alone, induced neither CPP nor place aversion, but blocked the acquisition of CPP when administered with 10mg/kg morphine. The highest dose of CCK-8 (1μg) administered before CPP testing increased CPP and, along with lower doses (0.1μg), reduced its extinction. In addition, the highest dose (1μg) of CCK-8 suppressed locomotor activity. Our study provides the first behavioral evidence for the inhibitory effects of exogenous CCK-8 on rewarding activity and reveals significant effects of exogenous CCK-8 on various stages of place preference and the development of opioid dependence. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Adenosine receptor distribution in Rhesus monkey ocular tissue.

PubMed

Beach, Krista M; Hung, Li-Fang; Arumugam, Baskar; Smith, Earl L; Ostrin, Lisa A

2018-05-21

Adenosine receptor (ADOR) antagonists, such as 7-methylxanthine (7-MX), have been shown to slow myopia progression in humans and animal models. Adenosine receptors are found throughout the body, and regulate the release of neurotransmitters such as dopamine and glutamate. However, the role of adenosine in eye growth is unclear. Evidence suggests that 7-MX increases scleral collagen fibril diameter, hence preventing axial elongation. This study used immunohistochemistry (IHC) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to examine the distribution of the four ADORs in the normal monkey eye to help elucidate potential mechanisms of action. Eyes were enucleated from six Rhesus monkeys. Anterior segments and eyecups were separated into components and flash-frozen for RNA extraction or fixed in 4% paraformaldehyde and processed for immunohistochemistry against ADORA1, ADORA2a, ADORA2b, and ADORA3. RNA was reverse-transcribed, and qPCR was performed using custom primers. Relative gene expression was calculated using the ΔΔCt method normalizing to liver expression, and statistical analysis was performed using Relative Expression Software Tool. ADORA1 immunostaining was highest in the iris sphincter muscle, trabecular meshwork, ciliary epithelium, and retinal nerve fiber layer. ADORA2a immunostaining was highest in the corneal epithelium, trabecular meshwork, ciliary epithelium, retinal nerve fiber layer, and scleral fibroblasts. ADORA2b immunostaining was highest in corneal basal epithelium, limbal stem cells, iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells and scattered scleral fibroblasts. ADORA3 immunostaining was highest in the iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells, and scleral fibroblasts. Compared to liver mRNA, ADORA1 mRNA was significantly higher in the brain, retina and choroid, and significantly lower in the iris/ciliary body. ADORA2a expression was higher in brain and retina, ADORA2b expression was higher in retina, and ADORA3 was higher in the choroid. In conclusion, immunohistochemistry and RT-qPCR indicated differential patterns of expression of the four adenosine receptors in the ocular tissues of the normal non-human primate. The presence of ADORs in scleral fibroblasts and the choroid may support mechanisms by which ADOR antagonists prevent myopia. The potential effects of ADOR inhibition on both anterior and posterior ocular structures warrant investigation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

PubMed

Zhan, Fei Xiang; Wang, Qin Hong; Jiang, Si Jing; Zhou, Yu Ling; Zhang, Gui Min; Ma, Yan He

2014-12-16

Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking. The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread. Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory properties of bread. Therefore, the expression yield of recombinant XYNZG can be further improved through engineered strain containing high copy numbers of the XYNZG, and optimized fermentation condition, making bread-baking application possible.

Galectins in the Pathogenesis of Rheumatoid Arthritis

PubMed Central

Li, Song; Yu, Yangsheng; Koehn, Christopher D; Zhang, Zhixin; Su, Kaihong

2013-01-01

Rheumatoid arthritis (RA) is a complex and common systemic autoimmune disease characterized by synovial inflammation and hyperplasia. Multiple proteins, cells, and pathways have been identified to contribute to the pathogenesis of RA. Galectins are a group of lectins that bind to β-galactoside carbohydrates on the cell surface and in the extracellular matrix. They are expressed in a wide variety of tissues and organs with the highest expression in the immune system. Galectins are potent immune regulators and modulate a range of pathological processes, such as inflammation, autoimmunity, and cancer. Accumulated evidence shows that several family members of galectins play positive or negative roles in the disease development of RA, through their effects on T and B lymphocytes, myeloid lineage cells, and fibroblast-like synoviocytes. In this review, we will summarize the function of different galectins in immune modulation and their distinct roles in RA pathogenesis. PMID:24416634

Toxicity of Diclofenac in the Fern Azolla filiculoides and the Lichen Xanthoria parietina.

PubMed

Vannini, Andrea; Paoli, Luca; Vichi, Marco; Bačkor, Martin; Bačkorová, Miriam; Loppi, Stefano

2018-03-01

This study investigated the occurrence of toxicity, expressed as damage to the photosynthetic apparatus, in the aquatic fern Azolla filiculoides and the lichen Xanthoria parietina following treatments with diclofenac at different concentrations (0.1, 1, 10 and 100 mg/L) and different exposure times (24, 48, 72 and 240 h). Measurements of photosynthetic efficiency, chlorophyll content and chlorophyll degradation indicated dose- and time-dependent toxicity, since significant differences with control samples as well as among treatments, emerged mainly for the highest concentration (100 mg/L) and the longest time (240 h). In addition, also the mycobiont of the lichen X. parietina showed similar toxic effects, expressed as ergosterol content. The absence of relevant alterations at the lowest concentration (0.1 mg/L) suggested a very limited susceptibility of these species to environmentally relevant levels of this pharmaceutical.

Relationship between levels of neuropeptide Substance P in periodontal disease and chronic pain: a literature review.

PubMed

de Avila, Erica Dorigatti; de Molon, Rafael Scaf; de Godoi Gonçalves, Daniela Aparecida; Camparis, Cinara Maria

2014-05-01

The aim of the current review was to investigate the relationship between levels of neuropeptide Substance P in periodontal disease and chronic pain. Substance P is a neuropeptide that is directly related with pain. In periodontal disease, it is expressed during the inflammatory process, and is one of the factors responsible for bone resorption. Studies have shown that Substance P levels are highest in the gingival crevicular fluid from sites with active periodontal disease and bone loss. The persistence of these substances could be sufficient to stimulate neurogenic inflammation in susceptible tissues, and cause pain. The scientific literature shows that Substance P expressed during periodontal disease can be a risk factor for patients with systemic inflammatory pathologies, such as chronic arthritis or rheumatoid arthritis. Additional research is needed to confirm the participation of this substance in the origin of some types of chronic pain. © 2014 Wiley Publishing Asia Pty Ltd.

Benzo(a)pyrene-induced cytochrome p4501A expression of four freshwater fishes (Oryzias latipes, Danio rerio, Cyprinus carpio, and Zacco platypus).

PubMed

Lee, Jin Wuk; Yoon, Hong-Gil; Lee, Sung Kyu

2015-05-01

Oryzias latipes, Danio rerio, Cyprinus carpio, and Zacco platypus are useful indicator species for CYP1A biomarker studies; however, comparative studies have not been performed. To compare susceptibility, dose- and time-dependent CYP1A induction at the mRNA and protein levels in response to benzo(a)pyrene (BaP) exposure was analyzed. At the mRNA level, a statistically significant difference was found among the four species; however, such was not observed at the protein level. C. carpio showed the highest CYP1A induction level and the steepest slope in the dose-response curve. To assess susceptibility, the difference in CYP1A mRNA induction among species must be considered, and C. carpio was the most sensitive species of the four evaluated in terms of CYP1A expression. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of HIF-1α and Markers of Angiogenesis Are Not Significantly Different in Triple Negative Breast Cancer Compared to Other Breast Cancer Molecular Subtypes: Implications for Future Therapy.

PubMed

Yehia, Lamis; Boulos, Fouad; Jabbour, Mark; Mahfoud, Ziyad; Fakhruddin, Najla; El-Sabban, Marwan

2015-01-01

Triple negative breast cancer lacks estrogen, progesterone and epidermal growth factor receptors rendering it refractory to available targetedtherapies. TNBC is associated with central fibrosis and necrosis, both indicators of tumor hypoxia. Hypoxia inducible factor 1α is up-regulated under hypoxia and its expression is associated with induction of angiogenesis resulting in proliferation, aggressive tumor phenotype and metastasis. In this study we evaluate the potential use of HIF-1α as aTNBC-specific marker. 62 TNBC, 64 HER2+, and 64 hormone-receptors positive breast cancer cases were evaluated for central fibrosis and necrosis, HIF-1α, HIF-1β, VEGFR3, CD31 expression and microvessel density. RNA extraction from paraffin-embedded samples, followed by quantitative real-time polymerase chain reaction (qRT-PCR) evaluation of HIF-1α and VEGF transcripts was performed on 54 cases (18 from each subtype). HIF-1α protein was expressed in 35.5% TNBC, 45.3% HER2+and 25.0% ER+/PR+ (p = 0.055; χ2 test). PCRanalysis of subgroup of breast cancers, 84.2% expressed HIF-1α protein and its transcripts, while only 66.7% expressed VEGF transcripts simultaneously with the HIF-1α protein and its transcripts. Central fibrosis and necrosis was highest in TNBC (p = 0.015; χ2 test), while MVD was comparable among all groups (p = 0.928; χ2 test). VEGFR3 was highest in TNBC expressing HIF-1α. HIF-1β protein was expressed in 32.0% of HIF-1α(+), and in (44.3%) of HIF-1α(-) breast cancer cases (p = 0.033; χ2 test). Moreover, HIF-1α expression in cases with central fibrosis and necrosis was highest in the HER2+ followed by the TNBC (p = 0.156; χ2 test). A proportion of TNBC express HIF-1α but not in a significantly different manner from other breast cancer subtypes. The potential of anti-HIF-1α targeted therapy is therefore not a candidate for exclusive use in TNBC, but should be considered in all breast cancers, especially in the setting of clinically aggressive or refractory disease.

