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Sample records for induced ucp3 gene

  1. Hypercaloric cafeteria-like diet induced UCP3 gene expression in skeletal muscle is impaired by hypothyroidism

    OpenAIRE

    2004-01-01

    The uncoupling protein UCP3 belongs to a family of mitochondrial carriers located in the inner mitochondrial membrane of certain cell types. It is expressed almost exclusively at high levels in skeletal muscle and its physiological role has not been fully determined in this tissue. In the present study we have addressed the possible interaction between a hypercaloric diet and thyroid hormone (T3), which are strong stimulators of UCP3 gene expression in skeletal muscle. Male Wistar rats weighi...

  2. Hypercaloric cafeteria-like diet induced UCP3 gene expression in skeletal muscle is impaired by hypothyroidism

    Directory of Open Access Journals (Sweden)

    Christoffolete M.A.

    2004-01-01

    Full Text Available The uncoupling protein UCP3 belongs to a family of mitochondrial carriers located in the inner mitochondrial membrane of certain cell types. It is expressed almost exclusively at high levels in skeletal muscle and its physiological role has not been fully determined in this tissue. In the present study we have addressed the possible interaction between a hypercaloric diet and thyroid hormone (T3, which are strong stimulators of UCP3 gene expression in skeletal muscle. Male Wistar rats weighing 180 ± 20 g were rendered hypothyroid by thyroidectomy and the addition of methimazole (0.05%; w/v to drinking water after surgery. The rats were fed a hypercaloric cafeteria diet (68% carbohydrates, 13% protein and 18% lipids for 10 days and sacrificed by decapitation. Subsequently, the gastrocnemius muscle was dissected, total RNA was isolated with Trizol? and UCP3 gene expression was determined by Northern blotting using a specific probe. Statistical analysis was performed by one-way analysis of variance (ANOVA followed by the Student-Newman-Keuls post-test. Skeletal muscle UCP3 gene expression was decreased by 60% in hypothyroid rats and UCP3 mRNA expression was increased 70% in euthyroid cafeteria-fed rats compared to euthyroid chow-fed animals, confirming previous studies. Interestingly, the cafeteria diet was unable to stimulate UCP3 gene expression in hypothyroid animals (40% lower as compared to euthyroid cafeteria-fed animals. The results show that a hypercaloric diet is a strong stimulator of UCP3 gene expression in skeletal muscle and requires T3 for an adequate action.

  3. Identification of putative PPAR response elements in and around the murine UCP3 gene

    DEFF Research Database (Denmark)

    Siersbæk, Majken

    in thermogenesis in unerring, the physiological functions of UCP2 and UCP3 are at present not fully understood. Synthetic agonists for the peroxisome proliferator-activated receptors (PPARs) as well as fatty acids have been shown to increase murine UCP2 and UCP3 mRNA expression but response elements and mechanisms...... differentiation. Results from sequencing of chromatin immunoprecipitated (ChIP) material from 3T3-L1 adipocytes revealed three PPAR and retinoid X receptor (RXR) binding sites in and around the murine UCP3 gene. The recruitment of PPAR and RXR to the three potential PPREs was determined by ChIP combined...

  4. Identification of putative PPAR response elements in and around the murine UCP3 gene

    DEFF Research Database (Denmark)

    Siersbæk, Majken

    in thermogenesis in unerring, the physiological functions of UCP2 and UCP3 are at present not fully understood. Synthetic agonists for the peroxisome proliferator-activated receptors (PPARs) as well as fatty acids have been shown to increase murine UCP2 and UCP3 mRNA expression but response elements and mechanisms...... are not yet characterized. The aim of this study was to investigate the transcriptional regulation of UCP3 by the PPARs. Results: The PPAR agonists increase UCP2 and UCP3 mRNA expression in skeletal muscle cells (C2C12). In addition, UCP2 and UCP3 mRNA expression is upregulated during 3T3-L1 adipocyte...... differentiation. Results from sequencing of chromatin immunoprecipitated (ChIP) material from 3T3-L1 adipocytes revealed three PPAR and retinoid X receptor (RXR) binding sites in and around the murine UCP3 gene. The recruitment of PPAR and RXR to the three potential PPREs was determined by ChIP combined...

  5. 4-Hydroxynonenal induces Nrf2-mediated UCP3 upregulation in mouse cardiomyocytes.

    Science.gov (United States)

    López-Bernardo, Elia; Anedda, Andrea; Sánchez-Pérez, Patricia; Acosta-Iborra, Bárbara; Cadenas, Susana

    2015-11-01

    4-Hydroxy-2-nonenal (HNE) is a highly cytotoxic product of lipid peroxidation. Nevertheless, at low concentrations, it is able to mediate cell signaling and to activate protective pathways, including that of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). In addition, HNE activates uncoupling proteins (UCPs), mitochondrial inner membrane proteins that mediate uncoupling of oxidative phosphorylation and have been proposed to protect against oxidative stress. It is not known, however, whether HNE might induce UCP expression via Nrf2 to cause mitochondrial uncoupling. We investigated the effects of HNE on UCP3 expression in mouse cardiomyocytes and the involvement of Nrf2. HNE induced the nuclear accumulation of Nrf2 and enhanced UCP3 expression, effects prevented by the antioxidant N-acetylcysteine. ChIP assays indicated that Nrf2 bound to the Ucp3 promoter after HNE treatment, increasing its expression. Cardiomyocytes treated with Nrf2- or UCP3-specific siRNA were less tolerant to HNE as reflected by increased cell death, and Nrf2 siRNA prevented HNE-induced UCP3 upregulation. The treatment with HNE greatly altered cardiomyocyte bioenergetics, increasing the proton leak across the inner mitochondrial membrane and severely decreasing the maximal respiratory capacity and the respiratory reserve capacity. These findings confirm that low HNE doses activate Nrf2 in cardiomyocytes and provide the first evidence of Nrf2 binding to the Ucp3 promoter in response to HNE, leading to increased protein expression. These results suggest that the upregulation of UCP3 mediated by Nrf2 in response to HNE might be important in the protection of the heart under conditions of oxidative stress such as ischemia-reperfusion.

  6. Characterization of UCP3 gene sequence and its expression in Hu lamb muscles%湖羊肌肉UCP3基因序列特征分析及其在肌肉中的表达

    Institute of Scientific and Technical Information of China (English)

    李隐侠; 乔永; 张俊; 钱勇; 孟春花; 王慧利; 钟声; 曹少先; 李齐发

    2015-01-01

    This study aims to obtain the full coding region of UCP3 gene in Hu sheep, characterize its sequence and investigate the mRNA level in sheep muscles. Each five male Hu lamb at 0 to 6 months ages were chosen and slaughtered, and total RNA from longissimus muscles, psoas muscles and front and rear leg biceps femoris muscles were extracted. The coding region of UCP3 gene of Hu sheep was cloned using TA cloning technoloty, and its conservation among different mam-mals was analyzed using DNAMAN software. Its mRNA level of different muscles of Hu sheep was investigated using real time PCR. The results showed that the coding region of UCP3 gene in Hu sheep was 936 bp, encoding 311 amino acid residues, and was relatively conserved in mammals. Phylogenetic tree constructed based on protein sequence of UCP3 showed that the genetic relationship between Hu sheep and cattle was the closest. Real-time PCR results revealed that UCP3 in different parts of muscles of differently-aged Hu sheep shared similar mRNA expression patterns, which were low at birth, and went up slightly and down afterwards over age. The expression level of UCP3 gene was relatively low in the rear leg biceps femoris muscles, and fluctuated in psoas muscles. UCP3 gene showed higher levels in 3-mouth-old sheep than those in the newborns and 5-mouth-old sheep.%为了获得湖羊UCP3基因编码区全序列并分析其序列特征,同时研究其在湖羊不同部位肌肉中的表达变化,选取初生与1、2、3、4、5和6月龄湖羊公羔各5只,提取屠宰后背最长肌、腰大肌、前腿肱二头肌和后腿股二头肌RNA,用TA克隆技术获得湖羊UCP3基因编码区序列,用DNAMAN软件分析UCP3基因在哺乳动物间的保守性,同时基于UCP3蛋白质序列用MEGA5.1软件构建系统发育树,用Real-time PCR检测湖羊不同肌肉部位UCP3基因的表达水平并分析其在不同发育时期的变化。结果显示:湖羊UCP3基因编码区全长936 bp,编码311个氨基酸残基

  7. A novel SP1/SP3 dependent intronic enhancer governing transcription of the UCP3 gene in brown adipocytes.

    Directory of Open Access Journals (Sweden)

    Christoph Hoffmann

    Full Text Available Uncoupling protein (UCP 3 is a mitochondrial inner membrane protein implicated in lipid handling and metabolism of reactive oxygen species. Its transcription is mainly regulated by peroxisome proliferator-activated receptors (PPAR, a family of nuclear hormone receptors. Employing bandshift assays, RNA interference and reporter gene assays we examine an intronic region in the UCP3 gene harboring a cis-element essential for expression in brown adipocytes. We demonstrate binding of SP1 and SP3 to this element which is adjacent to a direct repeat 1 element mediating activation of UCP3 expression by PPARγ agonists. Transactivation mediated by these elements is interdependent and indispensable for UCP3 expression. Systematic deletion uncovered a third binding element, a putative NF1 site, in close proximity to the SP1/3 and PPARγ binding elements. Data mining demonstrated binding of MyoD and Myogenin to this third element in C2C12 cells, and, furthermore, revealed recruitment of p300. Taken together, this intronic region is the main enhancer driving UCP3 expression with SP1/3 and PPARγ as the core factors required for expression.

  8. Fasting activates the gene expression of UCP3 independent of genes necessary for lipid transport and oxidation in skeletal muscle.

    Science.gov (United States)

    Tunstall, Rebecca J; Mehan, Kate A; Hargreaves, Mark; Spriet, Lawrence L; Cameron-Smith, David

    2002-06-07

    Fasting triggers a complex array of adaptive metabolic and hormonal responses including an augmentation in the capacity for mitochondrial fatty acid (FA) oxidation in skeletal muscle. This study hypothesized that this adaptive response is mediated by increased mRNA of key genes central to the regulation of fat oxidation in human skeletal muscle. Fasting dramatically increased UCP3 gene expression, by 5-fold at 15 h and 10-fold at 40 h. However the expression of key genes responsible for the uptake, transport, oxidation, and re-esterification of FA remained unchanged following 15 and 40 h of fasting. Likewise there was no change in the mRNA abundance of transcription factors. This suggests a unique role for UCP3 in the regulation of FA homeostasis during fasting as adaptation to 40 h of fasting does not require alterations in the expression of other genes necessary for lipid metabolism.

  9. AMP-activated protein kinase activates transcription of the UCP3 and HKII genes in rat skeletal muscle.

    Science.gov (United States)

    Stoppani, James; Hildebrandt, Audrey L; Sakamoto, Kei; Cameron-Smith, David; Goodyear, Laurie J; Neufer, P Darrell

    2002-12-01

    AMP-activated protein kinase (AMPK) has recently emerged as a key signaling protein in skeletal muscle, coordinating the activation of both glucose and fatty acid metabolism in response to increased cellular energy demand. To determine whether AMPK signaling may also regulate gene transcription in muscle, rats were given a single subcutaneous injection (1 mg/g) of the AMP analog 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR). AICAR injection activated (P < 0.05) AMPK-alpha 2 ( approximately 2.5-fold) and transcription of the uncoupling protein-3 (UCP3, approximately 4-fold) and hexokinase II (HKII, approximately 10-fold) genes in both red and white skeletal muscle. However, AICAR injection also elicited (P < 0.05) an acute drop (60%) in blood glucose and a sustained (2-h) increase in blood lactate, prompting concern regarding the specificity of AICAR on transcription. To maximize AMPK activation in muscle while minimizing potential systemic counterregulatory responses, a single-leg arterial infusion technique was employed in fully conscious rats. Relative to saline-infused controls, single-leg arterial infusion of AICAR (0.125, 0.5, and 2.5 micro g. g(-1). min(-1) for 60 min) induced a dose-dependent increase (2- to 4-fold, P < 0.05) in UCP3 and HKII transcription in both red and white skeletal muscle. Importantly, AICAR infusion activated transcription only in muscle from the infused leg and had no effect on blood glucose or lactate levels. These data provide evidence that AMPK signaling is linked to the transcriptional regulation of select metabolic genes in skeletal muscle.

  10. The contraction induced increase in gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1alpha), mitochondrial uncoupling protein 3 (UCP3) and hexokinase II (HKII) in primary rat skeletal muscle cells is dependent on reactive oxygen species

    DEFF Research Database (Denmark)

    Silveira, Leonardo R.; Pilegaard, Henriette; Kusuhara, Keiko

    2006-01-01

    We evaluated the role of reactive oxygen species (ROS) for the contraction induced increase in expression of PGC-1alpha, HKII and UCP3 mRNA. Rat skeletal muscle cells were subjected to acute or repeated electrostimulation in the presence and absence of antioxidants. Contraction of muscle cells le...

  11. [Relation of -55CT polymorphism of UCP3 gene with weight loss and metabolic changes after a high polyunsaturated fat diet in obese patients].

    Science.gov (United States)

    de Luis, D A; Aller, R; Izaola, O; GonzÁlez Sagrado, M; Conde, R; Ruiz Mambrilla, M

    2012-01-01

    The alteration in the protein expression of UCP3 could reduce energy consumption and increase energy storage as fat. The aim of our study was to investigate the influence of -55CT polymorphism of UCP3 gene in the metabolic response, weight loss and serum levels of adipokines following a hypocaloric diet rich in polyunsaturated fat in obese patients. A sample of 133 obese patients were analyzed prospectively for 3 months. The hypocaloric diet was 1459 kcal, 45.7% carbohydrate, 34.4% from 19.9% lipids and proteins. The fat distribution was, a 21.8% saturated fat, 55.5% monounsaturated and 22.7% of polyunsaturated fat (7 g per day of fatty acids w6, 2 g per day of w -3 and a ratio w6/w3 of 3.5). A total of 100 patients (28 males/72 females) (75.2%) had genotype - 55CC (wild genotype group) and 33 patients (8 males/25 females) (24.8%) -55CT genotype (group mutant genotype). In the wild genotype, body mass index (-2.5 ± 5.3 kg/m²), weight (-4.2 ± 3.7 kg), fat mass (-3,7 ± 3.3 kg), waist circumference (-4.1 ± 2.9 cm), systolic blood pressure (-4.9 ± 10.1 mmHg), total cholesterol levels (- 16.1 ± 23.6 mg / dl), LDL cholesterol (-11.1 ± 26.8 mg/dl), triglycerides (-12.0 ± 46.8 mg/dl), insulin (-1.8 ± 4.5 IU/L), HOMA-R (-0.6 ± 1.5) and leptin (-6.2 ± 8.4 ng/ml) decreased. In the mutant genotype anthropometric parameters were significantly decreased without significant changes in biochemical parameters. The T allele carriers of -55CT UCP3 polymorphism exhibit no metabolic response to weight loss induced by a hypocaloric diet rich in polyunsaturated fatty acids.

  12. Single nucleotide polymorphisms linked to mitochondrial uncoupling protein genes UCP2 and UCP3 affect mitochondrial metabolism and healthy aging in female nonagenarians.

    Science.gov (United States)

    Kim, Sangkyu; Myers, Leann; Ravussin, Eric; Cherry, Katie E; Jazwinski, S Michal

    2016-08-01

    Energy expenditure decreases with age, but in the oldest-old, energy demand for maintenance of body functions increases with declining health. Uncoupling proteins have profound impact on mitochondrial metabolic processes; therefore, we focused attention on mitochondrial uncoupling protein genes. Alongside resting metabolic rate (RMR), two SNPs in the promoter region of UCP2 were associated with healthy aging. These SNPs mark potential binding sites for several transcription factors; thus, they may affect expression of the gene. A third SNP in the 3'-UTR of UCP3 interacted with RMR. This UCP3 SNP is known to impact UCP3 expression in tissue culture cells, and it has been associated with body weight and mitochondrial energy metabolism. The significant main effects of the UCP2 SNPs and the interaction effect of the UCP3 SNP were also observed after controlling for fat-free mass (FFM) and physical-activity related energy consumption. The association of UCP2/3 with healthy aging was not found in males. Thus, our study provides evidence that the genetic risk factors for healthy aging differ in males and females, as expected from the differences in the phenotypes associated with healthy aging between the two sexes. It also has implications for how mitochondrial function changes during aging.

  13. Association and expression analyses of the Ucp2 and Ucp3 gene polymorphisms with body measurement and meat quality traits in Qinchuan cattle

    Indian Academy of Sciences (India)

    YANING WANG; WUCAI YANG; LINSHENG GUI; HONGBAO WANG; LINSEN ZAN

    2016-12-01

    The uncoupling proteins (UCPs) belong to the mitochondrial inner membrane anion carrier superfamily and play an important role in energy homeostasis. Genetic studies have demonstrated that Ucp2 and Ucp3 gene variants are involved in obesity and metabolic syndrome. The aim of this study was to identify associations between polymorphisms of Ucp2 and Ucp3 genes and economically-important traits in Qinchuan cattle. In the present study, one single-nucleotide polymorphism (SNP) in the 5'UTR region (SNP1: g.C-754G) of the Ucp2 gene was identified by direct sequencing of 441 Qinchuan cattle. Two SNPs in exon 3 (SNP2: g.G4877A; SNP3: g.C4902T) of the Ucp3 gene were identified by sequencing and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) among 441 Qinchuan cattle. Association analysis showed that SNP1 and SNP2 were associated with the meat quality traits (MQTs) including back fat thickness, loin muscle area and intramuscular fat content. SNP3 was found to be associated with part of the body measurement traits (BMTs) which referred to withers height and chest depth. In addition, QTL pyramiding analysis showed that individuals with diplotype P3P3 (GG–GG–CC) exhibited the best performance in terms of back fat thickness, loin muscle area, intramuscular fat content, rump length, hip width, chestdepth and chest circumference. With regard to the G4877A mutation, real time PCR analysis revealed that individuals with AA genotype of the Ucp3 gene expressed higher mRNA levels than those with GG genotype. These results suggest that thediplotype P3P3 (GG–GG–CC) could be used as a molecular marker of the combined genotypes for future selection of body measurement traits and meat quality traits in Qinchuan cattle.

  14. Variation in the UCP2 and UCP3 genes associates with abdominal obesity and serum lipids: The Finnish Diabetes Prevention Study

    Directory of Open Access Journals (Sweden)

    Ilanne-Parikka Pirjo

    2009-09-01

    Full Text Available Abstract Background We explored the associations of three variants in the uncoupling protein 2 (UCP2 gene, one variant in the UCP2-UCP3 intergenic region and five variants in the uncoupling protein 3 (UCP3 gene with obesity and diabetes related traits in subjects with impaired glucose tolerance participating in Finnish Diabetes Prevention Study. Altogether 507 overweight individuals (body mass index: 31.2 ± 4.5 kg/m2, age: 55 ± 7 years for whom DNA was available were randomized to either an intensified diet and physical activity group or to a conventional care control group. Methods We analysed the data from the baseline and annual follow-up visits from years 1, 2 and 3. Measurements of anthropometry, plasma glucose and serum insulin in oral glucose tolerance test, serum total cholesterol, HDL-cholesterol and triglycerides were included. The median follow-up time for type 2 diabetes incidence was 7 years. Genetic variants were screened by restriction fragment length polymorphism or Illumina method. Results UCP3 gene variant rs3781907 was associated with increased serum total and LDL-cholesterol levels, at baseline and during the follow-up period. The same variant was associated with a higher risk of type 2 diabetes. Variants rs1726745, rs11235972 and rs1800849 in the UCP3 gene associated with serum total and LDL-cholesterol at baseline. Haploblock including variants rs659366, rs653529, rs15763, and rs1726745 was associated with measures of abdominal obesity at baseline and in the longitudinal analysis. The haplotype comprising alleles rs659366-G, rs653529-A, rs15763-G and rs1726745-A was associated with higher waist-to-hip ratio, and haplotype comprising alleles rs3781907-G, rs11235972-A, and rs1800849-T was associated with increased serum total and LDL-cholesterol concentrations. Conclusion Genetic variation in the UCP2-UCP3 gene cluster may act as a modifier increasing serum lipid levels and indices of abdominal obesity, and may thereby also

  15. Relation of -55CT polymorphism of UCP3 gene with weight loss and metabolic changes after a high monounsaturated fat diet in obese non diabetic patients.

    Science.gov (United States)

    De Luis, D A; Aller, R; Izaola, O; De La Fuente, B; Conde, R; Eiros Bouza, J M

    2013-10-01

    The aim of our study was to investigate the influence of -55CT polymorphism of UCP3 gene on metabolic response, weight loss and serum adipokine levels to a high monounsaturated fat hypocaloric diet in obese patients. A sample of 128 obese patients was analyzed in a prospective way during 3 months. Eighty eight patients (21 males/67 females) (68.8%) had the genotype 55CC (wild genotype group) and 40 patients (8 males/32 females) (31.3%) 55CT (mutant genotype group). In wild genotype group, BMI (-1.6±1.3 kg/m2), weight (-4.3±3.7 kg), fat mass (-3.5±3.3 kg), waist circumference (-5.1±2.9 cm), total cholesterol (-7.2±10.6 mg/dl), LDL cholesterol (-5.3±12.8 mg/dl) and leptin (-4.7±10.1 ng/ml) decreased. In mutant genotype group, BMI (1.3±2.2 kg/m2), weight (-3.0±1.4 kg), fat mass (-2.5±1.1 kg), waist circumference (-2.8±3.1 cm) and leptin (-5.8±10.7 decreased. In patients with -55CC UCP3 genotype, a high mono-unsaturated hypocaloric diet reduced BMI, weight, waist circumference, waist to hip ratio, fat mass, LDL-cholesterol, total cholesterol and leptin levels. Carriers of T allele had a different response than -55CC patients, with a significant decrease of the same antropometric parameters, but lower than in the wild genotype group, and without significant changes in cholesterol levels.

  16. A prevalent polymorphism in the promoter of the UCP3 gene and its relationship to body mass index and long term body weight change in the Danish population

    DEFF Research Database (Denmark)

    Dalgaard, L T; Sørensen, Thomas; Drivsholm, T;

    2001-01-01

    Variability of the uncoupling protein 3 (UCP3) promoter has been associated with increased body mass index (BMI) and altered lipid profiles. Here we tested the hypothesis that variation of the UCP3 promoter is associated with either juvenile or maturity-onset obesity or body weight change over a 26...... of 744 obese Danish men who at the draft board examinations had a body mass index (BMI) of at least 31 kg/m(2), 2) a randomly selected control group consisting of 857 draftees, 3) 258 middle-aged subjects, and 4) 409 60-yr-old subjects. The frequency of the T allele was 26.0% (95% confidence interval, 23......-old subjects. The polymorphism was not associated with increased BMI or percent body fat in these 2 groups. It is concluded that this variant does not play a major role in the development of common obesity among Danish subjects....

  17. Relación del polimorfismo -55CT del gen UCP3 con la pérdida de peso y los cambios metabólicos tras una dieta hipocalórica rica en ácidos grasos poliinsaturados en pacientes obesos Relation of -55CT polymorphism of UCP3 gene with weight loss and metabolic changes after a high polyunsaturated fat diet in obese patients

    Directory of Open Access Journals (Sweden)

    D. A. de Luis

    2012-08-01

    alteration in the protein expression of UCP3 could reduce energy consumption and increase energy storage as fat. The aim of our study was to investigate the influence of -55CT polymorphism of UCP3 gene in the metabolic response, weight loss and serum levels of adipokines following a hypocaloric diet rich in polyunsaturated fat in obese patients. Design: A sample of 133 obese patients were analyzed prospectively for 3 months. The hypocaloric diet was 1459 kcal, 45.7% carbohydrate, 34.4% from 19.9% lipids and proteins. The fat distribution was, a 21.8% saturated fat, 55.5% monounsaturated and 22.7% of polyunsaturated fat (7 g per day of fatty acids w6, 2 g per day of w -3 and a ratio w6/w3 of 3.5. Results: A total of 100 patients (28 males/72 females (75.2% had genotype - 55CC (wild genotype group and 33 patients (8 males/25 females (24.8% -55CT genotype (group mutant genotype. In the wild genotype, body mass index (-2.5 ± 5.3 kg/m², weight (-4.2 ± 3.7 kg, fat mass (-3,7 ± 3.3 kg, waist circumference (-4.1 ± 2.9 cm, systolic blood pressure (-4.9 ± 10.1 mmHg, total cholesterol levels (- 16.1 ± 23.6 mg / dl, LDL cholesterol (-11.1 ± 26.8 mg/dl, triglycerides (-12.0 ± 46.8 mg/dl, insulin (-1.8 ± 4.5 IU/L, HOMA-R (-0.6 ± 1.5 and leptin (-6.2 ± 8.4 ng/ml decreased. In the mutant genotype anthropome-tric parameters were significantly decreased without significant changes in biochemical parameters. Conclusion: The T allele carriers of -55CT UCP3 polymorphism exhibit no metabolic response to weight loss induced by a hypocaloric diet rich in polyunsaturated fatty acids.

  18. Associations of Polymorphisms of IGF-IR,CAPN Ⅱ and UCP 3 Genes in Nanyang Cattle with Its Growth Traits%南阳牛IGF-IR、CAPNⅡ和UCP 3基因多态性与生长性状的相关分析

    Institute of Scientific and Technical Information of China (English)

    张润锋; 陈宏; 支立峰; 严镇钧; 顾勇; 徐平

    2010-01-01

    对南阳牛胰岛素样生长因子-Ⅰ受体(ICF-IR)、钙激活中性蛋白Ⅱ(CAPN Ⅱ)和解偶联蛋白3(UCP 3)3个位点PCR-RFLP与生长性状进行相关分析.在所研究群体中,IGF-IR/Taq Ⅰ多态位点上BB型个体仅检测到2头,IGF-IR/Taq Ⅰ位点等位基因A和CAPN Ⅱ/Hha Ⅰ位点等位基因B为优势等位基因.南阳牛在IGF-IR/Taq Ⅰ和CAPN Ⅱ/Hha Ⅰ位点处于Hardy-Weinberg平衡状态,在UCP 3/Bgl Ⅰ位点偏离HardyWeinberg平衡状态.相关分析表明,IGF-IR/Taq Ⅰ位点不同基因型对南阳牛生长性状影响不显著.CAPN Ⅱ/Hha Ⅰ位点对6月龄的生长性状影响显著(P<0.05);对不同生长阶段的体协长和胸围影响显著(P<0.05或P<0.01).UCP 3/Bgl Ⅰ位点对6月龄的体重、体高和体协长,24月龄体高、体协长和胸围影响显著(P<0.05).在CAPN Ⅱ/Hha Ⅰ和UCP 3/Bgl Ⅰ位点上,南阳牛AB型个体生长性状表现最好.CAPN Ⅱ/Hha Ⅰ和UCP 3/Bgl Ⅰ多态可以作为南阳牛早期选育的有效标记.

  19. UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion

    Energy Technology Data Exchange (ETDEWEB)

    Hirasaka, Katsuya, E-mail: hirasaka@nagasaki-u.ac.jp [Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki (Japan); Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima (Japan); Mills, Edward M. [Division of Pharmacology/Toxicology, University of Texas at Austin, Austin, TX (United States); Haruna, Marie; Bando, Aki; Ikeda, Chika; Abe, Tomoki [Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima (Japan); Kohno, Shohei; Nowinski, Sara M. [Division of Pharmacology/Toxicology, University of Texas at Austin, Austin, TX (United States); Lago, Cory U. [Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD (United States); Akagi, Ken-ichi [Section of Laboratory Equipment, National Institute of Biomedical Innovation, Osaka (Japan); Tochio, Hidehito [Graduate School of Engineering, Kyoto University, Kyoto (Japan); Ohno, Ayako; Teshima-Kondo, Shigetada [Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima (Japan); Okumura, Yuushi [Department of Nutrition and Health, Sagami Woman' s University, Kanagawa (Japan); Nikawa, Takeshi [Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima (Japan)

    2016-03-25

    Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca{sup 2+}. The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca{sup 2+}, suggesting that the C-terminal domain of Hax-1 underwent a Ca{sup 2+}-induced conformational change. In the Ca{sup 2+}-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca{sup 2+} binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca{sup 2+}. - Highlights: • UCP3 interacts with Hax-1. • The interaction of UCP3 and Hax-1 occurs in a calcium-dependent manner. • The C-terminal domain of Hax-1 undergoes a calcium-induced conformational change.

  20. A novel intronic peroxisome proliferator-activated receptor gamma enhancer in the uncoupling protein (UCP) 3 gene as a regulator of both UCP2 and -3 expression in adipocytes

    DEFF Research Database (Denmark)

    Bugge, Anne Skovsø; Siersbaek, Majken; Madsen, Maria S

    2010-01-01

    Uncoupling Proteins (UCPs) are integral ion channels residing in the inner mitochondrial membrane. UCP2 is ubiquitously expressed, while UCP3 is found primarily in muscles and adipose tissue. Although the exact molecular mechanism of action is controversial, it is generally agreed that both...

  1. A Novel Intronic Peroxisome Proliferator-activated Receptor γ Enhancer in the Uncoupling Protein (UCP) 3 Gene as a Regulator of Both UCP2 and -3 Expression in Adipocytes*

    OpenAIRE

    2010-01-01

    Uncoupling Proteins (UCPs) are integral ion channels residing in the inner mitochondrial membrane. UCP2 is ubiquitously expressed, while UCP3 is found primarily in muscles and adipose tissue. Although the exact molecular mechanism of action is controversial, it is generally agreed that both homologues function to facilitate mitochondrial fatty acid oxidation. UCP2 and -3 expression is activated by the peroxisome proliferator-activated receptors (PPARs), but so far no PPAR response element has...

  2. 解偶联蛋白1、2和3基因在中国西门塔尔牛组织器官中的表达水平及其与胴体品质关系分析%Analyze the ucp1,ucp2,ucp3 Genes Expression Level in Tissues/Organs and the Relationship with the Carcass Traits in Chinese Simmental Cattle

    Institute of Scientific and Technical Information of China (English)

    孙国权; 高树新; 吴慧光; 李俊雅; 丽春; 王景山; 王玉泉; 王国富

    2014-01-01

    The objective is to obtain expression profile of Chinese Simmental cattle ucp1,ucp2,ucp3 genes,to reveals the relationship between the ucp1,ucp2,ucp3 gene and energy metabolism ,to explore the expression rule of key gene of fat deposition ,and to screen the candidate gene affecting fat deposition .Total RNA were extracted from fifteen tissues(liver,lymph,intercostals muscle,shoulder muscle,spleen,kidney,rib eye muscle,lung,heart,gut fat, greater omental fat,testicle,small intestine,pancreas and backfat)of twenty animals from Chinese Simmental cattle . The primers of ucp1,ucp2,ucp3 genes were designed and synthesized ,and the data were normalized using the β-ac-tin gene as internal control .The specific expression of ucp1,ucp2,ucp3 genes in different tissues were investigated by real-time quantitative PCR.There were different degree expression of ucp1,ucp2,ucp3 genes mRNA in the tissues and organs of testicle ,small intestine ,heart,pancreas and so on .The expression of ucp1 was highest in the backfat , middle in the pancreas,small intestine,testicle,greater omental fat,gut fat,and heart,and lowest in the other tis-sues.The highest level expression of ucp2 was detected in Testicle ,and there were also higher expression of ucp2 in the lung,liver,and backfat than in other tissues .The highest level expression of ucp3 was in rib eye muscle,Inter-costal muscle ,and shoulder muscle ,middle in the lung ,and the lowest in other tissues .Correlation analyses showed that the expression level of ucp3 gene in rib eye had no significant relation with intramuscular fat and backfat thick-ness,but had positive relation to eye lean area(r=0.788).It is concluded that the expression level of ucp1,ucp2, ucp3 genes had tissue specific might be related to the various functions on different tissues of cattle .The ucp3 gene can be used as a candidate gene related to carcass traits .%为获得中国西门塔尔牛解偶联蛋白( Uncoupling proteins ,UCPs) ucp1、ucp2、ucp3基因

  3. Role of -55CT polymorphism of UCP3 gene on non alcoholic fatty liver disease and insulin resistance in patients with obesity Efecto del polimorfismo -55CT sobre el hígado graso y la resistencia a la insulina en pacientes con sobrepeso

    Directory of Open Access Journals (Sweden)

    R. Aller

    2010-08-01

    Full Text Available Background and aims: Some studies have pointed to a role of UCP3 in the regulation of biochemical and fat parameters in overweight patients. The aim of our study was to investigate the influence of -55CT polymorphism of UCP3 gene (rs1800849 on histological changes and insulin resistance in patients with non-alcoholic fatty liver disease (NAFLD. Material and methods: A population of 39 patients with NAFLD was recruited in a cross sectional study. The inclusion criterion was the presence of biopsy-proven NAFLD. A biochemical analysis of serum (lipid profile, and adipocytokines was measured. An anthropometric analysis was assessed, too. Genotype of UCP3 gene -55CT was studied. Results: Nine patients (23% had the genotype 55CC (mutant type group and 30 patients (77% 55CT (wild type group.TT genotype was not detected. Insulin levels and HOMA were higher in mutant type group (insulin: 17.7 ± 10.9 mUI/L vs 11.9 ± 4.7 mUI/L/; p Introducción y objetivos: Algunos trabajos han señalado una relación entre el polimorfismo de UCP3 y parámetros bioquímicos y antropométricos en pacientes con sobrepeso. El objetivo de nuestro trabajo es valorar la influencia del polimorfismo -55CT del gen UCP3 (rs1800849 en parámetros histológicos y resistencia a la insulina en paciente con esteatohepatitis no alcohólica (EHNA y sobrepeso. Material y métodos: Se seleccionó una muestra de 39 pacientes con EHNA. El criterio de inclusión fue la presencia de EHNA comprobado con biopsia hepática. Se realizó una evaluación analítica (lípidos y adipocitoquinas, así como una evaluación antropométrica. Se evaluó el genotipo de UCP3 -55CT. Resultados: Un total de 9 pacientes (23% presentaron el genotipo mutado 55CC y 30 pacientes (77% 55CT (genotipo salvaje.El genotipo TT no fue detectado. Los niveles de insulina y de resistencia a la insulina (HOMA fueron más elevados en los pacientes con genotipo mutado (insulina: 17,7 ± 10,9 mUI/L vs 11,9 ± 4,7 mUI/L/; p < 0

  4. The Preservation of in vivo Phosphorylated and Activated Uncoupling Protein 3 (UCP3) in Isolated Skeletal Muscle Mitochondria following Administration of 3,4-Methylenedioxymethamphetamine (MDMA aka Ecstasy) to Rats/Mice

    OpenAIRE

    2012-01-01

    PUBLISHED Previous researchers have demonstrated that 3,4-methylenedioxymethamphetamine (MDMA) induced hyperthermia, in skeletal muscle of animals, is uncoupling protein 3 (UCP3) dependent. In light of our investigations that in vivo phosphorylation of UCP1 is augmented under conditions of cold-acclimation, we set out to investigate whether (a) UCP3 was phosphorylated in vivo and (b) whether in vivo phosphorylation of UCP3 resulted in increased proton leak following MDMA administration to ...

  5. BAIBA Does Not Regulate UCP-3 Expression in Human Skeletal Muscle as a Response to Aerobic Exercise.

    Science.gov (United States)

    Morales, Flor E; Forsse, Jeffrey S; Andre, Thomas L; McKinley-Barnard, Sarah K; Hwang, Paul S; Anthony, Ian G; Tinsley, Grant M; Spillane, Mike; Grandjean, Peter W; Ramirez, Alejandro; Willoughby, Darryn S

    2017-01-01

    β-Aminoisobutyric acid (BAIBA) has shown to modulate uncoupling protein (UCP)-1 expression, which is mainly expressed in white adipose tissue; however, no studies to date have analyzed its potential effect on the main uncoupling protein of skeletal muscle, UCP-3. The main goal of this study was to assess the potential effect of acute aerobic exercise on serum BAIBA and skeletal muscle UCP-3. The secondary goal was to assess the potential involvement of the transcription factors proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor alpha (PPARα), as well as free fatty acids (FFAs) in UCP-3 expression. A tertiary goal of the study was to evaluate the potential effect of consuming a preexercise meal on the outcome of the first 2 objectives. In a randomized crossover design, untrained participants performed 2 acute cycling sessions (350 kcal at 70% of their VO2peak) after 2 experimental conditions: (1) consumption of a multi-macronutrient shake and (2) a fasting period of 8 hours. Blood samples were taken at baseline, preexercise, postexercise, 1 hour, and 4 hours postexercise, and muscle biopsies were taken at the last 4 time points. UCP-3 protein concentration and expression, as well as the mRNA expression of PGC-1α and PPARα, were measured in muscle, and BAIBA, glucose, and FFA were measured in serum. Aerobic exercise failed to induce a significant effect on serum BAIBA, PGC-1α, and PPARα regardless on the feeding condition. Despite the lack of effect of exercise on the previous variables, UCP-3 expression and protein concentration significantly increased in the shake condition. The expression of human skeletal muscle UCP-3 as a result of exercise might be controlled by factors other than BAIBA.

  6. UCP3 Regulates Single-Channel Activity of the Cardiac mCa1.

    Science.gov (United States)

    Motloch, Lukas J; Gebing, Tina; Reda, Sara; Schwaiger, Astrid; Wolny, Martin; Hoppe, Uta C

    2016-08-01

    Mitochondrial Ca(2+) uptake (mCa(2+) uptake) is thought to be mediated by the mitochondrial Ca(2+) uniporter (MCU). UCP2 and UCP3 belong to a superfamily of mitochondrial ion transporters. Both proteins are expressed in the inner mitochondrial membrane of the heart. Recently, UCP2 was reported to modulate the function of the cardiac MCU related channel mCa1. However, the possible role of UCP3 in modulating cardiac mCa(2+) uptake via the MCU remains inconclusive. To understand the role of UCP3, we analyzed cardiac mCa1 single-channel activity in mitoplast-attached single-channel recordings from isolated murine cardiac mitoplasts, from adult wild-type controls (WT), and from UCP3 knockout mice (UCP3(-/-)). Single-channel registrations in UCP3(-/-) confirmed a murine voltage-gated Ca(2+) channel, i.e., mCa1, which was inhibited by Ru360. Compared to WT, mCa1 in UCP3(-/-) revealed similar single-channel characteristics. However, in UCP3(-/-) the channel exhibited decreased single-channel activity, which was insensitive to adenosine triphosphate (ATP) inhibition. Our results suggest that beyond UCP2, UCP3 also exhibits regulatory effects on cardiac mCa1/MCU function. Furthermore, we speculate that UCP3 might modulate previously described inhibitory effects of ATP on mCa1/MCU activity as well.

  7. UCP-2 and UCP-3 Proteins Are Differentially Regulated in Pancreatic Beta-Cells

    Science.gov (United States)

    Li, Yunfeng; Maedler, Kathrin; Shu, Luan; Haataja, Leena

    2008-01-01

    Background Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets. Methodology Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry. Principal Findings Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased ∼2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by ∼40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion. Conclusions UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes. PMID:18167556

  8. UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Yunfeng Li

    Full Text Available BACKGROUND: Increased uncoupling protein-2 (UCP-2 expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets. METHODOLOGY: Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry. PRINCIPAL FINDINGS: Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05. UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05. Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased approximately 2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by approximately 40% (p<0.05. UCP-3 overexpression improved glucose-stimulated insulin secretion. CONCLUSIONS: UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.

  9. UCP-2 and UCP-3 Proteins Are Differentially Regulated in Pancreatic Beta-Cells

    OpenAIRE

    2008-01-01

    BACKGROUND: Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets. METHODOLOGY: Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP...

  10. UCP 3基因多态性与DR关系的研究%The Relationship Between UCP 3 Genetic Polymorphisms and Diabetic Retinopathy in Local Population

    Institute of Scientific and Technical Information of China (English)

    杨玉芝; 李贤厚; 冯琨; 朱巍; 段滨红; 王亦薇; 王丹; 杜馥曼

    2007-01-01

    目的:探讨解偶联蛋白(UCP)3基因G304A多态性与糖尿病视网膜病变(diabetic retinopathy, DR)的关系.方法:201例糖尿病患者(分为伴有DR组45例,不伴有DR的糖尿病患者作为对照组156例),检测所有患者的基因组,用聚合酶链反应(PCR)对UCP 3基因进行扩增,记录各组UCP 3等位基因及基因型频率并进行比较.结果:UCP 3基因G304A多态性和基因频率在各组间无显著性差异(P>0.05).结论:解偶联蛋白(UCP)3基因G304A多态性与中国人糖尿病视网膜病变无关系.

  11. Functional and Activity Analysis of Cattle UCP3 Promoter with MRFs-Related Factors

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2016-05-01

    Full Text Available Uncoupling protein 3 (UCP3 is mainly expressed in muscle. It plays an important role in muscle, but less research on the regulation of cattle UCP3 has been performed. In order to elucidate whether cattle UCP3 can be regulated by muscle-related factors, deletion of cattle UCP3 promoter was amplified and cloned into pGL3-basic, pGL3-promoter and PEGFP-N3 vector, respectively, then transfected into C2C12 myoblasts cells and UCP3 promoter activity was measured using the dual-Luciferase reporter assay system. The results showed that there is some negative-regulatory element from −620 to −433 bp, and there is some positive-regulatory element between −433 and −385 bp. The fragment (1.08 kb of UCP3 promoter was cotransfected with muscle-related transcription factor myogenic regulatory factors (MRFs and myocyte-specific enhancer factor 2A (MEF2A. We found that UCP3 promoter could be upregulated by Myf5, Myf6 and MyoD and downregulated by MyoG and MEF2A.

  12. Glutathionylation Acts as a Control Switch for Uncoupling Proteins UCP2 and UCP3*

    OpenAIRE

    2011-01-01

    The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactiv...

  13. Endurance training attenuates the bioenergetics alterations of rat skeletal muscle mitochondria submitted to acute hypoxia:Role of ROS and UCP3%耐力训练抑制急性低氧时骨骼肌线粒体生物能学变化:ROS和UCP3的作用

    Institute of Scientific and Technical Information of China (English)

    薄海; 王义和; 李海英; 赵娟; 张红英; 佟长青

    2008-01-01

    The physiological significance of skeletal muscle mitochondrial uncoupling protein 3(UCP3)in hypoxia is elusive.In the current study,UCP3 mRNA and protein expressions were investigated along with mitochondrial respiratory function,reactive oxygen species(ROS)generation,as well as manganese superoxide dismutase(MnSOD)expression in rat skeletal muscle with or without endurance training after an acute and severe hypobaric hypoxia exposure for different time.Acute hypoxia induced a series of impairments in skeletal muscle mitochondrial bioenergetics.In untrained rats,UCP3 protein content increased by 60%above resting level at 4 h hypoxia,whereas MnSOD protein content and activity were unaltered.UCP3 upregulation increased mitochondrial uncoupling respiration thus reducing 02 generation,but inevitably decreased ATP production.Training decreased acute hypoxia-induced upregulation of UCP3 protein(67% vs 42%)in rat skeletal muscle.ROS production in trained rats also showed a dramatic decrease at 2 h,4 h and 6 h,respectively,compared with that in untrained rats.MnSOD protein contents and activities were significantly(50%and 34%)higher in trained than those in untrained rats.Training adaptation of MnSOD may enhance the mitochondrial tolerante to ROS production,and reduce UCP3 activation during severe hypoxia.thus maintaining the efficiency of oxidative phosphorylation.In trained rats,mitochondrial respiratory control(RCR)and P/O ratios were maintained relatively constant despite severe hypoxia.whereas in untrained rats RCR and P/O ratios were significantly decreased.These results indicate that(1)UCP3 mRNA and protein expression in rat skeletal muscle are upregulated during acute and severe hypobaric hypoxia,which may reduce the increased cross-membrane potential(△Ψ)and thus ROS production;(2)Endurance training can blunt hypoxia-induced UCP3 upregulation,and improve mitochondrial efficiency of oxidative phosphorylation due to increased removal of ROS.%骨

  14. UCP3 polymorphisms, hand grip performance and survival at old age: association analysis in two Danish middle aged and elderly cohorts.

    Science.gov (United States)

    Dato, Serena; Soerensen, Mette; Montesanto, Alberto; Lagani, Vincenzo; Passarino, Giuseppe; Christensen, Kaare; Christiansen, Lene

    2012-08-01

    An efficient uncoupling process is generally considered to have a protective effect on the aging muscle by slowing down its age-related decay. Genetic polymorphisms in the Uncoupling Protein 3 (UCP3) gene, whose product is mainly expressed in skeletal muscle, were suggested to be associated with hand grip (HG) performances in elderly populations. Considering the population specificity of the quality of aging, we aimed to add further support to this evidence by analyzing the association between four SNPs in the UCP3 gene and relative haplotypes in two large cohorts of middle aged (N=708) and oldest old Danes (N=908). We found that the variability at rs1685354 and rs11235972 was associated with HG levels both at single and haplotypic level in both cohorts. Furthermore, taking advantage of large cohort and period survival data of the oldest cohort, we tested the association of each SNP with survival at 10years from the baseline visit. Interestingly, we found that allele A at rs11235972, associated in this cohort with lowest HG scores, influences also the survival patterns, with people carrying this allele showing higher mortality rates. On the whole, our work supports the role of UCP3 gene in functional status and survival at old age.

  15. UCP2 -866G/A, Ala55Val and UCP3 -55C/T polymorphisms in association with obesity susceptibility - a meta-analysis study.

    Directory of Open Access Journals (Sweden)

    Li Qian

    Full Text Available AIMS/HYPOTHESIS: Variants of UCP2 and UCP3 genes have been reported to be associated with obesity, but the available data on the relationship are inconsistent. A meta-analysis was performed to determine whether there are any associations between the UCP2 -866G/A, Ala55Val, and UCP3 -55C/T polymorphisms and obesity susceptibility. METHODS: The PubMed, Embase, Web of Science and CNKI, CBMdisc databases were searched for all relevant case-control studies. The fixed or random effect pooled measure was determined on the bias of heterogeneity test among studies. Publication bias was examined by the modified Begg's and Egger's test. RESULTS: Twenty-two published articles with thirty-two outcomes were included in the meta-analysis: 12 studies with a total of 7,390 cases and 9,860 controls were analyzed for UCP2 -866G/A polymorphism with obesity, 9 studies with 1,483 cases and 2,067 controls for UCP2 Ala55Val and 8 studies with 2,180 cases and 2,514 controls for UCP3 -55C/T polymorphism. Using an additive model, the UCP2 -866G/A polymorphism showed no significant association with obesity risk in Asians (REM OR = 0.81, 95% CI: 0.65-1.01. In contrast, a statistically significant association was observed in subjects of European descent (FEM OR = 1.06, 95% CI: 1.01-1.12. But neither the UCP2 Ala55Val nor the UCP3 -55C/T polymorphism showed any significant association with obesity risk in either subjects of Asian (REM OR = 0.84, 95% CI: 0.67-1.06 for Ala55Val; REM OR = 0.94, 95% CI: 0.55-1.28 for -55C/T or of European descent (REM OR = 1.04, 95% CI: 0.80-1.36 for Ala55Val; FEM OR = 1.08, 95% CI: 0.97-1.20 for -55C/T. CONCLUSIONS AND INTERPRETATION: Our meta-analysis revealed that the UCP2 -866G/A polymorphism may be a risk factor for susceptibility to obesity in subjects of European descent, but not in individuals of Asian descent. And our results did not support the association between UCP2 Ala55Val, UCP3 -55C/T polymorphisms and

  16. Impaired cytosolic NADH shuttling and elevated UCP3 contribute to inefficient citric acid cycle flux support of postischemic cardiac work in diabetic hearts.

    Science.gov (United States)

    Banke, Natasha H; Lewandowski, E Douglas

    2015-02-01

    Diabetic hearts are subject to more extensive ischemia/reperfusion (ISC/REP) damage. This study examined the efficiency of citric acid cycle (CAC) flux and the transfer of cytosolic reducing equivalents into the mitochondria for oxidative support of cardiac work following ISC/REP in hearts of c57bl/6 (NORM) and type 2 diabetic, db/db mouse hearts. Flux through the CAC and malate-aspartate shuttle (MA) were monitored via dynamic (13)C NMR of isolated hearts perfused with (13)C palmitate+glucose. MA flux was lower in db/db than NORM. Oxoglutarate malate carrier (OMC) was elevated in the db/db heart, suggesting a compensatory response to low NADHc. Baseline CAC flux per unit work (rate-pressure-product, RPP) was similar between NORM and db/db, but ISC/REP reduced the efficiency of CAC flux/RPP by 20% in db/db. ISC/REP also increased UCP3 transcription, indicating potential for greater uncoupling. Therefore, ISC/REP induces inefficient carbon utilization through the CAC in hearts of diabetic mice due to the combined inefficiencies in NADHc transfer per OMC content and increased uncoupling via UCP3. Ischemia and reperfusion exacerbated pre-existing mitochondrial defects and metabolic limitations in the cytosol of diabetic hearts. These limitations and defects render diabetic hearts more susceptible to inefficient carbon fuel utilization for oxidative energy metabolism. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Exercise attenuates the fasting-induced transcriptional activation of metabolic genes in skeletal muscle.

    Science.gov (United States)

    Hildebrandt, A L; Neufer, P D

    2000-06-01

    Fasting elicits a progressive increase in lipid metabolism within skeletal muscle. To determine the effects of fasting on the transcriptional regulation of genes important for metabolic control in skeletal muscle composed of different fiber types, nuclei from control and fasted (24 and 72 h) rats were subjected to nuclear run-on analysis using an RT-PCR-based technique. Fasting increased (P < 0.05) transcription rate of the muscle-specific uncoupling protein-3 gene (UCP3) 14.3- to 21.1-fold in white gastrocnemius (WG; fast-twitch glycolytic) and 5.5- to 7.5-fold in red gastrocnemius (RG; fast-twitch oxidative) and plantaris (PL; mixed) muscles. No change occurred in soleus (slow-twitch oxidative) muscle. Fasting also increased transcription rate of the lipoprotein lipase (LPL), muscle carnitine palmitoyltransferase I (CPT I), and long-chain acyl-CoA dehydrogenase (LCAD) genes 1.7- to 3.7-fold in WG, RG, and PL muscles. Transcription rate responses were similar after 24 and 72 h of fasting. Surprisingly, increasing metabolic demand during the initial 8 h of starvation (two 2-h bouts of treadmill running) attenuated the 24-h fasting-induced transcriptional activation of UCP3, LPL, CPT I, and LCAD in RG and PL muscles, suggesting the presence of opposing regulatory mechanisms. These data demonstrate that fasting elicits a fiber type-specific coordinate increase in the transcription rate of several genes involved in and/or required for lipid metabolism and indicate that exercise may attenuate the fasting-induced transcriptional activation of specific metabolic genes.

  18. Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle.

    Science.gov (United States)

    Jørgensen, Sebastian B; Wojtaszewski, Jørgen F P; Viollet, Benoit; Andreelli, Fabrizio; Birk, Jesper B; Hellsten, Ylva; Schjerling, Peter; Vaulont, Sophie; Neufer, P Darrell; Richter, Erik A; Pilegaard, Henriette

    2005-07-01

    We tested the hypothesis that 5'AMP-activated protein kinase (AMPK) plays an important role in regulating the acute, exercise-induced activation of metabolic genes in skeletal muscle, which were dissected from whole-body alpha2- and alpha1-AMPK knockout (KO) and wild-type (WT) mice at rest, after treadmill running (90 min), and in recovery. Running increased alpha1-AMPK kinase activity, phosphorylation (P) of AMPK, and acetyl-CoA carboxylase (ACC)beta in alpha2-WT and alpha2-KO muscles and increased alpha2-AMPK kinase activity in alpha2-WT. In alpha2-KO muscles, AMPK-P and ACCbeta-P were markedly lower compared with alpha2-WT. However, in alpha1-WT and alpha1-KO muscles, AMPK-P and ACCbeta-P levels were identical at rest and increased similarly during exercise in the two genotypes. The alpha2-KO decreased peroxisome-proliferator-activated receptor gamma coactivator (PGC)-1alpha, uncoupling protein-3 (UCP3), and hexokinase II (HKII) transcription at rest but did not affect exercise-induced transcription. Exercise increased the mRNA content of PGC-1alpha, Forkhead box class O (FOXO)1, HKII, and pyruvate dehydrogenase kinase 4 (PDK4) similarly in alpha2-WT and alpha2-KO mice, whereas glucose transporter GLUT 4, carnitine palmitoyltransferase 1 (CPTI), lipoprotein lipase, and UCP3 mRNA were unchanged by exercise in both genotypes. CPTI mRNA was lower in alpha2-KO muscles than in alpha2-WT muscles at all time-points. In alpha1-WT and alpha1-KO muscles, running increased the mRNA content of PGC-1alpha and FOXO1 similarly. The alpha2-KO was associated with lower muscle adenosine 5'-triphosphate content, and the inosine monophosphate content increased substantially at the end of exercise only in alpha2-KO muscles. In addition, subcutaneous injection of 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) increased the mRNA content of PGC-1alpha, HKII, FOXO1, PDK4, and UCP3, and alpha2-KO abolished the AICAR-induced increases in PGC-1alpha and HKII mRNA. In

  19. Acute starvation in C57BL/6J mice increases myocardial UCP2 and UCP3 protein expression levels and decreases mitochondrial bio-energetic function.

    Science.gov (United States)

    Wang, Chun-Ming; Almsherqi, Zakaria A; McLachlan, Craig S; Matthews, Slade; Ramachandran, Malarmathy; Tay, Stacey Kh; Deng, Yuru

    2011-01-01

    Associations between uncoupling protein (UCP) expression and functional changes in myocardial mitochondrial bio-energetics have not been well studied during periods of starvation stress. Our aim was to study the effects of acute starvation, for 24 or 48 h, on combined cardiac mitochondrial function and UCP expression in mice. Isolated heart mitochondria from female mice starved for 48 h compared to that from mice fed revealed a significantly (p bio-energetic functional changes were associated with increases in mitochondrial UCP2 and UCP3 protein expression. In conclusion, our findings suggest that increased UCP2 and UCP3 levels may contribute to decreased myocardial mitochondrial bio-energetic function due to starvation.

  20. Regulação da expressão gênica das UCP2 e UCP3 pela restrição energética,jejum e exercício físico UCP2 and UCP3 genic expression: regulation by food restriction, fasting and physical exercise

    Directory of Open Access Journals (Sweden)

    Renata Polessi Boschini

    2005-12-01

    Full Text Available O tecido adiposo marrom, onde se localiza a proteína desacopladora 1 (UCP1 - uncoupling protein 1, é um tecido termogênico presente somente nos pequenos mamíferos e neonatos, com função de manter temperatura e peso corporal estáveis quando da exposição ao frio ou consumo de dietas hipercalóricas. Como a UCP1 está localizada exclusivamente no tecido adiposo marrom, tecido pouco expressado em adultos, os estudos dão ênfase às proteínas desacopladoras 2 e 3 (UCP2 e UCP3, proteínas homólogas à UCP1, expressas em múltiplos tecidos e nos músculos esqueléticos, respectivamente. A atividade física provoca aumento do RNAm da UCP2 e UCP3, questiona-se, porém, se este aumento é devido a mudanças no metabolismo de gordura ou a mudanças no metabolismo energético. Durante a restrição energética ou jejum, há depleção de gordura corporal e aumento da concentração plasmática de ácidos graxos livres, com regulação positiva da UCP2 e da UCP3 no músculo e aumento da oxidação lipídica. A concentração elevada de ácidos graxos representa sinal intracelular importante na indução da expressão das UCP no músculo, o que pode estar ligado à sua utilização como combustível até que ocorra aumento da demanda do organismo para dissipação da energia. No entanto, discute-se se a UCP2 e a UCP3 no músculo esquelético têm como função mediar a termogênese ou regular a oxidação de lipídios.The brown adipose tissue, where the uncoupling protein 1 (UCP1 is located, is a thermogenetic tissue only present in small mammals and neonates, with the function of keeping the temperature and body weight stable when exposed to either cold or a hypercaloric diet. Since the UCP1 is located exclusively in brown adipose tissue, and this tissue has little expression in human adults, the studies have emphasized the UCP2 and UCP3, homologous proteins to the UCP1, expressed in multiple tissues and skeletal muscle, respectively. Physical

  1. Influence of pre-exercise muscle glycogen content on exercise-induced transcriptional regulation of metabolic genes.

    Science.gov (United States)

    Pilegaard, Henriette; Keller, Charlotte; Steensberg, Adam; Helge, Jørn Wulff; Pedersen, Bente Klarlund; Saltin, Bengt; Neufer, P Darrell

    2002-05-15

    Transcription of metabolic genes is transiently induced during recovery from exercise in skeletal muscle of humans. To determine whether pre-exercise muscle glycogen content influences the magnitude and/or duration of this adaptive response, six male subjects performed one-legged cycling exercise to lower muscle glycogen content in one leg and then, the following day, completed 2.5 h low intensity two-legged cycling exercise. Nuclei and mRNA were isolated from biopsies obtained from the vastus lateralis muscle of the control and reduced glycogen (pre-exercise glycogen = 609 +/- 47 and 337 +/- 33 mmol kg(-1) dry weight, respectively) legs before and after 0, 2 and 5 h of recovery. Exercise induced a significant (P 6-fold) than in the control (< 3-fold) trial. Induction of PDK4 and UCP3 mRNA in response to exercise was also significantly higher in the low glycogen (11.4- and 3.5-fold, respectively) than in the control (5.0- and 1.7-fold, respectively) trial. These data indicate that low muscle glycogen content enhances the transcriptional activation of some metabolic genes in response to exercise, raising the possibility that signalling mechanisms sensitive to glycogen content and/or FFA availability may be linked to the transcriptional control of exercise-responsive genes.

  2. Estudio funcional de la proteína desacopladora mitocondrial UCP3 en relación con la apoptosis y las especies reactivas de oxígeno

    OpenAIRE

    Cámara Navarro, Yolanda

    2005-01-01

    El presente trabajo de tesis doctoral se ha centrado en la determinación de la función fisiológica de la proteína desacopladora mitocondrial UCP3 en relación con la apoptosis y las especies reactivas de oxígeno (ROS). UCP3 es un miembro de la familia de transportadores mitocondriales que se expresa preferencialmente en el músculo esquelético. UCP3 es una proteína homóloga a la termogenina o proteína desacopladota UCP1. Ésta se expresa exclusivamente en tejido adiposo marrón dónde desempeña un...

  3. Rol de las proteinas desacoplantes UCP1, UCP2 y UCP3 en el gasto energetico, diabetes tipo 2 y obesidad: Sinergismo con la tiroides Role of uncoupling proteins UCP1, UCP2 and UCP3 in energy balance, type 2 diabetes and obesity: Synergism with the thyroid

    Directory of Open Access Journals (Sweden)

    Ángel A. Zaninovich

    2005-04-01

    Full Text Available La formación de tejido graso resulta del balance entre la ingestión y el consumo de energía, lo cual destaca la importancia del estudio de los factores que controlan el gasto energético. La hormona tiroidea es conocida desde hace tiempo como el principal regulador del metabolismo basal, a través de la estimulación del consumo de oxígeno en las células. El descubrimiento de la grasa parda y de la proteína desacoplante-1 (UCP1 demostró la importancia de este tejido para la regulación del consumo energético en mamíferos. La proteína desacoplante-2 (UCP2 se expresa en muchos tejidos y tendría una acción protectora de la función celular, al preservar el potencial de membrana afectado por el superóxido. La proteína desacoplante-3 (UCP3 estaría vinculada a la producción de calor, facilitando la combustión de ácidos grasos en la cadena respiratoria mitocondrial, pero no parece participar en el control del gasto energético. El exceso de UCP3 en ratones transgénicos disminuyó la grasa corporal y aumentó la sensibilidad a la insulina seguido de hipoglucemia, sugiriendo así un futuro, hipotético uso de esta proteína en la diabetes 2 y en la obesidad. Los estudios que se realizan sobre estas proteínas y sobre hormonas del tejido adiposo blanco como la leptina, adiponectina, resistina, de péptidos hipotalámicos como neuropéptido Y, CRF, hormona alfa-melanocítica y péptidos regulados por cocaína y anfetamina (CART, muestran resultados promisorios para una futura aplicación en el control del gasto energético en humanos y con ello en la prevención o el tratamiento de la obesidad y la diabetes tipo 2.Accumulation of fat in the tissues results from the balance between energy intake and expenditure. The thyroid hormones have long been known to be the main regulators of basal metabolism through its stimulation of oxygen consumption in cells. The discovery of brown adipose tissue (BAT and its unique activity of heat production

  4. CAPSAICIN SUPPLEMENTATION FAILS TO MODULATE AUTONOMIC AND CARDIAC ELECTROPHYSIOLOGIC ACTIVITY DURING EXERCISE IN THE OBESE: WITH VARIANTS OF UCP2 AND UCP3 POLYMORPHISM

    Directory of Open Access Journals (Sweden)

    Ki Ok Shin

    2008-09-01

    Full Text Available We investigated the effects of capsaicin supplementation (150mg on alterations of autonomic nervous system (ANS activity associated with adverse effects of cardiac depolarization-repolarization intervals during aerobic exercise in obese humans. Nine obese males (26.1 ± 1.5 yrs volunteered between study designed. The cardiac ANS activities evaluated by means of heart rate variability of power spectral analysis and cardiac QT interval were continuously measured during 5-min rest and 30-min exercise at 50% of maximal ventilation threshold (50%VTmax on stationary ergometer with placebo (CON or capsaicin (CAP oral administration chosen at random. The uncoupling protein (UCP 2 and UCP 3 genetic variants of the subjects were analyzed by noninvasive genotyping method from collecting buccal mucosa cells. The results indicated that there were no significant differences in cardiac ANS activities during rest and exercise between CON and CAP trials. Although no significant difference, A/A allele of UCP2 polymorphism showed a reduced sympathetic nervous system (SNS index activity compared to G/G + G/A allele during exercise intervention in our subjects. On the other hand, the data on cardiac QT interval showed no significant difference, indicating that oral administration of capsaicin did not cause any adverse effect on cardiac depolarization-repolarization. In conclusion, our results suggest that capsaicin supplementation 1 h before exercise intervention has no effect on cardiac ANS activities and cardiac electrical stability during exercise in obese individuals. Further studies should also consider genetic variants for exercise efficacy against obesity

  5. Feeding conditions and breed affect the level of DNA methylation of the mitochondrial uncoupling protein 3 gene in chicken breast muscle.

    Science.gov (United States)

    Gao, G L; Wang, H W; Zhao, X Z; Li, Qin; Li, J; Li, Q R; Wang, Q G

    2015-04-01

    To investigate the effects of feed condition and breed on the level of DNA methylation for the uncoupling protein 3 (UCP3) gene, which is an important candidate gene for regulating intramuscular fat (IMF) content in chicken breast muscle, breast muscle of Daninghe (DNH) and Qingjiaoma (QJM) chickens under scatter-feed and captivity-feed conditions was analyzed. Using RNA sequencing, 47 and 113 candidate genes were determined to be related to feed conditions and breed, respectively, and 7 differentially expressed genes were confirmed by real-time quantitative PCR, including UCP3. The mRNA levels of UCP3 were significantly different between the 2 feed conditions. The DNA region from bp +1700 to +2459 of the UCP3 gene was studied using the bisulfite sequencing method and contained 46 methylation sites and 3 CpG islands. The results showed that the methylation level of this UCP3 region was lower in DNH chickens (0.77% to 0.88%, P = 0.012) and QJM chickens (0.88% to 0.91%, P = 0.20) under scatter-feed conditions than under captivity-feed conditions. The mean methylation level of UCP3 in DNH chickens was lower than that in QJM chickens under scatter-feed conditions (DNH to QJM, 0.77% to 0.88%, P = 0.007), which suggests that breed affects the mean methylation level of UCP3 under scatter-feed conditions. In summary, our findings suggest that feed condition and breed affect the methylation of UCP3 in chicken breast muscle.

  6. Radiation-induced gene responses

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E.; Paunesku, T.; Shearin-Jones, P.; Oryhon, J.

    1996-12-31

    In the process of identifying genes that are differentially regulated in cells exposed to ultraviolet radiation (UV), we identified a transcript that was repressed following the exposure of cells to a combination of UV and salicylate, a known inhibitor of NF-kappaB. Sequencing this band determined that it has identify to lactate dehydrogenase, and Northern blots confirmed the initial expression pattern. Analysis of the sequence of the LDH 5` region established the presence of NF-kappaB, Sp1, and two Ap-2 elements; two partial AP- 1; one partial RE, and two halves of E-UV elements were also found. Electromobility shift assays were then performed for the AP-1, NF- kappaB, and E-UV elements. These experiments revealed that binding to NF-kappaB was induced by UV but repressed with salicylic acid; UV did not affect AP-1 binding, but salicylic acid inhibited it alone or following UV exposure; and E-UV binding was repressed by UV, and salicylic acid had little effect. Since the binding of no single element correlated with the expression pattern of LDH, it is likely that multiple elements govern UV/salicylate-mediated expression.

  7. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  8. Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise.

    Science.gov (United States)

    Pilegaard, H; Ordway, G A; Saltin, B; Neufer, P D

    2000-10-01

    Exercise training elicits a number of adaptive changes in skeletal muscle that result in an improved metabolic efficiency. The molecular mechanisms mediating the cellular adaptations to exercise training in human skeletal muscle are unknown. To test the hypothesis that recovery from exercise is associated with transcriptional activation of specific genes, six untrained male subjects completed 60-90 min of exhaustive one-legged knee extensor exercise for five consecutive days. On day 5, nuclei were isolated from biopsies of the vastus lateralis muscle of the untrained and the trained leg before exercise and from the trained leg immediately after exercise and after 15 min, 1 h, 2 h, and 4 h of recovery. Transcriptional activity of the uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase 4 (PDK4), and heme oxygenase-1 (HO-1) genes (relative to beta-actin) increased by three- to sevenfold in response to exercise, peaking after 1-2 h of recovery. Increases in mRNA levels followed changes in transcription, peaking between 2 and 4 h after exercise. Lipoprotein lipase and carnitine pamitoyltransferase I gene transcription and mRNA levels showed similar but less dramatic induction patterns, with increases ranging from two- to threefold. In a separate study, a single 4-h bout of cycling exercise (n = 4) elicited from 5 to >20-fold increases in UCP3, PDK4, and HO-1 transcription, suggesting that activation of these genes may be related to the duration or intensity of exercise. These data demonstrate that exercise induces transient increases in transcription of metabolic genes in human skeletal muscle. Moreover, the findings suggest that the cumulative effects of transient increases in transcription during recovery from consecutive bouts of exercise may represent the underlying kinetic basis for the cellular adaptations associated with exercise training.

  9. Association Studies of 3 Candidate Genes with Type 2 Diabetes Mellitus in a Chinese Population

    Institute of Scientific and Technical Information of China (English)

    鲁一兵; 缪珩; 王华; 何戎华; 马立隽; 金卫新; 华子春

    2002-01-01

    Objectives To explore the relationship between the polymorphisms of the select-ed short tandem repeats (STRs) of the candidate genes and type 2 diabetes mellitus (DM) in a Chinesepopulation, the role of genetic and environmental factors in the development of type 2 diabetes. Meth-ods STRs including D11S916 of uncoupling protein 3 (UCP3) gene,binucleotide repeat (CA). with-in intron 6 [HSLi6 (CA)n] of hormone- sensitive lipase(HSL) gene and D20S501 of protein tyrosinephosphatase- 1B (PTP-1B) gene polymorphisms were detected by polymerase chain reaction (PCR) , poly-acrylamiie gel electrophoresis and silver staining in 106 patients with type 2 DM and 102 control sub-jects. Results The allele distribution of UCP3 and HSL gene differed significantly between patientswith type 2 diabetes and control subjects (χ2 = 26. 12, P<0.005; χ2=10. 33, P<0. 005, respec-tively). For UCP3 and HSL gene,the frequencies of alleles A6,A7 ,A8 and allele B9 were much high-er in diabetic patients than in control subjects (0. 090 vs 0. 020,P<0. 005; 0. 109 vs 0. 015,P<0. 005; 0. 033 υs 0. 000,P<0.05; 0. 033 υs 0. 005,P<0. 05,respectively),while the fre-quencies of allele A1 and allele B5 were lower in diabetic patients than in control subjects (0. 090 vs0. 206,P<0. 005; 0. 057 vs 0. 118,P<0. 05,respectively). At D20S501 locus,The allele dis-tribution of PTP-1B gene had no significant difference in two groups (χ2=3. 77 ,P>0. 05). Multi-variate logistic regression analysis showed positive correlation between alleles A 6,A 7 of UCP3 gene,sys-tolic blood pressure , apolipoprotein B , lipoprotein (a) and type 2 diabetes. Conclusion Our datashow that D11S916 of UCP3 gene and HSLi6 (CA), of HSL gene polymorphisms are associated withtype 2 diabetes in Chinese suggesting that UCP3 and HSL might represent susceptibility genes for type 2diabetes. D20S501 of PTP-1B gene polymorphism isnot associated uith type 2 diabetes in Chinese. AllelesA6, A7 of UCP3 gene, systolic blood

  10. A Study on the Variance of Uncoupling Protein 3 Gene in Shanghai Han Race%上海地区汉族人解偶联蛋白3基因的变异

    Institute of Scientific and Technical Information of China (English)

    张蓉; 郑以漫; 项坤三; 贾伟平; 方启晨

    2001-01-01

    Objective To investigatce the relationship between ⅣS6 g→a + 1 variance of un- coupling protein 3 gene (UCP3) and obesity as well as diabetes in Chinese Hans. Methods The geno- type of this variance was determined by the PCR-RFLP method in 306 unrelated Shanghai Chinese Hans and 66 normal Caucasians. Results No ⅣS6 g→a + 1 variance of UCP3 gene was found in this group of Shanghai Chinese Hans and 66 Caucasians. Conclusion This variance of UCP3 gene is not a major predisposing genetic factor for obesity and diabetes in Chinese Hans.%目的探讨解偶联蛋白P3(UCP3)基因Ⅳ6G→a+1变异与中国汉族人肥胖及糖尿病的关系。方法选取306例无亲缘关系的中国人和66例白种人,运用PCR-RFLP方法检测UCP3基因Ⅳ两Pa+1的变异情况。结果中国人与白种人中均未发现这种变异。结论该基因变异不是中国汉族人肥胖与糖尿病发生的重要遗传因素。

  11. Tetracycline inducible gene manipulation in serotonergic neurons.

    Directory of Open Access Journals (Sweden)

    Tillmann Weber

    Full Text Available The serotonergic (5-HT neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA mouse line (TPH2-tTA that allows temporal and spatial control of tetracycline (Ptet controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ. In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox. Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20 were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We

  12. Gravity-Induced Gene Expression in Plants.

    Science.gov (United States)

    Sederoff, Heike; Heber, Steffen; Howard, Brian; Myburg-Nichols, Henrietta; Hammond, Rebecca; Salinas-Mondragon, Raul; Brown, Christopher S.

    Plants sense changes in their orientation towards the vector of gravity and respond with directional growth. Several metabolites in the signal transduction cascade have been identified. However, very little is known about the interaction between these sensing and signal transduction events and even less is known about their role in the differential growth response. Gravity induced changes in transcript abundance have been identified in Arabidopsis whole seedlings and root apices (Moseyko et al. 2002; Kimbrough et al. 2004). Gravity induced transcript abundance changes can be observed within less than 1 min after stimulation (Salinas-Mondragon et al. 2005). Gene expression however requires not only transcription but also translation of the mRNA. Translation can only occur when mRNA is associated with ribosomes, even though not all mRNA associated with ribosomes is actively translated. To approximate translational capacity we quantified whole genome transcript abundances in corn stem pulvini during the first hour after gravity stimulation in total and poly-ribosomal fractions. As in Arabidopsis root apices, transcript abundances of several clusters of genes responded to gravity stimulation. The vast majority of these transcripts were also found to associate with polyribosomes in the same temporal and quantitative pattern. These genes are transcriptionally regulated by gravity stimulation, but do not exhibit translational regulation. However, a small group of genes showed increased transcriptional regulation after gravity stimulation, but no association with polysomes. These transcripts likely are translationally repressed. The mechanism of translational repression for these transcripts is unknown. Based on the hypothesis that the genes essential for gravitropic responses should be expressed in most or all species, we compared the temporal gravity induced expression pattern of all orthologs identified between maize and Arabidopsis. A small group of genes showed high

  13. Photorhabdus luminescens genes induced upon insect infection

    Directory of Open Access Journals (Sweden)

    Jung Kirsten

    2008-05-01

    Full Text Available Abstract Background Photorhabdus luminescens is a Gram-negative luminescent enterobacterium and a symbiote to soil nematodes belonging to the species Heterorhabditis bacteriophora. P.luminescens is simultaneously highly pathogenic to insects. This bacterium exhibits a complex life cycle, including one symbiotic stage characterized by colonization of the upper nematode gut, and a pathogenic stage, characterized by release from the nematode into the hemocoel of insect larvae, resulting in rapid insect death caused by bacterial toxins. P. luminescens appears to sense and adapt to the novel host environment upon changing hosts, which facilitates the production of factors involved in survival within the host, host-killing, and -exploitation. Results A differential fluorescence induction (DFI approach was applied to identify genes that are up-regulated in the bacterium after infection of the insect host Galleria mellonella. For this purpose, a P. luminescens promoter-trap library utilizing the mCherry fluorophore as a reporter was constructed, and approximately 13,000 clones were screened for fluorescence induction in the presence of a G. mellonella larvae homogenate. Since P. luminescens has a variety of regulators that potentially sense chemical molecules, like hormones, the screen for up-regulated genes or operons was performed in vitro, excluding physicochemical signals like oxygen, temperature or osmolarity as variables. Clones (18 were obtained exhibiting at least 2.5-fold induced fluorescence and regarded as specific responders to insect homogenate. In combination with a bioinformatics approach, sequence motifs were identified in these DNA-fragments that are similar to 29 different promoters within the P. luminescens genome. By cloning each of the predicted promoters upstream of the reporter gene, induction was verified for 27 promoters in vitro, and for 24 promoters in viable G. mellonella larvae. Among the validated promoters are some known

  14. Effect of short-term fasting and refeeding on transcriptional regulation of metabolic genes in human skeletal muscle.

    Science.gov (United States)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-03-01

    During short-term fasting, substrate utilization in skeletal muscle shifts from predominantly carbohydrate to fat as a means of conserving glucose. To examine the potential influence of short-term fasting and refeeding on transcriptional regulation in skeletal muscle, muscle biopsies were obtained from nine male subjects at rest, after 20 h of fasting, and 1 h after consuming either a high-carbohydrate (CHO trial) or a low-carbohydrate (FAT trial) meal. Fasting induced an increase in transcription of the pyruvate dehydrogenase kinase 4 (PDK4) (10-fold), lipoprotein lipase (LPL) ( approximately 2-fold), uncoupling protein 3 (UCP3) ( approximately 5-fold), and carnitine palmitoyltransferase I (CPT I) ( approximately 2.5-fold) genes. Surprisingly, transcription of PDK4 and LPL increased further in response to refeeding (both trials) to more than 50-fold and 6- to 10-fold, respectively, over prefasting levels. However, responses varied among subjects with two subjects in particular displaying far greater activation of PDK4 (>100-fold) and LPL (>20-fold) than the other subjects (mean approximately 8-fold and approximately 2-fold, respectively). Transcription of UCP3 decreased to basal levels after the CHO meal but remained elevated after the FAT meal, whereas CPT I remained elevated after both refeeding meals. The present findings demonstrate that short-term fasting/refeeding in humans alters the transcription of several genes in skeletal muscle related to lipid metabolism. Marked heterogeneity in the transcriptional response to the fasting/refeeding protocol suggests that individual differences in genetic profile may play an important role in adaptive molecular responses to metabolic challenges.

  15. Heat-inducible RNAi for gene functional analysis in plants.

    Science.gov (United States)

    Masclaux, Frédéric; Galaud, Jean-Philippe

    2011-01-01

    Controlling gene expression during plant development is an efficient method to explore gene function and RNA interference (RNAi) is now considered as a powerful technology for gene functional analysis. However, constitutive gene silencing cannot be used with genes involved in fundamental processes such as embryo viability or plant growth and alternative silencing strategies avoiding these limitations should be preferred. Tissue-specific and inducible promoters, able to control gene expression at spatial and/or temporal level, can be used to circumvent viability problems. In this chapter, after a rapid overview of the inducible promoters currently used for transgenic approaches in plants, we describe a method we have developed to study gene function by heat-inducible RNAi. This system is easy to use and complementary to those based on chemical gene inducer treatments and might be useful for both research and biotechnological applications.