Structural characterization and expression analysis of a beta-thymosin homologue (Tβ) in disk abalone, Haliotis discus discus.

PubMed

Kasthuri, Saranya Revathy; Premachandra, H K A; Umasuthan, Navaneethaiyer; Whang, Ilson; Lee, Jehee

2013-09-15

Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus. The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon-2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates. Copyright © 2013 Elsevier B.V. All rights reserved.

Rosuvastatin protects against angiotensin II-induced renal injury in a dose-dependent fashion.

PubMed

Park, Joon-Keun; Mervaala, Eero Ma; Muller, Dominik N; Menne, Jan; Fiebeler, Anette; Luft, Friedrich C; Haller, Hermann

2009-03-01

We showed earlier that statin treatment ameliorates target-organ injury in a transgenic model of angiotensin (Ang) II-induced hypertension. We now test the hypothesis that rosuvastatin (1, 10, and 50 mg/kg/day) influences leukocyte adhesion and infiltration, prevents induction of inducible nitric oxide synthase (iNOS), and ameliorates target-organ damage in a dose-dependent fashion. We treated rats harboring the human renin and human angiotensinogen genes (dTGR) from week 4 to 8 (n = 20 per group). Untreated dTGR developed severe hypertension, cardiac hypertrophy, and renal damage, with a 100-fold increased albuminuria and focal cortical necrosis. Mortality of untreated dTGR at age 8 weeks was 59%. Rosuvastatin treatment decreased mortality dose-dependently. Blood pressure was not affected. Albuminuria was reduced dose-dependently. Interstitial adhesion molecule (ICAM)-1 expression was markedly reduced by rosuvastatin, as were neutrophil and monocyte infiltration. Immunohistochemistry showed an increased endothelial and medial iNOS expression in small vessels, infiltrating cells, afferent arterioles, and glomeruli of dTGR. Immunoreactivity was stronger in cortex than medulla. Rosuvastatin markedly reduced the iNOS expression in both cortex and medulla. Finally, matrix protein (type IV collagen, fibronectin) expression was also dose- dependently reduced by rosuvastatin. Our findings indicate that rosuvastatin dose- dependently ameliorates angiotensin II-induced-organ damage and almost completely prevents inflammation at the highest dose. The data implicate 3-hydroxy-3-methylglutaryl coenzyme A function in signaling events leading to target-organ damage.

Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

PubMed

Kölbl, Alexandra C; Hiller, Roman A; Ilmer, Mathias; Liesche, Friederike; Heublein, Sabine; Schröder, Lennard; Hutter, Stefan; Friese, Klaus; Jeschke, Udo; Andergassen, Ulrich

2015-08-01

Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

Role of the POZ Zinc Finger Transcription Factor FBI-1 in Human and Murine Adipogenesis

PubMed Central

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J.; Considine, Robert V.; Sethi, Jaswinder K.; Vidal-Puig, Antonio; O’Rahilly, Stephen

2015-01-01

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2–4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation. PMID:14701838

TUSC2(FUS1)-erlotinib Induced Vulnerabilities in Epidermal Growth Factor Receptor(EGFR) Wildtype Non-small Cell Lung Cancer(NSCLC) Targeted by the Repurposed Drug Auranofin.

PubMed

Xiaobo, Cao; Majidi, Mourad; Feng, Meng; Shao, Ruping; Wang, Jing; Zhao, Yang; Baladandayuthapani, Veerabhadran; Song, Juhee; Fang, Bingliang; Ji, Lin; Mehran, Reza; Roth, Jack A

2016-11-15

Expression of the TUSC2/FUS1 tumor suppressor gene in TUSC2 deficient EGFR wildtype lung cancer cells increased sensitivity to erlotinib. Microarray mRNA expression analysis of TUSC2 inducible lung cancer cells treated with erlotinib uncovered defects in the response to oxidative stress suggesting that increasing reactive oxygen species (ROS) would enhance therapeutic efficacy. Addition of the thioredoxin reductase 1 inhibitor (TXNRD1) auranofin (AF) to NSCLC cells treated with combination of TUSC2 forced expression with erlotinib increased tumor cell apoptosis and inhibited colony formation. TXNRD1 overexpression rescued tumors from AF-TUSC2-erlotinib induced apoptosis. Neutralizing ROS with nordihydroguaiaretic acid (NDGA) abrogated cell death induced by AF-TUSC2-erlotinib, indicating a regulatory role for ROS in the efficacy of the three drug combination. Isobologram-based statistical analysis of this combination demonstrated superior synergism, compared with each individual treatment at lower concentrations. In NSCLC tumor xenografts, tumor growth was markedly inhibited and animal survival was prolonged over controls by AF-TUSC2-erlotinib. Microarray mRNA expression analysis uncovered oxidative stress and DNA damage gene signatures significantly upregulated by AF-TUSC2-erlotinib compared to TUSC2-erlotinib. Pathway analysis showed the highest positive z-score for the NRF2-mediated oxidative stress response. Taken together these findings show that the combination of TUSC2-erlotinib induces additional novel vulnerabilities that can be targeted with AF.

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

PubMed

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

Aldehyde dehydrogenase 7A1 (ALDH7A1) is a novel enzyme involved in cellular defense against hyperosmotic stress.

PubMed

Brocker, Chad; Lassen, Natalie; Estey, Tia; Pappa, Aglaia; Cantore, Miriam; Orlova, Valeria V; Chavakis, Triantafyllos; Kavanagh, Kathryn L; Oppermann, Udo; Vasiliou, Vasilis

2010-06-11

Mammalian ALDH7A1 is homologous to plant ALDH7B1, an enzyme that protects against various forms of stress, such as salinity, dehydration, and osmotic stress. It is known that mutations in the human ALDH7A1 gene cause pyridoxine-dependent and folic acid-responsive seizures. Herein, we show for the first time that human ALDH7A1 protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes. Human ALDH7A1 expression in Chinese hamster ovary cells attenuated osmotic stress-induced apoptosis caused by increased extracellular concentrations of sucrose or sodium chloride. Purified recombinant ALDH7A1 efficiently metabolized a number of aldehyde substrates, including the osmolyte precursor, betaine aldehyde, lipid peroxidation-derived aldehydes, and the intermediate lysine degradation product, alpha-aminoadipic semialdehyde. The crystal structure for ALDH7A1 supports the enzyme's substrate specificities. Tissue distribution studies in mice showed the highest expression of ALDH7A1 protein in liver, kidney, and brain, followed by pancreas and testes. ALDH7A1 protein was found in the cytosol, nucleus, and mitochondria, making it unique among the aldehyde dehydrogenase enzymes. Analysis of human and mouse cDNA sequences revealed mitochondrial and cytosolic transcripts that are differentially expressed in a tissue-specific manner in mice. In conclusion, ALDH7A1 is a novel aldehyde dehydrogenase expressed in multiple subcellular compartments that protects against hyperosmotic stress by generating osmolytes and metabolizing toxic aldehydes.

Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels

PubMed Central

Iannotti, Fabio Arturo; Barrese, Vincenzo; Formisano, Luigi; Miceli, Francesco; Taglialatela, Maurizio

2013-01-01

Changes in the expression of potassium (K+) channels is a pivotal event during skeletal muscle differentiation. In mouse C2C12 cells, similarly to human skeletal muscle cells, myotube formation increased the expression of Kv7.1, Kv7.3, and Kv7.4, the last showing the highest degree of regulation. In C2C12 cells, Kv7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In Kv7.4-silenced cells, the differentiation-promoting effect of the Kv7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for Kv7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5′ untranslated region and in the first intron of the Kv7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that Kv7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K+ channel subunit. PMID:23242999

Differential relationships between D1 and D2 dopamine receptor expression in the medial preoptic nucleus and sexually-motivated song in male European starlings (Sturnus vulgaris)

PubMed Central

DeVries, M. S.; Cordes, M.A.; Stevenson, S.A.; Riters, L.V.

2015-01-01

Converging data in songbirds support a central role for the medial preoptic nucleus (POM) in motivational aspects of vocal production. Recent data suggest that dopamine in the POM plays a complex modulatory role in the production of sexually-motivated song and that an optimal level of dopamine D1 receptor stimulation is required to facilitate singing behavior. To further explore this possibility, we used quantitative real time PCR to examine relationships between mRNA expression of D1 as well as D2 receptors in the POM (and also the lateral septum and Area X) and sexually-motivated singing behavior in male European starlings. Results showed that both males with the highest and lowest D1 expression in the POM sang significantly less than males with intermediate levels of expression. Furthermore, singing behavior rose linearly in association with increasing levels of D1 expression in POM but dropped abruptly, such that individuals with D1 expression values higher than the mean sang very little. Analysis of birds with low and intermediate levels of D1 expression in POM revealed strong positive correlations between D1 expression and song but negative relationships between D2 receptor expression and song. These findings support prior work suggesting an optimal level of POM D1 receptor stimulation best facilitates sexually-motivated singing behavior. Results also suggest that D2 receptors may work in opposition to D1 receptors in POM to modify vocal production. PMID:26079111

Biomonitoring of heavy metals contamination by mosses and lichens around Slovinky tailing pond (Slovakia).

PubMed

Demková, Lenka; Bobul'ská, Lenka; Árvay, Július; Jezný, Tomáš; Ducsay, Ladislav

2017-01-02

Three moss (Pleurozium spp., Polytrichum spp., and Rhytidiadelphus spp.) and two lichen (Hypogymnia physodes and Pseudevernia furfuracea) taxons covered in the bags were used to monitor air quality. Bags were exposed at the different distances from the tailing pond because of insufficient security and source of heavy metal pollution. Moss/lichen bags were exposed for six weeks at 0-, 50-, 100-, 150- and 200-m distances from Slovinky tailing pond, in the main wind direction (down the valley). Accumulation ability of heavy metals expressed by relative accumulation factor (RAF) increases in the order of Polytrichum spp.

Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia

USDA-ARS?s Scientific Manuscript database

Expression of dsRNA fragments of rust pathogen genes in wheat seedlings through the barley stripe mosaic virus (BSMV) based host-induced gene silencing (HIGS) system can reduce the expression of the corresponding genes in the rust fungus. The highest levels of suppression have generally been observe...

Expression profile of human tissue kallikrein 15 provides preliminary insights into its roles in the prostate and testis.

PubMed

Filippou, Panagiota S; Ren, Annie H; Soosaipillai, Antoninus; Papaioannou, Michail-Dimitrios; Korbakis, Dimitrios; Safar, Roaa; Diamandis, Eleftherios P; Conner, James

2018-06-26

Human tissue kallikrein 15 (KLK15) is the latest member of the kallikrein-related peptidase family. Little is known about the pathophysiological roles of KLK15. Previous studies implied a role of KLK15 in prostate cancer. In the present study, we examined KLK15 protein expression using a new immunoassay (ELISA) and immunohistochemistry (IHC). Highest KLK15 levels were detected in the testis and seminal fluid, whereas lower levels were observed in prostate and other tissues. Immunohistochemical analysis of testis suggests that KLK15 is strongly expressed in mature spermatids, but not in immature germ cells. KLK15 displayed predominantly nuclear localization in the basal cell layer of the prostatic epithelium. We also measured KLK15 in supernatants of various cell lines. Highest KLK15 levels were primarily detected in prostate cancer cell lines and KLK15 expression was hormone-independent, in contrast to KLK3. Collectively, our data provide insights into the localization and possible role of KLK15 in human physiology. Copyright © 2018. Published by Elsevier Inc.

Androgens Exert a Cysticidal Effect upon Taenia crassiceps by Disrupting Flame Cell Morphology and Function

PubMed Central

Ambrosio, Javier R.; Valverde-Islas, Laura; Nava-Castro, Karen E.; Palacios- Arreola, M. Isabel; Ostoa-Saloma, Pedro; Reynoso-Ducoing, Olivia; Escobedo, Galileo; Ruíz-Rosado, Azucena; Dominguez-Ramírez, Lenin; Morales-Montor, Jorge

2015-01-01

The effects of testosterone (T4) and dihydrotestosterone (DHT) on the survival of the helminth cestode parasite Taenia crassiceps, as well as their effects on actin, tubulin and myosin expression and their assembly into the excretory system of flame cells are described in this paper. In vitro evaluations on parasite viability, flow cytometry, confocal microscopy, video-microscopy of live flame cells, and docking experiments of androgens interacting with actin, tubulin, and myosin were conducted. Our results show that T4 and DHT reduce T. crassiceps viability in a dose- and time-dependent fashion, reaching 90% of mortality at the highest dose used (40 ng/ml) and time exposed (10 days) in culture. Androgen treatment does not induce differences in the specific expression pattern of actin, tubulin, and myosin isoforms as compared with control parasites. Confocal microscopy demonstrated a strong disruption of the parasite tegument, with reduced assembly, shape, and motion of flame cells. Docking experiments show that androgens are capable of affecting parasite survival and flame cell morphology by directly interacting with actin, tubulin and myosin without altering their protein expression pattern. We show that both T4 and DHT are able to bind actin, tubulin, and myosin affecting their assembly and causing parasite intoxication due to impairment of flame cell function. Live flame cell video microscopy showing a reduced motion as well changes in the shape of flame cells are also shown. In summary, T4 and DHT directly act on T. crassiceps cysticerci through altering parasite survival as well as the assembly and function of flame cells. PMID:26076446

Condensin-driven remodelling of X chromosome topology during dosage compensation

NASA Astrophysics Data System (ADS)

Crane, Emily; Bian, Qian; McCord, Rachel Patton; Lajoie, Bryan R.; Wheeler, Bayly S.; Ralston, Edward J.; Uzawa, Satoru; Dekker, Job; Meyer, Barbara J.

2015-07-01

The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (~1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.

Characterization of a Digestive α-Amylase in the Midgut of Pieris brassicae L. (Lepidoptera: Pieridae)

PubMed Central

Sharifloo, Ali; Zibaee, Arash; Sendi, Jalal J.; Jahroumi, Khalil Talebi

2016-01-01

The current study deals with a digestive α-amylase in the larvae of Pieris brassicae L. through purification, enzymatic characterization, gene expression, and in vivo effect of a specific inhibitor, Acarbose. Although α-amylase activity was the highest in the whole gut homogenate of larvae but compartmentalization of amylolytic activity showed an equal activity in posterior midgut (PM) and anterior midgut (AM). A three step purification using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose Fast flow revealed an enzyme with a specific activity of 5.18 U/mg, recovery of 13.20, purification fold of 19.25 and molecular weight of 88 kDa. The purified α-amylase had the highest activity at optimal pH and temperature of 8 and 35°C. Also, the enzyme had Vmax values of 4.64 and 3.02 U/mg protein and Km values of 1.37 and 1.74% using starch and glycogen as substrates, respectively. Different concentrations of acarbose, ethylenediamine tetraacetic acid, and ethylene glycol-bis (β-aminoethylether) N, N, N′, N′-tetraacetic acid significantly decreased activity of the purified α-amylase. The 4th instar larvae of P. brassicae were fed on the treated leaves of Raphanus sativus L. with 0.22 mM of Acarbose to find in vivo effects on nutritional indices, α-amylase activity, and gene expression. The significant differences were only found in conversion efficiency of digested food, relative growth rate, and metabolic cost of control and fed larvae on Acarbose. Also, amylolytic activity significantly decreased in the treated larvae by both biochemical and native-PAGE experiments. Results of RT-PCR revealed a gene with 621 bp length responsible for α-amylase expression that had 75% identity with Papilio xuthus and P. polytes. Finally, qRT-PCR revealed higher expression of α-amylase in control larvae compared to acarbose-fed ones. PMID:27014094

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor 1 (PAC1) in the human infant brain and changes in the Sudden Infant Death Syndrome (SIDS).

PubMed

Huang, J; Waters, K A; Machaalani, R

2017-07-01

Pituitary adenylate cyclase activating polypeptide (PACAP) and its complementary receptor, PAC1, are crucial in central respiratory control. PACAP Knockout (KO) mice exhibit a SIDS-like phenotype, with an inability to overcome noxious insults, compression of baseline ventilation, and death in the early post-neonatal period. PAC1 KO demonstrate similar attributes to PACAP-null mice, but with the addition of increased pulmonary artery pressure, consequently leading to heart failure and death. This study establishes a detailed interpretation of the neuroanatomical distribution and localization of both PACAP and PAC1 in the human infant brainstem and hippocampus, to determine whether any changes in expression are evident in infants who died of Sudden Infant Death Syndrome (SIDS) and any relationships to risk factors of SIDS including smoke exposure and sleep related parameters. Immunohistochemistry for PACAP and PAC1 was performed on formalin fixed and paraffin embedded human infant brain tissue of SIDS (n=32) and non-SIDS (n=12). The highest expression of PACAP was found in the hypoglossal (XII) of the brainstem medulla and lowest expression in the subiculum of the hippocampus. Highest expression of PAC1 was also found in XII of the medulla and lowest in the midbrain dorsal raphe (MBDR) and inferior colliculus. SIDS compared to non-SIDS had higher PACAP in the MBDR (p<0.05) and lower PAC1 in the medulla arcuate nucleus (p<0.001). Correlations were found between PACAP and PAC1 with the risk factors of smoke exposure, bed sharing, upper respiratory tract infection (URTI) and seasonal temperatures. The findings of this study show for the first time that some abnormalities of the PACAP system are evident in the SIDS brain and could contribute to the mechanisms of infants succumbing to SIDS. Copyright © 2017 Elsevier Inc. All rights reserved.

Condensin-Driven Remodeling of X-Chromosome Topology during Dosage Compensation

PubMed Central

Crane, Emily; Bian, Qian; McCord, Rachel Patton; Lajoie, Bryan R.; Wheeler, Bayly S.; Ralston, Edward J.; Uzawa, Satoru; Dekker, Job; Meyer, Barbara J.

2015-01-01

The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure1,2. Here we perform genome-wide chromosome conformation capture analysis, FISH, and RNA-seq to obtain comprehensive 3D maps of the Caenorhabditis elegans genome and to dissect X-chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half3–7. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes5,6. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (~1 Mb) resembling mammalian Topologically Associating Domains (TADs)8,9. TADs on X have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X while regulating gene expression chromosome wide. PMID:26030525

Analysis of 320 gastroenteropancreatic neuroendocrine tumors identifies TS expression as independent biomarker for survival.

PubMed

Lee, Hye Seung; Chen, Min; Kim, Ji Hun; Kim, Woo Ho; Ahn, Soyeon; Maeng, Kyungah; Allegra, Carmen J; Kaye, Frederic J; Hochwald, Steven N; Zajac-Kaye, Maria

2014-07-01

Thymidylate synthase (TS), a critical enzyme for DNA synthesis and repair, is both a potential tumor prognostic biomarker as well as a tumorigenic oncogene in animal models. We have now studied the clinical implications of TS expression in gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) and compared these results to other cell cycle biomarker genes. Protein tissue arrays were used to study TS, Ki-67, Rb, pRb, E2F1, p18, p21, p27 and menin expression in 320 human GEP-NETs samples. Immunohistochemical expression was correlated with univariate and multivariate predictors of survival utilizing Kaplan Meier and Cox proportional hazards models. Real time RT-PCR was used to validate these findings. We found that 78 of 320 GEP-NETs (24.4%) expressed TS. NETs arising in the colon, stomach and pancreas showed the highest expression of TS (47.4%, 42.6% and 37.3%, respectively), whereas NETs of the appendix, rectum and duodenum displayed low TS expression (3.3%, 12.9% and 15.4%, respectively). TS expression in GEP-NETs was associated with poorly differentiated endocrine carcinoma, angiolymphatic invasion, lymph node metastasis and distant metastasis (p < 0.05). Patients with TS-positive NETs had markedly worse outcomes than TS-negative NETs as shown by univariate (p < 0.001) and multivariate (p = 0.01) survival analyses. Expression of p18 predicted survival in TS-positive patients that received chemotherapy (p = 0.015). In conclusion, TS protein expression was an independent prognostic biomarker for GEP-NETs. The strong association of increased TS expression with aggressive disease and early death supports the role of TS as a cancer promoting agent in these tumors. © 2013 UICC.