  16. Repeat-induced gene silencing in mammals.

    Science.gov (United States)

    Garrick, D; Fiering, S; Martin, D I; Whitelaw, E

    1998-01-01

    In both plants and Drosophila melanogaster, expression from a transgenic locus may be silenced when repeated transgene copies are arranged as a concatameric array. This repeat-induced gene silencing is frequently manifested as a decrease in the proportion of cells that express the transgene, resulting in a variegated pattern of expression. There is also some indication that, in transgenic mammals, the number of transgene copies within an array can exert a repressive influence on expression, with several mouse studies reporting a decrease in the level of expression per copy as copy number increases. However, because these studies compare different sites of transgene integration as well as arrays with different numbers of copies, the expression levels observed may be subject to varying position effects as well as the influence of the multicopy array. Here we describe use of the lox/Cre system of site-specific recombination to generate transgenic mouse lines in which different numbers of a transgene are present at the same chromosomal location, thereby eliminating the contribution of position effects and allowing analysis of the effect of copy number alone on transgene silencing. Reduction in copy number results in a marked increase in expression of the transgene and is accompanied by decreased chromatin compaction and decreased methylation at the transgene locus. These findings establish that the presence of multiple homologous copies of a transgene within a concatameric array can have a repressive effect upon gene expression in mammalian systems.

  17. Overexpression of uncoupling protein 3 in skeletal muscle protects against fat-induced insulin resistance

    Science.gov (United States)

    Choi, Cheol Soo; Fillmore, Jonathan J.; Kim, Jason K.; Liu, Zhen-Xiang; Kim, Sheene; Collier, Emily F.; Kulkarni, Ameya; Distefano, Alberto; Hwang, Yu-Jin; Kahn, Mario; Chen, Yan; Yu, Chunli; Moore, Irene K.; Reznick, Richard M.; Higashimori, Takamasa; Shulman, Gerald I.

    2007-01-01

    Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCθ in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1– (IRS-1–) and IRS-2–associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCθ activity in whole-body fat–matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus. PMID:17571165

  18. Estudio del papel protector de la proteína desacoplante UCP3 frente al extrés oxidativo en mitocondrias de músculo esquelético y corazón de ratones tratados con endotoxina y en cardiomiocitos de rata

    OpenAIRE

    Aguirre Laso, Enara

    2011-01-01

    En el presente trabajo de tesis doctoral se ha intentado determinar el posible papel protector de la proteína UCP3 frente al estrés oxidativo en el músculo esquelético y el corazón de ratón a través de la medida de la conductancia de la membrana mitocondrial interna a los protones en presencia del radical superóxido y del compuesto 4-hidroxinonenal (HNE), ambos descritos previamente como activadores de las proteínas desacoplantes. Estas medidas se realizaron en mitocondrias aislad...

  19. Estudio del papel protector de la proteína desacoplante UCP3 frente al extrés oxidativo en mitocondrias de músculo esquelético y corazón de ratones tratados con endotoxina y en cardiomiocitos de rata

    OpenAIRE

    Aguirre Laso, Enara

    2009-01-01

    En el presente trabajo de tesis doctoral se ha intentado determinar el posible papel protector de la proteína UCP3 frente al estrés oxidativo en el músculo esquelético y el corazón de ratón a través de la medida de la conductancia de la membrana mitocondrial interna a los protones en presencia del radical superóxido y del compuesto 4-hidroxinonenal (HNE), ambos descritos previamente como activadores de las proteínas desacoplantes. Estas medidas se realizaron en mitocondrias aislad...

  20. Expression of genes related to mitochondrial function in Nellore cattle divergently ranked on residual feed intake.

    Science.gov (United States)

    Fonseca, Larissa Fernanda Simielli; Gimenez, Daniele Fernanda Jovino; Mercadante, Maria Eugênia Zerlotti; Bonilha, Sarah Figueiredo Martins; Ferro, Jesus Aparecido; Baldi, Fernando; de Souza, Fábio Ricardo Pablos; de Albuquerque, Lucia Galvão

    2015-02-01

    Several measures have been proposed to investigate and improve feed efficiency in cattle. One of the most commonly used measure of feed efficiency is residual feed intake (RFI), which is estimated as the difference between actual feed intake and expected feed intake based on the animal's average live weight. This measure permits to identify and select the most efficient animals without selecting for higher mature weight. Mitochondrial function has been indicated as a major factor that influences RFI. The analysis of genes involved in mitochondrial function is therefore an alternative to identify molecular markers associated with higher feed efficiency. This study analyzed the expression of PGC1α, TFAM, UCP2 and UCP3 genes by quantitative real-time PCR in liver and muscle tissues of two groups of Nellore cattle divergently ranked on RFI values in order to evaluate the relationship of these genes with RFI. In liver tissue, higher expression of TFAM and UCP2 genes was observed in the negative RFI group. Expression of PGC1α gene did not differ significantly between the two groups, whereas UCP3 gene was not expressed in liver tissue. In muscle tissue, higher expression of TFAM gene was observed in the positive RFI group. Expression of PGC1α, UCP2 and UCP3 genes did not differ significantly between the two groups. These results suggest the use of TFAM and UCP2 as possible candidate gene markers in breeding programs designed to increase the feed efficiency of Nellore cattle.

  1. Virus-induced gene silencing in soybean and common bean.

    Science.gov (United States)

    Zhang, Chunquan; Whitham, Steven A; Hill, John H

    2013-01-01

    Plant viral vectors are useful for transient gene expression as well as for downregulation of gene expression via virus-induced gene silencing (VIGS). When used in reverse genetics approaches, VIGS offers a convenient way of transforming genomic information into knowledge of gene function. Efforts to develop and improve plant viral vectors have expanded their applications and have led to substantial advances needed to facilitate gene function studies in major row crops. Here, we describe a DNA-based Bean pod mottle virus (BPMV) vector system for both gene expression and VIGS in soybean and common bean.

  2. Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing

    NARCIS (Netherlands)

    Romero, I.; Tikunov, Y.M.; Bovy, A.G.

    2011-01-01

    Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through agroinfiltrat

  3. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Science.gov (United States)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  4. A riboswitch-based inducible gene expression system for mycobacteria.

    Directory of Open Access Journals (Sweden)

    Jessica C Seeliger

    Full Text Available Research on the human pathogen Mycobacterium tuberculosis (Mtb would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

  5. Associations between UCP1 -3826A/G, UCP2 -866G/A, Ala55Val and Ins/Del, and UCP3 -55C/T polymorphisms and susceptibility to type 2 diabetes mellitus: case-control study and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Bianca M de Souza

    Full Text Available BACKGROUND: Some studies have reported associations between five uncoupling protein (UCP 1-3 polymorphisms and type 2 diabetes mellitus (T2DM. However, other studies have failed to confirm the associations. This paper describes a case-control study and a meta-analysis conducted to attempt to determine whether the following polymorphisms are associated with T2DM: -3826A/G (UCP1; -866G/A, Ala55Val and Ins/Del (UCP2 and -55C/T (UCP3. METHODS: The case-control study enrolled 981 T2DM patients and 534 nondiabetic subjects, all of European ancestry. A literature search was run to identify all studies that investigated associations between UCP1-3 polymorphisms and T2DM. Pooled odds ratios (OR were calculated for allele contrast, additive, recessive, dominant and co-dominant inheritance models. Sensitivity analyses were performed after stratification by ethnicity. RESULTS: In the case-control study the frequencies of the UCP polymorphisms did not differ significantly between T2DM and nondiabetic groups (P>0.05. Twenty-three studies were eligible for the meta-analysis. Meta-analysis results showed that the Ala55Val polymorphism was associated with T2DM under a dominant model (OR = 1.27, 95% CI 1.03-1.57; while the -55C/T polymorphism was associated with this disease in almost all genetic models: allele contrast (OR = 1.17, 95% CI 1.02-1.34, additive (OR = 1.32, 95% CI 1.01-1.72 and dominant (OR = 1.18, 95% CI 1.02-1.37. However, after stratification by ethnicity, the UCP2 55Val and UCP3 -55C/T alleles remained associated with T2DM only in Asians (OR = 1.25, 95% CI 1.02-1.51 and OR = 1.22, 95% CI 1.04-1.44, respectively; allele contrast model. No significant association of the -3826A/G, -866G/A and Ins/Del polymorphisms with T2DM was observed. CONCLUSIONS: In our case-control study of people with European ancestry we were not able to demonstrate any association between the UCP polymorphisms and T2DM; however, our meta-analysis detected a significant

  6. Regulatory systems for hypoxia-inducible gene expression in ischemic heart disease gene therapy.

    Science.gov (United States)

    Kim, Hyun Ah; Rhim, Taiyoun; Lee, Minhyung

    2011-07-18

    Ischemic heart diseases are caused by narrowed coronary arteries that decrease the blood supply to the myocardium. In the ischemic myocardium, hypoxia-responsive genes are up-regulated by hypoxia-inducible factor-1 (HIF-1). Gene therapy for ischemic heart diseases uses genes encoding angiogenic growth factors and anti-apoptotic proteins as therapeutic genes. These genes increase blood supply into the myocardium by angiogenesis and protect cardiomyocytes from cell death. However, non-specific expression of these genes in normal tissues may be harmful, since growth factors and anti-apoptotic proteins may induce tumor growth. Therefore, tight gene regulation is required to limit gene expression to ischemic tissues, to avoid unwanted side effects. For this purpose, various gene expression strategies have been developed for ischemic-specific gene expression. Transcriptional, post-transcriptional, and post-translational regulatory strategies have been developed and evaluated in ischemic heart disease animal models. The regulatory systems can limit therapeutic gene expression to ischemic tissues and increase the efficiency of gene therapy. In this review, recent progresses in ischemic-specific gene expression systems are presented, and their applications to ischemic heart diseases are discussed.

  7. Screening of hypoxia-inducible genes in sporadic ALS.

    LENUS (Irish Health Repository)

    Cronin, Simon

    2008-10-01

    Genetic variations in two hypoxia-inducible angiogenic genes, VEGF and ANG, have been linked with sporadic amyotrophic lateral sclerosis (SALS). Common variations in these genes may reduce the levels or functioning of their products. VEGF and ANG belong to a larger group of angiogenic genes that are up-regulated under hypoxic conditions. We hypothesized that common genetic variation across other members of this group may also predispose to sporadic ALS. To screen other hypoxia-inducible angiogenic genes for association with SALS, we selected 112 tagging single nucleotide polymorphisms (tgSNPs) that captured the common genetic variation across 16 VEGF-like and eight ANG-like hypoxia-inducible genes. Screening for association was performed in 270 Irish individuals with typical SALS and 272 ethnically matched unrelated controls. SNPs showing association in the Irish phase were genotyped in a replication sample of 281 Swedish sporadic ALS patients and 286 Swedish controls. Seven markers showed association in the Irish. The one modest replication signal observed in the Swedish replication sample, at rs3801158 in the gene inhibin beta A, was for the opposite allele vs. the Irish cohort. We failed to detect association of common variation across 24 candidate hypoxia-inducible angiogenic genes with SALS.

  8. Effects of insulin replacements, inhibitors of angiotensin, and PKCbeta's actions to normalize cardiac gene expression and fuel metabolism in diabetic rats.

    Science.gov (United States)

    Arikawa, Emi; Ma, Ronald C W; Isshiki, Keiji; Luptak, Ivan; He, Zhiheng; Yasuda, Yutaka; Maeno, Yasuhiro; Patti, Mary Elizabeth; Weir, Gordon C; Harris, Robert A; Zammit, Victor A; Tian, Rong; King, George L

    2007-05-01

    High-density oligonucleotide arrays were used to compare gene expression of rat hearts from control, untreated diabetic, and diabetic groups treated with islet cell transplantation (ICT), protein kinase C (PKC)beta inhibitor ruboxistaurin, or ACE inhibitor captopril. Among the 376 genes that were differentially expressed between untreated diabetic and control hearts included key metabolic enzymes that account for the decreased glucose and increased free fatty acid utilization in the diabetic heart. ICT or insulin replacements reversed these gene changes with normalization of hyperglycemia, dyslipidemia, and cardiac PKC activation in diabetic rats. Surprisingly, both ruboxistaurin and ACE inhibitors improved the metabolic gene profile (confirmed by real-time RT-PCR and protein analysis) and ameliorated PKC activity in diabetic hearts without altering circulating metabolites. Functional assessments using Langendorff preparations and (13)C nuclear magnetic resonance spectroscopy showed a 36% decrease in glucose utilization and an impairment in diastolic function in diabetic rat hearts, which were normalized by all three treatments. In cardiomyocytes, PKC inhibition attenuated fatty acid-induced increases in the metabolic genes PDK4 and UCP3 and also prevented fatty acid-mediated inhibition of basal and insulin-stimulated glucose oxidation. Thus, PKCbeta or ACE inhibitors may ameliorate cardiac metabolism and function in diabetes partly by normalization of fuel metabolic gene expression directly in the myocardium.

  9. Mechanisms of radiation-induced gene responses

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E.; Paunesku, T.

    1996-10-01

    In the process of identifying genes differentially expressed in cells exposed ultraviolet radiation, we have identified a transcript having a 26-bp region that is highly conserved in a variety of species including Bacillus circulans, yeast, pumpkin, Drosophila, mouse, and man. When the 5` region (flanking region or UTR) of a gene, the sequence is predominantly in +/+ orientation with respect to the coding DNA strand; while in the coding region and the 3` region (UTR), the sequence is most frequently in the +/-orientation with respect to the coding DNA strand. In two genes, the element is split into two parts; however, in most cases, it is found only once but with a minimum of 11 consecutive nucleotides precisely depicting the original sequence. The element is found in a large number of different genes with diverse functions (from human ras p21 to B. circulans chitonase). Gel shift assays demonstrated the presence of a protein in HeLa cell extracts that binds to the sense and antisense single-stranded consensus oligomers, as well as to the double- stranded oligonucleotide. When double-stranded oligomer was used, the size shift demonstrated as additional protein-oligomer complex larger than the one bound to either sense or antisense single-stranded consensus oligomers alone. It is speculated either that this element binds to protein(s) important in maintaining DNA is a single-stranded orientation for transcription or, alternatively that this element is important in the transcription-coupled DNA repair process.

  10. Salmonella induces prominent gene expression in the rat colon

    Directory of Open Access Journals (Sweden)

    Roosing Susanne

    2007-09-01

    Full Text Available Abstract Background Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point. As fructo-oligosaccharides (FOS affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. Results Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase, antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2, inflammation (e.g. calprotectin, oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2 and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9. Furthermore, Salmonella translocation increased serum IFNγ and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap, showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. Conclusion We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in

  11. Inducible gene expression and environmentally regulated genes in lactic acid bacteria

    NARCIS (Netherlands)

    Kok, Jan

    1996-01-01

    Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transc

  12. Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum

    NARCIS (Netherlands)

    Peters, Dorien J.M.; Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van; Spek, Wouter; Schaap, Pauline

    1989-01-01

    We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl.

  13. Differential transcriptional activation of select metabolic genes in response to variations in exercise intensity and duration.

    Science.gov (United States)

    Hildebrandt, Audrey L; Pilegaard, Henriette; Neufer, P Darrell

    2003-11-01

    Cellular adaptations to endurance training are influenced by the intensity and duration of exercise. To examine the impact of exercise intensity and duration on the acute transcriptional regulation of metabolic genes in red (RG) and white (WG) gastrocnemius muscle, rats completed either low-intensity [ approximately 50% maximal O2 uptake (VO2 max)] treadmill exercise (LIE) for 45 min, LIE for 180 min, or high-intensity ( approximately 75% VO2 max) exercise (HIE) for 45 min. LIE for 45 min activated (P 30-fold) and HKII transcription (>25-fold). In WG, extending LIE for 180 min induced a much greater and prolonged (through 2- to 4-h recovery) activation of PDK4, UCP3 (both >200-fold), and HO-1 (>10-fold). HIE elicited a similar pattern of gene activation to LIE in both RG and WG, with the exception that HIE triggered >10-fold activation of HO-1 in WG. These data provide evidence that both the intensity and the duration of exercise affect the transcriptional regulation of metabolic genes in muscle in a fiber type-specific manner, possibly reflecting the relative stress imposed by the exercise bout.

  14. Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

    Science.gov (United States)

    Canuto, K. S.; Sergio, L. P. S.; Paoli, F.; Mencalha, A. L.; Fonseca, A. S.

    2016-03-01

    Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

  15. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David George James [ORNL; McKnight, Timothy E [ORNL; Mcpherson, Jackson [University of Tennessee, Knoxville (UTK); Hoyt, Peter R [ORNL; Melechko, Anatoli Vasilievich [ORNL; Simpson, Michael L [ORNL; Sayler, Gary Steven [ORNL

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and introduced alongside the yfp marker gene into Chinese hamster ovary cells using spatially indexed vertically aligned carbon nanofiber arrays (VACNFs) in a gene delivery process termed impalefection. The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. 24 hours after nanofiber-mediated delivery, 53.1% 10.4% of the cells that expressed the yfp marker gene were also fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  16. Transcription dynamics of inducible genes modulated by negative regulations.

    Science.gov (United States)

    Li, Yanyan; Tang, Moxun; Yu, Jianshe

    2015-06-01

    Gene transcription is a stochastic process in single cells, in which genes transit randomly between active and inactive states. Transcription of many inducible genes is also tightly regulated: It is often stimulated by extracellular signals, activated through signal transduction pathways and later repressed by negative regulations. In this work, we study the nonlinear dynamics of the mean transcription level of inducible genes modulated by the interplay of the intrinsic transcriptional randomness and the repression by negative regulations. In our model, we integrate negative regulations into gene activation process, and make the conventional assumption on the production and degradation of transcripts. We show that, whether or not the basal transcription is temporarily terminated when cells are stimulated, the mean transcription level grows in the typical up and down pattern commonly observed in immune response genes. With the help of numerical simulations, we clarify the delicate impact of the system parameters on the transcription dynamics, and demonstrate how our model generates the distinct temporal gene-induction patterns in mouse fibroblasts discerned in recent experiments.

  17. Roles of factorial noise in inducing bimodal gene expression

    Science.gov (United States)

    Liu, Peijiang; Yuan, Zhanjiang; Huang, Lifang; Zhou, Tianshou

    2015-06-01

    Some gene regulatory systems can exhibit bimodal distributions of mRNA or protein although the deterministic counterparts are monostable. This noise-induced bimodality is an interesting phenomenon and has important biological implications, but it is unclear how different sources of expression noise (each source creates so-called factorial noise that is defined as a component of the total noise) contribute separately to this stochastic bimodality. Here we consider a minimal model of gene regulation, which is monostable in the deterministic case. Although simple, this system contains factorial noise of two main kinds: promoter noise due to switching between gene states and transcriptional (or translational) noise due to synthesis and degradation of mRNA (or protein). To better trace the roles of factorial noise in inducing bimodality, we also analyze two limit models, continuous and adiabatic approximations, apart from the exact model. We show that in the case of slow gene switching, the continuous model where only promoter noise is considered can exhibit bimodality; in the case of fast switching, the adiabatic model where only transcriptional or translational noise is considered can also exhibit bimodality but the exact model cannot; and in other cases, both promoter noise and transcriptional or translational noise can cooperatively induce bimodality. Since slow gene switching and large protein copy numbers are characteristics of eukaryotic cells, whereas fast gene switching and small protein copy numbers are characteristics of prokaryotic cells, we infer that eukaryotic stochastic bimodality is induced mainly by promoter noise, whereas prokaryotic stochastic bimodality is induced primarily by transcriptional or translational noise.

  18. Perfluorooctane sulfonate induces neuronal and oligodendrocytic differentiation in neural stem cells and alters the expression of PPARγ in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Wan Ibrahim, Wan Norhamidah, E-mail: hamidah@science.upm.edu.my [Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm (Sweden); Tofighi, Roshan, E-mail: Roshan.Tofighi@ki.se [Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm (Sweden); Onishchenko, Natalia, E-mail: Natalia.Onishchenko@ki.se [Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm (Sweden); Rebellato, Paola, E-mail: Paola.Rebellato@ki.se [Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm (Sweden); Bose, Raj, E-mail: Raj.Bose@ki.se [Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm (Sweden); Uhlén, Per, E-mail: Per.Uhlen@ki.se [Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm (Sweden); Ceccatelli, Sandra, E-mail: Sandra.Ceccatelli@ki.se [Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm (Sweden)

    2013-05-15

    Perfluorinated compounds are ubiquitous chemicals of major concern for their potential adverse effects on the human population. We have used primary rat embryonic neural stem cells (NSCs) to study the effects of perfluorooctane sulfonate (PFOS) on the process of NSC spontaneous differentiation. Upon removal of basic fibroblast growth factor, NSCs were exposed to nanomolar concentrations of PFOS for 48 h, and then allowed to differentiate for additional 5 days. Exposure to 25 or 50 nM concentration resulted in a lower number of proliferating cells and a higher number of neurite-bearing TuJ1-positive cells, indicating an increase in neuronal differentiation. Exposure to 50 nM also significantly increased the number of CNPase-positive cells, pointing to facilitation of oligodendrocytic differentiation. PPAR genes have been shown to be involved in PFOS toxicity. By q-PCR we detected an upregulation of PPARγ with no changes in PPARα or PPARδ genes. One of the downstream targets of PPARs, the mitochondrial uncoupling protein 2 (UCP2) was also upregulated. The number of TuJ1- and CNPase-positive cells increased after exposure to PPARγ agonist rosiglitazone (RGZ, 3 μM) and decreased after pre-incubation with the PPARγ antagonist GW9662 (5 μM). RGZ also upregulated the expression of PPARγ and UCP2 genes. Meanwhile GW9662 abolished the UCP2 upregulation and decreased Ca{sup 2+} activity induced by PFOS. Interestingly, a significantly higher expression of PPARγ and UCP3 genes was also detected in mouse neonatal brain after prenatal exposure to PFOS. These data suggest that PPARγ plays a role in the alteration of spontaneous differentiation of NSCs induced by nanomolar concentrations of PFOS. - Highlights: • PFOS decreases proliferation of neural stem cells (NSCs). • PFOS induces neuronal and oligodendrocytic differentiation in NSCs. • PFOS alters expression of PPARγ and UCP2 in vitro. • PFOS alters expression of PPARγ and UCP3 in vivo. • Block of PPAR

  19. A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts

    Indian Academy of Sciences (India)

    Ramkumar Sambasivan; Grace K Pavlath; Jyotsna Dhawan

    2008-03-01

    Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent genetrap lines revealed arrest-dependent induction of gal activity, confirming the efficacy of the FACS screen. The locus of integration was identified in 15 lines. In three lines, insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY – a BRCA2-interacting protein, p8/com1– a p300HAT-binding protein and MLL5 – a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.

  20. Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls.

    Science.gov (United States)

    Yoshioka, R; Soga, K; Wakabayashi, K; Takeba, G; Hoson, T

    2003-01-01

    Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the alpha-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.

  1. Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

    Energy Technology Data Exchange (ETDEWEB)

    Huang Qihong; Jin Xidong; Gaillard, Elias T.; Knight, Brian L.; Pack, Franklin D.; Stoltz, James H.; Jayadev, Supriya; Blanchard, Kerry T

    2004-05-18

    Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1 mg/kg per day for 1, 7 and 14 days), methapyrilene (100 mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and

  2. Differential Gene Expression in Chemically Induced Mouse Lung Adenomas

    Directory of Open Access Journals (Sweden)

    Ruisheng Yao

    2003-01-01

    Full Text Available Because of similarities in histopathology and tumor progression stages between mouse and human lung adenocarcinomas, the mouse lung tumor model with lung adenomas as the endpoint has been used extensively to evaluate the efficacy of putative lung cancer chemopreventive agents. In this study, a competitive cDNA library screening (CCLS was employed to determine changes in the expression of mRNA in chemically induced lung adenomas compared with paired normal lung tissues. A total of 2555 clones having altered expression in tumors were observed following competitive hybridization between normal lung and lung adenomas after primary screening of over 160,000 clones from a mouse lung cDNA library. Among the 755 clones confirmed by dot blot hybridization, 240 clones were underexpressed, whereas 515 clones were overexpressed in tumors. Sixty-five clones with the most frequently altered expression in six individual tumors were confirmed by semiquantitative RT-PCR. When examining the 58 known genes, 39 clones had increased expression and 19 had decreased expression, whereas the 7 novel genes showed overexpression. A high percentage (>60% of overexpressed or underexpressed genes was observed in at least two or three of the lesions. Reproducibly overexpressed genes included ERK-1, JAK-1, surfactant proteins A, B, and C, NFAT1, α-1 protease inhibitor, helix-loop-helix ubiquitous kinase (CHUK, α-adaptin, α-1 PI2, thioether S-methyltransferase, and CYP2C40. Reproducibly underexpressed genes included paroxanase, ALDH II, CC10, von Ebner salivary gland protein, and α- and β-globin. In addition, CCLS identified several novel genes or genes not previously associated with lung carcinogenesis, including a hypothetical protein (FLJ11240 and a guanine nucleotide exchange factor homologue. This study shows the efficacy of this methodology for identifying genes with altered expression. These genes may prove to be helpful in our understanding of the genetic basis of

  3. Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing.

    Science.gov (United States)

    Nagamatsu, Atsushi; Masuta, Chikara; Senda, Mineo; Matsuura, Hideyuki; Kasai, Atsushi; Hong, Jin-Sung; Kitamura, Keisuke; Abe, Jun; Kanazawa, Akira

    2007-11-01

    Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase (CHS) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase (F3'H) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.

  4. Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley Stripe Mosaic Virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes afte...

  5. INDUCIBLE RNAi-MEDIATED GENE SILENCING USING NANOSTRUCTURED GENE DELIVERY ARRAYS

    Energy Technology Data Exchange (ETDEWEB)

    Mann, David George James [ORNL; McKnight, Timothy E [ORNL; Mcpherson, Jackson [University of Tennessee, Knoxville (UTK); Hoyt, Peter R [ORNL; Melechko, Anatoli Vasilievich [ORNL; Simpson, Michael L [ORNL; Sayler, Gary Steven [ORNL

    2008-01-01

    RNA interference has become a powerful biological tool over the last decade. In this study, a tetracycline-inducible shRNA vector system was designed for silencing CFP expression and delivered alongside the yfp marker gene into Chinese hamster ovary cells using impalefection on spatially indexed vertically aligned carbon nanofiber arrays (VACNFs). The VACNF architecture provided simultaneous delivery of multiple genes, subsequent adherence and proliferation of interfaced cells, and repeated monitoring of single cells over time. Following impalefection and tetracycline induction, 53.1% 10.4% of impalefected cells were fully silenced by the inducible CFP-silencing shRNA vector. Additionally, efficient CFP-silencing was observed in single cells among a population of cells that remained CFP-expressing. This effective transient expression system enables rapid analysis of gene silencing effects using RNAi in single cells and cell populations.

  6. The dilp2/5 genes control diapause inducibility

    OpenAIRE

    Schiesari, Luca

    2015-01-01

    Many holometabolous insects hibernate by triggering diapause, an “actively-induced” dormancy that blocks developmental functions. Yet, the nature of signals enhancing the plasticity of developmental system and underlying diapause inducibility is still elusive. We show that the “Insulin/IGF” dilp2/5 genes, encoding for developmental hormones, antagonize diapause switch in D. melanogaster and their modulation is pivotal in sensitizing the developmental system to environmental perturbations. Fun...

  7. Screening Helicobacter pylori genes induced during infection of mouse stomachs

    Institute of Scientific and Technical Information of China (English)

    Aparna Singh; Nathaniel Hodgson; Ming Yan; Jungsoo Joo; Lei Gu; Hong Sang; Emmalena Gregory-Bryson

    2012-01-01

    AIM:To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H.pylori) as it relates to its survival in the host.METHODS:In vivo expression technology (IVET) systems are used to identify microbial virulence genes.We modified the IVET-transcriptional fusion vector,pIVET8,which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors,pIVET11 and pIVET12.Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H.pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase.Additionally,each vector contains a kanamycin resistance gene.We used a mouse macrophage cell line,RAW 264.7 and mice,as selective media to identify specific genes that H.pylori expresses in vivo.Gene expression studies were conducted by infecting RAW 264.7 cells with H.pylori.This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes.RESULTS:In this study,we have identified 31 in vivo induced (ivi) genes in the initial screens.These 31 genes belong to several functional gene families,including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs.Virulence factors,vacA and cagA,were found in this screen and are known to play important roles in H.pylori infection,colonization and pathogenesis.Their detection validates the efficacy of these screening systems.Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H.pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae.Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H.pylori RNA isolated from H.pylori infected RAW 264.7 macrophages.We compared the expression profile of H.pylori and RAW 264.7 coculture with that of H.pylori only

  8. Cold acclimation induced genes of trifoliate orange (Poncirus trifoliata).

    Science.gov (United States)

    Zhang, Can-kui; Lang, Ping; Dane, Fenny; Ebel, Robert C; Singh, Narendra K; Locy, Robert D; Dozier, William A

    2005-03-01

    Commercial citrus varieties are sensitive to low temperature. Poncirus trifoliata is a close relative of Citrus species and has been widely used as a cold-hardy rootstock for citrus production in low-temperature environments. mRNA differential display-reverse transcription (DDRT)-PCR and quantitative relative-RT-PCR were used to study gene expression of P. trifoliata under a gradual cold-acclimation temperature regime. Eight up-regulated cDNA fragments were isolated and sequenced. These fragments showed high similarities at the amino acid level to the following genes with known functions: betaine/proline transporter, water channel protein, aldo-keto reductase, early light-induced protein, nitrate transporter, tetratricopeptide-repeat protein, F-box protein, and ribosomal protein L15. These cold-acclimation up-regulated genes in P. trifoliata are also regulated by osmotic and photo-oxidative signals in other plants.

  9. Virus-induced Gene Silencing in Eggplant (Solanum melongena)

    Institute of Scientific and Technical Information of China (English)

    HaipingLiu; Daqi Fu; Benzhong Zhu; Huaxue Yan; Xiaoying Shen; Jinhua Zuo; Yi Zhu; Yunbo Luo

    2012-01-01

    Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions.The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays.Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses.In this paper,TRV-mediated VIGS was successfully elicited in eggplant.We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene.Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation,indicating that VIGS can be used to silence genes in eggplant.To further illustrate the reliability of VIGS in eggplant,we selected Chl H,Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method.Suppression of Chl H and Su led to yellow leaves,while the depletion of CLA1 resulted in albino.In conclusion,four genes,PDS,Chl H,Su (Sulfur),CLA1,were down-regulated significantly by VIGS,indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

  10. Octylphenol induced gene expression in testes of Frog, Rana chensinensis.

    Science.gov (United States)

    Li, Xinyi; Liu, Jia; Zhang, Yuhui

    2016-06-01

    Octylphenol (OP) is an endocrine-disrupting chemical (EDC), which can disrupt the reproductive system. To understand the effect of OP, a subtractive cDNA library was constructed using suppression subtractive hybridization (SSH) to identify alterations of gene transcription in the testes of the frog Rana chensinensis after OP exposure. Two hundred positive clones were selected and 134 sequences of gene fragments were produced from the subtractive library randomly. These genes were identified to be involved in metabolic process, cellular process, biological regulation, stimulus, immune system and female pregnancy process. In order to verify the efficiency of the subtractive cDNA library, PSG9 and PAPP-A were analyzed further as two representatives of differentially expressed transcription genes using semi-quantitative RT-PCR. Our result was the first successful construction of the subtractive cDNA library in frog testes after OP treatment. Based on this cDNA library, OP was shown to affect multiple physiological processes including inducing immune response, disrupting the steroid hormone synthesis and influencing spermatogenesis in the testis by up-regulation of specific genes.

  11. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    Directory of Open Access Journals (Sweden)

    Lan-Huan Meng

    Full Text Available Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS and Chlorophyll H subunit (ChlH of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

  12. Efficient Virus-Induced Gene Silencing in Solanum rostratum

    Science.gov (United States)

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a “super weed” that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum. PMID:27258320

  13. Efficient Virus-Induced Gene Silencing in Solanum rostratum.

    Science.gov (United States)

    Meng, Lan-Huan; Wang, Rui-Heng; Zhu, Ben-Zhong; Zhu, Hong-Liang; Luo, Yun-Bo; Fu, Da-Qi

    2016-01-01

    Solanum rostratum is a "super weed" that grows fast, is widespread, and produces the toxin solanine, which is harmful to both humans and other animals. To our knowledge, no study has focused on its molecular biology owing to the lack of available transgenic methods and sequence information for S. rostratum. Virus-induced gene silencing (VIGS) is a powerful tool for the study of gene function in plants; therefore, in the present study, we aimed to establish tobacco rattle virus (TRV)-derived VIGS in S. rostratum. The genes for phytoene desaturase (PDS) and Chlorophyll H subunit (ChlH) of magnesium protoporphyrin chelatase were cloned from S. rostratum and used as reporters of gene silencing. It was shown that high-efficiency VIGS can be achieved in the leaves, flowers, and fruit of S. rostratum. Moreover, based on our comparison of three different types of infection methods, true leaf infection was found to be more efficient than cotyledon and sprout infiltration in long-term VIGS in multiple plant organs. In conclusion, the VIGS technology and tomato genomic sequences can be used in the future to study gene function in S. rostratum.

  14. Bitumen fume-induced gene expression profile in rat lung.

    Science.gov (United States)

    Gate, Laurent; Langlais, Cristina; Micillino, Jean-Claude; Nunge, Hervé; Bottin, Marie-Claire; Wrobel, Richard; Binet, Stéphane

    2006-08-15

    Exposure to bitumen fumes during paving and roofing activities may represent an occupational health risk. To date, most of the studies performed on the biological effect of asphalt fumes have been done with regard to their content in carcinogenic polycyclic aromatic hydrocarbons (PAH). In order to gain an additional insight into the mechanisms of action of bitumen fumes, we studied their pulmonary effects in rodents following inhalation using the microarray technology. Fisher 344 rats were exposed for 5 days, 6 h/day to bitumen fumes generated at road paving temperature (170 degrees C) using a nose-only exposition device. With the intention of studying the early transcriptional events induced by asphalt fumes, lung tissues were collected immediately following exposure and gene expression profiles in control and exposed rats were determined by using oligonucleotide microarrays. Data analysis revealed that genes involved in lung inflammatory response as well as genes associated with PAH metabolization and detoxification were highly expressed in bitumen-exposed animals. In addition, the expression of genes related to elastase activity and its inhibition which are associated with emphysema was also modulated. More interestingly genes coding for monoamine oxidases A and B involved in the metabolism of neurotransmitters and xenobiotics were downregulated in exposed rats. Altogether, these data give additional information concerning the bitumen fumes biological effects and would allow to better review the health effects of occupational asphalt fumes exposure.

  15. The relationship of host-mediated induced resistance to polymorphism in gene-for-gene relationships.

    Science.gov (United States)

    Tellier, Aurélien; Brown, James K M

    2008-01-01

    Gene-for-gene relationships are a common feature of plant-parasite interactions. Polymorphism at host resistance and parasite avirulence loci is maintained if there is negative, direct frequency-dependent selection on alleles of either gene. More specifically, selection of this kind is generated when the disease is polycyclic with frequent auto-infection. When an incompatible interaction occurs between a resistant host and an avirulent parasite, systemic defenses are triggered, rendering the plant more resistant to a later attack by another parasite. However, induced resistance (IR) incurs a fitness cost to the plant. Here, the effect of IR on polymorphism in gene-for-gene interactions is investigated. First, in an infinite population model in which parasites have two generations per host generation, increasing the fitness cost of IR increases selection for susceptible plants at low disease severity, while increasing the effectiveness of IR against further parasite attacks enhances selection for resistant plants at high disease severity. This reduces the possibility of polymorphism being maintained in host and parasite populations. In finite population models, the number of plants varies over time as a function of the disease burden of the population. Polymorphism in gene-for-gene relationships is then more stable at high disease prevalence and severity if IR reactions are more costly when there is competition for resources between plants.

  16. Inducible gene manipulations in brain serotonergic neurons of transgenic rats.

    Directory of Open Access Journals (Sweden)

    Tillmann Weber

    Full Text Available The serotonergic (5-HT system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP, in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system.

  17. Anti-Obesity Effects of Aster spathulifolius Extract in High-Fat Diet-Induced Obese Rats.

    Science.gov (United States)

    Kim, Sa-Jic; Bang, Chae-Young; Guo, Yuan-Ri; Choung, Se-Young

    2016-04-01

    The aim of this study was to investigate the anti-obesity and antihyperlipidemic efficacy and molecular mechanisms of Aster spathulifolius Maxim extract (ASE) in rats with high-fat diet (HFD)-induced obesity. Rats were separately fed a normal diet or a HFD for 8 weeks, then they were treated with ASE (62.5, 125, or 250 mg/kg) for another 4.5 weeks. The ASE supplementation significantly lowered body weight gain, visceral fat pad weights, serum lipid levels, as well as hepatic lipid levels in HFD-induced obese rats. Histological analysis showed that the ASE-treated group showed lowered numbers of lipid droplets and smaller size of adipocytes compared to the HFD group. To understand the mechanism of action of ASE, the expression of genes and proteins involved in obesity were measured in liver and skeletal muscle. The expression of fatty acid oxidation and thermogenesis-related genes (e.g., PPAR-α, ACO, CPT1, UCP2, and UCP3) of HFD-induced obese rats were increased by ASE treatment. On the other hand, ASE treatment resulted in decreased expression of fat intake-related gene ACC2 and lipogenesis-related genes (e.g., SREBP-1c, ACC1, FAS, SCD1, GPATR, AGPAT, and DGAT). Furthermore, ASE treatment increased the level of phosphorylated AMPKα in obese rats. Similarly, the level of phosphorylated ACC, a target protein of AMPKα in ASE groups, was increased by ASE treatment compared with the HFD group. These results suggest that ASE attenuated visceral fat accumulation and improved hyperlipidemia in HFD-induced obese rats by increasing lipid metabolism through the regulation of AMPK activity and the expression of genes and proteins involved in lipolysis and lipogenesis.

  18. Putting the diet back into diet-induced obesity: diet-induced hypothalamic gene expression.

    Science.gov (United States)

    Mercer, Julian G; Archer, Zoë A

    2008-05-06

    A wealth of detailed mechanistic information relating to obesity and body weight regulation has emerged from study of single gene mutation models, and continues to be generated by engineered rodent models targeting specific genes. However, as an early step in translational research, many researchers are turning to models of diet-induced obesity. Interpretation of data generated from such models is not aided by the variety of diets and rodent strains employed in these studies and a strong case could be made for rationalisation. Differences in experimental protocol, which may deploy a single obligatory solid diet, a choice of solid diets, or liquid/solid combinations, and which may or may not allow a preferred macronutrient composition to be selected, mean that different models of diet-induced obesity achieve that obesity by different routes. The priority should be to mimic the palatability- and choice-driven over-consumption that probably underlies the majority of human obesity. Some of the hypothalamic energy balance genes apparently 'recognise' developing diet-induced obesity as indicated by counter-regulatory changes in expression levels. However, substantial changes in gene expression on long-term exposure to obesogenic diets are not able to prevent weight gain. Forebrain reward systems are widely assumed to be overriding hypothalamic homeostatic energy balance systems under these circumstances. More mechanism-based research at the homeostatic/reward/diet interface may enable diets to be manipulated with therapeutic benefit, or define the contribution of these interactions to susceptibility to diet-induced obesity.

  19. Tomato leaf spatial expression of stress-induced Asr genes.

    Science.gov (United States)

    Maskin, Laura; Maldonado, Sara; Iusem, Norberto D

    2008-12-01

    Asr1 and Asr2 are water stress-inducible genes belonging to the Asr gene family, which transcriptionally regulate a sugar transporter gene, at least in grape. Using an in situ RNA hybridization methodology, we determined that, in basal conditions, expression of Asr2 in tomato leaves is detected in the phloem tissue, particularly in companion phloem cells. When plants are exposed to water stress, Asr2 expression is contained in companion cells but expands occasionally to mesophyll cells. In contrast, Asr1 transcript localization seems to be sparse in leaf vascular tissue under both non-stress and stress conditions. The occurrence of Asr transcripts precisely in companion cells is in accordance with the cell type specificity reported for hexose-transporter protein molecules in grape encoded by the only Asr-target gene known to date. The results are discussed in light of the reported scarcity of plasmodesmata between companion cells and the rest of leaf tissue in the family Solanaceae.

  20. Virus-induced gene silencing and transient gene expression in soybean using Bean pod mottle virus infectious clones

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200-300 base pair size range. The recombinant viruses are used to ...

  1. Inducible gene expression and environmentally regulated genes in lactic acid bacteria.

    Science.gov (United States)

    Kok, J

    1996-10-01

    Relatively recently, a number of genes and operons have been identified in lactic acid bacteria that are inducible and respond to environmental factors. Some of these genes/operons had been isolated and analysed because of their importance in the fermentation industry and, consequently, their transcription was studied and found to be regulatable. Examples are the lactose operon, the operon for nisin production, and genes in the proteolytic pathway of Lactococcus lactis, as well as xylose metabolism in Lactobacillus pentosus. Some other operons were specifically targetted with the aim to compare their mode of regulation with known regulatory mechanisms in other well-studied bacteria. These studies, dealing with the biosynthesis of histidine, tryptophan, and of the branched chain amino acids in L. lactis, have given new insights in gene regulation and in the occurrence of auxotrophy in these bacteria. Also, nucleotide sequence analyses of a number of lactococcal bacteriophages was recently initiated to, among other things, specifically learn more about regulation of the phage life cycle. Yet another approach in the analysis of regulated genes is the 'random' selection of genetic elements that respond to environmental stimuli and the first of such sequences from lactic acid bacteria have been identified and characterized. The potential of these regulatory elements in fundamental research and practical (industrial) applications will be discussed.

  2. Virus-induced gene silencing in detached tomatoes and biochemical effects of phytoene desaturase gene silencing.

    Science.gov (United States)

    Romero, Irene; Tikunov, Yury; Bovy, Arnaud

    2011-07-01

    Virus-induced gene silencing (VIGS) is a technology that has rapidly emerged for gene function studies in plants. Many advances have been made in applying this technique in an increasing number of crops. Recently, VIGS has been successfully used to silence genes in tomato fruit through agroinfiltration of fruit attached to the plant. The phytoene desaturase (Pds) gene has been widely used as a reporter gene in VIGS experiments, although little is known about the changes that occur due to its silencing in plants. In this paper, we describe the efficient silencing of the Pds gene through the VIGS approach in detached tomato fruits, which makes the VIGS procedure even more versatile and applicable. After 16 days of agroinfiltration, approximately 75% of the tomatoes showed Pds silencing symptoms, although the distribution of silenced areas was variable among fruits. To study the potential effects caused by Pds silencing in detached tomatoes, carotenoids and other semi-polar secondary metabolites were analyzed using Liquid Chromatography-Mass Spectrometry. In addition, potential differences in primary metabolites were analyzed using Gas Chromatography-Mass Spectrometry. The results indicated that the yellow phenotype observed in Pds-silenced fruit was mainly due to the lack of the red-colored lycopene and therefore to a more pronounced contribution of the yellow-orange carotenoids (lutein, violaxanthin, and zeaxanthin) to the final color of the fruits. Furthermore, the biochemical changes observed in Pds-silenced detached tomatoes suggested that carotenoid and other pathways, e.g. leading to alkaloids and flavonoids, might be affected by the silencing of this reporter gene, and this should be taken into consideration for future experimental designs.

  3. Virus-induced silencing of a tobacco deoxyhypusine synthase gene

    Institute of Scientific and Technical Information of China (English)

    WANG Hongzhi; MA Rongcai; LI Ruifen; WANG Guoying; WEI Jianhua

    2005-01-01

    A cDNA fragment corresponding to deoxyhypusine synthase gene NbDHS was isolated and cloned into potato virus X (PVX) vector for functional analysis in Nicotiana benthamiana by using virus-induced gene silencing (VIGS). Plants agroinfected with recombinant virus vector PVX-NbDHS exhibited an increase in leaf biomass, delay in natural leaf senescence and flowering time, and decrease in leaf chlorophyll content. Semi-quantitative RT-PCR and Northern analysis showed that the transcript level of DHS was significantly lower in PVX-NbDHS infected plants. At the same time, the expression for eIF-5A, the target proteins of DHS in N. benthamiana, was concomitantly suppressed by semi-quantitative RT-PCR and Western analysis. From the phenotypic feature of the infected plants and the reduced expression abundance of DHS and eIF-5A, we concluded that NbDHS plays important roles in plant growth, development and senescence. The possible application of DHS gene in genetic modification of crops and horticultural plants was discussed.

  4. L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes

    Science.gov (United States)

    Lobel, Lior; Burg-Golani, Tamar; Sigal, Nadejda; Rose, Jessica; Livnat-Levanon, Nurit; Lewinson, Oded; Herskovits, Anat A.

    2017-01-01

    The high environmental adaptability of bacteria is contingent upon their ability to sense changes in their surroundings. Bacterial pathogen entry into host poses an abrupt and dramatic environmental change, during which successful pathogens gauge multiple parameters that signal host localization. The facultative human pathogen Listeria monocytogenes flourishes in soil, water and food, and in ~50 different animals, and serves as a model for intracellular infection. L. monocytogenes identifies host entry by sensing both physical (e.g., temperature) and chemical (e.g., metabolite concentrations) factors. We report here that L-glutamine, an abundant nitrogen source in host serum and cells, serves as an environmental indicator and inducer of virulence gene expression. In contrast, ammonia, which is the most abundant nitrogen source in soil and water, fully supports growth, but fails to activate virulence gene transcription. We demonstrate that induction of virulence genes only occurs when the Listerial intracellular concentration of L-glutamine crosses a certain threshold, acting as an on/off switch: off when L-glutamine concentrations are below the threshold, and fully on when the threshold is crossed. To turn on the switch, L-glutamine must be present, and the L-glutamine high affinity ABC transporter, GlnPQ, must be active. Inactivation of GlnPQ led to complete arrest of L-glutamine uptake, reduced type I interferon response in infected macrophages, dramatic reduction in expression of virulence genes, and attenuated virulence in a mouse infection model. These results may explain observations made with other pathogens correlating nitrogen metabolism and virulence, and suggest that gauging of L-glutamine as a means of ascertaining host localization may be a general mechanism. PMID:28114430

  5. Activation of pregnane X receptor by pregnenolone 16 α-carbonitrile prevents high-fat diet-induced obesity in AKR/J mice.

    Science.gov (United States)

    Ma, Yongjie; Liu, Dexi

    2012-01-01

    Pregnane X receptor (PXR) is known to function as a xenobiotic sensor to regulate xenobiotic metabolism through selective transcription of genes responsible for maintaining physiological homeostasis. Here we report that the activation of PXR by pregnenolone 16α-carbonitrile (PCN) in AKR/J mice can prevent the development of high-fat diet-induced obesity and insulin resistance. The beneficial effects of PCN treatment are seen with reduced lipogenesis and gluconeogenesis in the liver, and lack of hepatic accumulation of lipid and lipid storage in the adipose tissues. RT-PCR analysis of genes involved in gluconeogenesis, lipid metabolism and energy homeostasis reveal that PCN treatment on high-fat diet-fed mice reduces expression in the liver of G6Pase, Pepck, Cyp7a1, Cd36, L-Fabp, Srebp, and Fas genes and slightly enhances expression of Cyp27a1 and Abca1 genes. RT-PCR analysis of genes involved in adipocyte differentiation and lipid metabolism in white adipose tissue show that PCN treatment reduces expression of Pparγ2, Acc1, Cd36, but increases expression of Cpt1b and Pparα genes in mice fed with high-fat diet. Similarly, PCN treatment of animals on high-fat diet increases expression in brown adipose tissue of Pparα, Hsl, Cpt1b, and Cd36 genes, but reduces expression of Acc1 and Scd-1 genes. PXR activation by PCN in high-fat diet fed mice also increases expression of genes involved in thermogenesis in brown adipose tissue including Dio2, Pgc-1α, Pgc-1β, Cidea, and Ucp-3. These results verify the important function of PXR in lipid and energy metabolism and suggest that PXR represents a novel therapeutic target for prevention and treatment of obesity and insulin resistance.

  6. Activation of pregnane X receptor by pregnenolone 16 α-carbonitrile prevents high-fat diet-induced obesity in AKR/J mice.

    Directory of Open Access Journals (Sweden)

    Yongjie Ma

    Full Text Available Pregnane X receptor (PXR is known to function as a xenobiotic sensor to regulate xenobiotic metabolism through selective transcription of genes responsible for maintaining physiological homeostasis. Here we report that the activation of PXR by pregnenolone 16α-carbonitrile (PCN in AKR/J mice can prevent the development of high-fat diet-induced obesity and insulin resistance. The beneficial effects of PCN treatment are seen with reduced lipogenesis and gluconeogenesis in the liver, and lack of hepatic accumulation of lipid and lipid storage in the adipose tissues. RT-PCR analysis of genes involved in gluconeogenesis, lipid metabolism and energy homeostasis reveal that PCN treatment on high-fat diet-fed mice reduces expression in the liver of G6Pase, Pepck, Cyp7a1, Cd36, L-Fabp, Srebp, and Fas genes and slightly enhances expression of Cyp27a1 and Abca1 genes. RT-PCR analysis of genes involved in adipocyte differentiation and lipid metabolism in white adipose tissue show that PCN treatment reduces expression of Pparγ2, Acc1, Cd36, but increases expression of Cpt1b and Pparα genes in mice fed with high-fat diet. Similarly, PCN treatment of animals on high-fat diet increases expression in brown adipose tissue of Pparα, Hsl, Cpt1b, and Cd36 genes, but reduces expression of Acc1 and Scd-1 genes. PXR activation by PCN in high-fat diet fed mice also increases expression of genes involved in thermogenesis in brown adipose tissue including Dio2, Pgc-1α, Pgc-1β, Cidea, and Ucp-3. These results verify the important function of PXR in lipid and energy metabolism and suggest that PXR represents a novel therapeutic target for prevention and treatment of obesity and insulin resistance.

  7. Saponins from stems and leaves of Panax ginseng prevent obesity via regulating thermogenesis, lipogenesis and lipolysis in high-fat diet-induced obese C57BL/6 mice.

    Science.gov (United States)

    Chen, Guilin; Li, Haijun; Zhao, Yan; Zhu, Hongyan; Cai, Enbo; Gao, Yugang; Liu, Shuangli; Yang, He; Zhang, Lianxue

    2017-08-01

    In this study, high-fat diet (HFD)-induced obesity in mouse model was used to evaluate the dietary effect of saponins from stems and leaves of Panax ginseng (SLG), and to explore its mechanism of action in producing anti-obesity effects. The results indicate that SLG showed significant anti-obesity effects in diet-induced obese mice, represented by decreased serum levels of free fatty acids (FFA), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL)-cholesterol, glucose, leptin and insulin, as well as a reduction in overall body and liver weight, epididymal adipose tissue weight, and food efficiency, and inhibition of abnormal increases in acyl carnitine levels normally caused by an HFD. Additionally, the down-regulated expression of PPARγ, FAS, CD36, FATP2 and up-regulated expression of CPT-1, UCP-2, PPARα, HSL, and ATGL in liver tissue was induced by SLG. In addition, the SLG groups showed decreased PPARγ, aP2 and leptin mRNA levels and increased expression of PPARα, PGC-1α, UCP-1 and UCP-3 genes in adipose tissues, compared with the HFD group. In short, SLG may play a key role in producing anti-obesity effects in mice fed an HFD, and its mechanism may be related to regulation of thermogenesis, lipogenesis and lipolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Relationship between type 2 diabetes mellitus complicated by periodontal infection and uncoupling protein 3 gene%2型糖尿病并发牙周感染与解偶联蛋白3基因的关系研究

    Institute of Scientific and Technical Information of China (English)

    周逸丹; 张萍萍; 黄爱华

    2011-01-01

    目的 探讨2型糖尿病并发牙周感染与解偶联蛋白3(UCP3)基因的关系.方法 将80例2型糖尿病患者根据是否并发牙周感染分为两组,用聚合酶链反应(pCR)法对两组UCP3基因多态性进行分析并比较.结果 并发感染组II、ID、DD的基因频率分别为39.0%、51.2%、9.8%,与非并发感染组比较,差异无统计学意义;等位基因频率两组间亦差异无统计学意义.结论 2型糖尿病并发牙周感染与解偶联蛋白3基因之间未见明显的关系.%OBJECTIVE To explore the relationship between type 2 diabetes complicated by periodontal infection and uncoupling protein 3 (UCP3) gene.METHODS 80 patients with type 2 diabetes were divided into two groups according to whether complicated by periodontal infection.PCR method was used to analyze the polymorphism of UCP3 gene and the results were compared.RESULTS The gene frequencie of Ⅱ, ID, DD of the infection group was 39.0 %, 51.2 %, 9.8 %, respectively.There was no significant difference between the two groups(P>0.05).The allele frequency between the two groups had no significant difference (P>0.05).CONCLUSION It shows no significant relationship between type 2 diabetes complicated by periodontal infection and uncoupling protein 3 genes.

  9. Chemical memory reactions induced bursting dynamics in gene expression.

    Science.gov (United States)

    Tian, Tianhai

    2013-01-01

    Memory is a ubiquitous phenomenon in biological systems in which the present system state is not entirely determined by the current conditions but also depends on the time evolutionary path of the system. Specifically, many memorial phenomena are characterized by chemical memory reactions that may fire under particular system conditions. These conditional chemical reactions contradict to the extant stochastic approaches for modeling chemical kinetics and have increasingly posed significant challenges to mathematical modeling and computer simulation. To tackle the challenge, I proposed a novel theory consisting of the memory chemical master equations and memory stochastic simulation algorithm. A stochastic model for single-gene expression was proposed to illustrate the key function of memory reactions in inducing bursting dynamics of gene expression that has been observed in experiments recently. The importance of memory reactions has been further validated by the stochastic model of the p53-MDM2 core module. Simulations showed that memory reactions is a major mechanism for realizing both sustained oscillations of p53 protein numbers in single cells and damped oscillations over a population of cells. These successful applications of the memory modeling framework suggested that this innovative theory is an effective and powerful tool to study memory process and conditional chemical reactions in a wide range of complex biological systems.

  10. Fetal Globin Gene Inducers: Novel Agents & New Potential

    Science.gov (United States)

    Perrine, Susan P.; Castaneda, Serguei A.; Chui, David H.; Faller, Douglas V.; Berenson, Ronald J.; Fucharoen, Suthat

    2013-01-01

    Inducing expression of endogenous fetal globin (γ-globin) gene expression to 60-70% of alpha globin synthesis produces β-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced γ-globin expression in beta thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as 7 years. However, prior generations were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic sodium 2,2-dimethylbutyrate is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of 3 major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used world-wide. PMID:20712788

  11. Deoxynivalenol-Induced Proinflammatory Gene Expression: Mechanisms and Pathological Sequelae

    Directory of Open Access Journals (Sweden)

    James J. Pestka

    2010-06-01

    Full Text Available The trichothecene mycotoxin deoxynivalenol (DON is commonly encountered in human cereal foods throughout the world as a result of infestation of grains in the field and in storage by the fungus Fusarium. Significant questions remain regarding the risks posed to humans from acute and chronic DON ingestion, and how to manage these risks without imperiling access to nutritionally important food commodities. Modulation of the innate immune system appears particularly critical to DON’s toxic effects. Specifically, DON induces activation of mitogen-activated protein kinases (MAPKs in macrophages and monocytes, which mediate robust induction of proinflammatory gene expression—effects that can be recapitulated in intact animals. The initiating mechanisms for DON-induced ribotoxic stress response appear to involve the (1 activation of constitutive protein kinases on the damaged ribosome and (2 autophagy of the chaperone GRP78 with consequent activation of the ER stress response. Pathological sequelae resulting from chronic low dose exposure include anorexia, impaired weight gain, growth hormone dysregulation and aberrant IgA production whereas acute high dose exposure evokes gastroenteritis, emesis and a shock-like syndrome. Taken together, the capacity of DON to evoke ribotoxic stress in mononuclear phagocytes contributes significantly to its acute and chronic toxic effects in vivo. It is anticipated that these investigations will enable the identification of robust biomarkers of effect that will be applicable to epidemiological studies of the human health effects of this common mycotoxin.

  12. Host-Induced Gene Silencing of Rice Blast Fungus Magnaporthe oryzae Pathogenicity Genes Mediated by the Brome Mosaic Virus.

    Science.gov (United States)

    Zhu, Lin; Zhu, Jian; Liu, Zhixue; Wang, Zhengyi; Zhou, Cheng; Wang, Hong

    2017-09-26

    Magnaportheoryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M.oryzae-derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M.oryzae was examined by targeting three predicted pathogenicity genes, MoABC1,MoMAC1 and MoPMK1. Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M.oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi.

  13. BRD4 regulates fructose-inducible lipid accumulation-related genes in the mouse liver.

    Science.gov (United States)

    Yamada, Aki; Honma, Kazue; Mochizuki, Kazuki; Goda, Toshinao

    2016-10-01

    Fructose intake induces hepatic steatosis by activating fat synthesis. In this study, we searched for genes that showed acute induction in the livers of mice force-fed with fructose, and examined how this induction is regulated. We identified genes induced at 6h after the fructose force-feeding using a microarray and quantitative real-time RT-PCR. Histone acetylation and an acetylated histone binding protein bromodomain containing (BRD)4 binding around the fructose-inducible genes were examined using a chromatin immunoprecipitation assay. We examined whether (+)-JQ1, an inhibitor of the binding between the BRD4 and acetylated histones, inhibited the expressions of fructose-inducible genes, histone acetylation and BRD4 binding around the genes. We identified upregulated genes related to lipid accumulation, such as Cyp8b1, Dak and Plin5, in mice force-fed with fructose compared with those force-fed with glucose. Acetylation of histones H3 and H4, and BRD4 binding around the transcribed region of those fructose-inducible genes, were enhanced by fructose force-feeding. Meanwhile, (+)-JQ1 treatment reduced expressions of fructose-inducible genes, histone acetylation and BRD4 binding around these genes. Acute induction of genes related to lipid accumulation in the livers of mice force-fed with fructose is associated with the induction of histone acetylation and BRD4 binding around these genes. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene

    NARCIS (Netherlands)

    Kop, D.A.M. van der; Schuyer, M.; Pinas, J.E.; Zaal, B.J. van der; Hooykaas, P.J.J.

    1999-01-01

    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the β-glucuronidase (gusA) reporter gene. Subsequently, seeds were tr

  15. Identification of differentially expressed radiation-induced genes in cervix carcinoma cells using suppression subtractive hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun Sang; Lee, Young Sook; Lee, Jeung Hoon; Lee, Woong Hee; Seo, Eun Young; Cho, Moon June [Chungnam National University, Daejeon (Korea, Republic of)

    2005-03-15

    A number of genes and their products are induced early or late following exposure of cells to ionizing radiation. These radiation-induced genes have various effects of irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to identify the differentially expressed genes by radiation in cervix carcinoma cells. Total RNA and poly (A){sup +} mRNA were isolated from irradiated and non-irradiated HeLa cells. Forward-and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Of the 176 clones, 10 genes in the forward-subtracted library and 9 genes in the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. We identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

  16. Protocol: using virus-induced gene silencing to study the arbuscular mycorrhizal symbiosis in Pisum sativum

    DEFF Research Database (Denmark)

    Grønlund, Mette; Olsen, Anne; Johansen, Elisabeth

    2010-01-01

    Virus-induced gene silencing (VIGS) is an alternative reverse genetics tool for silencing of genes in some plants, which are difficult to transform. The pea early-browning virus (PEBV) has been developed as a VIGS vector and used in pea for functional analysis of several genes. However, the avail......Virus-induced gene silencing (VIGS) is an alternative reverse genetics tool for silencing of genes in some plants, which are difficult to transform. The pea early-browning virus (PEBV) has been developed as a VIGS vector and used in pea for functional analysis of several genes. However......, the available PEBV-VIGS protocols are inadequate for studying genes involved in the symbiosis with arbuscular mycorrhizal fungi (AMF). Here we describe a PEBV-VIGS protocol suitable for reverse genetics studies in pea of genes involved in the symbiosis with AMF and show its effectiveness in silencing genes...... involved in the early and late stages of AMF symbiosis....

  17. Activation-Induced Cytidine Deaminase Contributes to Pancreatic Tumorigenesis by Inducing Tumor-Related Gene Mutations.

    Science.gov (United States)

    Sawai, Yugo; Kodama, Yuzo; Shimizu, Takahiro; Ota, Yuji; Maruno, Takahisa; Eso, Yuji; Kurita, Akira; Shiokawa, Masahiro; Tsuji, Yoshihisa; Uza, Norimitsu; Matsumoto, Yuko; Masui, Toshihiko; Uemoto, Shinji; Marusawa, Hiroyuki; Chiba, Tsutomu

    2015-08-15

    Pancreatic ductal adenocarcinoma (PDAC) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDAC development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemical findings revealed AID protein expression in human acinar ductal metaplasia, pancreatic intraepithelial neoplasia, and PDAC. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDAC tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc, and Smad4 mutations, with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDAC.

  18. Cyclic AMP-inducible genes respond uniformly to seasonal lighting conditions in the rat pineal gland.

    Science.gov (United States)

    Spessert, R; Gupta, B B P; Rohleder, N; Gerhold, S; Engel, L

    2006-12-01

    The encoding of photoperiodic information ensues in terms of the daily profile in the expression of cyclic AMP (cAMP)-inducible genes such as the arylalkylamine N-acetyltransferase (AA-NAT) gene that encodes the rate-limiting enzyme in melatonin formation. In the present study, we compared the influence of the photoperiodic history on the cAMP-inducible genes AA-NAT, inducible cyclic AMP early repressor (ICER), fos-related antigen-2 (FRA-2), mitogen-activated protein kinase phosphatase-1 (MKP-1), nerve growth factor inducible gene-A (NGFI-A) and nerve growth factor inducible gene-B (NGFI-B) in the pineal gland of rats. For this purpose, we monitored the daily profiles of each gene in the same pineal gland under a long (light/dark 16:8) and a short (light/dark 8:16) photoperiod by measuring the respective mRNA amounts by real-time polymerase chain reaction analysis. We found that, for all genes under investigation, the duration of increased nocturnal expression is lengthened and, in relation to light onset, the nocturnal rise is earlier under the long photoperiod (light/dark 16:8). Furthermore, with the exception of ICER, all other cAMP-inducible genes tend to display higher maximum expression under light/dark 8:16 than under light/dark 16:8. Photoperiod-dependent changes persist for all of the cAMP-inducible genes when the rats are kept for two cycles under constant darkness. Therefore, all cAMP-inducible genes are also influenced by the photoperiod of prior entrained cycles. Our study indicates that, despite differences regarding the expressional control and the temporal phasing of the daily profile, cAMP-inducible genes are uniformly influenced by photoperiodic history in the rat pineal gland.

  19. Dexamethasone-Inducible Green Fluorescent Protein Gene Expression in Transgenic Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Wei Tang; Hilary Collver; Katherine Kinken

    2004-01-01

    Genomic research has made a large number of sequences of novel genes or expressed sequence tags available. To investigate functions of these genes, a system for conditional control of gene expression would be a useful tool. Inducible transgene expression that uses green fluorescent protein gene (gfp) as a reporter gene has been investigated in transgenic cell lines of cotton (COT; Gossypium hirsutum L.), Fraser fir [FRA; Abies fraseri (Pursh) Poir], Nordmann fir (NOR; Abies nordmanniana Lk.), and rice (RIC; Oryza sativa L. Cv. Radon). Transgenic cell lines were used to test the function of the chemical inducer dexamethasone. Inducible transgene expression was observed with fluorescence and confocal microscopy, and was confirmed by northern blot analyses. Dexamethasone at 5 mg/L induced gfp expression to the nearly highest level 48 h after treatment in COT, FRA, NOR, and RIC. Dexamethasone at 10 mg/L inhibited the growth of transgenic cells in FRA and NOR, but not COT and RIC. These results demonstrated that concentrations of inducer for optimum inducible gene expression system varied among transgenic cell lines. The inducible gene expression system described here was very effective and could be valuable in evaluating the function of novel gene.

  20. Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis.

    Directory of Open Access Journals (Sweden)

    Sara Tengvall

    Full Text Available Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA. Targeting the expression of the collagen type II (CII to antigen presenting cells (APCs induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1 increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases.

  1. Coordinate gene regulation by fimbriae-induced signal transduction

    DEFF Research Database (Denmark)

    Schembri, Mark; Klemm, Per

    2001-01-01

    whether fimbriae expression can affect expression of other genes, Analysis of gene expression in two E.coli strains, differing in the fim locus, indicated the flu gene to be affected. The flu gene encodes the antigen 43 (Ag43) surface protein, specifically involved in bacterial aggregation...... of Ag43 production. No effect was observed in an oxyR mutant. We conclude that fimbriae expression per se constitutes a signal transduction mechanism that affects a number of unrelated genes via the thiol-disulfide status of OxyR. Thus, phase variation in fimbrial expression is coordinated...

  2. Glomerulonephritis-induced changes in kidney gene expression in rats

    Directory of Open Access Journals (Sweden)

    Mira Pavkovic

    2015-12-01

    Full Text Available We investigated a glomerulonephritis (GN model in rats induced by nephrotoxic serum (NTS which contains antibodies against the glomerular basement membrane (GBM. The anti-GBM GN model in rats is widely used since its biochemical and histopathological characteristics are similar to crescentic nephritis and Goodpasture's disease in humans (Pusey, 2003 [2]. Male Wistar Kyoto (WKY and Sprague–Dawley (SD rats were dosed once with 1, 2.5 and 5 ml/kg nephrotoxic serum (NTS or 1.5 and 5 ml/kg NTS, respectively. GN and tubular damage were observed histopathologically in all treated rats after 14 days. To obtain insight into molecular processes during GN pathogenesis, mRNA expression was investigated in WKY and SD kidneys using Affymetrix's GeneChip Rat genome 230_2.0 arrays (GSE64265. The immunopathological processes during GN are still not fully understood and likely involve both innate and adaptive immunity. In the present study, several hundred mRNAs were found deregulated, which functionally were mostly associated with inflammation and regeneration. The β-chain of the major histocompatibility complex class II RT1.B (Rt1-Bb and complement component 6 (C6 were identified as two mRNAs differentially expressed between WKY and SD rat strains which could be related to known different susceptibilities to NTS of different rat strains; both were increased in WKY and decreased in SD rats (Pavkovic et al., 2015 [1]. Increased Rt1-Bb expression in WKY rats could indicate a stronger and more persistent cellular reaction of the adaptive immune system in this strain, in line with findings indicating adaptive immune reactions during GN. The complement cascade is also known to be essential for GN development, especially terminal cascade products like C6.

  3. Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene.

    Science.gov (United States)

    van der Kop, D A; Schuyer, M; Pinas, J E; van der Zaal, B J; Hooykaas, P J

    1999-03-01

    Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the beta-glucuronidase (gusA) reporter gene. Subsequently, seeds were treated with EMS to obtain mutants in which both reporter gene fusions were up-regulated. Northern analysis showed that the mRNA level of a related, endogenous auxin-inducible GST gene of Arabidopsis was increased in some of these mutants as well. Two of the gup (GST up-regulated) mutants were characterized in more detail and roughly mapped. Both had epinastic cotyledons and leaves, a phenotype that turned out to be linked to the gup mutation.

  4. RNA splicing regulates the temporal order of TNF-induced gene expression.

    Science.gov (United States)

    Hao, Shengli; Baltimore, David

    2013-07-16

    When cells are induced to express inflammatory genes by treatment with TNF, the mRNAs for the induced genes appear in three distinct waves, defining gene groups I, II, and III, or early, intermediate, and late genes. To examine the basis for these different kinetic classes, we have developed a PCR-based procedure to distinguish pre-mRNAs from mRNAs. It shows that the three groups initiate transcription virtually simultaneously but that delays in splicing characterize groups II and III. We also examined the elongation times, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production.

  5. Relationship between gene responses and symptoms induced by Rice grassy stunt virus

    Directory of Open Access Journals (Sweden)

    Kouji eSatoh

    2013-10-01

    Full Text Available Rice grassy stunt virus (RGSV is a serious threat to rice production in Southeast Asia. RGSV is a member of the genus Tenuivirus, and it induces leaf yellowing, stunting, and excess tillering on rice plants. Here we examined gene responses of rice to RGSV infection to gain insight into the gene responses which might be associated with the disease symptoms. The results indicated that 1 many genes related to cell wall synthesis and chlorophyll synthesis were predominantly suppressed by RGSV infection; 2 RGSV infection induced genes associated with tillering process; 3 RGSV activated genes involved in inactivation of gibberellic acid and indole-3-acetic acid ; and 4 the genes for strigolactone signaling were suppressed by RGSV. These results suggest that these gene responses to RGSV infection account for the excess tillering specific to RGSV infection as well as other symptoms by RGSV, such as stunting and leaf chlorosis.

  6. Rationale for developing new virus vectors to analyze gene function in grasses through virus-induced gene silencing.

    Science.gov (United States)

    Ramanna, Hema; Ding, Xin Shun; Nelson, Richard S

    2013-01-01

    The exploding availability of genome and EST-based sequences from grasses requires a technology that allows rapid functional analysis of the multitude of genes that these resources provide. There are several techniques available to determine a gene's function. For gene knockdown studies, silencing through RNAi is a powerful tool. Gene silencing can be accomplished through stable transformation or transient expression of a fragment of a target gene sequence. Stable transformation in rice, maize, and a few other species, although routine, remains a relatively low-throughput process. Transformation in other grass species is difficult and labor-intensive. Therefore, transient gene silencing methods including Agrobacterium-mediated and virus-induced gene silencing (VIGS) have great potential for researchers studying gene function in grasses. VIGS in grasses already has been used to determine the function of genes during pathogen challenge and plant development. It also can be used in moderate-throughput reverse genetics screens to determine gene function. However, the number of viruses modified to serve as silencing vectors in grasses is limited, and the silencing phenotype induced by these vectors is not optimal: the phenotype being transient and with moderate penetration throughout the tissue. Here, we review the most recent information available for VIGS in grasses and summarize the strengths and weaknesses in current virus-grass host systems. We describe ways to improve current virus vectors and the potential of other grass-infecting viruses for VIGS studies. This work is necessary because VIGS for the foreseeable future remains a higher throughput and more rapid system to evaluate gene function than stable transformation.

  7. Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

    2002-01-01

    Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

  8. CTCF-mediated chromatin loops enclose inducible gene regulatory domains

    NARCIS (Netherlands)

    Oti, M.O.; Falck, J.; Huynen, M.A.; Zhou, Huiqing

    2016-01-01

    BACKGROUND: The CCTC-binding factor (CTCF) protein is involved in genome organization, including mediating three-dimensional chromatin interactions. Human patient lymphocytes with mutations in a single copy of the CTCF gene have reduced expression of enhancer-associated genes involved in response to

  9. Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression

    DEFF Research Database (Denmark)

    Pinto, Rita; Hansen, Lars; Hintze, John Birger Hjalmar

    2017-01-01

    Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven...... to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii......) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide...

  10. Identification of novel light-induced genes in the suprachiasmatic nucleus

    Directory of Open Access Journals (Sweden)

    Piontkivska Helen

    2007-11-01

    Full Text Available Abstract Background The transmission of information about the photic environment to the circadian clock involves a complex array of neurotransmitters, receptors, and second messenger systems. Exposure of an animal to light during the subjective night initiates rapid transcription of a number of immediate-early genes in the suprachiasmatic nucleus of the hypothalamus. Some of these genes have known roles in entraining the circadian clock, while others have unknown functions. Using laser capture microscopy, microarray analysis, and quantitative real-time PCR, we performed a comprehensive screen for changes in gene expression immediately following a 30 minute light pulse in suprachiasmatic nucleus of mice. Results The results of the microarray screen successfully identified previously known light-induced genes as well as several novel genes that may be important in the circadian clock. Newly identified light-induced genes include early growth response 2, proviral integration site 3, growth-arrest and DNA-damage-inducible 45 beta, and TCDD-inducible poly(ADP-ribose polymerase. Comparative analysis of promoter sequences revealed the presence of evolutionarily conserved CRE and associated TATA box elements in most of the light-induced genes, while other core clock genes generally lack this combination of promoter elements. Conclusion The photic signalling cascade in the suprachiasmatic nucleus activates an array of immediate-early genes, most of which have unknown functions in the circadian clock. Detected evolutionary conservation of CRE and TATA box elements in promoters of light-induced genes suggest that the functional role of these elements has likely remained the same over evolutionary time across mammalian orders.

  11. Transposon-induced nuclear mutations that alter chloroplast gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Barkan, A.

    1992-01-01

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  12. Ebola virus infection induces irregular dendritic cell gene expression.

    Science.gov (United States)

    Melanson, Vanessa R; Kalina, Warren V; Williams, Priscilla

    2015-02-01

    Filoviruses subvert the human immune system in part by infecting and replicating in dendritic cells (DCs). Using gene arrays, a phenotypic profile of filovirus infection in human monocyte-derived DCs was assessed. Monocytes from human donors were cultured in GM-CSF and IL-4 and were infected with Ebola virus Kikwit variant for up to 48 h. Extracted DC RNA was analyzed on SuperArray's Dendritic and Antigen Presenting Cell Oligo GEArray and compared to uninfected controls. Infected DCs exhibited increased expression of cytokine, chemokine, antiviral, and anti-apoptotic genes not seen in uninfected controls. Significant increases of intracellular antiviral and MHC I and II genes were also noted in EBOV-infected DCs. However, infected DCs failed to show any significant difference in co-stimulatory T-cell gene expression from uninfected DCs. Moreover, several chemokine genes were activated, but there was sparse expression of chemokine receptors that enabled activated DCs to home to lymph nodes. Overall, statistically significant expression of several intracellular antiviral genes was noted, which may limit viral load but fails to stop replication. EBOV gene expression profiling is of vital importance in understanding pathogenesis and devising novel therapeutic treatments such as small-molecule inhibitors.

  13. Pregnancy-induced gene expression changes in vivo among women with rheumatoid arthritis

    DEFF Research Database (Denmark)

    Goin, Dana E; Smed, Mette Kiel; Pachter, Lior

    2017-01-01

    of Gene Ontology processes and protein networks. RESULTS: A total of 1296 genes were differentially expressed between T3 and T0 among the 8 pregDASimproved women, with 161 genes showing at least two-fold change (FC) in expression by T3. The majority (108 of 161 genes) were also differentially expressed......BACKGROUND: Little is known about gene expression changes induced by pregnancy in women with rheumatoid arthritis (RA) and healthy women because the few studies previously conducted did not have pre-pregnancy samples available as baseline. We have established a cohort of women with RA and healthy...... women followed prospectively from a pre-pregnancy baseline. In this study, we tested the hypothesis that pregnancy-induced changes in gene expression among women with RA who improve during pregnancy (pregDASimproved) overlap substantially with changes observed among healthy women and differ from changes...

  14. Роль генов nos, UCP2 и UCP3 в предрасположенности к занятиям академической греблей

    OpenAIRE

    2011-01-01

    В статье исследованы ассоциации полиморфизмов генов NOS, UCP2 и UCP3 с предрасположенностью к занятиям академической греблей. Молекулярногенетический анализ данного комплекса генов можно рекомендовать в качестве критерия при отборе в данный вид спорта.

  15. Elicitor and fusarium-induced expression of NPR-1 like genes in banana

    CSIR Research Space (South Africa)

    Endah, R

    2008-11-01

    Full Text Available NPR1 is an essential positive regulator of salicylic acid-induced PR gene expression and systemic acquired resistance. Two novel full-length NPR1-like genes; MNPR1A and MNPR1B, were isolated by application of the PCR and RACE techniques. The two...

  16. Alcohol-induced histone acetylation reveals a gene network involved in alcohol tolerance.

    Directory of Open Access Journals (Sweden)

    Alfredo Ghezzi

    Full Text Available Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol.

  17. Effect of electro-acupuncture on gene expression in heart of rats with stress-induced pre-hypertension based on gene chip technology

    National Research Council Canada - National Science Library

    Guo Yan Xie Xiaojia Guo Changqing Wang Zhaoyang Liu Qingguo

    2015-01-01

    OBJECTIVE:To explore electro-acupuncture's(EA's)effect on gene expression in heart of rats with stress-induced pre-hypertension and try to reveal its biological mechanism based on gene chip...