Cloning and functional expression of a cDNA encoding stearoyl-ACP Δ9-desaturase from the endosperm of coconut (Cocos nucifera L.).

PubMed

Gao, Lingchao; Sun, Ruhao; Liang, Yuanxue; Zhang, Mengdan; Zheng, Yusheng; Li, Dongdong

2014-10-01

Coconut (Cocos nucifera L.) is an economically tropical fruit tree with special fatty acid compositions. The stearoyl-acyl carrier protein (ACP) desaturase (SAD) plays a key role in the properties of the majority of cellular glycerolipids. In this paper, a full-length cDNA of a stearoyl-acyl carrier protein desaturase, designated CocoFAD, was isolated from cDNA library prepared from the endosperm of coconut (C. nucifera L.). An 1176 bp cDNA from overlapped PCR products containing ORF encoding a 391-amino acid (aa) protein was obtained. The coded protein was virtually identical and shared the homology to other Δ9-desaturase plant sequences (greater than 80% as similarity to that of Elaeis guineensis Jacq). The real-time fluorescent quantitative PCR result indicated that the yield of CocoFAD was the highest in the endosperm of 8-month-old coconut and leaf, and the yield was reduced to 50% of the highest level in the endosperm of 15-month-old coconut. The coding region showed heterologous expression in strain INVSc1 of yeast (Saccharomyces cerevisiae). GC-MS analysis showed that the levels of palmitoleic acid (16:1) and oleic acid (18:1) were improved significantly; meanwhile stearic acid (18:0) was reduced. These results indicated that the plastidial Δ9 desaturase from the endosperm of coconut was involved in the biosynthesis of hexadecenoic acid and octadecenoic acid, which was similar with other plants. These results may be valuable for understanding the mechanism of fatty acid metabolism and the genetic improvement of CocoFAD gene in palm plants in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

Selection of reference genes for gene expression studies in heart failure for left and right ventricles.

PubMed

Li, Mengmeng; Rao, Man; Chen, Kai; Zhou, Jianye; Song, Jiangping

2017-07-15

Real-time quantitative reverse transcriptase-PCR (qRT-PCR) is a feasible tool for determining gene expression profiles, but the accuracy and reliability of the results depends on the stable expression of selected housekeeping genes in different samples. By far, researches on stable housekeeping genes in human heart failure samples are rare. Moreover the effect of heart failure on the expression of housekeeping genes in right and left ventricles is yet to be studied. Therefore we aim to provide stable housekeeping genes for both ventricles in heart failure and normal heart samples. In this study, we selected seven commonly used housekeeping genes as candidates. By using the qRT-PCR, the expression levels of ACTB, RAB7A, GAPDH, REEP5, RPL5, PSMB4 and VCP in eight heart failure and four normal heart samples were assessed. The stability of candidate housekeeping genes was evaluated by geNorm and Normfinder softwares. GAPDH showed the least variation in all heart samples. Results also indicated the difference of gene expression existed in heart failure left and right ventricles. GAPDH had the highest expression stability in both heart failure and normal heart samples. We also propose using different sets of housekeeping genes for left and right ventricles respectively. The combination of RPL5, GAPDH and PSMB4 is suitable for the right ventricle and the combination of GAPDH, REEP5 and RAB7A is suitable for the left ventricle. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

PubMed Central

Subramaniam, R; Reinold, S; Molitor, E K; Douglas, C J

1993-01-01

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues. PMID:8108506

The matricellular protein periostin contributes to proper collagen function and is downregulated during skin aging.

PubMed

Egbert, M; Ruetze, M; Sattler, M; Wenck, H; Gallinat, S; Lucius, R; Weise, J M

2014-01-01

Periostin is a secreted 90kDa matricellular protein, which is predominantly expressed in collagen-rich tissues. Collagen is the most abundant protein in mammals and has great tensile strength. Recent investigations have shown that periostin influences collagen fibrillogenesis and biomechanical properties of murine connective tissues. We investigated the function of periostin concerning collagen homeostasis during intrinsic and extrinsic skin aging. For this purpose, human skin samples of young and old donors as well as samples of photoaged and sun-protected skin areas were analyzed for periostin expression. Using in vitro models, we determined the cell types responsible for periostin expression and performed functional analyses with periostin knockdown cells. TaqMan Real-Time PCR, UV irradiation, knockdown experiments, immunostaining, electron microscopy, collagen degradation assay, collagen crosslink analysis. Periostin expression is highest in the papillary dermis and downregulated during skin aging. Fibroblasts and non-follicular skin derived precursors were identified as main source for periostin expression in human skin. Periostin knockdown in fibroblasts has no effect on collagen expression, but results in an increased fibril diameter and aberrant collagen structure. This leads to an increased susceptibility of collagen toward proteases, whereas recombinant periostin protects collagen fibrils from degradation. Our data show that periostin plays an important role for proper collagen assembly and homeostasis. During skin aging periostin expression decreases and contributes to the phenotype of aged skin. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli.

PubMed

Mohammadinezhad, Rezvan; Farahmand, Hamid; Jalali, Seyed Amir Hossein; Mirvaghefi, Alireza

2018-05-01

The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.

Tissue-specific changes in OGG1 and SOD mRNA expression caused by NaOCl exposure in black seabream ( Acanthopagrus schlegelii)

NASA Astrophysics Data System (ADS)

Park, Ho-Ra; Kim, Yong; Yeo, Won-Jun; Kim, Ji-Hye; Han, Kyung-Nam

2017-09-01

The DNA-damage defense mechanism was studied in black seabreams after oxidative stress caused by exposure to sodium hypochlorite (NaOCl). Liver, muscle, and brain tissues were obtained after different NaOCl-exposure times (0, 24, 48, 72, and 96 h) and concentrations (0.5, 1, 1.5, 2, and 3 mg/L), after which oxoguanine glycosylase (OGG1) and superoxide dismutase (SOD) mRNA-expression levels were analyzed. At all NaOCl concentrations tested, liver OGG1 expression increased to a maximum in a time-dependent manner after NaOCl exposure and then decreased. In muscles, OGG1 expression increased over time following exposure to a low concentration of NaOCl (0.5, 1, and 1.5 mg/L), whereas it showed a mixed pattern (both increases and decreases observed) in the high-concentration groups (2 and 3 mg/L). SOD mRNA expression increased over time, both in the liver and muscles. In the brain, both OGG1 and SOD mRNA expression levels were highest after exposure to the lowest NaOCl concentration (0.5 mg/L), whereas basal levels were maintained over time at higher concentrations. These results indicate that OGG1 and SOD provide resistance to oxidative stress in black seabreams. In addition, continuous exposure to oxidative stress can suppress enzyme expression, suggesting a risk for long-term exposure to NaOCl.

Identification and expression analysis of starch branching enzymes involved in starch synthesis during the development of chestnut (Castanea mollissima Blume) cotyledons

PubMed Central

Li, Zhi; Zhao, Yanyan; Jiang, Yichen; Zhang, Qing; Cao, Qingqin; Fang, Kefeng; Xing, Yu; Qin, Ling

2017-01-01

Chinese chestnut (Castanea mollissima Blume) is native to China and distributes widely in arid and semi-arid mountain area with barren soil. As a perennial crop, chestnut is an alternative food source and acts as an important commercial nut tree in China. Starch is the major metabolite in nuts, accounting for 46 ~ 64% of the chestnut dry weight. The accumulation of total starch and amylopectin showed a similar increasing trend during the development of nut. Amylopectin contributed up to 76% of the total starch content at 80 days after pollination (DAP). The increase of total starch mainly results from amylopectin synthesis. Among genes associated with starch biosynthesis, CmSBEs (starch branching enzyme) showed significant increase during nut development. Two starch branching enzyme isoforms, CmSBE I and CmSBE II, were identified from chestnut cotyledon using zymogram analysis. CmSBE I and CmSBE II showed similar patterns of expression during nut development. The accumulations of CmSBE transcripts and proteins in developing cotyledons were characterized. The expressions of two CmSBE genes increased from 64 DAP and reached the highest levels at 77 DAP, and SBE activity reached its peak at 74 DAP. These results suggested that the CmSBE enzymes mainly contributed to amylopectin synthesis and influenced the amylopectin content in the developing cotyledon, which would be beneficial to chestnut germplasm selection and breeding. PMID:28542293

Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of Olea europaea L. genotypes with different cold hardiness

PubMed Central

Matteucci, M.; D'Angeli, S.; Errico, S.; Lamanna, R.; Perrotta, G.; Altamura, M. M.