  18. An insulin-induced DNA-binding protein for the human growth hormone gene.

    OpenAIRE

    Prager, D; Gebremedhin, S; Melmed, S

    1990-01-01

    The control of gene transcription is usually mediated by transacting transcriptional factors that bind to upstream regulatory elements. As insulin regulates transcription of the growth hormone (GH) gene, we tested nuclear extracts from unstimulated and insulin-stimulated Chinese hamster ovarian (CHO) cells for binding to four human GH (hGH) gene promoter oligonucleotide fragments identified as target-binding sequences by DNAse I footprinting. Using a mobility shift assay, an insulin-induced D...

  19. A virus-induced gene silencing method to study soybean cyst nematode parasitism in Glycine max

    OpenAIRE

    Kandoth, Pramod K; Heinz, Robert; Yeckel, Greg; Gross, Nathan W; Juvale, Parijat S; Hill, John; Whitham, Steven A.; Baum, Thomas J.; Mitchum, Melissa G.

    2013-01-01

    Background Bean pod mottle virus (BPMV) based virus-induced gene silencing (VIGS) vectors have been developed and used in soybean for the functional analysis of genes involved in disease resistance to foliar pathogens. However, BPMV-VIGS protocols for studying genes involved in disease resistance or symbiotic associations with root microbes have not been developed. Findings Here we describe a BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots. We use this method for anal...

  20. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

    OpenAIRE

    Sang-Ho Park; Hoseong Choi; Semin Kim; Won Kyong Cho; Kook-Hyung Kim

    2016-01-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the n...

  1. Probiotic bacteria change Echherichia coli-induced gene expression in cultured colonocytes: Implications in intestinal pathophysiology

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate the change in eukaryotic gene expression profile in Caco-2 cells after infection with strains of Escherichia coli and commensal probiotic bacteria.METHODS: A 19200 gene/expressed sequence tag gene chip was used to examine expression of genes after infection of Caco-2 cells with strains of normal flora E.coli, Lactobacillus plantarum, and a combination of the two.RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of 177 genes by E. coli. L. plantarum up-regulated 45 and down-regulated 36 genes. During mixed infection, 27 genes were upregulated and 59 were down-regulated, with nullification of stimulatory/inhibitory effects on most of the genes. Expression of several new genes was noted in this group.CONCLUSION: The commensal bacterial strains used in this study induced the expression of a large number of genes in colonocyte-like cultured cells and changed the expression of several genes involved in important cellular processes such as regulation of transcription, protein biosynthesis, metabolism, cell adhesion, ubiquitination,and apoptosis. Such changes induced by the presence of probiotic bacteria may shape the physiologic and pathologic responses they trigger in the host.

  2. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    DEFF Research Database (Denmark)

    Weiss, Gudrun Margarethe; Rasmussen, Simon; Fink, Lisbeth Nielsen

    2010-01-01

    cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium...

  3. RNA-Seq analysis of high NaCl-induced gene expression.

    Science.gov (United States)

    Izumi, Yuichiro; Yang, Wenjing; Zhu, Jun; Burg, Maurice B; Ferraris, Joan D

    2015-10-01

    High extracellular NaCl is known to change expression of numerous genes, many of which are regulated by the osmoprotective transcription factor nuclear factor of activated T cells-5 (NFAT5). In the present study we employed RNA-Seq to provide a comprehensive, unbiased account of genes regulated by high NaCl in mouse embryonic fibroblast cells (MEFs). To identify genes regulated by NFAT5 we compared wild-type MEFs (WT-MEFs) to MEFs in which mutation of the NFAT5 gene inhibits its transcriptional activity (Null-MEFs). In WT-MEFs adding NaCl to raise osmolality from 300 to 500 mosmol/kg for 24 h increases expression of 167 genes and reduces expression of 412. Raising osmolality through multiple passages (adapted cells) increases expression of 196 genes and reduces expression of 528. In Null-MEFs, after 24 h of high NaCl, expression of 217 genes increase and 428 decrease, while in adapted Null-MEFs 143 increase and 622 decrease. Fewer than 10% of genes are regulated in common between WT- and null-MEFs, indicating a profound difference in regulation of high-NaCl induced genes induced by NFAT5 compared with those induced in the absence of NFAT5. Based on our findings we suggest a mechanism for this phenomenon, which had previously been unexplained. The NFAT5 DNA-binding motif (osmotic response element) is overrepresented in the vicinity of genes that NFAT5 upregulates, but not genes that it downregulates. We used Gene Ontology and manual curation to determine the function of the genes targeted by NFAT5, revealing many novel consequences of NFAT5 transcriptional activity.

  4. Lipopolysaccharide triggers nuclear import of Lpcat1 to regulate inducible gene expression in lung epithelia

    Institute of Scientific and Technical Information of China (English)

    Bryon; Ellis; Leah; Kaercher; Courtney; Snavely

    2012-01-01

    AIM:To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene tran-scription. METHODS:Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus influenza and Escherichia coli infection was analyzed by employing quantitative Reverse Transcription Polymerase Chain Reaction techniques. Nucleofection was used to deliver Lenti-viral system to express or knock down Lpcat1 in MLE cells. Subcellular protein fractionation and Western blotting were utilized to study Lpcat1 nuclear relocation. RESULTS:Lpcat1 translocates into the nucleus from thecytoplasm in murine lung epithelia (MLE) after LPS treatment. Haemophilus influenza and Escherichia coli , two LPS-containing pathogens that cause pneumonia, triggered Lpcat1 nuclear translocation from the cytoplasm. The LPS inducible gene expression profile was determined by quantitative reverse transcription polymerase chain reaction after silencing Lpcat1 or overexpression of the enzyme in MLE cells. We detected that 17 out of a total 38 screened genes were upregulated, 14 genes were suppressed, and 7 genes remained unchanged in LPS treated cells in comparison to controls. Knockdown of Lpcat1 by shRNA dramatically changed the spectrum of the LPS inducible gene transcription, as 18 genes out of 38 genes were upregulated, of which 20 genes were suppressed or unchanged. Notably, in Lpcat1 overex-pressed cells, 25 genes out of 38 genes were reduced in the setting of LPS treatment.CONCLUSION:These observations suggest that Lpcat1 relocates into the nucleus in response to bacterial infection to differentially regulate gene transcriptional repression.

  5. Analysis of target genes induced by IL-13 cytotoxin in human glioblastoma cells.

    Science.gov (United States)

    Han, Jing; Yang, Liming; Puri, Raj K

    2005-03-01

    IL-13 cytotoxin comprised of IL-13 and a mutated form of Pseudomonas exotoxin (fusion protein termed IL-13-PE38QQR) has been shown to inhibit protein synthesis leading to necrotic and apoptotic cell death in glioblastoma cells that express high levels of interleukin-13 receptors (IL-13R). To identify target genes of cell death and other cellular genes with IL-13 receptors in glioblastoma cells, we utilized the cDNA microarrays to analyze global gene expression profiles after IL-13 cytotoxin and IL-13 treatment. IL-13 cytotoxin mediated cytotoxicity to U251 cells in a dose-dependent manner. Hierarchical cluster analysis of differentially expressed genes in U251 glioma cells at different time points after IL-13 cytotoxin treatment showed three major groups, each representing a specific expression pattern. Randomly selected differentially expressed genes from each group were confirmed by RT-PCR analysis. Most down-regulated genes belong to cell adhesion, motility, angiogenesis, DNA repair, and metabolic pathways. While up-regulated genes belong to cell cycle arrest, apoptosis, signaling and various metabolic pathways. Unexpectedly, at early time points, both IL-13 and IL-13 cytotoxin induced several genes belonging to different pathways most notably IL-8, DIO2, END1, and ALDH1A3 indicating that these genes are early response genes and their products may be associated with IL-13R. In addition, IL-13 cytotoxin induced IL-13Ralpha2 mRNA expression during the treatment in glioma cells. Our results indicate that novel cellular genes are involved with IL-13 receptors and that IL-13 cytotoxin induced cell death involves various target genes in human glioblastoma cells. On going studies will determine the role of associated genes and their products in the IL-13R functions in glioma cells.

  6. Fast and sensitive detection of indels induced by precise gene targeting

    DEFF Research Database (Denmark)

    Yang, Zhang; Steentoft, Catharina; Hauge, Camilla

    2015-01-01

    The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect...... and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect...

  7. Protective effects of L-selenomethionine on space radiation induced changes in gene expression.

    Science.gov (United States)

    Stewart, J; Ko, Y-H; Kennedy, A R

    2007-06-01

    Ionizing radiation can produce adverse biological effects in astronauts during space travel. Of particular concern are the types of radiation from highly energetic, heavy, charged particles known as HZE particles. The aims of our studies are to characterize HZE particle radiation induced biological effects and evaluate the effects of L-selenomethionine (SeM) on these adverse biological effects. In this study, microarray technology was used to measure HZE radiation induced changes in gene expression, as well as to evaluate modulation of these changes by SeM. Human thyroid epithelial cells (HTori-3) were irradiated (1 GeV/n iron ions) in the presence or in the absence of 5 microM SeM. At 6 h post-irradiation, all cells were harvested for RNA isolation. Gene Chip U133Av2 from Affymetrix was used for the analysis of gene expression, and ANOVA and EASE were used for a determination of the genes and biological processes whose differential expression is statistically significant. Results of this microarray study indicate that exposure to small doses of radiation from HZE particles, 10 and 20 cGy from iron ions, induces statistically significant differential expression of 196 and 610 genes, respectively. In the presence of SeM, differential expression of 77 out of 196 genes (exposure to 10 cGy) and 336 out of 610 genes (exposure to 20 cGy) is abolished. In the presence or in the absence of SeM, radiation from HZE particles induces differential expression of genes whose products have roles in the induction of G1/S arrest during the mitotic cell cycle, as well as heat shock proteins. Some of the genes, whose expressions were affected by radiation from HZE particles and were unchanged in irradiated cells treated with SeM, have been shown to have altered expression levels in cancer cells. The conclusions of this report are that radiation from HZE particles can induce differential expression of many genes, some of which are known to play roles in the same processes that have

  8. Copper induces the expression of cholesterogenic genes in human macrophages.

    Science.gov (United States)

    Svensson, Per Arne; Englund, Mikael C O; Markström, Emilia; Ohlsson, Bertil G; Jernås, Margareta; Billig, Håkan; Torgerson, Jarl S; Wiklund, Olov; Carlsson, Lena M S; Carlsson, Björn

    2003-07-01

    Accumulation of lipids and cholesterol by macrophages and subsequent transformation into foam cells are key features in development of atherosclerosis. Serum copper concentrations have been shown to be associated with cardiovascular disease. However, the mechanism behind the proatherogenic effect of copper is not clear. We used DNA microarrays to define the changes in gene expression profile in response to copper exposure of human macrophages. Expression monitoring by DNA microarray revealed 91 genes that were regulated. Copper increased the expression of seven cholesterogenic genes (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, IPP isomerase, squalene synthase, squalene epoxidase, methyl sterol oxidase, H105e3 mRNA and sterol-C5-desaturase) and low-density lipoprotein receptor (LDL-R), and decreased the expression of CD36 and lipid binding proteins. The expression of LDL-R and HMG CoA reductase was also investigated using real time PCR. The expression of both of these genes was increased after copper treatment of macrophages (Pmechanism for the association between copper and atherosclerosis. The effect of copper on cholesterogenic genes may also have implications for liver steatosis in early stages of Wilson's disease.

  9. Fluoroquinolone-induced gene transfer in multidrug-resistant Salmonella

    Science.gov (United States)

    Fluoroquinolones are broad spectrum antibiotics that inhibit bacterial DNA gyrase and topoisomerase activity. Bacterial exposure to fluoroquinolones can cause DNA damage and induce a bacterial SOS response to stimulate repair of damaged DNA. Certain prophages (integrated in bacterial chromosomes) ...

  10. Radiation-induced gene expression in the nematode caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, G.A.; Jones, T.A.; Chesnut, A.; Smith, A.L. [Loma Linda Univ., CA (United States)

    2002-12-01

    We used the nematode C. elegans to characterize the genotoxic and cytotoxic effects of ionizing radiation in a simple animal model emphasizing the unique effects of charged particle radiation. Here we demonstrate by reverse transcription polymerase chain reaction (RT-PCR) differential display and whole genome microarray hybridization experiments that gamma rays, accelerated protons and iron ions at the same physical dose lead to unique transcription profiles. 599 of 17871 genes analyzed (3.4%) showed differential expression 3 hrs after exposure to 3 Gy of radiation. 193 were up-regulated, 406 were down-regulated and 90% were affected only by a single species of radiation. A novel statistical clustering technique identified the regulatory relationships between the radiation-modulated genes and showed that genes affected by each radiation species were associated with unique regulatory clusters. This suggests that independent homeostatic mechanisms are activated in response to radiation exposure as a function of track structure or ionization density. (author)

  11. Radiation-induced gene expression in the nematode Caenorhabditis elegans

    Science.gov (United States)

    Nelson, Gregory A.; Jones, Tamako A.; Chesnut, Aaron; Smith, Anna L.

    2002-01-01

    We used the nematode C. elegans to characterize the genotoxic and cytotoxic effects of ionizing radiation in a simple animal model emphasizing the unique effects of charged particle radiation. Here we demonstrate by RT-PCR differential display and whole genome microarray hybridization experiments that gamma rays, accelerated protons and iron ions at the same physical dose lead to unique transcription profiles. 599 of 17871 genes analyzed (3.4%) showed differential expression 3 hrs after exposure to 3 Gy of radiation. 193 were up-regulated, 406 were down-regulated and 90% were affected only by a single species of radiation. A novel statistical clustering technique identified the regulatory relationships between the radiation-modulated genes and showed that genes affected by each radiation species were associated with unique regulatory clusters. This suggests that independent homeostatic mechanisms are activated in response to radiation exposure as a function of track structure or ionization density.

  12. Substrate-induced gene expression screening: a method for high-throughput screening of metagenome libraries.

    Science.gov (United States)

    Uchiyama, Taku; Miyazaki, Kentaro

    2010-01-01

    The SIGEX (substrate-induced gene expression) method is a novel approach for the screening of gene (genome) libraries. In addition to the commonly used function- and sequence-driven approaches to screening, SIGEX provides a third option; in SIGEX, positives are identified using a reporter gene, and the library is constructed using an "operon-trap" vector. This vector contains the reporter gene immediately downstream of the cloning site for the genomic insert so that the expression of the inserted gene(s) is coupled with that of the reporter gene. This system is especially suitable for screening catabolic genes that are induced in response to metabolically relevant compounds, such as substrates. If expression of the inserted gene(s) is activated in response to the addition of these compounds, then positive clones can be identified based on the reporter signal. The most effective selection is obtained by the use of a FACS (fluorescence-activated cell sorter) in conjunction with a FACS-compatible fluorescent reporter protein, such as GFP (green fluorescent protein). Activity-based screening of metagenomic libraries often suffers from low sensitivity and low throughput. In contrast, the high throughput, high sensitivity, and versatility of SIGEX make it a particularly suitable method for screening metagenomic libraries.

  13. Modulation of Gene Expression Networks underlying Realgar-Induced Differentiation of Acute Promyelocytic Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    王怀宇; 刘陕西

    2002-01-01

    Objective: To elucidate the molecular mechanism of the differentiation of acute promyelocytic leukemia (APL) cell line NB4 induced by realgar. Methods: The response of NB4 cell to realgar was explored with a cDNA microarray representing 1003 different human genes. Results: The analysis of gene expression profiles indicated that 8 genes were up-regulated and 33 genes were down-regulated 48 hrs after realgar treatment. Among the 8 up-regulated genes, 2 genes were involved in ubiquitin proteasome degradation pathway. Some genes related to RNA processing, protein synthesis and signal transduction were down-regulated. Conclusion: The ubiquitin-proteasome degradation pathway may play an important role in the degradation of PML/RAR α fusion protein and the differentiation of NB4 cells.

  14. Differentially Gene Expression Profile Related to Inflammation in Endometrial Cells Induce by Lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Li-hua QIN; Ruo-guang WANG; Sheng LI; Chun-mei LI

    2009-01-01

    Objective To investigate differentially expressed genes related to inflammation in endometrial cells induced by Lipopolysaccharide(LPS).Methods Normal endometrium in the proliferative phase of specimen from 3 cases for the experiment was collected. The LPS group were treated with 50 μg/ml LPS. Total RNA was extracted using Trizol reagent from cells. RNA quality was assessed by determining the OD260/280 ratio by agarose gel electrophoresis, the chip was scanned by laser scanner. The acquired was analyzed. Results A total of differentially expressed genes were found, these genes were relative to many aspects. Among them, the expression of genes involved in inflammation were up-regulated by LPS, such as overexpression of lL-lβ, 8, etc.Conclusion The results indicates that inflammation-related genes may be one of the mechanisms of abnormal uterine bleeding by LPS-induced.

  15. Identification of in vivo-induced genes during infection of chickens with Salmonella enterica serovar Enteritidis.

    Science.gov (United States)

    Geng, Shizhong; Liu, Zhicheng; Lin, Zhijie; Barrow, Paul; Pan, Zhiming; Li, Qiuchun; Jiao, Xinan

    2015-06-01

    Chickens are an important source of food worldwide and are often infected with food-poisoning serovars of Salmonella enterica, frequently Salmonella Enteritidis (SE), without exhibiting clinical signs of disease. Ivi (in vivo induced) genes identified using in vivo-induced antigen technology (IVIAT) are expressed only during bacterial infection and have the potential value of identifying epidemic strains and antigens which can form the basis for sub-unit vaccine development. We applied IVIAT to SE strain 50041 and identified 42 ivi genes. Eight representative ivi genes were further confirmed by qRT-PCR as being expressed only in vivo within 48 h of infection compared with that of in vitro-cultured. Although our results indicated that the identified ivi genes are expressed only in vivo, further research is needed to elucidate the exact roles of these genes during infection and pathogenesis.

  16. Coordinate gene regulation by fimbriae-induced signal transduction

    DEFF Research Database (Denmark)

    Schembri, Mark; Klemm, Per

    2001-01-01

    of Ag43 production. No effect was observed in an oxyR mutant. We conclude that fimbriae expression per se constitutes a signal transduction mechanism that affects a number of unrelated genes via the thiol-disulfide status of OxyR. Thus, phase variation in fimbrial expression is coordinated...

  17. Identification of genes induced by salt stress from Medicago ...

    African Journals Online (AJOL)

    hope&shola

    2010-11-08

    Nov 8, 2010 ... Local Alignment Search Tool (BLAST) analysis of deduced protein sequences revealed that 35 .... uni-ESTs may be either new genes or segments unique to .... Yang QC, Wu MS, Wang QP, Kang JM (2005). Cloning ... Page 7 ...

  18. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    Science.gov (United States)

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  19. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    Science.gov (United States)

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  20. Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense.

    Directory of Open Access Journals (Sweden)

    Jinhuan Pang

    Full Text Available Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species. These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum.

  1. Changes in gene expression induced by Micro-Immunotherapy

    Directory of Open Access Journals (Sweden)

    Maurice Jeaner

    2012-09-01

    Full Text Available Background: Metabolic syndrome (MS is a metabolic disorder associated with obesity, type-II diabetes, and “low grade inflammation”, with the concomitant increased risk of cardiovascular events. As a chronic inflammatory process, MS results in a dysregulation of the cytokine profile. 2L®INFLAM, a Micro-immunotherapy (MI medication formulated with highly diluted cytokines, is currently prescribed in Belgium for inflammatory diseases and potentially may be helpful for MS patients. Aims: To investigate the impact of 2L®INFLAM on selected gene expression markers (mRNA in patients suffering from MS, in addition to biological and clinical parameters. Methodology: Four well characterized MS adult patients with stabilized body-weight were advised to take one capsule of 2L®INFLAM per day (by sublingual-oral route for 6 months (composition in table 1. Concomitantly to biological and clinical examination, genes expression status was assessed by a DNA microarray technology (Oxygen™ comprising 200 genes involved mainly in oxidative stress and inflammation. Whole blood collection was performed before and after treatment (3-6 months and mRNA levels measured. Gene expression was classified in 3 series (normally expressed, up or down-regulated and genes related to diabetes predisposition were scored by using a proprietary Diascore (Probiox. Results: Before MI medication, a significant percentage of dysregulated genes (median: 16.3% as well as a positive Diascore (median: 1.6 were noticed. Impressive correction of dysregulated genes (reaching 90% for one patient was observed after 3 months of treatment (median: 2.3% in addition to an improvement of Diascore in 3 MS patients out of 4 (median: 0.5. During the same period, both clinical and biological parameters remained unchanged. Conclusions: MS patients showing a high level of gene dysregulation efficiently normalized after 3 months of 2L

  2. Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5.

    Science.gov (United States)

    Buxadé, Maria; Lunazzi, Giulia; Minguillón, Jordi; Iborra, Salvador; Berga-Bolaños, Rosa; Del Val, Margarita; Aramburu, José; López-Rodríguez, Cristina

    2012-02-13

    Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.

  3. System for stable β-estradiol-inducible gene expression in the moss Physcomitrella patens.

    Science.gov (United States)

    Kubo, Minoru; Imai, Akihiro; Nishiyama, Tomoaki; Ishikawa, Masaki; Sato, Yoshikatsu; Kurata, Tetsuya; Hiwatashi, Yuji; Reski, Ralf; Hasebe, Mitsuyasu

    2013-01-01

    Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrellapatens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with β-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing β-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This β-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.

  4. Interferon induced IFIT family genes in host antiviral defense

    Science.gov (United States)

    Secretion of interferons (IFNs) from virus-infected cells is a hallmark of host antiviral immunity and in fact, IFNs exert their antiviral activities through the induction of antiviral proteins. The IFN-induced protein with tetratricopeptide repeats (IFITs) family is among hundreds of IF stimulated ...

  5. Thermally induced osteocyte damage initiates pro-osteoclastogenic gene expression in vivo.

    Science.gov (United States)

    Dolan, Eimear B; Tallon, David; Cheung, Wing-Yee; Schaffler, Mitchell B; Kennedy, Oran D; McNamara, Laoise M

    2016-06-01

    Bone is often subject to harsh temperatures during orthopaedic procedures resulting in thermally induced bone damage, which may affect the healing response. Postsurgical healing of bone is essential to the success of surgery, therefore, an understanding of the thermally induced responses of bone cells to clinically relevant temperatures in vivo is required. Osteocytes have been shown to be integrally involved in the bone remodelling cascade, via apoptosis, in micro-damage systems. However, it is unknown whether this relationship is similar following thermal damage. Sprague-Dawley rat tibia were exposed to clinically relevant temperatures (47°C or 60°C) to investigate the role of osteocytes in modulating remodelling related factors. Immunohistochemistry was used to quantify osteocyte thermal damage (activated caspase-3). Thermally induced pro-osteoclastogenic genes (Rankl, Opg and M-csf), in addition to genes known to mediate osteoblast and osteoclast differentiation via prostaglandin production (Cox2), vascularization (Vegf) and inflammatory (Il1a) responses, were investigated using gene expression analysis. The results demonstrate that heat-treatment induced significant bone tissue and cellular damage. Pro-osteoclastogenic genes were upregulated depending on the amount of temperature elevation compared with the control. Taken together, the results of this study demonstrate the in vivo effect of thermally induced osteocyte damage on the gene expression profile.

  6. Virus-induced opposite effect on Bombyx mori gene transcriptions

    Directory of Open Access Journals (Sweden)

    Y Yin

    2016-09-01

    Full Text Available Bombyx mori bidensovirus (BmBDV and Bombyx mori nucleopolyhedrovirus (BmNPV are serious pathogens of Bombyx mori. In this study, we reported the changes of transcription level of several immune genes, including bmi, argo, dicer, cap1, cap3 and car, in Bombyx mori midgut after exposure to BmBDV or BmNPV. Silkworm strains 798 (anti-BmBDV and 306 (susceptible to BmBDV were subjected to BmBDV infection, and NB (anti-BmNPV and HUABA (35 (susceptible to BmNPV were subjected to BmNPV infection. The results showed that the transcription levels differ largely among different silkworm strains, and that the extent to which the gene transcriptions were affected by the viruses was different. However, both BmNPV and BmBDV viruses can reverse the transcription patterns of these genes when the silkworms were administered with the viruses compared with those control groups. The transcript levels of bmi and dicer were decreased in 798 and 306 strains that were inoculated with BmBDV compared with their respective controls, but were increased in NB and HUABA (35 inoculated with BmNPV. The transcript levels of argo and cap3 were risen in 798, 306 and NB strains when inoculated with their respective viruses, but were decreased in HUABA (35 strain. The transcript levels of cap1 were risen in all silkworm strains, while the levels of car were decreased in 798, 306 and HUABA (35 strains, and increased in NB strain when inoculated with their respective viruses. These findings may contribute to more in-depth understanding on functions of these genes in virus infection and proliferation.

  7. HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Hongzhu LU; Jianhua ZHOU

    2008-01-01

    The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

  8. Exploring Mycobacterium tuberculosis infection-induced alterations in gene expression in macrophage by microarray hybridization

    Institute of Scientific and Technical Information of China (English)

    XIE; Jianping; (谢建平); LI; Yao; (李; 瑶); YUE; Jun; (乐; 军); XU; Yongzhong; (徐永忠); HUANG; Daqiang; (黄达蔷); LIANG; Li; (梁; 莉); WANG; Honghai; (王洪海)

    2003-01-01

    Tuberculosis remains a serious threat to public health. Its causative agent Mycobacte- rium tuberculosis is an intracellular pathogen which survives and replicates within cells of the host immune system, primarily macrophages. Knowledge of the bacteria-macrophage interaction can help to develop novel measures to combat the disease. The global gene expression of macro- phage following invasion by and growth of M. tuberculosis was studied by cDNA microarray. Of the 12800 human genes analyzed, totally 473 (3.7%) macrophage genes were differentially expressed after being infected by M. tuberculosis, among which, only 25 (5.2%, corresponding to less than 0.2% of the 12800 genes) genes were up-regulated, while others (94.8%) were down-regulated against the control. Of the 473 genes, 376 genes are registered in the GenBank, and 97 are novel genes. Expression of 5 up-regulated genes has been induced by more than 3-fold. 25 genes were down-regulated by more than 3-fold. Syndecan binding protein has been down-regu- lated up to 12.5-fold. The data gave an insight into the early gene expression in macrophage ensuing M. tuberculosis infection and a basis for further study.

  9. Myostatin gene mutated mice induced with tale nucleases.

    Science.gov (United States)

    Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

    2015-01-01

    Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

  10. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    asy gene is a novel apoptosis-inducing gene,but its mechanism is unclear.To investigate the mechanism of asy inducing apoptosis,a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system.The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels.Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end,and share a common open reading frame encoding a polypeptide of 236 amino acids.Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence.Two highly hydrophobic regions encoding potentially two transmembrane domains are present.The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu).Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells.Compared with asy gene,overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.

  11. Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes.

    Directory of Open Access Journals (Sweden)

    David Proud

    Full Text Available Human rhinovirus (HRV infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.

  12. Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish.

    Directory of Open Access Journals (Sweden)

    Hiroyasu Kamei

    Full Text Available BACKGROUND: Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1. IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker. CONCLUSIONS/SIGNIFICANCE: These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic

  13. A comparative study examining the cytotoxicity of inducible gene expression system ligands in different cell types.

    Science.gov (United States)

    Xie, Jinger; Nair, Ayyappan; Hermiston, Terry W

    2008-02-01

    Inducible gene expression systems are being used in many in vitro and in vivo applications for target discovery, target validation and as components in exploratory therapeutic agents. Ideally, the ligands, which activate the systems, are benign so that the effects can be strictly attributed to the induced protein. As a first step to defining the potential effects of these inducers, we tested three of them, doxycycline, muristerone A and mifepristone (for tet-, ecdysone- and progesterone antagonist-inducible systems respectively), for toxicity across a panel of normal cells and cancer cell lines. In contrast to both muristerone A and mifepristone that showed no significant toxicity on any of the tested cells, we observed that doxycycline induced cell death in selected cancer and primary cell lines. The different susceptibility of cell lines to the ligands commonly used in these inducible systems suggests that it is important to consider the effects of the inducers prior to their use in experimental in vitro cell culture systems.

  14. Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

    Science.gov (United States)

    Morlino, G B; Tizzani, L; Fleer, R; Frontali, L; Bianchi, M M

    1999-11-01

    Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

  15. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

    Science.gov (United States)

    Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

    2014-10-17

    The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

  16. Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

    Science.gov (United States)

    Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

    2016-03-01

    Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. Copyright © 2016

  17. Identification of antiviralrelevant genes in the cultured fish cells induced by UV-inactivated virus

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (crucian carp (Carassius auratus L.) blastulae) cells, and thus defend host cells against the virus invasion. The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antivi-ral-relevant genes. In this study, suppressive subtractive hy-bridization is applied to constructing a subtracted cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells. 272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library. Sequencing analysis reveals 69 genes, including 46 known gene homologues, and 23 unknown putative genes. The known genes include the genes involved in interferon signaling pathways, such as Stat1 and Jak1, the antiviral genes, such as Mx and Viperin, and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknown putative genes contain AU-rich ele-ment in their sequences. Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR. The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells, but also leads to the expression of a series of antiviral-relevant genes or immune-relevant genes, and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.

  18. Systemic virus-induced gene silencing allows functional characterization of maize genes during biotrophic interaction with Ustilago maydis.

    Science.gov (United States)

    van der Linde, Karina; Kastner, Christine; Kumlehn, Jochen; Kahmann, Regine; Doehlemann, Gunther

    2011-01-01

    Infection of maize (Zea mays) plants with the corn smut fungus Ustilago maydis leads to the formation of large tumors on the stem, leaves and inflorescences. In this biotrophic interaction, plant defense responses are actively suppressed by the pathogen, and previous transcriptome analyses of infected maize plants showed massive and stage-specific changes in host gene expression during disease progression. To identify maize genes that are functionally involved in the interaction with U. maydis, we adapted a virus-induced gene silencing (VIGS) system based on the brome mosaic virus (BMV) for maize. Conditions were established that allowed successful U. maydis infection of BMV-preinfected maize plants. This set-up enabled quantification of VIGS and its impact on U. maydis infection using a quantitative real-time PCR (qRT-PCR)-based readout. In proof-of-principle experiments, an U. maydis-induced terpene synthase was shown to negatively regulate disease development while a protein involved in cell death inhibition was required for full virulence of U. maydis. The results suggest that this system is a versatile tool for the rapid identification of maize genes that determine compatibility with U. maydis.

  19. Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews

    Institute of Scientific and Technical Information of China (English)

    Yuan Li; Dan Luo; Hui-Fen Yue; Li-Sheng Zhang; Jian-Ren Gu; Da-Fang Wan; Jian-Jia Su; Ji Cao; Chao Ou; Xiao-Kun Qiu; Ke-Chen Ban; Chun Yang; Liu-Liang Qin

    2004-01-01

    AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1),to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism.METHODS: Tree shrews ( 7upaia belangeri chinensis)were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding noncancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of AtlasTM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared.RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as "important genes" because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC,genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC.CONCLUSION: A considerable number of genes could change

  20. Gene expression in rat skin induced by irritating chemicals.

    Science.gov (United States)

    Rogers, James V; Garrett, Carol M; McDougal, James N

    2003-01-01

    Occupational skin disease is the second most significant cause of occupational disease, after accidents. Irritation from occupational chemicals such as solvents, hydrocarbons, and surfactants are one cause of this disease. Gene expression studies provide useful information about normal processes in the skin and responses of the skin to exogenous chemicals. We exposed rats, cutaneously, to sodium lauryl sulfate (SLS, 1% and 10% aqueous solution), m-xylene (pure liquid), and d-limonene (pure liquid) for 1 h and measured transcriptional responses at the end of the exposure and 3 h later for comparison with untreated skin samples. Total skin RNA was isolated and analyzed using the Affymetrix RatTox U34 array. Using the Affymetrix software, we found that 234 of approximately 850 genes were detected as present in at least 80% of the normal skin samples. The largest number of these genes was related to metabolism, oxidative/cellular stress, and signal transduction. Limonene caused the largest change in mRNA levels with a total of 34 increased transcripts and 4 decreased transcripts. Xylene treatment resulted in 6 increased transcripts and 14 decreased transcripts, while 10% SLS caused 5 transcripts to increase and 17 to decrease. Only two transcripts were observed to change in skin following a 1% SLS exposure. Sodium lauryl sulfate transcript changes increased with dose and were maximum at 4 h. Limonene transcript changes were more numerous at 1 h than at 4 h. The observed differences may reflect different mechanisms of irritation. Copyright 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:123-137, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10079

  1. Gene expression profiles reveal effect of a high-fat diet on the development of white and brown adipose tissues.

    Science.gov (United States)

    Kim, Hyeng-Soo; Ryoo, Zae Young; Choi, Sang Un; Lee, Sanggyu

    2015-07-01

    Because of the recent discovery of brown adipose tissues tissue in adult humans, brown adipose tissues have garnered additional attention. Many studies have attempted to transform the precursor cells within the white adipocyte cultures to Brite (brown-in-white) cells by using genomic modification or pharmacological activation in order to determine the therapeutic effect of obesity. However, genome-scale analysis of the genetic factors governing the development of white and brown adipose tissues remains incomplete. In order to identify the key genes that regulate the development of white and brown adipose tissues in mice, a transcriptome analysis was performed on the adipose tissues. Network analysis of differentially expressed genes indicated that Trim30 and Ucp3 play pivotal roles in energy balance and glucose homeostasis. In addition, it was discovered that identical biological processes and pathways in the white and brown adipose tissues might be regulated by different genes. Trim30 and Ucp3 might be used as genetic markers to precisely represent the stage of obesity during the early and late stages of adipose tissue development, respectively. These results may provide a stepping-stone for future obesity-related studies. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Host-Induced Silencing of Pathogenicity Genes Enhances Resistance to Fusarium oxysporum Wilt in Tomato.

    Science.gov (United States)

    Bharti, Poonam; Jyoti, Poonam; Kapoor, Priya; Sharma, Vandana; Shanmugam, V; Yadav, Sudesh Kumar

    2017-08-01

    This study presents a novel approach of controlling vascular wilt in tomato by RNAi expression directed to pathogenicity genes of Fusarium oxysporum f. sp. lycopersici. Vascular wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici leads to qualitative and quantitative loss of the crop. Limitation in the existing control measures necessitates the development of alternative strategies to increase resistance in the plants against pathogens. Recent findings paved way to RNAi, as a promising method for silencing of pathogenicity genes in fungus and provided effective resistance against fungal pathogens. Here, two important pathogenicity genes FOW2, a Zn(II)2Cys6 family putative transcription regulator, and chsV, a putative myosin motor and a chitin synthase domain, were used for host-induced gene silencing through hairpinRNA cassettes of these genes against Fusarium oxysporum f. sp. lycopersici. HairpinRNAs were assembled in appropriate binary vectors and transformed into tomato plant targeting FOW2 and chsV genes, for two highly pathogenic strains of Fusarium oxysporum viz. TOFOL-IHBT and TOFOL-IVRI. Transgenic tomatoes were analyzed for possible attainment of resistance in transgenic lines against fungal infection. Eight transgenic lines expressing hairpinRNA cassettes showed trivial disease symptoms after 6-8 weeks of infection. Hence, the host-induced posttranscriptional gene silencing of pathogenicity genes in transgenic tomato plants has enhanced their resistance to vascular wilt disease caused by Fusarium oxysporum.

  3. Identification of Candida albicans genes induced during thrush offers insight into pathogenesis.

    Science.gov (United States)

    Cheng, Shaoji; Clancy, Cornelius J; Checkley, Mary Ann; Handfield, Martin; Hillman, Jeffrey D; Progulske-Fox, Ann; Lewin, Alfred S; Fidel, Paul L; Nguyen, M Hong

    2003-06-01

    Candida albicans causes a wide spectrum of diseases, ranging from mucocutaneous infections like oral thrush to disseminated candidiasis. Screening for C. albicans genes expressed within infected hosts might advance understanding of candidal pathogenesis, but is impractical using existing techniques. In this study, we used an antibody-based strategy to identify C. albicans genes expressed during thrush. We adsorbed sera from HIV-infected patients with thrush against candidal cells grown in vitro and screened a C. albicans genomic expression library. We identified 10 genes encoding immunogenic antigens and used reverse transcription-polymerase chain reaction to verify that they were induced within thrush pseudomembranes recovered from a patient. The in vivo induced genes are involved in diverse functions, including regulation of yeast-hyphal morphogenesis, adhesion to host cells, nutrient uptake, phospholipid biosynthesis and amino acid catabolism. Four genes encode known virulence determinants (HWP1, CST20, CPP1 and RBF1). Another gene, LPD1, for which a role in candidal pathogenesis is unknown, encodes a protein homologous to a bacterial virulence determinant. Most importantly, disruption of CaNOT5, a newly identified gene, conferred defects in morphogenesis, decreased adherence to human buccal epithelial cells and attenuated mortality during murine disseminated candidiasis, proving that our strategy can identify genes encoding novel virulence determinants.

  4. Identification of Differently Expressed Genes in Chemical Carcinogen-induced Rat Bladder Cancers

    Institute of Scientific and Technical Information of China (English)

    Guangfu CHEN; Franky L. CHAN; Xu ZHANG; Peter S.F. CHAN

    2009-01-01

    Possible altered gene expression patterns in bladder turnout carcinogenesis in rat bladder cancers induced by BBN [N-butyl-N-(4-hydroxybutyl)nitrosamine] was examined by cDNA microarray analysis of gene expression profiles.Thirty Sprague-Dawley rats were given drinking water containing 0.05% BBN ad libitum for 24 to 28-weeks.Equal numbers of control rats were given tap water without BBN.After treatment,the rat bladders were excised for RNA extraction and histopathological examinations.Total RNAs were extracted from rat transitional cell carcinoma (TCC) tissues and micro-dissected normal rat bladder epithelia.The atlas glass rat microarray was used,which included oligonucleotides of 1081 rat genes.Some of the up-regulated genes in rat bladder TCCs were further confirmed by Northern blotting.Our results showed that the transcriptions of 30 genes were significantly elevated in the rat bladder TCCs,and these included fly proto-oncogene,Lipocortin 2,COX Ⅳ,COX Ⅴ a,and cathepsin D.Also,15 genes were significantly down-regulated in the rat bladder TCCs and they included B7.1,TNFrl,APOAI and VHL.The resuits of cDNA microarray analysis demonstrated that normal rat bladder epithelia and bladder TCC exhibited different and specific gene statement profiles.The increased expressions of the identified genes may play an important role in the chemically induced bladder carcinogenesis.

  5. TIMP-1 gene deficiency increases tumour cell sensitivity to chemotherapy-induced apoptosis

    DEFF Research Database (Denmark)

    Davidsen, Marie Louise; Würts, S.Ø.; Rømer, Maria Unni Koefoed;

    2006-01-01

    in cancer. In this regard, several studies have demonstrated an antiapoptotic effect of TIMP-1 in a number of different cell types. Since chemotherapy works by inducing apoptosis in cancer cells, we raised the hypothesis that TIMP-1 promotes resistance against chemotherapeutic drugs. In order to investigate...... this hypothesis, we have established TIMP-1 gene-deficient and TIMP-1 wild-type fibrosarcoma cells from mouse lung tissue. We have characterised these cells with regard to TIMP-1 genotype, TIMP-1 expression, malignant transformation and sensitivity to chemotherapy-induced apoptosis. We show that TIMP-1 gene...... deficiency increases the response to chemotherapy considerably, confirming that TIMP-1 protects the cells from apoptosis. This is to our knowledge the first study investigating TIMP-1 and chemotherapy-induced apoptosis employing a powerful model system comprising TIMP-1 gene-deficient cells...

  6. The model of defense gene expression induced by signaling molecule β-ocimene

    Institute of Scientific and Technical Information of China (English)

    LIU Chunlin; RUAN Ying; GUAN Chunyun

    2004-01-01

    @@ β-ocimene, a kind of monoterpene, was found recently as a plant communication signal molecule[1]. It has two isomeric forms in nature: cis-β-ocimene and trans-β- ocimene. According to recent reports, all investigated plants, such as corn, cotton, lima bean, potato, tobacco, arabidopsis, and Mediterranean pine, could release the chemical component β-ocimene after fed by arthropod herbivores[2-5], suggesting thatβ-ocimene is an important functioal component in the herbivore-induced volatile. Nowadays, we know that β-ocimene can induce the expression of defense genes relative to salicylic acid in detatched leaves. But many problems of β-ocimene, for example, whether β-ocimene can induce the defense gene expression in intact plants, what role it can play in the expression model of defense genes, are elusive[1,6].

  7. Muscle Contraction Induces Acute Hydroxymethylation of the Exercise-Responsive Gene Nr4a3

    DEFF Research Database (Denmark)

    Pattamaprapanont, Pattarawan; Garde, Christian; Fabre, Odile

    2016-01-01

    promoters. Exercise induces dynamic DNA demethylation at gene promoters; however, the contribution of the demethylation precursor hydroxymethylcytosine is unknown. Given the evanescent nature of hydroxymethylcytosine, a muscle contraction model that allows for the collection of samples that are repeatedly...... stimulated over time is required to determine whether contraction-induced demethylation is preceded by changes in the hydroxymethylcytosine level. Here, we established an acute skeletal muscle contraction model to mimic the effects of acute exercise on gene expression. We used this model to investigate...... the effect of muscle contraction on DNA demethylation and hydroxymethylation. First, we performed an acute exercise study in healthy humans to identify an exercise-responsive gene that we could study in culture. We identified the nuclear receptor subfamily 4 group A member 3 (Nr4a3) gene with the highest...

  8. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes.

    Science.gov (United States)

    Kato, Lucia; Begum, Nasim A; Burroughs, A Maxwell; Doi, Tomomitsu; Kawai, Jun; Daub, Carsten O; Kawaguchi, Takahisa; Matsuda, Fumihiko; Hayashizaki, Yoshihide; Honjo, Tasuku

    2012-02-14

    Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.

  9. The Agricultural Antibiotic Carbadox Induces Phage-mediated Gene Transfer in Salmonella

    Directory of Open Access Journals (Sweden)

    Bradley L. Bearson

    2014-02-01

    Full Text Available Antibiotics are used for disease therapeutic or preventative effects in humans and animals, as well as for enhanced feed conversion efficiency in livestock. Antibiotics can also cause undesirable effects in microbial populations, including selection for antibiotic resistance, enhanced pathogen invasion, and stimulation of horizontal gene transfer. Carbadox is a veterinary antibiotic used in the U.S. during the starter phase of swine production for improved feed efficiency and control of swine dysentery and bacterial swine enteritis. Carbadox has been shown in vitro to induce phage-encoded Shiga toxin in Shiga toxin-producing Escherichia coli and a phage-like element transferring antibiotic resistance genes in Brachyspira hyodysenteriae, but the effect of carbadox on prophages in other bacteria is unknown. This study examined carbadox exposure on prophage induction and genetic transfer in Salmonella enterica serovar Typhimurium, a human foodborne pathogen that frequently colonizes swine without causing disease. S. Typhimurium LT2 exposed to carbadox induced prophage production, resulting in bacterial cell lysis and release of virions that were visible by electron microscopy. Carbadox induction of phage-mediated gene transfer was confirmed by monitoring the transduction of a sodCIII::neo cassette in the Fels-1 prophage from LT2 to a recipient Salmonella strain. Furthermore, carbadox frequently induced generalized transducing phages in multidrug-resistant phage type DT104 and DT120 isolates, resulting in the transfer of chromosomal and plasmid DNA that included antibiotic resistance genes. Our research indicates that exposure of Salmonella to carbadox induces prophages that can transfer virulence and antibiotic resistance genes to susceptible bacterial hosts. Carbadox-induced, phage-mediated gene transfer could serve as a contributing factor in bacterial evolution during animal production, with prophages being a reservoir for bacterial fitness

  10. Reduced Tyk2 gene expression in β-cells due to natural mutation determines susceptibility to virus-induced diabetes

    OpenAIRE

    Izumi, Kenichi; Mine, Keiichiro; Inoue, Yoshitaka; Teshima, Miho; Ogawa, Shuichiro; Kai, Yuji; Kurafuji, Toshinobu; Hirakawa, Kanako; Miyakawa, Daiki; Ikeda, Haruka; Inada, Akari; Hara, Manami; Yamada, Hisakata; Akashi, Koichi; Niho, Yoshiyuki

    2015-01-01

    Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 backgrou...

  11. Stable oncogenic transformation induced by microcell-mediated gene transfer

    Institute of Scientific and Technical Information of China (English)

    吕有勇; Donald G.Blair

    1995-01-01

    Oncogenes have been identified using DNA-mediated transfection, but the size of the transferable and unrearranged DNA, gene rearrangement and amplification which occur during the transfection process limit the use of the techniques. We have evaluated microcell-mediated gene transfer techniques for the transfer and analysis of dominant oncogenes. MNNG-HOS, a transformed human cell line which contained the met oncogene mapping to human chromosome 7 was infected with retroviruses carrying drug resistance markers and used to optimize microcell preparation and transfer. Stable and drug-resistant hybrids containing single human chromosomes as well as the foci of the transformed cells containing the activated met oncogene and intact hitman chromosomes were obtained. Hybridization analysis with probes (i.e. collA2, pJ3.11) mapping up to 1 Mb away from met shows that the cells from the individual focr contain different amounts of apparently unrearranged human DNA associated with the oncogene, and the microcell-g

  12. Differential expression of genes during aflatoxin B1-induced hepatocarcinogenesis in tree shrews

    Science.gov (United States)

    Li, Yuan; Wan, Da-Fang; Su, Jian-Jia; Cao, Ji; Ou, Chao; Qiu, Xiao-Kun; Ban, Ke-Chen; Yang, Chun; Qin, Liu-Liang; Luo, Dan; Yue, Hui-Fen; Zhang, Li-Sheng; Gu, Jian-Ren

    2004-01-01

    AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B1 (AFB1), to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism. METHODS: Tree shrews (Tupaia belangeri chinensis) were treated with or without AFB1 for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding non-cancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of AtlasTM cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared. RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as “important genes” because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC, genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC. CONCLUSION: A considerable number of genes could

  13. ohr, Encoding an Organic Hydroperoxide Reductase, Is an In Vivo-Induced Gene in Actinobacillus pleuropneumoniae

    OpenAIRE

    Shea, Robin J.; Mulks, Martha H.

    2002-01-01

    Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a disease characterized by pulmonary necrosis and hemorrhage caused in part by neutrophil degranulation. In an effort to understand the pathogenesis of this disease, we have developed an in vivo expression technology (IVET) system to identify genes that are specifically up-regulated during infection. One of the genes that we have identified as being induced in vivo is ohr, encoding organic hydroperoxide reducta...

  14. Glucose ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat oxidative genes.

    Science.gov (United States)

    Civitarese, Anthony E; Hesselink, Matthijs K C; Russell, Aaron P; Ravussin, Eric; Schrauwen, Patrick

    2005-12-01

    Ingestion of carbohydrate during exercise may blunt the stimulation of fat oxidative pathways by raising plasma insulin and glucose concentrations and lowering plasma free fatty acid (FFA) levels, thereby causing a marked shift in substrate oxidation. We investigated the effects of a single 2-h bout of moderate-intensity exercise on the expression of key genes involved in fat and carbohydrate metabolism with or without glucose ingestion in seven healthy untrained men (22.7 +/- 0.6 yr; body mass index: 23.8 +/- 1.0 kg/m(2); maximal O(2) consumption: 3.85 +/- 0.21 l/min). Plasma FFA concentration increased during exercise (P glucose ingestion, whereas fat oxidation (indirect calorimetry) was higher in the fasted state vs. glucose feeding (P expression of pyruvate dehydrogenase kinase-4 (P glucose ingestion during exercise produced minimal effects on the expression of genes involved in carbohydrate utilization. However, glucose ingestion resulted in a decrease in the expression of genes involved in fatty acid transport and oxidation (CD36, carnitine palmitoyltransferase-1, uncoupling protein 3, and 5'-AMP-activated protein kinase-alpha(2); P glucose ingestion during exercise decreases the expression of genes involved in lipid metabolism rather than increasing genes involved in carbohydrate metabolism.

  15. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  16. Construction of an inducible cell-communication system that amplifies Salmonella gene expression in tumor tissue.

    Science.gov (United States)

    Dai, Yumei; Toley, Bhushan J; Swofford, Charles A; Forbes, Neil S

    2013-06-01

    Bacterial therapies have the potential to overcome resistances that cause chemotherapies to fail. When using bacteria to produce anticancer agents in tumors, triggering gene expression is necessary to prevent systemic toxicity. The use of chemical triggers, however, is hampered by poor delivery of inducing molecules, which reduces the number of activated bacteria. To solve this problem, we created a cell-communication system that enables activated bacteria to induce inactive neighbors. We hypothesized that introducing cell communication into Salmonella would improve direct triggering strategies by increasing protein production, increasing sensitivity to inducer molecules, and enabling expression in tumor tissue. To test these hypotheses we integrated the PBAD promoter into the quorum-sensing machinery from Vibrio fischeri. The expression of a fluorescent reporter gene was compared to expression from non-communicating controls. Function in three-dimensional tissue was tested in a tumor-on-a-chip device. Bacterial communication increased fluorescence 40-fold and increased sensitivity to inducer molecules more than 10,000-fold. The system enabled bacteria to activate neighbors and increased the time-scale of protein production. Gene expression was controllable and tightly regulated. At the optimal inducing signal, communicating bacteria produced 350 times more protein than non-communicating bacteria. The cell-communication system created in this study has uses beyond cancer therapy, including protein manufacturing, bioremediation and biosensing. It would enable amplified induction of gene expression in any environment that limits availability of inducer molecules. Ultimately, because inducible cellular communication enables gene expression in tissue, it will be a critical component of bacterial anticancer therapies.

  17. Importance of interferon inducible trans-membrane proteins and retinoic acid inducible gene I for influenza virus replication: A review.

    Science.gov (United States)

    Suo, Siqingaowa; Ren, Xiaofeng

    2016-01-01

    Understanding the interplay between Influenza viruses and host cells is key to elucidating the pathogenesis of these viruses. Several host factors have been identified that exert antiviral functions; however, influenza viruses continue to replicate utilizing host cell machinery. Herein, we review the mechanisms of action of two host-derived proteins on conferring cellular resistance to the influenza virus; (1) the interferon inducible trans-membrane proteins, 1, 2 and 3, a recently identified family of early restriction factors; and (2) retinoic acid inducible gene I, a key mediator of antiviral immunity. These data may contribute to the design of novel and efficient anti-influenza treatments.

  18. Gene expression in rats with Barrett's esophagus and esophageal adenocarcinoma induced by gastroduodenoesophageal reflux

    Institute of Scientific and Technical Information of China (English)

    Peng Cheng; Jun Gong; Tao Wang; Jie Chen; Gui-Sheng Liu; Ru Zhang

    2005-01-01

    AIM: To study the different gene expression profiles in rats with Barrett's esophagus (BE) and esophageal adenocarcinoma (EA) induced by gastro-duodenoesophageal reflux.METHODS: Esophagoduodenostomy was performed in 8-wk old Sprague-Dawley rats to induce gastro-duodenoesophageal reflux, and a group of rats that received sham operation served as control. Esophageal epithelial pathological tissues were dissected and frozen in liquid nitrogen immediately. The expression profiles of 4 096genes in EA and BE tissues were compared to normal esophagus epithelium in normal control (NC) by cDNA microarray.RESULTS: Four hundred and forty-eight genes in BE were more than three times different from those in NC, including 312 upregulated and 136 downregulated genes. Three hundred and seventy-seven genes in EA were more than three times different from those in NC, including 255upregulated and 142 downregulated genes. Compared to BE, there were 122 upregulated and 156 downregulated genes in EA. In the present study, the interested genes were those involved in carcinogenesis. Among them, the upregulated genes included cathepsin C, aminopeptidase M, arachidonic acid epoxygenase, tryptophan-2,3-dioxygenase, ubiquitin-conjugating enzyme, cyclic GMP-stimulated phosphodiesterase, tissue inhibitor of metalloproteinase-1, betaine-homocysteine methyltransferase, lysozyme, complement 4b binding protein,complement 9 protein, insulin-like growth factor binding protein, UDP-glucuronosyltransferase, tissue inhibitor of metalloproteinase-3, aldolase B, retinoid X receptor gamma, carboxylesterase and testicular cell adhesion molecule 1. The downregulated genes included glutathione synthetase, lecithin-cholesterol acyltransferase, p55CDC,heart fatty acid binding protein, cell adhesion regulator and endothelial cell selectin ligand.CONCLUSION: Esophageal epithelium exposed excessively to harmful ingredients of duodenal and gastric reflux may develop into BE and even EA gradually. The gene

  19. Effect of electro-acupuncture on gene expression in heart of rats with stress-induced pre-hypertension based on gene chip technology.

    Science.gov (United States)

    Guo, Yan; Xie, Xiaojia; Guo, Changqing; Wang, Zhaoyang; Liu, Qingguo

    2015-06-01

    To explore electro-acupuncture's (EA's) effect on gene expression in heart of rats with stress-induced pre-hypertension and try to reveal its biological mechanism based on gene chip technology. Twenty-seven Wistar male rats were randomly divided into 3 groups. The stress-induced hypertensive rat model was prepared by electric foot-shocks combined with generated noise. Molding cycle lasted for 14 days and EA intervene was applied,on rats in model + EA group during model preparation. Rat Gene 2.0 Sense Target Array technology was used for the determination of gene expression profiles and the screened key genes were verified by real-time quantitative polymerase chain reaction (RT-PCR) method. Compared with blank control group, 390 genes were changed in model group; compared with model control group, 330 genes were changed in model+EA group. Significance analysis of gene function showed that the differentially expressed genes are those involved in biological process, molecular function and cellular components. RT-PCR result of the screened key genes is consistent with that of gene chip test. EA could significantly lower blood pressure of stress-induced pre-hypertension rats and affect its gene expression profile in heart. Genes that related to the contraction of vascular smooth muscle may be involved in EA's anti-hypertensive mechanism.

  20. Functional connections and pathways of coenzyme Q10-inducible genes: an in-silico study.

    Science.gov (United States)

    Schmelzer, Constance; Lindner, Inka; Vock, Christina; Fujii, Kenji; Döring, Frank

    2007-10-01

    Coenzyme Q10 (CoQ10, ubiquinone) is an essential cofactor in the electron transport chain, serves as a potent antioxidant in mitochondria and lipid membranes, and is often used as a dietary supplement for a number of diseases including cardiovascular diseases. Recently, we obtained evidence that CoQ10 (Kaneka Q10) affects the expression of hundreds of human genes. To decipher the functional and regulatory connections of these genes, a literature search combined with transcription factor binding site analysis was performed using Genomatix BiblioSphere and MatInspector. This in-silico analysis revealed 17 CoQ10-inducible genes which are functionally connected by signalling pathways of G-protein coupled receptors, JAK/STAT, integrin, and beta-arrestin. Promoter analysis of these CoQ10-inducible genes showed one group of NF B-regulated genes, namely IL5, thrombin, vitronectin receptor and C-reactive protein (CRP). Furthermore, a common promoter framework containing binding sites of the transcription factor families EVI1, HOXF, HOXC, and CLOX was identified in the promoters of IL5, CRP, and vitronectin receptor. The identified CoQ10-inducible genes and pathways play an important role in inflammatory response. Since these effects are based on an in-vitro study, the effect of CoQ10 on vascular health in vivo needs to be addressed in further animal and/or human intervention studies.

  1. A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

    Science.gov (United States)

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

    2015-07-28

    West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.

    Science.gov (United States)

    Gómez-Santos, Nuria; Treuner-Lange, Anke; Moraleda-Muñoz, Aurelio; García-Bravo, Elena; García-Hernández, Raquel; Martínez-Cayuela, Marina; Pérez, Juana; Søgaard-Andersen, Lotte; Muñoz-Dorado, José

    2012-04-01

    Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.

  3. Cutin monomer induces expression of the rice OsLTP5 lipid transfer protein gene.

    Science.gov (United States)

    Kim, Tae Hyun; Park, Jong Ho; Kim, Moon Chul; Cho, Sung Ho

    2008-01-01

    Treatment with the cutin monomer 16-hydroxypalmitic acid (HPA), a major component of cutin, elicited the synthesis of hydrogen peroxide (H2O2) in rice leaves and induced the expression of the lipid transfer protein gene OsLTP5. Treatment with HPA also induced expression of OsLTP1, OsLTP2, and the pathogen-related PR-10 genes to a lesser extent. The OsLTP5 transcript was expressed prominently in stems and flowers, but was barely detectable in leaves. Expression of OsLTP5 was induced in shoots in response to ABA and salicylic acid. It is proposed that HPA is perceived by rice as a signal, inducing defense reactions.

  4. In Vivo Imaging of Local Gene Expression Induced by Magnetic Hyperthermia

    Science.gov (United States)

    Sandre, Olivier; Genevois, Coralie; Garaio, Eneko; Adumeau, Laurent; Mornet, Stéphane; Couillaud, Franck

    2017-01-01

    The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release. PMID:28208731

  5. In Vivo Imaging of Local Gene Expression Induced by Magnetic Hyperthermia

    Directory of Open Access Journals (Sweden)

    Olivier Sandre

    2017-02-01

    Full Text Available The present work aims to demonstrate that colloidal dispersions of magnetic iron oxide nanoparticles stabilized with dextran macromolecules placed in an alternating magnetic field can not only produce heat, but also that these particles could be used in vivo for local and noninvasive deposition of a thermal dose sufficient to trigger thermo-induced gene expression. Iron oxide nanoparticles were first characterized in vitro on a bio-inspired setup, and then they were assayed in vivo using a transgenic mouse strain expressing the luciferase reporter gene under transcriptional control of a thermosensitive promoter. Iron oxide nanoparticles dispersions were applied topically on the mouse skin or injected subcutaneously with Matrigel™ to generate so-called pseudotumors. Temperature was monitored continuously with a feedback loop to control the power of the magnetic field generator and to avoid overheating. Thermo-induced luciferase expression was followed by bioluminescence imaging 6 h after heating. We showed that dextran-coated magnetic iron oxide nanoparticle dispersions were able to induce in vivo mild hyperthermia compatible with thermo-induced gene expression in surrounding tissues and without impairing cell viability. These data open new therapeutic perspectives for using mild magnetic hyperthermia as noninvasive modulation of tumor microenvironment by local thermo-induced gene expression or drug release.

  6. Degranulation of rat cerebellum induces selective variations in gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Eliyahu, D.; Soreq, H.

    1982-02-01

    Selective variations in the composition of poly(A)-containing mRNA were found to be induced in the rat cerebellum by X-irradiation. mRNA populations prepared from normal and X-irradiated rat cerebella at different stages of their development displayed equal efficiencies when translated in vitro in reticulocyte lysates. Specific differences were revealed, however, when the labeled translation products of both mRNA preparations were subjected to two-dimensional gel electrophoresis followed by fluorography of the dried gels. Of more than 100 polypeptide products, several showed marked intensity differences, indicating changes in the abundance of their directing mRNA species. These differences appear both in developing and in mature cerebellar mRNA, and the extent of modification in mRNA is much higher than the consequent changes in the composition of proteins in the irradiated cerebellum. The degranulation-induced modifications in levels of specific cerebellar mRNA species can be used to identify proteins whose biosynthesis depends on the presence of interneurons.

  7. Molecular cloning and functional analyses of low-temperature induced genes from Ammopiptanthus mongolicus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Studies on the cold-responsive genes and cold signaling of woody species drop far behind in comparison to herbaceous plants.Due to similar lignified structure,perennial characteristic,and enhanced tolerance,it seems much easier to find strongly antifreeze genes and obtain effective results in transgenic woody plants.In this study,Ammopiptanthus mongolicus,an evergreen,broadleaf and cold-resist leguminous shrub growing in the desert of Inner Mongolia,was used as a material for low-temperature induced gene is...

  8. [Osmotic shock induces expression of Vibrio fischeri lux genes in Escherichia coli cells].

    Science.gov (United States)

    Zavil'gel'skiĭ, G B; Kotova, V Iu

    2003-04-01

    The effect of osmotic shock on the expression of genes in the lux regulon of marine bacteria Vibrio fischeri was studied in cells of Escherichia coli. Bioluminescence of cells was shown to drastically increase, when cells were exposed to osmotic shock at the early logarithmic growth phase. The expression of lux genes induced by osmotic shock is determined by the two-component regulatory system RcsC-RcsB. A nucleotide sequence in the regulatory region of the luxR gene homologous to the RcsB-box consensus of E. coli is assumed to be a primary site for this system.

  9. Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes

    DEFF Research Database (Denmark)

    Ronchel, M.C.; Molina, L.; Witte, A.;

    1998-01-01

    Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides, The physiological effects that lead to cell death...... upon the induction of expression of two different heterologous killing genes in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed, P. putida CMC4 and CMC12 carry in their chromosomes a fusion of the PAl-04/03 promoter to the Escherichia coli gef gene and the phi X174 lysis gene E......, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of P. putida CMC12 cells mediated by phi X174 lysis protein E was faster than that observed for P. putida CMC4, for which the Gef...

  10. Identification of genes regulated during mechanical load-induced cardiac hypertrophy

    Science.gov (United States)

    Johnatty, S. E.; Dyck, J. R.; Michael, L. H.; Olson, E. N.; Abdellatif, M.; Schneider, M. (Principal Investigator)

    2000-01-01

    Cardiac hypertrophy is associated with both adaptive and adverse changes in gene expression. To identify genes regulated by pressure overload, we performed suppressive subtractive hybridization between cDNA from the hearts of aortic-banded (7-day) and sham-operated mice. In parallel, we performed a subtraction between an adult and a neonatal heart, for the purpose of comparing different forms of cardiac hypertrophy. Sequencing more than 100 clones led to the identification of an array of functionally known (70%) and unknown genes (30%) that are upregulated during cardiac growth. At least nine of those genes were preferentially expressed in both the neonatal and pressure over-load hearts alike. Using Northern blot analysis to investigate whether some of the identified genes were upregulated in the load-independent calcineurin-induced cardiac hypertrophy mouse model, revealed its incomplete similarity with the former models of cardiac growth. Copyright 2000 Academic Press.

  11. Identification of genes regulated during mechanical load-induced cardiac hypertrophy

    Science.gov (United States)

    Johnatty, S. E.; Dyck, J. R.; Michael, L. H.; Olson, E. N.; Abdellatif, M.; Schneider, M. (Principal Investigator)

    2000-01-01

    Cardiac hypertrophy is associated with both adaptive and adverse changes in gene expression. To identify genes regulated by pressure overload, we performed suppressive subtractive hybridization between cDNA from the hearts of aortic-banded (7-day) and sham-operated mice. In parallel, we performed a subtraction between an adult and a neonatal heart, for the purpose of comparing different forms of cardiac hypertrophy. Sequencing more than 100 clones led to the identification of an array of functionally known (70%) and unknown genes (30%) that are upregulated during cardiac growth. At least nine of those genes were preferentially expressed in both the neonatal and pressure over-load hearts alike. Using Northern blot analysis to investigate whether some of the identified genes were upregulated in the load-independent calcineurin-induced cardiac hypertrophy mouse model, revealed its incomplete similarity with the former models of cardiac growth. Copyright 2000 Academic Press.

  12. Gene Analysis of Arsenic Trioxide—induced Apoptosis of Lymphoma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANGZidong; LIWeiyu; 等

    2002-01-01

    Objective The effect of arsenic trioxide on apoptosis gene expression of Raji cell was explored when Raji cells were incubated with 0.5μmol/L of arsenic trioxide for 6h。Methods Cell culture,extraction and isolation of mRNA,preparation of probes labeled with fluorescence,hybridization technique of DNA chip(each chip containing 200 apoptosis genes,Chinese Shanghai Biostar,In.)were used.Results Arsenic trioxide induced significant changes in 10%(20/200 genes)of the apoptosis genes:18 genes were downregulated,only two upregulated.In particular,inhibitors of apoptosis protein,such as X-linked inhibitor of apoptosis protein,were significantly downregulated.P53 and the other apoptosis genes were also downregulatec.Of the upregulated genes,high expression of heat-shock protein could promote apoptosis of Raji cells.Conclusion The inhibitors of apoptosis protein play an important role in the process of arsenic trioxide-induced apoptosis of Raji cells.

  13. Acute ozone-induced differential gene expression profiles in rat lung.

    Science.gov (United States)

    Nadadur, Srikanth S; Costa, Daniel L; Slade, Ralph; Silbjoris, Robert; Hatch, Gary E

    2005-12-01

    Ozone is an oxidant gas that can directly induce lung injury. Knowledge of the initial molecular events of the acute O3 response would be useful in developing biomarkers of exposure or response. Toward this goal, we exposed rats to toxic concentrations of O3 (2 and 5 ppm) for 2 hr and the molecular changes were assessed in lung tissue 2 hr postexposure using a rat cDNA expression array containing 588 characterized genes. Gene array analysis indicated differential expression in almost equal numbers of genes for the two exposure groups: 62 at 2 ppm and 57 at 5 ppm. Most of these genes were common to both exposure groups, suggesting common roles in the initial toxicity response. However, we also identified the induction of nine genes specific to 2-ppm (thyroid hormone-beta receptor c-erb-A-beta; and glutathione reductase) or 5-ppm exposure groups (c-jun, induced nitric oxide synthase, macrophage inflammatory protein-2, and heat shock protein 27). Injury markers in bronchoalveolar lavage fluid (BALF) were used to assess immediate toxicity and inflammation in rats similarly exposed. At 2 ppm, injury was marked by significant increases in BALF total protein, N-acetylglucosaminidase, and lavageable ciliated cells. Because infiltration of neutrophils was observed only at the higher 5 ppm concentration, the distinctive genes suggested a potential amplification role for inflammation in the gene profile. Although the specific gene interactions remain unclear, this is the first report indicating a dose-dependent direct and immediate induction of gene expression that may be separate from those genes involved in inflammation after acute O3 exposure.

  14. Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation.

    Science.gov (United States)

    Obayashi, Shinya; Tabunoki, Hiroko; Kim, Seung U; Satoh, Jun-ichi

    2009-05-01

    Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

  15. Regulation of mouse hepatic genes in response to diet induced obesity, insulin resistance and fasting induced weight reduction

    Directory of Open Access Journals (Sweden)

    Mantzoros Christos

    2005-06-01

    Full Text Available Abstract Background Obesity is associated with insulin resistance that can often be improved by caloric restriction and weight reduction. Although many physiological changes accompanying insulin resistance and its treatment have been characterized, the genetic mechanisms linking obesity to insulin resistance are largely unknown. We used DNA microarrys and RT-PCR to investigate significant changes in hepatic gene transcription in insulin resistant, diet-induced obese (DIO-C57/BL/6J mice and DIO-C57/BL/6J mice fasted for 48 hours, whose weights returned to baseline levels during these conditions. Results Transcriptional profiling of hepatic mRNA revealed over 1900 genes that were significantly perturbed between control, DIO, and fasting/weight reduced DIO mice. From this set, our bioinformatics analysis identified 41 genes that rigorously discriminate these groups of mice. These genes are associated with molecular pathways involved in signal transduction, and protein metabolism and secretion. Of particular interest are genes that participate in pathways responsible for modulating insulin sensitivity. DIO altered expression of genes in directions that would be anticipated to antagonize insulin sensitivity, while fasting/ weight reduction partially or completely normalized their levels. Among these discriminatory genes, Sh3kbp1 and RGS3, may have special significance. Sh3kbp1, an endogenous inhibitor of PI-3-kinase, was upregulated by high-fat feeding, but normalized to control levels by fasting/weight reduction. Because insulin signaling occurs partially through PI-3-kinase, increased expression of Sh3kbp1 by DIO mice may contribute to hepatic insulin resistance via inhibition of PI-3-kinase. RGS3, a suppressor of G-protein coupled receptor generation of cAMP, was repressed by high-fat feeding, but partially normalized by fasting/weight reduction. Decreased expression of RGS3 may augment levels of cAMP and thereby contribute to increased, cAMP-induced

  16. Bifidobacterium bifidum Actively Changes the Gene Expression Profile Induced by Lactobacillus acidophilus in Murine Dendritic Cells

    DEFF Research Database (Denmark)

    Weiss, Gudrun Margarethe; Rasmussen, Simon; Fink, Lisbeth Nielsen;

    2010-01-01

    Dendritic cells (DC) play a pivotal regulatory role in activation of both the innate as well as the adaptive immune system by responding to environmental microorganisms. We have previously shown that Lactobacillus acidophilus induces a strong production of the pro-inflammatory and Th1 polarizing...... cytokine IL-12 in DC, whereas bifidobacteria do not induce IL-12 but inhibit the IL-12 production induced by lactobacilli. In the present study, genome-wide microarrays were used to investigate the gene expression pattern of murine DC stimulated with Lactobacillus acidophilus NCFM and Bifidobacterium...

  17. Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Jørgensen, Sebastian Beck; Wojtaszewski, Jørgen; Viollet, Benoit

    2005-01-01

    We tested the hypothesis that 5'AMP-activated protein kinase (AMPK) plays an important role in regulating the acute, exercise-induced activation of metabolic genes in skeletal muscle, which were dissected from whole-body a2- and a1-AMPK knockout (KO) and wild-type (WT) mice at rest, after treadmi...

  18. Exercise induces transient transcriptional activation of the PGC-1a gene in human skeletal muscle

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P. Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1a (PGC-1a) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell...

  19. Transcriptomic sequencing reveals a set of unique genes activated by butyrate-induced histone modification

    Science.gov (United States)

    Butyrate is a nutritional element with strong epigenetic regulatory activity as an inhibitor of histone deacetylases (HDACs). Based on the analysis of differentially expressed genes induced by butyrate in the bovine epithelial cell using deep RNA-sequencing technology (RNA-seq), a set of unique gen...

  20. Abrogation of p53-induced apoptosis by the hepatitis B virus X gene.

    NARCIS (Netherlands)

    X.W. Wang (Xin Wei); M.K. Gibson (Michael); W. Vermeulen (Wim); H. Yeh; K. Forrester; H.-W. Stürzbecher; J.H.J. Hoeijmakers (Jan); C.C. Harris

    1996-01-01

    textabstractThe p53 tumor suppressor gene product is a transcriptional transactivator and a potent apoptotic inducer. The fact that many of the DNA tumor virus oncoproteins bind to p53 and affect these p53 functions indicates that this interaction is an important step in oncogenic transformation. We

  1. Gene expression profiling and pathway analysis of hepatotoxicity induced by triptolide in Wistar rats.

    Science.gov (United States)

    Wang, Jiaying; Jiang, Zhenzhou; Ji, Jinzi; Wang, Xinzhi; Wang, Tao; Zhang, Yun; Tai, Ting; Chen, Mi; Sun, Lixin; Li, Xia; Zhang, Luyong

    2013-08-01

    Triptolide (TP), a major component of TWHF, is widely used to treat rheumatoid arthritis, systemic lupus erythematosus, nephritis and leprosy. However, its clinical use is limited by hepatotoxicity. To further elucidate the underlying mechanism of its hepatotoxic effects, hepatic gene expression profiles were analyzed. TP (1000 and 300 μg/kg) was orally administered to Wistar rats for 14 days. Current study indicated that female rats were more sensitive to TP-induced hepatotoxicity than males. Genome-wide microarray analyses identified 3329 differentially expressed genes in liver of female rats. Analyses of these genes identified over-represented functions associated with insulin signaling pathway, glucose metabolism, cell cycle, oxidative stress and apoptosis, which were consistent with the results of significant increase of Caspase-3 activity and reduction of serum glucose, GSH/GSSG ratio, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities, liver glycogen. In addition, it was observed for the first time that glucocorticoids and IGF1 might get involved in TP-induced hepatotoxicity. These data suggest that TP treatment could alter the hepatic redox status, reduce serum glucose and induce hepatocyte apoptosis, consistent with the differential expression of genes involved in insulin signaling pathway, glucose metabolism pathway and cell stress pathway, all of which might contribute to the overall TP-induced hepatotoxicity.

  2. An in planta induced gene of Phytophthora infestans codes for ubiquitin

    NARCIS (Netherlands)

    Pieterse, C.M.J.; Risseeuw, E.P.; Davidse, L.C.

    1991-01-01

    An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was selected from a genomic library by differential hybridization using labelled cDNA derived from poly(A)+ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P. infestans interaction

  3. An in planta induced gene of Phytophthora infestans codes for ubiquitin

    NARCIS (Netherlands)

    Pieterse, C.M.J.; Risseeuw, E.P.; Davidse, L.C.

    1991-01-01

    An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was isolated from a genomic library by differential hybridization using labelled cDNA derived from poly(A)⁺ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P,

  4. Gene expression array analyses predict increased proto-oncogene expression in MMTV induced mammary tumors.

    Science.gov (United States)

    Popken-Harris, Pamela; Kirchhof, Nicole; Harrison, Ben; Harris, Lester F

    2006-08-01

    Exogenous infection by milk-borne mouse mammary tumor viruses (MMTV) typically induce mouse mammary tumors in genetically susceptible mice at a rate of 90-95% by 1 year of age. In contrast to other transforming retroviruses, MMTV acts as an insertional mutagen and under the influence of steroid hormones induces oncogenic transformation after insertion into the host genome. As these events correspond with increases in adjacent proto-oncogene transcription, we used expression array profiling to determine which commonly associated MMTV insertion site proto-oncogenes were transcriptionally active in MMTV induced mouse mammary tumors. To verify our gene expression array results we developed real-time quantitative RT-PCR assays for the common MMTV insertion site genes found in RIII/Sa mice (int-1/wnt-1, int-2/fgf-3, int-3/Notch 4, and fgf8/AIGF) as well as two genes that were consistently up regulated (CCND1, and MAT-8) and two genes that were consistently down regulated (FN1 and MAT-8) in the MMTV induced tumors as compared to normal mammary gland. Finally, each tumor was also examined histopathologically. Our expression array findings support a model whereby just one or a few common MMTV insertions into the host genome sets up a dominant cascade of events that leave a characteristic molecular signature.

  5. Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

    Science.gov (United States)

    Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei

    2016-05-01

    Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

  6. Identification and characterization of the inducible murine mast cell gene, imc-415.

    Science.gov (United States)

    Cho, S H; Cho, J J; Kim, I S; Vliagoftis, H; Metcalfe, D D; Oh, C K

    1998-11-01

    Activation of mast cells results in the generation and release of bioactive mediators which in turn initiate allergic inflammation. Mast cell function is enhanced following stimulation in part because of the induction of specific genes and their products. To identify additional genes induced in mast cells that support this process, we thus constructed an activation-specific mast cell subtraction library. To date, we have isolated 26 novel inducible murine mast cell (imc) cDNA clones. Among them, a full-coding region of the murine gene imc-415 was found to have a greater than 90% nucleotide sequence homology and a 97.5% amino acid sequence homology to both a human beta4 integrin-binding protein (p27(BBP)) and a human translation initiation factor 6 (eIF6), which in turn are identical. In vitro translation of the imc-415 gene yielded a band of an approximately 26 kDa. This is the same as the calculated molecular weight of murine IMC-415 protein based on the predicted amino acid sequence and is the molecular weight of p27(BBP)/eIF6. Murine imc-415 message was also induced in inflamed lung tissues in a mouse model of asthma. These results suggest a role for murine imc-415 in allergic inflammation where it may enhance protein synthesis. Human eIF6/p27(BBP) may also play a role in allergic diseases based on the similarities in sequence and in gene expression patterns.

  7. Cloning of murine BRI3 gene and study on its function for inducing cell death

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.

  8. Gene-gun DNA vaccination aggravates respiratory syncytial virus-induced pneumonitis

    DEFF Research Database (Denmark)

    Bartholdy, Christina; Olszewska, Wieslawa; Stryhn, Anette

    2004-01-01

    elicited with recombinant vaccinia virus expressing the complete RSV M2 protein, but stronger than those induced by a similar DNA construct without the beta2m gene. DNA vaccination led to enhanced pulmonary disease after RSV challenge, with increased weight loss and cell recruitment to the lung. Depletion......A CD8+ T-cell memory response to respiratory syncytial virus (RSV) was generated by using a DNA vaccine construct encoding the dominant Kd-restricted epitope from the viral transcription anti-terminator protein M2 (M2(82-90)), linked covalently to human beta2-microglobulin (beta2m). Cutaneous gene......+ T-cell responses were not induced. Thus, in addition to specific CD8+ T cell-mediated immunopathology, gene-gun DNA vaccination causes non-specific enhancement of RSV disease without affecting virus clearance....