2011-01-01

The olive tree lacks dormancy and is low temperature sensitive, with differences in cold tolerance and oil quality among genotypes. The oil is produced in the drupe, and the unsaturated fatty acids contribute to its quality. The aim of the present research was to investigate the relationship among development, cold response, expression of fatty acid desaturase (FAD) genes, and unsaturated fatty acid composition in drupes belonging to genotypes differing in leaf cold tolerance, but producing good oil (i.e. the non-hardy Moraiolo, the semi-hardy Frantoio, and the hardy Canino). In all genotypes, cold sensitivity, evaluated by cold-induced transient increases in cytosolic calcium, was high in the epi-mesocarp cells before oil body formation, and decreased during oil biogenesis. However, genotype-dependent differences in cold sensitivity appeared at the end of oil production. Genotype-dependent differences in FAD2.1, FAD2.2, FAD6, and FAD7 expression levels occurred in the epi-mesocarp cells during the oleogenic period. However, FAD2.1 and FAD7 were always the highest in the first part of this period. FAD2.2 and FAD7 increased after cold applications during oleogenesis, independently of the genotype. Unsaturated fatty acids increased in the drupes of the non-hardy genotype, but not in those of the hardy one, after cold exposure at the time of the highest FAD transcription. The results show a direct relationship between FAD expression and lipid desaturation in the drupes of the cold-sensitive genotype, and an inverse relationship in those of the cold-resistant genotype, suggesting that drupe cold acclimation requires a fine FAD post-transcriptional regulation. Hypotheses relating FAD desaturation to storage and membrane lipids, and genotype cold hardiness are discussed. PMID:21357772

Domain retention in transcription factor fusion genes and its biological and clinical implications: a pan-cancer study

PubMed Central

Kim, Pora; Ballester, Leomar Y.; Zhao, Zhongming

2017-01-01

Genomic rearrangements involving transcription factors (TFs) can form fusion proteins resulting in either enhanced, weakened, or even loss of TF activity. Functional domain (FD) retention is a critical factor in the activity of transcription factor fusion genes (TFFGs). A systematic investigation of FD retention in TFFGs and their outcome (e.g. expression changes) in a pan-cancer study has not yet been completed. Here, we examined the FD retention status in 386 TFFGs across 13 major cancer types and identified 83 TFFGs involving 67 TFs that retained FDs. To measure the potential biological relevance of TFs in TFFGs, we introduced a Major Active Isofusion Index (MAII) and built a prioritized TFFG network using MAII scores and the observed frequency of fusion positive samples. Interestingly, the four TFFGs (PML-RARA, RUNX1-RUNX1T1, TMPRSS2-ERG, and SFPQ-TFE3) with the highest MAII scores showed 50 differentially expressed target genes (DETGs) in fusion-positive versus fusion-negative cancer samples. DETG analysis revealed that they were involved in tumorigenesis-related processes in each cancer type. PLAU, which encodes plasminogen activator urokinase and serves as a biomarker for tumor invasion, was found to be consistently activated in the samples with the highest MAII scores. Among the 50 DETGs, 21 were drug targetable genes. Fourteen of these 21 DETGs were expressed in acute myeloid leukemia (AML) samples. Accordingly, we constructed an AML-specific TFFG network, which included 38 DETGs in RUNX1-RUNX1T1 or PML-RARA positive samples. In summary, this study revealed several TFFGs and their potential target genes, and provided insights into the clinical implications of TFFGs. PMID:29299133

Multiple genes of mevalonate and non-mevalonate pathways contribute to high aconites content in an endangered medicinal herb, Aconitum heterophyllum Wall.

PubMed

Malhotra, Nikhil; Kumar, Varun; Sood, Hemant; Singh, Tiratha Raj; Chauhan, Rajinder Singh

2014-12-01

Aconitum heterophyllum Wall, popularly known as Atis or Patis, is an important medicinal herb of North-Western and Eastern Himalayas. No information exists on molecular aspects of aconites biosynthesis, including atisine- the major chemical constituent of A. heterophyllum. Atisine content ranged from 0.14% to 0.37% and total alkaloids (aconites) from 0.20% to 2.49% among 14 accessions of A. heterophyllum. Two accessions contained the highest atisine content with 0.30% and 0.37% as well as the highest alkaloids content with 2.22% and 2.49%, respectively. No atisine was detected in leaves and shoots of A. heterophyllum, thereby, suggesting that the biosynthesis and accumulation of aconite alkaloids occur mainly in roots. Quantitative expression analysis of 15 genes of MVA/MEP pathways in roots versus shoots, differing for atisine content (0-2.2 folds) showed 11-100 folds increase in transcript amounts of 4 genes of MVA pathway; HMGS, HMGR, PMK, IPPI, and 4 genes of MEP pathway; DXPS, ISPD, HDS, GDPS, respectively. The overall expression of 8 genes decreased to 5-12 folds after comparative expression analysis between roots of high (0.37%) versus low (0.14%) atisine content accessions, but their relative transcript amounts remained higher in high content accessions, thereby implying their role in atisine biosynthesis and accumulation. PCA analysis revealed a positive correlation between MVA/MEP pathways genes and alkaloids content. The current study provides first report wherein partial sequences of 15 genes of MVA/MEP pathways have been cloned and studied for their possible role in aconites biosynthesis. The outcome of study has potential applications in the genetic improvement of A. heterophyllum. Copyright © 2014 Elsevier Ltd. All rights reserved.

Association of zygotic piRNAs derived from paternal P elements with hybrid dysgenesis in Drosophila melanogaster.

PubMed

Wakisaka, Keiko Tsuji; Ichiyanagi, Kenji; Ohno, Seiko; Itoh, Masanobu

2018-01-01

P -element transposition in the genome causes P-M hybrid dysgenesis in Drosophila melanogaster . Maternally deposited piRNAs suppress P -element transposition in the progeny, linking them to P-M phenotypes; however, the role of zygotic piRNAs derived from paternal P elements is poorly understood. To elucidate the molecular basis of P -element suppression by zygotic factors, we investigated the genomic constitution and P -element piRNA production derived from fathers. As a result, we characterized males of naturally derived Q, M' and P strains, which show different capacities for the P -element mobilizations introduced after hybridizations with M-strain females. The amounts of piRNAs produced in ovaries of F1 hybrids varied among the strains and were influenced by the characteristics of the piRNA clusters that harbored the P elements. Importantly, while both the Q- and M'-strain fathers restrict the P -element mobilization in ovaries of their daughters, the Q-strain fathers supported the production of the highest piRNA expression in the ovaries of their daughters, and the M' strain carries KP elements in transcriptionally active regions directing the highest expression of KP elements in their daughters. Interestingly, the zygotic P -element piRNAs, but not the KP element mRNA, contributed to the variations in P transposition immunity in the granddaughters. The piRNA-cluster-embedded P elements and the transcriptionally active KP elements from the paternal genome are both important suppressors of P element activities that are co-inherited by the progeny. Expression levels of the P -element piRNA and KP -element mRNA vary among F1 progeny due to the constitution of the paternal genome, and are involved in phenotypic variation in the subsequent generation.

A Nematode Calreticulin, Rs-CRT, Is a Key Effector in Reproduction and Pathogenicity of Radopholus similis

PubMed Central

Li, Yu; Wang, Ke; Xie, Hui; Wang, Yan-Tao; Wang, Dong-Wei; Xu, Chun-Lin; Huang, Xin; Wang, De-Sen

2015-01-01

Radopholus similis is a migratory plant-parasitic nematode that causes severe damage to many agricultural and horticultural crops. Calreticulin (CRT) is a Ca2+-binding multifunctional protein that plays key roles in the parasitism, immune evasion, reproduction and pathogenesis of many animal parasites and plant nematodes. Therefore, CRT is a promising target for controlling R. similis. In this study, we obtained the full-length sequence of the CRT gene from R. similis (Rs-crt), which is 1,527-bp long and includes a 1,206-bp ORF that encodes 401 amino acids. Rs-CRT and Mi-CRT from Meloidogyne incognita showed the highest similarity and were grouped on the same branch of the phylogenetic tree. Rs-crt is a multi-copy gene that is expressed in the oesophageal glands and gonads of females, the gonads of males, the intestines of juveniles and the eggs of R. similis. The highest Rs-crt expression was detected in females, followed by juveniles, eggs and males. The reproductive capability and pathogenicity of R. similis were significantly reduced after treatment with Rs-crt dsRNA for 36 h. Using plant-mediated RNAi, we confirmed that Rs-crt expression was significantly inhibited in the nematodes, and resistance to R. similis was significantly improved in transgenic tomato plants. Plant-mediated RNAi-induced silencing of Rs-crt could be effectively transmitted to the F2 generation of R. similis; however, the silencing effect of Rs-crt induced by in vitro RNAi was no longer detectable in F1 and F2 nematodes. Thus, Rs-crt is essential for the reproduction and pathogenicity of R. similis. PMID:26061142

Isolation and characterization of a novel glycosyltransferase that converts phloretin to phlorizin, a potent antioxidant in apple.

PubMed

Jugdé, Hélène; Nguy, Danny; Moller, Isabel; Cooney, Janine M; Atkinson, Ross G

2008-08-01

The dihydrochalcone phlorizin (phloretin 2'-glucoside) contributes to the flavor, color and health benefits of apple fruit and processed products. A genomics approach was used to identify the gene MdPGT1 in apple (Malus x domestica) with homology to the UDP-glycosyltransferase 88 family of uridine diphosphate glycosyltransferases that show specificity towards flavonoid substrates. Expressed sequence tags for MdPGT1 were found in all tissues known to produce phlorizin including leaf, flower and fruit. However, the highest expression was measured by quantitative PCR in apple root tissue. The recombinant MdPGT1 enzyme expressed in Escherichia coli glycosylated phloretin in the presence of [(3)H]-UDP-glucose, but not other apple antioxidants, including quercetin, naringenin and cyanidin. The product of phloretin and UDP-glucose co-migrated with an authentic phlorizin standard. LC/MS indicated that MdPGT1 could glycosylate phloretin in the presence of three sugar donors: UDP-glucose, UDP-xylose and UDP-galactose. This is the first report of functional characterization of a UDP-glycosyltransferase that utilizes a dihydrochalcone as its primary substrate.