  9. Identification of LPS-inducible genes downregulated by ubiquinone in human THP-1 monocytes.

    Science.gov (United States)

    Schmelzer, Constance; Döring, Frank

    2010-01-01

    Coenzyme Q(10) (CoQ(10)) is an obligatory element in the respiratory chain and functions as a potent antioxidant of lipid membranes. More recently, anti-inflammatory effects as well as an impact of CoQ(10) on gene expression have been observed. To reveal putative effects of Q(10) on LPS-induced gene expression, whole genome expression analysis was performed in the monocytic cell line THP-1. Thousand one hundred twenty-nine and 710 probe sets have been identified to be significantly (P genes revealed a functional connection in the NFkappaB pathway and confirmed our applied in vitro stimulation model. Moreover, 33 LPS-sensitive genes have been identified to be significantly downregulated by Q(10)-treatment between a factor of 1.32 and 1.85. GeneOntology (GO) analysis revealed for the Q(10)-sensitve genes a primary involvement in protein metabolism (e.g., HERC1 and EPS15), cell proliferation (e.g., CCDC100 and SMURF1), and transcriptional processes (e.g., CNOT4 and STK4). Three genes were either related to NFkappaB transcription factor activity (ERC1), cytokinesis (DIAPH2), or modulation of oxidative stress (MSRA). In conclusion, our data provide evidence that Q(10) downregulates LPS-inducible genes in the monocytic cell line THP-1. Thus, the previously described effects of Q(10) on the reduction of proinflammatory mediators might be due to its antioxidant impact on gene expression.

  10. Gene expression in Barrett's esophagus and reflux esophagitis induced by gastroduodenoesophageal reflux in rats

    Institute of Scientific and Technical Information of China (English)

    Peng Cheng; Jun Gong; Tao Wang; Chen Jie; Gui-Sheng Liu; Ru Zhang

    2005-01-01

    AIM: To investigate the difference of gene expression profiles between Barrett's esophagus and reflux esophagitis induced by gastroduodenoesophageal reflux in rats.METHODS: Eight-week-old Sprague-Dawley rats were treated esophagoduodenostomy to produce gastroduodenoesophageal reflux, and another group received sham operation as control. Esophageal epithelial tissues were dissected and frozen in liquid nitrogen immediately for pathology 40 wk after surgery. The expression profiles of 4 096 genes in reflux esophagitis and Barrett's esophagus tissues were compared with normal esophageal epithelium by cDNA microarray.RESULTS: Four hundred and forty-eight genes in Barrett'sesophagus were more than three times different from those in normal esophageal epithelium, including 312 up regulated and 136 down-regulated genes. Two hundred and thirty-twogenes in RE were more than three times different from those in normal esophageal epithelium, 90up-regulated and 142 down-regulated genes. Compared to reflux esophagitis, there were 214 up-regulated and 142 down-regulated genes in Barrett's esophagus. CONCLUSION: Esophageal epithelium exposed excessively to harmful ingredients of duodenal and gastric reflux can develop esophagitis and Barrett's esophagus gradually.The gene expression level is different between reflux esophagitis and Barrett's esophagus and the differentially expressed genes might be related to the occurrence and development of Barrett's esophagus and the promotion or progression in adenocarcinoma.

  11. Combining Click Chemistry-Based Proteomics With Dox-Inducible Gene Expression.

    Science.gov (United States)

    Gebert, J; Schnölzer, M; Warnken, U; Kopitz, J

    2017-01-01

    Inactivating mutations in single genes can trigger, prevent, promote, or alleviate diseases. Identifying such disease-related genes is a main pillar of medical research. Since proteins play a crucial role in mediating these effects, their impact on the diseased cells' proteome including posttranslational modifications has to be elucidated for a detailed understanding of the role of these genes in the disease process. In complex disorders, like cancer, several genes contribute to the disease process, thereby hampering the assignment of a proteomic change to the corresponding causative gene. To enable comprehensive screening for the impact of inactivation of a gene, e.g., loss of a tumor suppressor in cancer, on the cellular proteome, we present a strategy based on combination of three technologies that is recombinase-mediated cassette exchange, click chemistry, and mass spectrometry. The methodology is exemplified by the analysis of the proteomic changes induced by the loss of a tumor suppressor gene in colorectal cancer cells. To demonstrate the applicability to screen for posttranslational modification changes, we also describe the analysis of protein glycosylation changes caused by the tumor suppressor inactivation. In principle, this strategy can be applied to analyze the effects of any gene of interest on protein expression as well as posttranslational modification by glycosylation. Moreover adaptation of the strategy to an appropriate cell culture model has the potential for application on a broad range of diseases where the disease-promoting mutations have been identified. © 2017 Elsevier Inc. All rights reserved.

  12. A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus.

    Science.gov (United States)

    Liscombe, David K; O'Connor, Sarah E

    2011-11-01

    The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003-0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.

  13. Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9.

    Science.gov (United States)

    Chen, Yuejun; Cao, Jingyuan; Xiong, Man; Petersen, Andrew J; Dong, Yi; Tao, Yunlong; Huang, Cindy Tzu-Ling; Du, Zhongwei; Zhang, Su-Chun

    2015-08-06

    Precise temporal control of gene expression or deletion is critical for elucidating gene function in biological systems. However, the establishment of human pluripotent stem cell (hPSC) lines with inducible gene knockout (iKO) remains challenging. We explored building iKO hPSC lines by combining CRISPR/Cas9-mediated genome editing with the Flp/FRT and Cre/LoxP system. We found that "dual-sgRNA targeting" is essential for biallelic knockin of FRT sequences to flank the exon. We further developed a strategy to simultaneously insert an activity-controllable recombinase-expressing cassette and remove the drug-resistance gene, thus speeding up the generation of iKO hPSC lines. This two-step strategy was used to establish human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines with iKO of SOX2, PAX6, OTX2, and AGO2, genes that exhibit diverse structural layout and temporal expression patterns. The availability of iKO hPSC lines will substantially transform the way we examine gene function in human cells.

  14. UVB-induced gene expression in the skin of Xiphophorus maculatus Jp 163 B.

    Science.gov (United States)

    Yang, Kuan; Boswell, Mikki; Walter, Dylan J; Downs, Kevin P; Gaston-Pravia, Kimberly; Garcia, Tzintzuni; Shen, Yingjia; Mitchell, David L; Walter, Ronald B

    2014-06-01

    Xiphophorus fish and interspecies hybrids represent long-standing models to study the genetics underlying spontaneous and induced tumorigenesis. The recent release of the Xiphophorus maculatus genome sequence will allow global genetic regulation studies of genes involved in the inherited susceptibility to UVB-induced melanoma within select backcross hybrids. As a first step toward this goal, we report results of an RNA-Seq approach to identify genes and pathways showing modulated transcription within the skin of X. maculatus Jp 163 B upon UVB exposure. X. maculatus Jp 163 B were exposed to various doses of UVB followed by RNA-Seq analysis at each dose to investigate overall gene expression in each sample. A total of 357 genes with a minimum expression change of 4-fold (p-adjbasal expression level of each transcript for each skin sample, (2) the changes in expression levels for each gene in the transcriptome upon exposure to increasing doses of UVB, and (3) clusters of genes that exhibit similar patterns of change in expression upon UVB exposure. These data provide a foundation for understanding the molecular genetic response of fish skin to UVB exposure.

  15. Laparotomy in mice induces blood cell expression of inflammatory and stress genes.

    Science.gov (United States)

    Ko, Fred; Isoda, Fumiko; Mobbs, Charles

    2015-04-01

    Surgical trauma induces immune and stress responses although its effects on postsurgical inflammatory and stress gene expression remain poorly characterized. This study sought to improve current scientific knowledge by investigating the effects of laparotomy on mouse blood cell inflammatory and stress gene expression. Three-month-old male C57BL/6J mice were subjected to 2% isoflurane or 2% isoflurane with laparotomy and sacrificed 4 h postintervention. Blood was collected and blood cell expression of 158 genes central to inflammatory and stress responses was assayed using quantitative polymerase chain reaction arrays. Mice subjected to isoflurane with laparotomy, compared with mice receiving isoflurane alone, had >2-fold upregulation of genes in inflammation (Osm, IL1rn, IL1b, and Csf1), oxidative stress (Hmox1), heat shock (Hspa1b), growth arrest (Cdkn1a), and DNA repair (Ugt1a2). These genes demonstrated similar expression patterns by Pearson correlation and cluster analysis. Thus, laparotomy induces coordinated, postsurgical blood cell expression of unique inflammatory and stress genes whose roles in influencing surgical outcomes need further investigation.

  16. ADHESION INDUCES MATRIX METALLOPROTEINASE-9 GENE EXPRESSION IN OVARIAN CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    田方; 颜春洪; 薛红; 肖凤君

    2002-01-01

    Objective: To investigate the expression of matrix metalloproteinase-9 (MMP-9) gene in cancer cells induced by adhesion with fibronectin and the underlying mechanism of cell invasion. Methods: Following adhesion of ovarian cancer cells A2780 to fibronectin, MMP mRNA expression was assayed by using reverse transcription-polymerase chain reaction (RT-PCR). MMP-9 promoter was cloned from genomic DNA of HT1080 cells with PCR. The MMP-9-pGL2 reporter gene vector was constructed and then transiently transfected into A2780 cells. Results: Adhesion could induce the expression of MMP-9 gene in A2780 cells, but did not affect longer theexpression of MMP-2 or TIMP-1 gene. The induction was enhanced with longer adhesion time. When the transfected cells were allowed to adhere and spread on FN-coated surface, the promoter activity of MMP-9 gene was also enhanced dramatically. Conclusion: adhesion of cells with ECM may stimulate the expression of MMP-9 gene through stimulating the promoter activity, thereby enhancing cancer cell invasion and metastasis.

  17. Characterization of chemically induced liver injuries using gene co-expression modules.

    Directory of Open Access Journals (Sweden)

    Gregory J Tawa

    Full Text Available Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk that may be hard to assess due to a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific damage and clinical outcomes via biomarkers or biomarker panels will provide the foundation for highly specific and robust diagnostic tests. Here, we have used DrugMatrix, a toxicogenomics database containing organ-specific gene expression data matched to dose-dependent chemical exposures and adverse clinical pathology assessments in Sprague Dawley rats, to identify groups of co-expressed genes (modules specific to injury endpoints in the liver. We identified 78 such gene co-expression modules associated with 25 diverse injury endpoints categorized from clinical pathology, organ weight changes, and histopathology. Using gene expression data associated with an injury condition, we showed that these modules exhibited different patterns of activation characteristic of each injury. We further showed that specific module genes mapped to 1 known biochemical pathways associated with liver injuries and 2 clinically used diagnostic tests for liver fibrosis. As such, the gene modules have characteristics of both generalized and specific toxic response pathways. Using these results, we proposed three gene signature sets characteristic of liver fibrosis, steatosis, and general liver injury based on genes from the co-expression modules. Out of all 92 identified genes, 18 (20% genes have well-documented relationships with liver disease, whereas the rest are novel and have not previously been associated with liver disease. In conclusion, identifying gene co-expression modules associated with chemically induced liver injuries aids in generating testable hypotheses and has the potential to identify putative biomarkers of adverse health effects.

  18. Characterization of Chemically Induced Liver Injuries Using Gene Co-Expression Modules

    Science.gov (United States)

    Tawa, Gregory J.; AbdulHameed, Mohamed Diwan M.; Yu, Xueping; Kumar, Kamal; Ippolito, Danielle L.; Lewis, John A.; Stallings, Jonathan D.; Wallqvist, Anders

    2014-01-01

    Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk that may be hard to assess due to a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific damage and clinical outcomes via biomarkers or biomarker panels will provide the foundation for highly specific and robust diagnostic tests. Here, we have used DrugMatrix, a toxicogenomics database containing organ-specific gene expression data matched to dose-dependent chemical exposures and adverse clinical pathology assessments in Sprague Dawley rats, to identify groups of co-expressed genes (modules) specific to injury endpoints in the liver. We identified 78 such gene co-expression modules associated with 25 diverse injury endpoints categorized from clinical pathology, organ weight changes, and histopathology. Using gene expression data associated with an injury condition, we showed that these modules exhibited different patterns of activation characteristic of each injury. We further showed that specific module genes mapped to 1) known biochemical pathways associated with liver injuries and 2) clinically used diagnostic tests for liver fibrosis. As such, the gene modules have characteristics of both generalized and specific toxic response pathways. Using these results, we proposed three gene signature sets characteristic of liver fibrosis, steatosis, and general liver injury based on genes from the co-expression modules. Out of all 92 identified genes, 18 (20%) genes have well-documented relationships with liver disease, whereas the rest are novel and have not previously been associated with liver disease. In conclusion, identifying gene co-expression modules associated with chemically induced liver injuries aids in generating testable hypotheses and has the potential to identify putative biomarkers of adverse health effects. PMID:25226513

  19. Dynamic gene expression in fish muscle during recovery growth induced by a fasting-refeeding schedule

    Directory of Open Access Journals (Sweden)

    Esquerré Diane

    2007-11-01

    Full Text Available Abstract Background Recovery growth is a phase of rapid growth that is triggered by adequate refeeding of animals following a period of weight loss caused by starvation. In this study, to obtain more information on the system-wide integration of recovery growth in muscle, we undertook a time-course analysis of transcript expression in trout subjected to a food deprivation-refeeding sequence. For this purpose complex targets produced from muscle of trout fasted for one month and from muscle of trout fasted for one month and then refed for 4, 7, 11 and 36 days were hybridized to cDNA microarrays containing 9023 clones. Results Significance analysis of microarrays (SAM and temporal expression profiling led to the segregation of differentially expressed genes into four major clusters. One cluster comprising 1020 genes with high expression in muscle from fasted animals included a large set of genes involved in protein catabolism. A second cluster that included approximately 550 genes with transient induction 4 to 11 days post-refeeding was dominated by genes involved in transcription, ribosomal biogenesis, translation, chaperone activity, mitochondrial production of ATP and cell division. A third cluster that contained 480 genes that were up-regulated 7 to 36 days post-refeeding was enriched with genes involved in reticulum and Golgi dynamics and with genes indicative of myofiber and muscle remodelling such as genes encoding sarcomeric proteins and matrix compounds. Finally, a fourth cluster of 200 genes overexpressed only in 36-day refed trout muscle contained genes with function in carbohydrate metabolism and lipid biosynthesis. Remarkably, among the genes induced were several transcriptional regulators which might be important for the gene-specific transcriptional adaptations that underlie muscle recovery. Conclusion Our study is the first demonstration of a coordinated expression of functionally related genes during muscle recovery growth

  20. Controlled Gene Expression Systems for Lactic Acid Bacteria : Transferable Nisin-Inducible Expression Cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

    NARCIS (Netherlands)

    Kleerebezem, Michiel; Beerthuyzen, Marke M.; Vaughan, Elaine E.; Vos, Willem M. de; Kuipers, Oscar P.

    1997-01-01

    A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lac

  1. Effects of modulation of calcium levels and calcium fluxes on ABA- induced gene expression in barley aleurone

    NARCIS (Netherlands)

    Meulen, R.M. van der; Visser, K.; Wang, M.

    1996-01-01

    We present data to elucidate the involvement of calcium ions in abscisic acid (ABA)-induced gene expression. Modulation of external calcium concentrations was able to affect ABA-induced specific RAB gene expression. At a constant ABA level with increasing extracellular calcium level, an increasing R

  2. Effects of modulation of calcium levels and calcium fluxes on ABA- induced gene expression in barley aleurone

    NARCIS (Netherlands)

    Meulen, R.M. van der; Visser, K.; Wang, M.

    1996-01-01

    We present data to elucidate the involvement of calcium ions in abscisic acid (ABA)-induced gene expression. Modulation of external calcium concentrations was able to affect ABA-induced specific RAB gene expression. At a constant ABA level with increasing extracellular calcium level, an increasing R

  3. Mitochondrial uncoupling proteins in human physiology and disease.

    Science.gov (United States)

    Hagen, T; Vidal-Puig, A

    2002-02-01

    Uncoupling proteins are mitochondrial carrier proteins that catalyse a regulated proton leak across the inner mitochondrial membrane, diverting free energy from ATP synthesis by the mitochondrial F0F1-ATP synthase to the production of heat. Uncoupling protein 1 (UCP1), which is exclusively expressed in brown adipose tissue, is the mediator of thermogenesis in response to beta-adrenergic stimulation. Using gene a knockout mouse model, UCP1 has been shown to be required for cold acclimation. Two homologues of UCP1, UCP2 and UCP3, have been identified recently and show a much wider tissue distribution. UCP2 and UCP3 have been postulated to play a role in the regulation of cold acclimation, energy expenditure and diet-induced thermogenesis in humans, who, in contrast to rodents, have very little brown fat in adult life. However, evidence is accumulating that thermogenesis and regulation of body weight may not be the physiological functions of UCP2 and UCP3. For instance, mice deficient for UCP2 or UCP3 are not cold-intolerant and do not develop obesity. Alternative functions were suggested, primarily based on findings in UCP2 and UCP3 gene knockout mice. Both UCP2- and UCP3-deficient mice were found to overproduce reactive oxygen species and UCP2-deficient mice to hypersecrete insulin. Thus, the UCP1 homologues may play a role in regulating mitochondrial production of reactive oxygen species and b-cell function. In this review, we discuss the role of UCP1, UCP2 and UCP3 in human physiology and disease, primarily based on findings from the various animal models that have been generated.

  4. Barcode Sequencing Screen Identifies SUB1 as a Regulator of Yeast Pheromone Inducible Genes

    Directory of Open Access Journals (Sweden)

    Anna Sliva

    2016-04-01

    Full Text Available The yeast pheromone response pathway serves as a valuable model of eukaryotic mitogen-activated protein kinase (MAPK pathways, and transcription of their downstream targets. Here, we describe application of a screening method combining two technologies: fluorescence-activated cell sorting (FACS, and barcode analysis by sequencing (Bar-Seq. Using this screening method, and pFUS1-GFP as a reporter for MAPK pathway activation, we readily identified mutants in known mating pathway components. In this study, we also include a comprehensive analysis of the FUS1 induction properties of known mating pathway mutants by flow cytometry, featuring single cell analysis of each mutant population. We also characterized a new source of false positives resulting from the design of this screen. Additionally, we identified a deletion mutant, sub1Δ, with increased basal expression of pFUS1-GFP. Here, in the first ChIP-Seq of Sub1, our data shows that Sub1 binds to the promoters of about half the genes in the genome (tripling the 991 loci previously reported, including the promoters of several pheromone-inducible genes, some of which show an increase upon pheromone induction. Here, we also present the first RNA-Seq of a sub1Δ mutant; the majority of genes have no change in RNA, but, of the small subset that do, most show decreased expression, consistent with biochemical studies implicating Sub1 as a positive transcriptional regulator. The RNA-Seq data also show that certain pheromone-inducible genes are induced less in the sub1Δ mutant relative to the wild type, supporting a role for Sub1 in regulation of mating pathway genes. The sub1Δ mutant has increased basal levels of a small subset of other genes besides FUS1, including IMD2 and FIG1, a gene encoding an integral membrane protein necessary for efficient mating.

  5. Contribution of genetic polymorphisms on functional status at very old age: a gene-based analysis of 38 genes (311 SNPs) in the oxidative stress pathway.

    Science.gov (United States)

    Dato, S; Soerensen, M; Lagani, V; Montesanto, A; Passarino, G; Christensen, K; Tan, Q; Christiansen, L

    2014-04-01

    Preservation of functional ability is a well-recognized marker of longevity. At a molecular level, a major determinant of the physiological decline occurring with aging is the imbalance between production and accumulation of oxidative damage to macromolecules, together with a decreased efficiency of stress response to avoid or repair such damage. In this paper we investigated the association of 38 genes (311 SNPs) belonging to the pro-antioxidant pathways with physical and cognitive performances, by analyzing single SNP and gene-based associations with Hand Grip strength (HG), Activities of Daily Living (ADL), Walking Speed (WS), Mini Mental State Examination (MMSE) and Composite Cognitive Score (CCS) in a Cohort of 1089 Danish nonagenarians. Moreover, for each gene analyzed in the pro-antioxidant pathway, we tested the influence on longitudinal survival. In the whole sample, nominal associations were found for TXNRD1 variability with ADL and WS, NDUFS1 and UCP3 with HG and WS, GCLC and UCP2 with WS (p<0.05). Stronger associations although not holding the multiple comparison correction, were observed between MMSE and NDUFV1, MT1A and GSTP1 variability (p<0.009). Moreover, we found that association between genetic variability in the pro-antioxidant pathway and functional status at old age is influenced by sex. In particular, most significant associations were observed in nonagenarian females, between HG scores and GLRX and UCP3 variability, between ADL levels and TXNRD1, MMSE and MT1A genetic variability. In males, a borderline statistically significant association with ADL level was found for UQCRFS1 gene. Nominally significant associations in relation to survival were found in the female sample only with SOD2, NDUFS1, UCP3 and TXNRD1 variability, the latter two confirming previous observations reported in the same cohort. Overall, our work supports the evidence that genes belonging to the pro-anti-oxidant pathway are able to modulate physical and cognitive

  6. Therapeutic angiogenesis induced by human hepatocyte growth factor (HGF) gene in rat myocardial ischemia models

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate the feasibility of myocardial ischemia gene therapy, we cloned human hepatocyte growth factor gene from human placenta cDNA library by the RT-PCR method. Recombination adenovirus Ad-HGF was constructed by the method of co-transfection and homologous recombination of plasmids in 293 cells. Ad-HGF was amplified in 293 cells and purified through CsCl density gradient centrifugation. Ad-HGF could be expressed in rat primary myocardial cells and HGF secreted into the culture media, which was tested by ELISA. The distribution and persistence of adenovirus in rat were investigated by green fluorescence protein as a report gene. In vivo we found that intramyocardial administration of Ad-HGF could induce angiogenesis in rat myocardium after ligation of coronary artery. The results suggested that Ad-HGF was effective in vitro and in vivo, and the data for designing human trial of gene therapy-- mediated cardiac angiogenesis were provided.

  7. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Montanez, Cecilia [Department of Genetics and Molecular Biology, Centre for Research and Advanced Studies (CINVESTAV), IPN, Mexico City 07360 (Mexico); Wong, Carlos [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Baeza, Isabel, E-mail: ibaeza@encb.ipn.mx [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico)

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  8. Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

    Science.gov (United States)

    Dupuy, Bruno; Mani, Nagraj; Katayama, Seiichi; Sonenshein, Abraham L

    2005-02-01

    Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

  9. Plant defense gene promoter enhances the reliability of shiva-1 gene-induced resistance to soft rot disease in potato.

    Science.gov (United States)

    Yi, Jung Yoon; Seo, Hyo Won; Yang, Moon Sik; Robb, E Jane; Nazar, Ross N; Lee, Shin Woo

    2004-11-01

    PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.

  10. Atypical antipsychotics induce both proinflammatory and adipogenic gene expression in human adipocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sárvári, Anitta K., E-mail: anittasarvari@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Veréb, Zoltán, E-mail: jzvereb@gmail.com [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Uray, Iván P., E-mail: ipuray@mdanderson.org [Clinical Cancer Prevention Department, The University of Texas, MD Anderson Cancer Center, Houston, TX (United States); Fésüs, László, E-mail: fesus@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); MTA DE Apoptosis, Genomics and Stem Cell Research Group of the Hungarian Academy of Sciences (Hungary); Balajthy, Zoltán, E-mail: balajthy@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary)

    2014-08-08

    Highlights: • Antipsychotics modulate the expression of adipogenic genes in human adipocytes. • Secretion of proinflammatory cytokine IL8 and MCP-1 is induced by antipsychotics. • Adipocyte-dependent inflammatory abnormality could develop during chronic treatment. • Infiltrated macrophages would further enhance proinflammatory cytokine production. - Abstract: Schizophrenia requires lifelong treatment, potentially causing systemic changes in metabolic homeostasis. In the clinical setting, antipsychotic treatment may differentially lead to weight gain among individual patients, although the molecular determinants of such adverse effects are currently unknown. In this study, we investigated changes in the expression levels of critical regulatory genes of adipogenesis, lipid metabolism and proinflammatory genes during the differentiation of primary human adipose-derived stem cells (ADSCs). These cells were isolated from patients with body mass indices <25 and treated with the second-generation antipsychotics olanzapine, ziprasidone, clozapine, quetiapine, aripiprazole and risperidone and the first-generation antipsychotic haloperidol. We found that antipsychotics exhibited a marked effect on key genes involved in the regulation of cell cycle, signal transduction, transcription factors, nuclear receptors, differentiation markers and metabolic enzymes. In particular, we observed an induction of the transcription factor NF-KB1 and NF-KB1 target genes in adipocytes in response to these drugs, including the proinflammatory cytokines TNF-α, IL-1β, IL-8 and MCP-1. In addition, enhanced secretion of both IL8 and MCP-1 was observed in the supernatant of these cell cultures. In addition to their remarkable stimulatory effects on proinflammatory gene transcription, three of the most frequently prescribed antipsychotic drugs, clozapine, quetiapine and aripiprazole, also induced the expression of essential adipocyte differentiation genes and the adipocyte hormones leptin

  11. Identification of rice (Oryza sativa L.) signal factors capable of inducing Agrobacterium vir gene expression

    Institute of Scientific and Technical Information of China (English)

    许东晖; 李宝健; 刘煜; 黄志纾; 古练权

    1996-01-01

    Two kinds of signal factors capable of inducing Agrobaorerium vir gene expression were purified and identified from leaf extracts of panicle-differentiating to flowering stage of rice (Oryza saliva L. cv. IR 72) detected by Agrobacterium vir(?) lacZ. fusion genes. The induction was similar to that observed with 5 μm actosyringone (AS). Based on the comprehensive analysis of the data by UV, IR, NMR, MS, HMQC and HMBC, the structures of these two signal factors are identified as 5, 7, 4’-trihydroxy-3’, 5’-dimethoxy-flavone (named tricin) and 5, 4’ -dihydroxy-3’, 5’ -dimethoxy-7- (β-D-glucosyloxy) -flavone, respectively. These results demonstrate that monocotyledonous plants do contain highly efficient vir gene inducing factors of Agrobacterium, and the reason why monocotyledonous plants are difficult to transform by Ayrobacterium is not due to absence of vir gene inducing factors, but due to the signal factors only produced in specific stage and tissue of monocotyledonous plants

  12. Regulated gene insertion by steroid-induced PhiC31 integrase.

    Science.gov (United States)

    Sharma, Nynne; Moldt, Brian; Dalsgaard, Trine; Jensen, Thomas G; Mikkelsen, Jacob Giehm

    2008-06-01

    Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and PhiC31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the PhiC31 integrase is documented. In addition, fusion of the PhiC31 integrase with the ER(T2) modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible PhiC31 integrase systems are important new tools in transgenesis and in safety studies of the PhiC31 integrase for gene therapy applications.

  13. Inducible gene silencing in podocytes: a new tool for studying glomerular function.

    Science.gov (United States)

    Bugeon, Laurence; Danou, Aliki; Carpentier, David; Langridge, Paul; Syed, Nelofer; Dallman, Margaret J

    2003-03-01

    Glomerular filtration is one of the primary functions of the kidney. Podocytes, a highly specialized cell type found in glomeruli, are believed to play a critical role in that function. Null mutations of genes expressed in podocytes like WT1, nephrin, and NEPH1 result in an embryo and perinatal lethal phenotype and therefore do not allow the functional analysis of these genes in the adult kidney. Here is describes the generation of a model that will allow such studies. We have engineered transgenic mice in which the disruption of targeted genes can be induced in a temporally controlled fashion in podocytes. For this, a transgene encoding the mutated estrogen receptor-Cre recombinase fusion protein was introduced into the mouse genome. Animals were crossed with Z/AP reporter mice to test for efficient and inducible recombination. We found that, after injection of inducer drug tamoxifen, Cre fusion protein translocates to the nuclei of podocytes, where it becomes active and mediates recombination of DNA carrying loxP target sequences. These animals provide for the first time a tool for silencing genes selectively in podocytes of adult animals.

  14. Oligonucleotide microarray identifies genes differentially expressed during tumorigenesis of DMBA-induced pancreatic cancer in rats.

    Directory of Open Access Journals (Sweden)

    Jun-Chao Guo

    Full Text Available The extremely dismal prognosis of pancreatic cancer (PC is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

  15. Effects of L-Theanine on Posttraumatic Stress Disorder Induced Changes in Rat Brain Gene Expression

    Directory of Open Access Journals (Sweden)

    Tomás Eduardo Ceremuga

    2014-01-01

    Full Text Available Posttraumatic stress disorder (PTSD is characterized by the occurrence of a traumatic event that is beyond the normal range of human experience. The future of PTSD treatment may specifically target the molecular mechanisms of PTSD. In the US, approximately 20% of adults report taking herbal products to treat medical illnesses. L-theanine is the amino acid in green tea primarily responsible for relaxation effects. No studies have evaluated the potential therapeutic properties of herbal medications on gene expression in PTSD. We evaluated gene expression in PTSD-induced changes in the amygdala and hippocampus of Sprague-Dawley rats. The rats were assigned to PTSD-stressed and nonstressed groups that received either saline, midazolam, L-theanine, or L-theanine + midazolam. Amygdala and hippocampus tissue samples were analyzed for changes in gene expression. One-way ANOVA was used to detect significant difference between groups in the amygdala and hippocampus. Of 88 genes examined, 17 had a large effect size greater than 0.138. Of these, 3 genes in the hippocampus and 5 genes in the amygdala were considered significant (P<0.05 between the groups. RT-PCR analysis revealed significant changes between groups in several genes implicated in a variety of disorders ranging from PTSD, anxiety, mood disorders, and substance dependence.

  16. Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

    Science.gov (United States)

    Lawo, N C; Griesser, M; Forneck, A

    Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir, we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C (V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

  17. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Keiko Miyoshi

    2015-01-01

    Full Text Available Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs, human dermal fibroblasts (hDFs, and hOF-derived induced pluripotent stem cells (hOF-iPSCs, linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.

  18. Oligonucleotide microarray identifies genes differentially expressed during tumorigenesis of DMBA-induced pancreatic cancer in rats.

    Science.gov (United States)

    Guo, Jun-Chao; Li, Jian; Yang, Ying-Chi; Zhou, Li; Zhang, Tai-Ping; Zhao, Yu-Pei

    2013-01-01

    The extremely dismal prognosis of pancreatic cancer (PC) is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA)-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR) and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes) were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

  19. RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding.

    Science.gov (United States)

    Younis, Adnan; Siddique, Muhammad Irfan; Kim, Chang-Kil; Lim, Ki-Byung

    2014-01-01

    RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties.

  20. MOLECULAR ANALYSIS OF RADIATION-INDUCED MUTATION IN EXON 7/8 OF RAT HPRT GENE

    Institute of Scientific and Technical Information of China (English)

    任晓庆; 黄定九; 黄钢; 王利民

    2003-01-01

    Objective To investigate the relationship between the radiation dose and the HPRT gene locus mutation in rat smooth muscle cells, and provide the molecular basis for prevention of restenosis after percutaneous transluminal coronary angioplasty (PTCA).MethodsThe smooth muscle cells cultured in vitro were irradiated by radionuclide 188Re in different doses. HPRT gene mutation colonies were selected and isolated by 6 thioguanine. Analysis of mutation in exon 7/8 of HPRT gene were accomplished by polymerase chain reaction and single strand conformation polymorphism.ResultsThe HPRT gene mutation frequency of rat smooth muscle cells that were irradiated by radionuclide 188Re ranged from 5.5×10-6 to 13×10-6. Of 91 HPRT gene mutation colonies, 13(14.3%) contained exon 7/8 deletion and 15(16.5%) had point mutation. The exon 7/8 mutation frequency was 30.8%. There were significant relationships between radiation dose and mutation frequency of HPRT gene and exon 7/8.ConclusionThe DNA damage and gene mutation induced by radiation has positive relationship with radiation dose, and is a basis of proliferation inhibition and apoptosis of smooth muscle cells.

  1. Oligonucleotide microarray analysis of dietary-induced hyperlipidemia gene expression profiles in miniature pigs.

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    Junko Takahashi

    Full Text Available BACKGROUND: Hyperlipidemia animal models have been established, but complete gene expression profiles of the transition from normal lipid levels have not been obtained. Miniature pigs are useful model animals for gene expression studies on dietary-induced hyperlipidemia because they have a similar anatomy and digestive physiology to humans, and blood samples can be obtained from them repeatedly. METHODOLOGY: Two typical dietary treatments were used for dietary-induced hyperlipidemia models, by using specific pathogen-free (SPF Clawn miniature pigs. One was a high-fat and high-cholesterol diet (HFCD and the other was a high-fat, high-cholesterol, and high-sucrose diet (HFCSD. Microarray analyses were conducted from whole blood samples during the dietary period and from white blood cells at the end of the dietary period to evaluate the transition of expression profiles of the two dietary models. PRINCIPAL FINDINGS: Variations in whole blood gene expression intensity within the HFCD or the HFCSD group were in the same range as the controls provide with normal diet at all periods. This indicates uniformity of dietary-induced hyperlipidemia for our dietary protocols. Gene ontology- (GO based functional analyses revealed that characteristics of the common changes between HFCD and HFCSD were involved in inflammatory responses and reproduction. The correlation coefficient between whole blood and white blood cell expression profiles at 27 weeks with the HFCSD diet was significantly lower than that of the control and HFCD diet groups. This may be due to the effects of RNA originating from the tissues and/or organs. CONCLUSIONS: No statistically significant differences in fasting plasma lipids and glucose levels between the HFCD and HFCSD groups were observed. However, blood RNA analyses revealed different characteristics corresponding to the dietary protocols. In this study, whole blood RNA analyses proved to be a useful tool to evaluate transitions in

  2. Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

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    Nuria Troyano-Suárez

    2015-01-01

    Full Text Available Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK, a scaffold protein at cell-extracellular matrix (ECM adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

  3. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    齐兵; 齐义鹏; Masuo; Yutsudo; 刘青珍

    2000-01-01

    asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5’ end and different 3’ end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of

  4. Isolation and Analysis of Drought-Induced Genes in Maize Roots

    Institute of Scientific and Technical Information of China (English)

    LI Hui-yong; HUANG Shu-hua; SHI Yun-su; SONG Yan-chun; ZHONG Zhong-bao; WANG Guo-ying; WANG Tian-yu; LI Yu

    2009-01-01

    Maize roots are important component for plant adaptation to soil water deficits because they are supposed to take up water and necessary solutes from the soil. In the present study, the drought-induced genes were isolated in maize roots. A suppression subtractive hybridization protocol was applied to construct a forward subtractive cDNA library from CN165 for drought-stressed maize roots and a number of drought-induced genes were isolated. Totally, 126 uniESTs (containing 82 singlets and 44 contigs) were obtained from 503 available ESTs sequences after macroarray hybridization. UniESTs were analyzed using BLASTN and BLASTX and the results showed that 92% of the uniESTs had homolgous sequences in maize nr database by BLASTN. About 89% of uniESTs appeared the homlogous amino acid sequences in rice protein database but not in maize protein database by BLASTX, implying that those genes are likely new functional genes in maize. Function analysis showed that those genes were involved in a broad spectrum of biological pathways, mainly in signaling and regulatory pathways related to stress tolerance.

  5. Characterization of Phototransduction Gene Knockouts Revealed Important Signaling Networks in the Light-Induced Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    Jayalakshmi Krishnan

    2008-01-01

    Full Text Available Understanding the molecular pathways mediating neuronal function in retinas can be greatly facilitated by the identification of genes regulated in the retinas of different mutants under various light conditions. We attempted to conduct a gene chip analysis study on the genes regulated during rhodopsin kinase (Rhok-/- and arrestin (Sag-/- knockout and double knockouts in mice retina. Hence, mice were exposed to constant illumination of 450 lux or 6,000 lux on dilated pupils for indicated periods. The retinas were removed after the exposure and processed for microarray analysis. Double knockout was associated with immense changes in gene expression regulating a number of apoptosis inducing transcription factors. Subsequently, network analysis revealed that during early exposure the transcription factors, p53, c-MYC, c-FOS, JUN, and, in late phase, NF-B, appeared to be essential for the initiation of light-induced retinal rod loss, and some other classical pro- and antipoptotic genes appeared to be significantly important as well.

  6. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

    Directory of Open Access Journals (Sweden)

    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  7. Hydrogen peroxide-induced gene expression across kingdoms: a comparative analysis.

    Science.gov (United States)

    Vandenbroucke, Korneel; Robbens, Steven; Vandepoele, Klaas; Inzé, Dirk; Van de Peer, Yves; Van Breusegem, Frank

    2008-03-01

    Cells react to oxidative stress conditions by launching a defense response through the induction of nuclear gene expression. The advent of microarray technologies allowed monitoring of oxidative stress-dependent changes of transcript levels at a comprehensive and genome-wide scale, resulting in a series of inventories of differentially expressed genes in different organisms. We performed a meta-analysis on hydrogen peroxide (H(2)O(2))-induced gene expression in the cyanobacterium Synechocystis PCC 6803, the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, the land plant Arabidopsis thaliana, and the human HeLa cell line. The H(2)O(2)-induced gene expression in both yeast species was highly conserved and more similar to the A. thaliana response than that of the human cell line. Based on the expression characteristics of genuine antioxidant genes, we show that the antioxidant capacity of microorganisms and higher eukaryotes is differentially regulated. Four families of evolutionarily conserved eukaryotic proteins could be identified that were H(2)O(2) responsive across kingdoms: DNAJ domain-containing heat shock proteins, small guanine triphosphate-binding proteins, Ca(2+)-dependent protein kinases, and ubiquitin-conjugating enzymes.