V-ATPase as an effective therapeutic target for sarcomas

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

Perut, Francesca, E-mail: francesca.perut@ior.it; Avnet, Sofia; Fotia, Caterina

2014-01-01

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles andmore » induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity.« less

Unravelling the contribution of the Penicillium expansum PeSte12 transcription factor to virulence during apple fruit infection.

PubMed

Sánchez-Torres, Paloma; Vilanova, Laura; Ballester, Ana Rosa; López-Pérez, Mario; Teixidó, Neus; Viñas, Inmaculada; Usall, Josep; González-Candelas, Luis; Torres, Rosario

2018-02-01

Blue mould disease caused by Penicillium expansum infection is one of the most important diseases of pome fruit accounting for important economic losses. In the present study, the PeSte12 transcription factor gene was identified, and deletant mutants were produced by gene replacement. Knockout mutants showed a significant decrease of virulence during apple fruit infection. Virulence was affected by the maturity stage of the fruit (immature, mature and over-mature), and disease severity was notably reduced when the apples were stored at 0 °C. The ΔPeSte12 mutants resulted defective in asexual reproduction, producing less conidia, but this characteristic did not correlate with differences in microscopic morphology. In addition, the ΔPeSte12 mutants produced higher quantity of hydrogen peroxide than the wild type strain. Gene expression analysis revealed that PeSte12 was induced over time during apple infection compared to axenic growth, particularly from 2 dpi, reinforcing its role in virulence. Analysis of transcriptional abundance of several genes in ΔPeSte12 mutants showed that in most of the evaluated genes, PeSte12 seemed to act as a negative regulator during axenic growth, as most of them exhibited an increasing expression pattern along the time period evaluated. The highest expression values corresponded to detoxification, ATPase activity, protein folding and basic metabolism. Gene expression analysis during apple infection showed that 3 out of 9 analysed genes were up regulated; thus, PeSte12 seemed to exert a positive control to particular type of aldolase. These results demonstrate the PeSte12 transcription factor could play an important role in P. expansum's virulence and asexual reproduction. Copyright © 2017 Elsevier Ltd. All rights reserved.

A novel HLA-A*0201 restricted peptide derived from cathepsin G is an effective immunotherapeutic target in acute myeloid leukemia.

PubMed

Zhang, Mao; Sukhumalchandra, Pariya; Enyenihi, Atim A; St John, Lisa S; Hunsucker, Sally A; Mittendorf, Elizabeth A; Sergeeva, Anna; Ruisaard, Kathryn; Al-Atrache, Zein; Ropp, Patricia A; Jakher, Haroon; Rodriguez-Cruz, Tania; Lizee, Gregory; Clise-Dwyer, Karen; Lu, Sijie; Molldrem, Jeffrey J; Glish, Gary L; Armistead, Paul M; Alatrash, Gheath

2013-01-01

Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients. CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL-mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development.

Baked corn (Zea mays L.) and bean (Phaseolus vulgaris L.) snack consumption lowered serum lipids and differentiated liver gene expression in C57BL/6 mice fed a high-fat diet by inhibiting PPARγ and SREBF2.

PubMed

Dominguez-Uscanga, Astrid; Loarca-Piña, Guadalupe; Gonzalez de Mejia, Elvira

2017-12-01

The aim was to determine the effect of consuming a baked white corn/bean snack (70/30% blend) on improving diet-induced dyslipidemia and liver differential gene expression in mice fed a high-fat diet (HFD). C57BL/6 mice were randomized into six groups and different doses of the snack (0.5-2.0 g/d) supplemented to a basal HFD for 12 weeks. Unsupplemented HFD and a standard diet were used as positive and negative controls, respectively. Groups receiving HFD1.0, HFD1.5 and HFD2.0 showed attenuation in body weight gain (20%). Serum cholesterol and triglycerides were reduced (P<.05), 29% and 31%, respectively. Blood glucose was also reduced (P<.05) in all groups receiving the snack. Histological analysis showed a reduction in adipocyte diameters (P<.05) suggesting an attenuation of lipid accumulation. Snack consumption induced differential expression of 529 genes in the liver; RGS16 was the highest up-regulated molecule (+15-fold change). Increased expression of this gene could have improved glucose metabolism in HFD2.0. Ingenuity Pathway Analysis downstream analysis showed a predicted inhibition of target genes of peroxisome PPARγ and key regulators of lipogenic genes in the liver. The results suggest that consumption of a white corn/bean snack (70%/30% blend) attenuates weight gain, fat mass accumulation, adipocyte size and nonalcoholic fatty liver disease in HFD-fed mice by inhibiting PPARγ and SREBF2. The study proposes that this type of product might be beneficial by preventing dyslipidemia, obesity and hepatic steatosis. Copyright © 2017 Elsevier Inc. All rights reserved.

A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes

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

Zhao, Nan; Ferrer, Jean-Luc; Moon, Hong S

2012-01-01

Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested formore » methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5 lM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.« less

MiR-132 regulated olfactory bulb proteins linked to olfactory learning in greater short-nosed fruit bat Cynopterus sphinx.

PubMed

Mukilan, Murugan; Rajathei, David Mary; Jeyaraj, Edwin; Kayalvizhi, Nagarajan; Rajan, Koilmani Emmanuvel

2018-05-30

Earlier, we showed that micro RNA-132 (miR-132) regulate the immediate early genes (IEGs) in the olfactory bulb (OB) of fruit bat Cynopterus sphinx during olfactory learning. This study was designed to examine whether the miR-132 regulate other proteins in OB during olfactory learning. To test this, miR-132 anti-sense oligodeoxynucleotide (AS-ODN) was delivered to the OB and then trained to novel odor. The 2-dimensional gel electrophoresis analysis showed that inhibition of miR-132 altered olfactory training induced expression of 321 proteins. Further, liquid chromatography-mass spectrometry (LC-MS/MS) analysis reveals the identity of differently expressed proteins such as phosphoribosyl transferase domain containing protein (PRTFDC 1), Sorting nexin-8 (SNX8), Creatine kinase B-type (CKB) and Annexin A11 (ANX A11). Among them PRTFDC 1 showing 189 matching peptides with highest sequence coverage (67.0%) and protein-protein interaction analysis showed that PRTFDC 1 is a homolog of hypoxanthine phosphoribosyltransferase-1 (HPRT-1). Subsequent immunohistochemical analysis (IHC) showed that inhibition of miR-132 down-regulated HPRT expression in OB of C. sphinx. In addition, western blot analysis depicts that HPRT, serotonin transporter (SERT), N-methyl-d-asparate (NMDA) receptors (2A,B) were down-regulated, but not altered in OB of non-sense oligodeoxynucleotide (NS-ODN) infused groups. These analyses suggest that miR-132 regulates the process of olfactory learning and memory formation through SERT and NMDA receptors signalling, which is possibly associated with the PRTFDC1-HPRT interaction. Copyright © 2017. Published by Elsevier B.V.

Expression of SIRT1 and oxidative stress in diabetic dry eye.

PubMed

Liu, Hao; Sheng, Minjie; Liu, Yu; Wang, Peng; Chen, Yihui; Chen, Li; Wang, Weifang; Li, Bing

2015-01-01

To explore the expression of SIRT1 with oxidative stress and observe physiological and pathological changes in the corneas as well as the association between SIRT1 and oxidative stress of diabetic dry eyes in mice. Forty-eight C57BL/6Jdb/db mice at eight weeks of age were divided randomly into two groups: the diabetic dry eye group and the diabetic group. An additional forty-eight C57BL/6J mice at eight weeks of age were divided randomly into two groups: the dry eye group and the control group. Every mouse in the dry eye groups (diabetic and normal) was injected with scopolamine hydrobromide three times daily, combined with low humidity to establish a dry eye model. After the intervention, phenol red cotton string tests and corneal fluorescein staining were performed. In addition, HE staining and immunofluorescence were done. Expression of SIRT1 in the cornea was examined by real-time PCR and Western Blot and expression of FOXO3 and MnSOD proteins was detected by Western Blot. At one, four, and eight weeks post intervention, all of the groups except the controls showed significant decreases in tear production and increases in the corneal fluorescein stain (P<0.05 vs control). Between the experimental groups, the diabetic dry eye group had the least tear production and the highest corneal fluorescein stain score (P<0.05). As the disease progressed, all of the experimental groups showed obviously pathological changes in HE staining, particularly the diabetic dry eye group. In the 1(st) and 4(th) week, the expression of SIRT1, FOXO3, and MnSOD were significantly higher in the diabetic DE and DM groups but lower in the DE group compared to the controls (P<0.05). In the 8(th) week, the expression of SIRT1, FOXO3, and MnSOD was significantly down-regulated in the diabetic DE group and the DM group (P<0.05). Immunofluorescence showed similar results. In the condition of diabetic dry eye, tear production declined markedly coupled with seriously wounded corneal epithelium. Oxidative stress in the cornea was enhanced significantly and the expression of SIRT1 was decreased.

Cloning, expression and characterization of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767

PubMed Central

2012-01-01

Background The white-rot fungus Phanerochaete chrysosporium is among the small group of fungi that can degrade lignin to carbon dioxide while leaving the crystalline cellulose untouched. The efficient lignin oxidation system of this fungus requires cyclic redox reactions involving the reduction of aryl-aldehydes to the corresponding alcohols by aryl-alcohol dehydrogenase. However, the biochemical properties of this enzyme have not been extensively studied. These are of most interest for the design of metabolic engineering/synthetic biology strategies in the field of biotechnological applications of this enzyme. Results We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus Phanerochaete chrysosporium, its expression in Escherichia coli and the biochemical characterization of the encoded GST and His6 tagged protein. The purified recombinant enzyme showed optimal activity at 37°C and at pH 6.4 for the reduction of aryl- and linear aldehydes with NADPH as coenzyme. NADH could also be the electron donor, while having a higher Km (220 μM) compared to that of NADPH (39 μM). The purified recombinant enzyme was found to be active in the reduction of more than 20 different aryl- and linear aldehydes showing highest specificity for mono- and dimethoxylated Benzaldehyde at positions 3, 4, 3,4 and 3,5. The enzyme was also capable of oxidizing aryl-alcohols with NADP + at 30°C and an optimum pH of 10.3 but with 15 to 100-fold lower catalytic efficiency than for the reduction reaction. Conclusions In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. We show that this enzyme functions in the reductive sense under physiological conditions and that it displays relatively large substrate specificity with highest activity towards the natural compound Veratraldehyde. PMID:22742413

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart

PubMed Central

Li, Ning; Csepe, Thomas A.; Hansen, Brian J.; Sul, Lidiya V.; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O.; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R.; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul ML; Biesiadecki, Brandon; Hummel, John D; Weiss, Raul; Fedorov, Vadim V.

2016-01-01

Background Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor (A1R) and its downstream GIRK channels (IK,Ado). Methods We applied bi-atrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and non-failing human hearts (n=37). Results Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100μM) produced more significant shortening of action potential durations (APD80) in RA (from 290±45ms to 239±41ms, 17.3±10.4%; p<0.01) than LA (from 307±24ms to 286±23ms, 6.7±6.6%; p<0.01). In ten hearts, adenosine induced AF (317±116 sec) that, when sustained (≥2 min), was primarily maintained by one/two localized reentrant drivers in lateral RA. Tertiapin (10–100nM), a selective GIRK channel blocker, counteracted adenosine-induced APD shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher A1R (2.7±1.7 fold; p<0.01) and GIRK4 (1.7±0.8 fold; p<0.05) protein expression than lateral/posterior LA. Conclusions This study revealed a three-fold RA-to-LA A1R protein expression gradient in the human heart, leading to significantly greater RA vs. LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest A1R/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. PMID:27462069

Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart.

PubMed

Li, Ning; Csepe, Thomas A; Hansen, Brian J; Sul, Lidiya V; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul M L; Biesiadecki, Brandon J; Hummel, John D; Weiss, Raul; Fedorov, Vadim V

2016-08-09

Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10-100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10-100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. © 2016 American Heart Association, Inc.

Biocontrol activity of an alkaline serine protease from Aureobasidium pullulans expressed in Pichia pastoris against four postharvest pathogens on apple.

PubMed

Banani, Houda; Spadaro, Davide; Zhang, Dianpeng; Matic, Slavica; Garibaldi, Angelo; Gullino, Maria Lodovica

2014-07-16

The yeast-like fungus Aureobasidium pullulans PL5 is a microbial antagonist against postharvest pathogens of fruits. The strain is able to produce hydrolases, including glucanases, chitinases and proteases. The alkaline serine protease gene ALP5 from A. pullulans was cloned, inserted into the vector pPIC9 to construct pPIC9/ALP5, and then expressed in Pichia pastoris strain KM71. ALP5 had a molecular mass of 42.9kDa after 5days growth with 1% methanol induction at 28°C. The recombinant protease expressed in P. pastoris showed its highest activity under alkaline conditions (at pH10) and a temperature of 50°C. The antifungal activity of the recombinant protease was investigated against Penicillium expansum, Botrytis cinerea, Monilinia fructicola and Alternaria alternata in vitro and on apple. The recombinant protease reduced significantly the spore germination and the germ tube length of the tested pathogens in PDB medium. The highest level of protease efficacy was observed against M. fructicola and B. cinerea, whereas a lower efficacy was observed against P. expansum and A. alternata indicating a possible effect of the pathogen cell wall composition on the proteolytic activity of the recombinant protease. The presence of protease was able to cause the swelling of the hyphae of B. cinerea, under an optical microscope. The recombinant protease expressed in P. pastoris was more active against the pathogens in vitro than the same enzyme expressed in E. coli in previous studies. The efficacy of ALP5 was also evaluated against the pathogens in vivo on cv Golden Delicious apples. The protease was more efficient in controlling M. fructicola, B. cinerea and P. expansum than A. alternata. However, the extent of the activity was dependent on the enzyme concentration and the length of fruit storage. This study demonstrated the capacity of the alkaline serine protease to keep its enzymatic activity for some days in the unfavorable environment of the fruit wounds. The alkaline serine protease could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short storage period. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of the effects of sdiA, a luxR homologue, on adherence and motility of Escherichia coli O157 : H7.

PubMed

Sharma, Vijay K; Bearson, Shawn M D; Bearson, Bradley L

2010-05-01

Quorum-sensing (QS) signalling pathways are important regulatory networks for controlling the expression of genes promoting adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to epithelial cells. A recent study has shown that EHEC O157 : H7 encodes a luxR homologue, called sdiA, which upon overexpression reduces the expression of genes encoding flagellar and locus of enterocyte effacement (LEE) proteins, thus negatively impacting on the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157 : H7 strain 86-24, and from a hha (a negative regulator of ler) mutant of this strain, enhanced bacterial adherence to HEp-2 epithelial cells of the sdiA mutant strains relative to the strains containing a wild-type copy of sdiA. Quantitative reverse transcription PCR showed that the expression of LEE-encoded genes ler, espA and eae in strains with the sdiA deletions was not significantly different from that of the strains wild-type for sdiA. Similarly, no additional increases in the expression of LEE genes were observed in a sdiA hha double mutant strain relative to that observed in the hha deletion mutant. While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the expression of fliC was reduced by several fold in the hha mutant strain, irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of fliC. The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant, suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes lpfA and fimA of the operons encoding long polar and type 1 fimbriae in the sdiA mutant strain. These data indicate that SdiA has no significant effect on the expression of LEE genes, but that it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157 : H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 epithelial cells.

Molecular profiling of subclinical inflammatory lesions in long-term surviving adult liver transplant recipients.

PubMed

Londoño, María-Carlota; Souza, Lara Neves; Lozano, Juan-José; Miquel, Rosa; Abraldes, Juan G; Llovet, Laura-Patricia; Quaglia, Alberto; Rimola, Antoni; Navasa, Miquel; Sánchez-Fueyo, Alberto

2018-04-28

Subclinical inflammatory changes are commonly described in long-term transplant recipients undergoing protocol liver biopsies. The pathogenesis of these lesions remains unclear. The aim of this study was to identify the key molecular pathways driving progressive subclinical inflammatory liver allograft damage. All liver recipients followed at Hospital Clínic Barcelona who were >10 years post-transplant were screened for participation in the study. Patients with recurrence of underlying liver disease, biliary or vascular complications, chronic rejection, and abnormal liver function tests were excluded. Sixty-seven patients agreed to participate and underwent blood and serological tests, transient elastography and a liver biopsy. Transcriptome profiling was performed on RNA extracted from 49 out of the 67 biopsies employing a whole genome next generation sequencing platform. Patients were followed for a median of 6.8 years following the index liver biopsy. Median time since transplantation to liver biopsy was 13 years (10-22). The most frequently observed histological abnormality was portal inflammation with different degrees of fibrosis, present in 45 biopsies (67%). Two modules of 102 and 425 co-expressed genes were significantly correlated with portal inflammation, interface hepatitis and portal fibrosis. These modules were enriched in molecular pathways known to be associated with T cell mediated rejection. Liver allografts showing the highest expression levels for the two modules recapitulated the transcriptional profile of biopsies with clinically apparent rejection and developed progressive damage over time, as assessed by non-invasive markers of fibrosis. A large proportion of adult liver transplant recipients who survive long-term exhibit subclinical histological abnormalities. The transcriptomic profile of these patients' liver tissue closely resembles that of T cell mediated rejection and may result in progressive allograft damage. A large proportion of adult liver transplant recipients who survive for a long time exhibit subclinical histological abnormalities. The expression profile (a measurement of the activity of genes) of liver tissue from a large fraction of these patients closely resembles the profile of T cell mediated rejection. Liver allografts showing the highest expression levels of rejection-related genes developed progressive damage over time. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Treatment with low-dose atorvastatin, losartan, and their combination increases expression of vasoactive-related genes in rat aortas.

PubMed

Lunder, Mojca; Drevenšek, Gorazd; Černe, Darko; Marc, Janja; Janić, Miodrag; Šabovič, Mišo

2013-03-01

Recently it has been shown that statins and angiotensin receptor blockers (ARBs) at low doses express beneficial pleiotropic vascular effects. We aimed to explore whether these drugs at low doses induce the expression of vasoactive-related genes. Sixty adult Wistar rats were treated with low-dose atorvastatin (2 mg/kg), low-dose losartan (5 mg/kg), their combination or saline daily for 4, 6, or 8 weeks. Expression of the vasoactive-related genes endothelin receptor type A (EDNRA), endothelial nitric oxide synthase 3 (NOS3), inducible nitric oxide synthase 2 (NOS2), and angiotensin II receptor type 1 (AGTRL1a) was measured in isolated thoracic aortas. Expression of EDNRA gradually decreased, the lowest values being obtained after 8 weeks (low-dose atorvastatin, losartan [1.6- and 1-7-fold vs controls, respectively; both P < .05], and the combination [2.3-fold vs control, P < .001]). The highest values of NOS3 were obtained after 6 weeks (low-dose atorvastatin, losartan, and their combination, 3.1-fold, P < .01; 3.4-fold, P < .001; and 3.6-fold, P < .001 vs controls, respectively) and then declined after 8 weeks. The combination was more effective in inducing total NOS3 expression when compared to the separate drugs (1.4-fold; P < .05). Importantly, expression of NOS3 was associated with increased plasma NO levels and positively correlated with thoracic aorta relaxation. No changes in expression of NOS2 and AGTRL1a were observed. We showed that low-dose atorvastatin or losartan and especially their combination increases the expression of NOS3 and decreases the expression of EDNRA. These findings are valuable in explaining the effectiveness of the "low-dose pharmacological approach" for improvement in arterial function.

The potential role of myocardial serotonin receptor 2B expression in canine dilated cardiomyopathy.

PubMed

Fonfara, Sonja; Hetzel, Udo; Oyama, Mark A; Kipar, Anja

2014-03-01

Serotonin signalling in the heart is mediated by receptor subtype 2B (5-HTR2B). A contribution of serotonin to valvular disease has been reported, but myocardial expression of 5-HTR2B and its role in canine dilated cardiomyopathy (DCM) is not known. The aim of the present study was to investigate myocardial 5-HTR2B mRNA expression in dogs with DCM and to correlate results with expression of markers for inflammation and remodelling. Myocardial samples from eight healthy dogs, four dogs with DCM, five with cardiac diseases other than DCM and six with systemic non-cardiac diseases were investigated for 5-HTR2B mRNA expression using quantitative PCR (qPCR). The results were compared to mRNA expression of selected cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP). Laser microdissection with subsequent qPCR and immunohistochemistry were employed to identify the cells expressing 5-HTR2B. The myocardium of control dogs showed constitutive 5-HTR2B mRNA expression. In dogs with DCM, 5-HTR2B mRNA values were significantly greater than in all other groups, with highest levels of expression in the left ventricle and right atrium. Myocytes were identified as the source of 5-HTR2B mRNA and protein. A significant positive correlation of 5-HTR2B mRNA with expression of several cytokines, MMPs and TIMPs was observed. The findings suggest that serotonin might play a role in normal cardiac structure and function and could contribute to myocardial remodelling and functional impairment in dogs with DCM. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dose-related gene expression changes in forebrain following acute, low-level chlorpyrifos exposure in neonatal rats

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

Ray, Anamika; Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078; Liu Jing

2010-10-15

Chlorpyrifos (CPF) is a widely used organophosphorus insecticide (OP) and putative developmental neurotoxicant in humans. The acute toxicity of CPF is elicited by acetylcholinesterase (AChE) inhibition. We characterized dose-related (0.1, 0.5, 1 and 2 mg/kg) gene expression profiles and changes in cell signaling pathways 24 h following acute CPF exposure in 7-day-old rats. Microarray experiments indicated that approximately 9% of the 44,000 genes were differentially expressed following either one of the four CPF dosages studied (546, 505, 522, and 3,066 genes with 0.1, 0.5, 1.0 and 2.0 mg/kg CPF). Genes were grouped according to dose-related expression patterns using K-means clusteringmore » while gene networks and canonical pathways were evaluated using Ingenuity Pathway Analysis (registered) . Twenty clusters were identified and differential expression of selected genes was verified by RT-PCR. The four largest clusters (each containing from 276 to 905 genes) constituted over 50% of all differentially expressed genes and exhibited up-regulation following exposure to the highest dosage (2 mg/kg CPF). The total number of gene networks affected by CPF also rose sharply with the highest dosage of CPF (18, 16, 18 and 50 with 0.1, 0.5, 1 and 2 mg/kg CPF). Forebrain cholinesterase (ChE) activity was significantly reduced (26%) only in the highest dosage group. Based on magnitude of dose-related changes in differentially expressed genes, relative numbers of gene clusters and signaling networks affected, and forebrain ChE inhibition only at 2 mg/kg CPF, we focused subsequent analyses on this treatment group. Six canonical pathways were identified that were significantly affected by 2 mg/kg CPF (MAPK, oxidative stress, NF{Kappa}B, mitochondrial dysfunction, arylhydrocarbon receptor and adrenergic receptor signaling). Evaluation of different cellular functions of the differentially expressed genes suggested changes related to olfactory receptors, cell adhesion/migration, synapse/synaptic transmission and transcription/translation. Nine genes were differentially affected in all four CPF dosing groups. We conclude that the most robust, consistent changes in differential gene expression in neonatal forebrain across a range of acute CPF dosages occurred at an exposure level associated with the classical marker of OP toxicity, AChE inhibition. Disruption of multiple cellular pathways, in particular cell adhesion, may contribute to the developmental neurotoxicity potential of this pesticide.« less

Analysis of gene expression profiles in tympanic membrane following perforation using PCR Array in rats--preliminary investigation.

PubMed

Hassmann-Poznańska, Elżbieta; Taranta, Andrzej; Bialuk, Izabela; Poznańska, Maria; Zajączkiewicz, Hanna; Winnicka, Maria Małgorzata

2013-10-01

The goal of this work was to identify genes, known to be involved in the skin wound healing, that express differentially in the healthy and injured tympanic membrane (TM), and designate the molecules potentially beneficial for treatment of TM perforation. The molecular mechanisms controlling the course of TM regeneration are far from being elucidated. Twenty rats had their tympanic membranes perforated, while four served as a control. Animals were sacrificed on either days 1, 2, 3, 5 and 10 post injury, and TMs were immediately dissected and frozen in liquid nitrogen. Total TM RNA was isolated and reversely transcribed. qPCR was performed using Rat Wound Healing RT(2) Profiler PCR Array (QIAGEN) containing primers for 84 genes. Statistically significant changes in the expression of 42 genes were found in various stages of TM healing. The increased expression of genes taking part in the inflammatory reaction (interleukin 6, granulocyte and macrophage chemotactic proteins) was observed from day 2. The expression of several genes of extracellular matrix components and their remodeling enzymes was also changed. Among growth factor genes: Vegfa, Igf1 and Hbegf showed increased expression at the beginning of the healing process, while Hgf expression was highest on day 3. Several changes in the expression of genes involved in remodeling of extracellular matrix point to important role of connective tissue in TM healing. The molecules accelerating this process, like HbEGF and HGF, seem to be good candidates for further evaluation of their possible use in clinical treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Genome-wide gene expression profiling of low-dose, long-term exposure of human osteosarcoma cells to bisphenol A and its analogs bisphenols AF and S.

PubMed

Fic, A; Mlakar, S Jurković; Juvan, P; Mlakar, V; Marc, J; Dolenc, M Sollner; Broberg, K; Mašič, L Peterlin

2015-08-01

The bisphenols AF (BPAF) and S (BPS) are structural analogs of the endocrine disruptor bisphenol A (BPA), and are used in common products as a replacement for BPA. To elucidate genome-wide gene expression responses, estrogen-dependent osteosarcoma cells were cultured with 10 nM BPA, BPAF, or BPS, for 8 h and 3 months. Genome-wide gene expression was analyzed using the Illumina Expression BeadChip. Three months exposure had significant effects on gene expression, particularly for BPS, followed by BPAF and BPA, according to the number of differentially expressed genes (1980, 778, 60, respectively), the magnitude of changes in gene expression, and the number of enriched biological processes (800, 415, 33, respectively) and pathways (77, 52, 6, respectively). 'Embryonic skeletal system development' was the most enriched bone-related process, which was affected only by BPAF and BPS. Interestingly, all three bisphenols showed highest down-regulation of genes related to the cardiovascular system (e.g., NPPB, NPR3, TXNIP). BPA only and BPA/BPAF/BPS also affected genes related to the immune system and fetal development, respectively. For BPAF and BPS, the 'isoprenoid biosynthetic process' was enriched (up-regulated genes: HMGCS1, PDSS1, ACAT2, RCE1, DHDDS). Compared to BPA, BPAF and BPS had more effects on gene expression after long-term exposure. These findings stress the need for careful toxicological characterization of BPA analogs in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

PubMed

Sun, Wan-Wei; Zhang, Xin-Xu; Wan, Wei-Song; Wang, Shu-Qi; Wen, Xiao-Bo; Zheng, Huai-Ping; Zhang, Yue-Ling; Li, Sheng-Kang

2017-02-01

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain. Copyright © 2016. Published by Elsevier Ltd.

Differential expression of chicken dimerization cofactor of hepatocyte nuclear factor-1 (DcoH) and its novel counterpart, DcoHalpha.

PubMed Central

Kim, H; You, S; Foster, L K; Farris, J; Choi, Y J; Foster, D N

2001-01-01

We have used differential display PCR to study altered gene expression in immortalized chicken embryo fibroblasts (CEFs) that have been established in our laboratory. This technique resulted in the cloning of a novel counterpart of the previously cloned chicken dimerization cofactor of hepatocyte nuclear factor (HNF)-1 (cDcoH), which was identified as cDcoHalpha. The steady-state mRNA levels of cDcoHalpha were up-regulated in all immortal CEFs tested compared with primary CEF cells. cDcoH and cDcoHalpha showed opposite patterns of mRNA expression due to differential regulation of transcription rates, but not mRNA half-lives, in primary and immortal CEFs. Expression of cDcoHalpha increased in the late G1 and early S phases of the cell cycle, while cDcoH mRNA increased in the late S and G2/M phases. In contrast with consistent expression of both genes in primary quiescent cells, cDcoH mRNA, but not cDcoHalpha mRNA, was dramatically decreased in primary senescent cells. The highest levels of cDcoHalpha mRNA were found in the kidney, liver, heart and ovarian follicles, while the major tissues expressing cDcoH were hypothalamus, kidney and liver. cDcoH and cDcoHalpha probes did not cross-hybridize to human hepatocyte mRNA. When transfected into human HepG2 cells, both cDcoH and cDcoHalpha showed similar functional activity as measured by increased expression of a reporter gene, as well as alpha-fetoprotein and albumin genes that both contain HNF-1 binding elements in their promoters. Our results suggest that the novel chicken DcoHalpha might function as a transcriptional cofactor for HNF-1 in specific cellular-environmental states. PMID:11237869