  8. Enriched Environment-induced Maternal Weight Loss Reprograms Metabolic Gene Expression in Mouse Offspring*

    Science.gov (United States)

    Wei, Yanchang; Yang, Cai-Rong; Wei, Yan-Ping; Ge, Zhao-Jia; Zhao, Zhen-Ao; Zhang, Bing; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2015-01-01

    The global prevalence of weight loss is increasing, especially in young women. However, the extent and mechanisms by which maternal weight loss affects the offspring is still poorly understood. Here, using an enriched environment (EE)-induced weight loss model, we show that maternal weight loss improves general health and reprograms metabolic gene expression in mouse offspring, and the epigenetic alterations can be inherited for at least two generations. EE in mothers induced weight loss and its associated physiological and metabolic changes such as decreased adiposity and improved glucose tolerance and insulin sensitivity. Relative to controls, their offspring exhibited improved general health such as reduced fat accumulation, decreased plasma and hepatic lipid levels, and improved glucose tolerance and insulin sensitivity. Maternal weight loss altered gene expression patterns in the liver of offspring with coherent down-regulation of genes involved in lipid and cholesterol biosynthesis. Epigenomic profiling of offspring livers revealed numerous changes in cytosine methylation depending on maternal weight loss, including reproducible changes in promoter methylation over several key lipid biosynthesis genes, correlated with their expression patterns. Embryo transfer studies indicated that oocyte alteration in response to maternal metabolic conditions is a strong factor in determining metabolic and epigenetic changes in offspring. Several important lipid metabolism-related genes have been identified to partially inherit methylated alleles from oocytes. Our study reveals a molecular and mechanistic basis of how maternal lifestyle modification affects metabolic changes in the offspring. PMID:25555918

  9. ROCK signalling induced gene expression changes in mouse pancreatic ductal adenocarcinoma cells

    Science.gov (United States)

    Rath, Nicola; Kalna, Gabriela; Clark, William; Olson, Michael F.

    2016-01-01

    The RhoA and RhoC GTPases act via the ROCK1 and ROCK2 kinases to promote actomyosin contraction, resulting in directly induced changes in cytoskeleton structures and altered gene transcription via several possible indirect routes. Elevated activation of the Rho/ROCK pathway has been reported in several diseases and pathological conditions, including disorders of the central nervous system, cardiovascular dysfunctions and cancer. To determine how increased ROCK signalling affected gene expression in pancreatic ductal adenocarcinoma (PDAC) cells, we transduced mouse PDAC cell lines with retroviral constructs encoding fusion proteins that enable conditional activation of ROCK1 or ROCK2, and subsequently performed RNA sequencing (RNA-Seq) using the Illumina NextSeq 500 platform. We describe how gene expression datasets were generated and validated by comparing data obtained by RNA-Seq with RT-qPCR results. Activation of ROCK1 or ROCK2 signalling induced significant changes in gene expression that could be used to determine how actomyosin contractility influences gene transcription in pancreatic cancer. PMID:27824338

  10. Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes.

    Science.gov (United States)

    Bianchi, M S; Bianchi, N O; de la Chapelle, A

    1990-09-01

    Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.

  11. Jasmonate signal induced expression of cystatin genes for providing resistance against Karnal bunt in wheat.

    Science.gov (United States)

    Dutt, Shriparna; Pandey, Dinesh; Kumar, Anil

    2011-06-01

    Two wheat varieties HD-29 (resistant, R) and WH-542 (susceptible, S) were pretreated with jasmonic acid (JA) or jasmonate and then artificially inoculated with sporidial suspension of Tilletia indica to study its influence in reducing Karnal bunt (KB) infection by regulating cystatin gene expression. JA was found to improve the plant defense against KB as its exogenous application resulted in decrease in coefficient of infection (CI) in both susceptible and resistant varieties following pathogen inoculation. Transcript profiling of wheat cystatin genes at different days after inoculation (DAI) showed that JA pretreatment positively induced cystatin gene expression in both varieties with greater induction of expression in resistant variety than the susceptible one (Pcystatin genes, WC2, WC3 and WCMD was observed with their increased expression at 1DAI in the boot emergence stage which is most susceptible to KB and then slowly declined gradually at 3, 7 and 15 DAI in both the varieties. Except WC2, higher expression of other two cystatins viz. WC3 and WCMD at 1DAI showed higher response (Pcystatin by inhibitor assay were found to be consistent with those of transcript profiling. These findings suggest that jasmonic acid (JA) may act as a potential activator of induced resistance against Karnal bunt of wheat by upregulating cystatin gene expression.

  12. Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island.

    Science.gov (United States)

    Ling, Jun; Wang, Hui; Wu, Ping; Li, Tao; Tang, Yu; Naseer, Nawar; Zheng, Huiming; Masson-Boivin, Catherine; Zhong, Zengtao; Zhu, Jun

    2016-11-29

    Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICE(Ac)) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICE(Ac)-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.

  13. DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks.

    Science.gov (United States)

    Tse, Margaret J; Chu, Brian K; Roy, Mahua; Read, Elizabeth L

    2015-10-20

    Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks.

  14. Molecular basis for effects of carcinogenic heavy metals on inducible gene expression.

    Science.gov (United States)

    Hamilton, J W; Kaltreider, R C; Bajenova, O V; Ihnat, M A; McCaffrey, J; Turpie, B W; Rowell, E E; Oh, J; Nemeth, M J; Pesce, C A; Lariviere, J P

    1998-08-01

    Certain forms of the heavy metals arsenic and chromium are considered human carcinogens, although they are believed to act through very different mechanisms. Chromium(VI) is believed to act as a classic and mutagenic agent, and DNA/chromatin appears to be the principal target for its effects. In contrast, arsenic(III) is considered nongenotoxic, but is able to target specific cellular proteins, principally through sulfhydryl interactions. We had previously shown that various genotoxic chemical carcinogens, including chromium (VI), preferentially altered expression of several inducible genes but had little or no effect on constitutive gene expression. We were therefore interested in whether these carcinogenic heavy metals might target specific but distinct sites within cells, leading to alterations in gene expression that might contribute to the carcinogenic process. Arsenic(III) and chromium(VI) each significantly altered both basal and hormone-inducible expression of a model inducible gene, phosphoenolpyruvate carboxykinase (PEPCK), at nonovertly toxic doses in the chick embryo in vivo and rat hepatoma H411E cells in culture. We have recently developed two parallel cell culture approaches for examining the molecular basis for these effects. First, we are examining the effects of heavy metals on expression and activation of specific transcription factors known to be involved in regulation of susceptible inducible genes, and have recently observed significant but different effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding. Second, we have developed cell lines with stably integrated PEPCK promoter-luciferase reporter gene constructs to examine effects of heavy metals on promoter function, and have also recently seen profound effects induced by both chromium(VI) and arsenic(III) in this system. These model systems should enable us to be able to identify the critical cis (DNA) and trans (protein) cellular targets of heavy metal exposure

  15. The symptom difference induced by Tobacco mosaic virus and Tomato mosaic virus in tobacco plants containing the N gene is determined by movement protein gene

    Institute of Scientific and Technical Information of China (English)

    YU; Cui; HU; Dongwei; DONG; Jiahong; CUI; Xiaofeng; WU; Jun

    2004-01-01

    Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.

  16. CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells.

    Science.gov (United States)

    Kim, Eun Ji; Kang, Ki Ho; Ju, Ji Hyeon

    2017-01-01

    Recent advances in genome editing with programmable nucleases have opened up new avenues for multiple applications, from basic research to clinical therapy. The ease of use of the technology-and particularly clustered regularly interspaced short palindromic repeats (CRISPR)-will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. Here, we highlight the progress made in correcting gene mutations in monogenic hereditary disorders and discuss various CRISPR-associated applications, such as cancer research, synthetic biology, and gene therapy using induced pluripotent stem cells. The challenges, ethical issues, and future prospects of CRISPR-based systems for human research are also discussed.

  17. Parallel logic gates in synthetic gene networks induced by non-Gaussian noise.

    Science.gov (United States)

    Xu, Yong; Jin, Xiaoqin; Zhang, Huiqing

    2013-11-01

    The recent idea of logical stochastic resonance is verified in synthetic gene networks induced by non-Gaussian noise. We realize the switching between two kinds of logic gates under optimal moderate noise intensity by varying two different tunable parameters in a single gene network. Furthermore, in order to obtain more logic operations, thus providing additional information processing capacity, we obtain in a two-dimensional toggle switch model two complementary logic gates and realize the transformation between two logic gates via the methods of changing different parameters. These simulated results contribute to improve the computational power and functionality of the networks.

  18. Plant protein kinase genes induced by drought, high salt and cold stresses

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Drought, high salt and cold are three different kinds of environment stresses that severely influence the growth, development and productivity of crops. They all decrease the water state of plant cells, and consequently result in the harm of plant from water deficit. Several genes encoding protein kinases and induced by drought, high salt and low temperature have been isolated from Arabidopsis. These protein kinases include receptor protein kinase (RPK), MAP kinases, ribosomal-protein kinases and transcription-regulation protein kinase. The expression features of these genes and the regulatory roles of these protein kinases in stress response and signal transduction are discussed.

  19. Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family.

    Science.gov (United States)

    Krenek, Sascha; Schlegel, Martin; Berendonk, Thomas U

    2013-02-21

    Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia

  20. Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD-inducible genes in human amniotic epithelial cells

    Directory of Open Access Journals (Sweden)

    Kokame Koichi

    2006-05-01

    Full Text Available Abstract Background Exposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD treated primary cultures of human amniotic epithelial cells. Methods Human amniotic epithelial cells were dispersed by trypsin from amniotic membranes and cultured in DME/Ham's F12 medium supplemented with 10% FBS. Two weeks after plating, cells were treated with 50 nM TCDD or DMSO (control, further incubated for 48 hrs, and the gene expression was analyzed by DNA microarray technology and quantitative real-time PCR. Results Thirty eight TCDD-inducible genes, including cytochromeP4501A1 and cytochromeP4501B1, were identified. One of the remarkable profiles of the gene expression was the prominent up-regulation of interferon-inducible genes. The genes involved in the interferon gene expression and interferon signaling pathways were also up-regulated. Furthermore, the expression of genes related to collagen synthesis or degradation was enhanced by TCDD. Conclusion Using DNA microarray and quantitative real-time PCR analyses, we identified TCDD-inducible genes, including interferon-inducible genes and genes related to collagen synthesis or degradation, in human amniotic epithelial cells.

  1. Osteogenic-related gene expression profiles of human dental follicle cells induced by dexamethasone

    Institute of Scientific and Technical Information of China (English)

    Zuo-lin JIN; Yong-kuan ZHANG; Hai-yan SUN; Zhu LIN; Ying-chun BI; Yin-zhong DUAN; Yin DING

    2008-01-01

    Aim:Human dental follicle cells (hDFC) have the ability to differentiate into mineralized tissue-forming cells during root and periodontal development or os-teogenic induction in vitro. The present study aimed to validate the osteogenic induction of hDFC by dexamethasone (DEX) and to explore the changes of related genes responsible for the osteogenic differentiation process. Methods: Passage-cultured hDFC were induced by DEX and analyzed for mineralization activity by morphological observation, alkaline phosphatase (ALP) activity, and alizarin red S staining. GEArray Q series human osteogenesis gene array was used to describe large-scale gene expression in treated hDFC compared to the control group. Quantitative real-time RT-PCR was performed to confirm the microarray data by analyzing the expression of 7 critical transcripts. Results: Osteogenic differentiation of hDFC was confirmed by morphological change, elevated ALP activity and calcified nodules. In 96 genes investigated through the microarray analysis, 20 genes were upregulated and 8 genes were downregn-lated more than 2-fold. The results of the real-time RT-PCR correlated with the microarray analysis. The expression of the transforming growth factor-β superfamily showed varying degrees of increase, and fibroblast growth factors exhibited a differential changing trend of expression. The expression of most types of collagen genes representative of extracellular matrixes increased under DEX treatment while small mothers against decapentaplegic 6 and 7 expressions significantly decreased. Conclusion: Our results demonstrated that hDFC dis-played osteoblastic features in both phenotypic and genotypic traits induced by DEX in vitro.

  2. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis.

    Science.gov (United States)

    Halliday, Gary M

    2005-04-01

    Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppression in the skin. These biological changes are responsible for photocarcinogenesis. UV radiation in sunlight is divided into two wavebands, UVB and UVA, both of which contribute to these biological changes, and therefore probably to skin cancer in humans and animal models. Oxidative damage caused by UV contributes to inflammation, gene mutation and immunosuppression. This article reviews evidence for the hypothesis that UV oxidative damage to these processes contributes to photocarcinogenesis. UVA makes a larger impact on oxidative stress in the skin than UVB by inducing reactive oxygen and nitrogen species which damage DNA, protein and lipids and which also lead to NAD+ depletion, and therefore energy loss from the cell. Lipid peroxidation induces prostaglandin production that in association with UV-induced nitric oxide production causes inflammation. Inflammation drives benign human solar keratosis (SK) to undergo malignant conversion into squamous cell carcinoma (SCC) probably because the inflammatory cells produce reactive oxygen species, thus increasing oxidative damage to DNA and the immune system. Reactive oxygen or nitrogen appears to cause the increase in mutational burden as SK progress into SCC in humans. UVA is particularly important in causing immunosuppression in both humans and mice, and UV lipid peroxidation induced prostaglandin production and UV activation of nitric oxide synthase is important mediators of this event. Other immunosuppressive events are likely to be initiated by UV oxidative stress. Antioxidants have also been shown to reduce photocarcinogenesis. While most of this evidence comes from studies in mice, there is supporting evidence in humans that UV-induced oxidative damage contributes to inflammation, gene mutation and immunosuppression. Available evidence implicates oxidative damage as an important contributor to sunlight-induced carcinogenesis in humans.

  3. Adventitial gene transfer of catalase attenuates angiotensin II-induced vascular remodeling.

    Science.gov (United States)

    Liu, Cun-Fei; Zhang, Jia; Shen, Kai; Gao, Ping-Jin; Wang, Hai-Ya; Jin, Xin; Meng, Chao; Fang, Ning-Yuan

    2015-04-01

    Vascular adventitia and adventitia‑derived reactive oxygen species (ROS) contribute to vascular remodeling following vascular injury. A previous ex vivo study in adventitial fibroblasts showed that catalase, one of most important anti‑oxide enzymes, was downregulated by angiotensin II (AngII). The aim of the present study was to investigate whether adventitial gene transfer of catalase affects AngII‑induced vascular remodeling in vivo. Adenoviruses co‑expressing catalase and enhanced green fluorescent protein (eGFP) or expressing eGFP only were applied to the adventitial surface of common carotid arteries of Sprague‑Dawley rats. Alzet minipumps administering AngII (0.75 mg/kg/day) were then implanted subcutaneously for 14 days. Systolic blood pressure and biological parameters of vascular remodeling were measured in each group. Adventitial fibroblasts were cultured and p38 mitogen‑activated protein kinase (MAPK) phosphorylation was measured using western blot analysis. The results showed that adventitial gene transfer of catalase had no effect on AngII‑induced systolic blood pressure elevation. However, catalase adenovirus transfection significantly inhibited AngII‑induced media hypertrophy compared with that of the control virus (Pcatalase transfection significantly attenuated AngII‑induced ROS generation, macrophage infiltration, collagen deposition and adventitial α‑smooth muscle actin expression. Furthermore, catalase transfection significantly inhibited the AngII‑induced increase in p38MAPK phosphorylation. In conclusion, the results of the present study demonstrated that adventitial gene transfer of catalase significantly attenuated AngII‑induced vascular remodeling in rats via inhibition of adventitial p38MAPK phosphorylation.

  4. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Halliday, Gary M. [Dermatology Research Laboratories, Division of Medicine, Melanoma and Skin Cancer Research Institute, Royal Prince Alfred Hospital at the University of Sydney, Sydney, NSW (Australia)]. E-mail: garyh@med.usyd.edu.au

    2005-04-01

    Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppression in the skin. These biological changes are responsible for photocarcinogenesis. UV radiation in sunlight is divided into two wavebands, UVB and UVA, both of which contribute to these biological changes, and therefore probably to skin cancer in humans and animal models. Oxidative damage caused by UV contributes to inflammation, gene mutation and immunosuppression. This article reviews evidence for the hypothesis that UV oxidative damage to these processes contributes to photocarcinogenesis. UVA makes a larger impact on oxidative stress in the skin than UVB by inducing reactive oxygen and nitrogen species which damage DNA, protein and lipids and which also lead to NAD+ depletion, and therefore energy loss from the cell. Lipid peroxidation induces prostaglandin production that in association with UV-induced nitric oxide production causes inflammation. Inflammation drives benign human solar keratosis (SK) to undergo malignant conversion into squamous cell carcinoma (SCC) probably because the inflammatory cells produce reactive oxygen species, thus increasing oxidative damage to DNA and the immune system. Reactive oxygen or nitrogen appears to cause the increase in mutational burden as SK progress into SCC in humans. UVA is particularly important in causing immunosuppression in both humans and mice, and UV lipid peroxidation induced prostaglandin production and UV activation of nitric oxide synthase is important mediators of this event. Other immunosuppressive events are likely to be initiated by UV oxidative stress. Antioxidants have also been shown to reduce photocarcinogenesis. While most of this evidence comes from studies in mice, there is supporting evidence in humans that UV-induced oxidative damage contributes to inflammation, gene mutation and immunosuppression. Available evidence implicates oxidative damage as an important contributor to sunlight-induced carcinogenesis in humans.

  5. Radiation-induced gene expression in human subcutaneous fibroblasts is predictive of radiation-induced fibrosis

    DEFF Research Database (Denmark)

    Rødningen, Olaug Kristin; Børresen-Dale, Anne-Lise; Alsner, Jan

    2008-01-01

    with variable risk of RIF (grouped into five classes from low to high risk) were irradiated with two different schemes: 1x3.5Gy with RNA isolated 2 and 24h after irradiation, and a fractionated scheme with 3x3.5Gy in intervals of 24h with RNA isolated 2h after the last dose. RNA was also isolated from non......BACKGROUND AND PURPOSE: Breast cancer patients show a large variation in normal tissue reactions after ionizing radiation (IR) therapy. One of the most common long-term adverse effects of ionizing radiotherapy is radiation-induced fibrosis (RIF), and several attempts have been made over the last...... years to develop predictive assays for RIF. Our aim was to identify basal and radiation-induced transcriptional profiles in fibroblasts from breast cancer patients that might be related to the individual risk of RIF in these patients. MATERIALS AND METHODS: Fibroblast cell lines from 31 individuals...

  6. Temporal gene expression profiling during rat femoral marrow ablation-induced intramembranous bone regeneration.

    Directory of Open Access Journals (Sweden)

    Joel K Wise

    -ablation were also identified. These data present the first temporal gene expression profiling analysis of the rat genome during intramembranous bone regeneration induced by femoral marrow ablation.

  7. Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves

    Science.gov (United States)

    Kurita, Akihiro; Matsunobu, Takeshi; Satoh, Yasushi; Ando, Takahiro; Sato, Shunichi; Obara, Minoru; Shiotani, Akihiro

    2011-09-01

    We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.

  8. Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing.

    Science.gov (United States)

    Cooper, Bret; Campbell, Kimberly B; McMahon, Michael B; Luster, Douglas G

    2013-01-01

    Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.

  9. Microarray analysis of genes differentially expressed in placentas of pregnancy-induced hypertension patients

    Institute of Scientific and Technical Information of China (English)

    李东红; 黄飞; 郑维国; 姜锋; 高平

    2003-01-01

    Objective: To uncover new clue for the research of the etiology of pregnancy-induced hypertension (PIH) by testing the gene expression difference between preeclamptic placentas and normal ones. Methods: mRNA level of 4 PIH placentas were examined using 4000 feature cDNA microarray in comparison with the pooled control consisting of total RNA from 4 cases of PIH placentas after the control cDNA and experimental cDNA were labeled by cy3 and cy5 respectively. Results: Fifty-eight to 131 genes were found down or up-regulated in 4 runs of hybridization. Among the differentially expressed genes, 22 genes, including genes encoding secreted protein ADRP, CYR61, EPI and HIF2, had the concordance in at least 2 cases were up-regulated or down-regulated. Conclusion: cDNA microarray is a high throughput and time-saving method to monitor the altered gene expression and the result could provide interesting clue and strategy for the etiological research of PIH.

  10. Exposure to ionizing radiation induced persistent gene expression changes in mouse mammary gland

    Directory of Open Access Journals (Sweden)

    Datta Kamal

    2012-12-01

    Full Text Available Abstract Background Breast tissue is among the most sensitive tissues to the carcinogenic actions of ionizing radiation and epidemiological studies have linked radiation exposure to breast cancer. Currently, molecular understanding of radiation carcinogenesis in mammary gland is hindered due to the scarcity of in vivo long-term follow up data. We undertook this study to delineate radiation-induced persistent alterations in gene expression in mouse mammary glands 2-month after radiation exposure. Methods Six to eight week old female C57BL/6J mice were exposed to 2 Gy of whole body γ radiation and mammary glands were surgically removed 2-month after radiation. RNA was isolated and microarray hybridization performed for gene expression analysis. Ingenuity Pathway Analysis (IPA was used for biological interpretation of microarray data. Real time quantitative PCR was performed on selected genes to confirm the microarray data. Results Compared to untreated controls, the mRNA levels of a total of 737 genes were significantly (p Conclusions Exposure to a clinically relevant radiation dose led to long-term activation of mammary gland genes involved in proliferative and metabolic pathways, which are known to have roles in carcinogenesis. When considered along with downregulation of a number of tumor suppressor genes, our study has implications for breast cancer initiation and progression after therapeutic radiation exposure.

  11. Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Ana M. Crane

    2015-04-01

    Full Text Available Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC lines. We then utilized zinc-finger nucleases (ZFNs, designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR. We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

  12. Gene Mutation as Biomarker of Radiation Induced Cell Injury and Genomic Instability

    Directory of Open Access Journals (Sweden)

    M Syaifudin

    2006-07-01

    Full Text Available Gene expression profiling and its mutation has become one of the most widely used approaches to identify genes and their functions in the context of identify and categorize genes to be used as radiation effect markers including cell and tissue sensitivities. Ionizing radiation produces genetic damage and changes in gene expression that may lead to cancer due to specific protein that controlling cell proliferation altered the function, its expression or both. P53 protein encoded by p53 gene plays an important role in protecting cell by inducing growth arrest and or cell suicide (apoptosis after deoxyribonucleic acid (DNA damage induced by mutagen such as ionizing radiation. The mutant and thereby dysfunctional of this gene was found in more than 50% of various human cancers, but it is as yet unclear how p53 mutations lead to neoplastic development. Wild-type p53 has been postulated to play a role in DNA repair, suggesting that expression of mutant forms of p53 might alter cellular resistance to the DNA damage caused by radiation. Moreover, p53 is thought to function as a cell cycle checkpoint after irradiation, also suggesting that mutant p53 might change the cellular proliferative response to radiation. p53 mutations affect the cellular response to DNA damage, either by increasing DNA repair processes or, possibly, by increasing cellular tolerance to DNA damage. The association of p53 mutations with increased radioresistance suggests that alterations in the p53 gene might lead to oncogenic transformation. Current attractive model of carcinogenesis also showed that p53 gene is the major target of radiation. The majority of p53 mutations found so far is single base pairchanges (point mutations, which result in amino acid substitutionsor truncated forms of the P53 protein, and are widely distributedthroughout the evolutionarily conserved regions of the gene. Examination of p53 mutations in human cancer also shows an association between particular

  13. Kinetics of gene expression and bone remodelling in the clinical phase of collagen induced arthritis

    DEFF Research Database (Denmark)

    Denninger, Katja Caroline Marie; Litman, Thomas; Marstrand, Troels

    2015-01-01

    Introduction: Pathological bone changes differ considerably between inflammatory arthritic diseases and most studies have focused on bone erosion. Collagen-induced arthritis (CIA) is a model for rheumatoid arthritis, which, in addition to bone erosion, demonstrates bone formation at the time...... osteoblast differentiation and function, which mirrored the histopathological bone changes. The differentially expressed genes belong to the bone morphogenetic pathway (BMP) and, in addition, include the osteoblast markers integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap1...

  14. Molecular mapping of a new induced gene for nuclear male sterility in sunflower (Helianthus annuus L.)

    Science.gov (United States)

    A new NMS line, NMS HA89-872, induced by mitomycin C and streptomycin carries a single recessive male-sterile gene ms6. An F2 population of 88 plants was obtained from a cross between nuclear male-sterile mutant NMS HA89-872 (msms) and male-fertile line RHA271 (MsMs). 225 SSR primers and 9 RFLP-deri...

  15. Delivery of antioxidant enzyme genes to protect against ischemia/reperfusion-induced injury to retinal microvasculature.

    Science.gov (United States)

    Chen, Baihua; Caballero, Sergio; Seo, Soojung; Grant, Maria B; Lewin, Alfred S

    2009-12-01

    Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice. I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated. Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration. Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease.

  16. Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells.

    Science.gov (United States)

    Geck, P; Szelei, J; Jimenez, J; Lin, T M; Sonnenschein, C; Soto, A M

    1997-01-01

    Androgens control cell numbers in the prostate through three separate pathways: (a) inhibition of cell death, (b) induction of cell proliferation (Step-1) and (c) inhibition of cell proliferation (Step-2, proliferative shutoff). The mechanisms underlying these phenomena are incompletely understood. The human prostate carcinoma LNCaP variants express these pathways as follows: LNCaP-FGC express both steps, LNCaP-LNO expresses Step-2, LNCaP-TAC expresses Step-1, and LNCaP-TJA cells express neither step. These cells facilitated the search for mediators of the androgen-induced proliferative shutoff pathway. Androgen exposure for 24 h or longer induced an irreversible proliferative shutoff in LNCaP-FGC cells. The Wang and Brown approach for identifying differentially expressed mRNAs was used to search for mediators of Step-2. Ten unique inserts were identified and from those ten, three genes were further studied. The basal expression of these genes in shutoff-negative variants was not affected by androgen exposure. They were induced by androgens in shutoff-positive LNCaP variants and the androgen receptor-transfected, shutoff-positive, MCF7-AR1 cells. These genes were induced only in the range of androgen concentrations that elicited the shutoff response. Time course analysis showed that their induction precedes the commitment point by 12-18 h. In addition, they were expressed in the normal prostate during proliferative shutoff. These features suggest that the candidate genes have a role in the regulation cascade for proliferative shutoff.

  17. Proinsulin misfolding and diabetes: mutant INS gene-induced diabetes of youth.

    Science.gov (United States)

    Liu, Ming; Hodish, Israel; Haataja, Leena; Lara-Lemus, Roberto; Rajpal, Gautam; Wright, Jordan; Arvan, Peter

    2010-11-01

    Type 1B diabetes (typically with early onset and without islet autoantibodies) has been described in patients bearing small coding sequence mutations in the INS gene. Not all mutations in the INS gene cause the autosomal dominant Mutant INS-gene Induced Diabetes of Youth (MIDY) syndrome, but most missense mutations affecting proinsulin folding produce MIDY. MIDY patients are heterozygotes, with the expressed mutant proinsulins exerting dominant-negative (toxic gain of function) behavior in pancreatic beta cells. Here we focus primarily on proinsulin folding in the endoplasmic reticulum, providing insight into perturbations of this folding pathway in MIDY. Accumulated evidence indicates that, in the molecular pathogenesis of the disease, misfolded proinsulin exerts dominant effects that initially inhibit insulin production, progressing to beta cell demise with diabetes.

  18. Analysis of human transforming growth factor β-induced gene mutation in corneal dystrophy

    Institute of Scientific and Technical Information of China (English)

    李杨; 孙旭光; 任慧媛; 董冰; 王智群; 孙秀英

    2004-01-01

    Background Corneal dystrophy is a group of inherited blinding diseases of the cornea. This study was to identify the mutations of the keratoepithelin (KE) gene for proper diagnosis of corneal dystrophy. Methods Three families with corneal dystrophy were analysed. Thirteen individuals at risk for corneal dystrophy in family A, the proband and her son in family B, and the proband in family C were examined after their blood samples were obtained. Mutation screening of human transforming growth factor β-induced gene (BIGH3 gene) was performed. Results Five individuals in family A were found by clinical evaluation to be affected with granular corneal dystrophy and carried the BIGH3 mutation W555R. However, both probands in families B and C, also diagnosed with granular corneal dystrophy, harboured the BIGH3 mutation R124H. Conclusion Molecular genetic analysis can improve accurate diagnosis of corneal dystrophy.

  19. Constitutive components and induced gene expression are involved in the desiccation tolerance of Selaginella tamariscina.

    Science.gov (United States)

    Liu, Mao-Sen; Chien, Ching-Te; Lin, Tsan-Piao

    2008-04-01

    Selaginella tamariscina, one of the most primitive vascular plants, can remain alive in a desiccated state and resurrect when water becomes available. To evaluate the nature of desiccation tolerance in this plant, we compared the composition of soluble sugars and saturation ratios of phospholipids (PLs) between hydrated and desiccated tissues of S. tamariscina using gas chromatography. In this study, differences in gene expression and ABA contents were also analyzed during dehydration. The results revealed that trehalose (at >130 mg g(-1) DW) was the major soluble sugar, and low saturated fatty acid content in PLs (0.31) was maintained in both hydrated and desiccated tissues. In addition, the ABA content of S. tamariscina increased 3-fold, and genes involved in ABA signaling and cellular protection were up-regulated while photosystem-related genes were down-regulated during dehydration. The biochemical and molecular findings suggest that both constitutive and inducible protective molecules contribute to desiccation tolerance of S. tamariscina.

  20. Stability of Barley stripe mosaic virus induced gene silencing in barley

    DEFF Research Database (Denmark)

    Bruun-Rasmussen, Marianne; Madsen, Christian Toft; Jessing, Stine

    2007-01-01

    Virus-induced gene silencing (VIGS) can be used as a powerful tool for functional genomics studies in plants. With this approach, it is possible to target most genes and downregulate the messenger (m)RNA in a sequence-specific manner. Barley stripe mosaic virus (BSMV) is an established VIGS vector...... for barley and wheat; however, silencing using this vector is generally transient, with efficient silencing often being confined to the first two or three systemically infected leaves. To investigate this further, part of the barley Phytoene desaturase (PDS) gene was inserted into BSMV and the resulting...... inoculation, although large parts of the insert had been lost from the virus vector. The instability of the insert, observed consistently throughout our experiments, offers an explanation for the transient nature of silencing when using BSMV as a VIGS vector....

  1. Immediate-early Inducible Function in Upstream Region of junB Gene

    Institute of Scientific and Technical Information of China (English)

    HONG WAN; HIROSHI ISHIHARA; IZUMI TANAKA

    2006-01-01

    Objective To analyze the upstream region of radiation-induced junB gene. Methods Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/β-actin measured by quantitative Northern blot hybridization. Results CAT mRNA expression containing 900 bp and 1560 bpjunB promoter remarkably and rapidly increased, and reached its peak 30 min after 2 Gy X-ray irradiation. Conclusions 590~900 bp fragments located in the upstream region ofjunB gene play an important role in the early process of cells against radiation.

  2. Time delay induced transition of gene switch and stochastic resonance in a genetic transcriptional regulatory model

    Science.gov (United States)

    2012-01-01

    Background Noise, nonlinear interactions, positive and negative feedbacks within signaling pathways, time delays, protein oligomerization, and crosstalk between different pathways are main characters in the regulatory of gene expression. However, only a single noise source or only delay time in the deterministic model is considered in the gene transcriptional regulatory system in previous researches. The combined effects of correlated noise and time delays on the gene regulatory model still remain not to be fully understood. Results The roles of time delay on gene switch and stochastic resonance are systematically explored based on a famous gene transcriptional regulatory model subject to correlated noise. Two cases, including linear time delay appearing in the degradation process (case I) and nonlinear time delay appearing in the synthesis process (case II) are considered, respectively. For case I: Our theoretical results show that time delay can induce gene switch, i.e., the TF-A monomer concentration shifts from the high concentration state to the low concentration state ("on"→"off"). With increasing the time delay, the transition from "on" to "off" state can be further accelerated. Moreover, it is found that the stochastic resonance can be enhanced by both the time delay and correlated noise intensity. However, the additive noise original from the synthesis rate restrains the stochastic resonance. It is also very interesting that a resonance bi-peaks structure appears under large additive noise intensity. The theoretical results by using small-delay time-approximation approach are consistent well with our numerical simulation. For case II: Our numerical simulation results show that time delay can also induce the gene switch, however different with case I, the TF-A monomer concentration shifts from the low concentration state to the high concentration state ("off"→"on"). With increasing time delay, the transition from "on" to "off" state can be further

  3. Involvement of multiple transcription factors for metal-induced spy gene expression in Escherichia coli.

    Science.gov (United States)

    Yamamoto, Kaneyoshi; Ogasawara, Hiroshi; Ishihama, Akira

    2008-01-20

    Bacteria are directly exposed to metals in environment. To maintain the intracellular metal homeostasis, Escherichia coli contain a number of gene regulation systems, each for response to a specific metal. A periplasmic protein Spy of E. coli was found to be induced upon short-exposure to copper ion in CpxAR-dependent manner. Transcription of the spy gene was also induced by long-exposure to zinc ion. This induction, however, depended on another two-component system BaeSR. Using DNase-I footprinting assay, we identified two BaeR-binding regions on the spy promoter with a direct repeat of the BaeR-box sequence, TCTNCANAA. The zinc-responsive BaeR-binding sites were separated from copper-responsive CpxR-binding site, implying that the spy promoter responds to two species of metal independently through different using sensor-response regulator systems. Since BaeSR-dependent zinc response requires longer time, the induction of spy gene transcription by external zinc may include multiple steps such as through sensing the zinc-induced envelope disorder by BaeSR.

  4. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  5. Comparison of gene expression changes induced by biguanides in db/db mice liver.

    Science.gov (United States)

    Heishi, Masayuki; Hayashi, Koji; Ichihara, Junji; Ishikawa, Hironori; Kawamura, Takao; Kanaoka, Masaharu; Taiji, Mutsuo; Kimura, Toru

    2008-08-01

    Large-scale clinical studies have shown that the biguanide drug metformin, widely used for type 2 diabetes, to be very safe. By contrast, another biguanide, phenformin, has been withdrawn from major markets because of a high incidence of serious adverse effects. The difference in mode of action between the two biguanides remains unclear. To gain insight into the different modes of action of the two drugs, we performed global gene expression profiling using the livers of obese diabetic db/db mice after a single administration of phenformin or metformin at levels sufficient to cause a significant reduction in blood glucose level. Metformin induced modest expression changes, including G6pc in the liver as previously reported. By contrast, phenformin caused changes in expression level of many additional genes. We used a knowledge-based bioinformatic analysis to study the effects of phenformin. Differentially expressed genes identified in this study constitute a large gene network, which may be related to cell death, inflammation or wound response. Our results suggest that the two biguanides show a similar hypoglycemic effect in db/db mice, but phenformin induces a greater stress on the liver even a short time after a single administration. These findings provide a novel insight into the cause of the relatively high occurrence of serious adverse effect after phenformin treatment.

  6. Arabidopsis FLOWERING LOCUS D influences systemic-acquiredresistance-induced expression and histone modifications of WRKY genes

    Indian Academy of Sciences (India)

    Vijayata Singh; Shweta Roy; Deepjyoti Singh; Ashis Kumar Nandi

    2014-03-01

    A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections – a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that FLOWERING LOCUS D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD’s involvement in epigenetic regulation of SAR.

  7. Insulin-induced gene 2 expression correlates with colorectal cancer metastasis and disease outcome.

    Science.gov (United States)

    Sun, Shengjie; Zhang, Guoqing; Sun, Qiong; Wu, Zhiyong; Shi, Weiwei; Yang, Bo; Li, Ying

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers worldwide accounting for ∼9% of cancer-related deaths, 90% of which are due to metastasis resulting from resistance to chemotherapeutic agents. Hence, it is imperative to develop novel biomarkers of CRC. Insulin-induced gene 2 (INSIG2) has been previously reported to be a negative regulator of cholesterol synthesis and was recently identified as a putative-positive prognostic biomarker for colon and pancreatic cancer prognosis. Even though it has been suggested as a colon cancer biomarker and as an inhibitor of Bax-mediated apoptosis, the role of INSIG2 in CRC is elusive. We initially validated that INSIG2 is a gene with univariate-negative prognostic capacity to discriminate human colon cancer survivorship and that if present along with adenomatous polyposis coli (APC) gene mutations further decrease overall survival. Gain- and loss-of-function studies of INSIG2 showed that the gene product is responsible for inducing migration and invasion and maintenance of the mesenchymal phenotype in vitro and metastasis in vivo. Interestingly, loss of INSIG2 did not affect tumorigenic potential per se, but affected hepatic invasion in a xenograft assay. Our findings reinforce that INSIG2 is a novel colon cancer biomarker, and suggest, for the first time, an exclusive connection between INSIG2 and metastatic dissemination without any effect on tumorigenesis. © 2015 IUBMB Life, 68(1):65-71, 2016.

  8. DELLA proteins regulate expression of a subset of AM symbiosis-induced genes in Medicago truncatula.

    Science.gov (United States)

    Floss, Daniela S; Lévesque-Tremblay, Véronique; Park, Hee-Jin; Harrison, Maria J

    2016-01-01

    The majority of the vascular flowering plants form symbiotic associations with fungi from the phylum Glomeromycota through which both partners gain access to nutrients, either mineral nutrients in the case of the plant, or carbon, in the case of the fungus. (1) The association develops in the roots and requires substantial remodeling of the root cortical cells where branched fungal hyphae, called arbuscules, are housed in a new membrane-bound apoplastic compartment. (2) Nutrient exchange between the symbionts occurs over this interface and its development and maintenance is critical for symbiosis. Previously, we showed that DELLA proteins, which are well known as repressors of gibberellic acid signaling, also regulate development of AM symbiosis and are necessary to enable arbuscule development. (3) Furthermore, constitutive overexpression of a dominant DELLA protein (della1-Δ18) is sufficient to induce transcripts of several AM symbiosis-induced genes, even in the absence of the fungal symbiont. (4) Here we further extend this approach and identify AM symbiosis genes that respond transcriptionally to constitutive expression of a dominant DELLA protein and also genes that do respond to this treatment. Additionally, we demonstrate that DELLAs interact with REQUIRED FOR ARBUSCULE DEVELOPMENT 1 (RAD1) which further extends our knowledge of GRAS factor complexes that have the potential to regulate gene expression during AM symbiosis.

  9. Biological Characterization of Gene Response to Insulin-Induced Hypoglycemia in Mouse Retina.

    Directory of Open Access Journals (Sweden)

    Martine Emery

    Full Text Available Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Chronic, exaggerated, glycemic excursions could lead to cardiovascular diseases, nephropathy, neuropathy and retinopathy. We recently showed that hypoglycemia induced retinal cell death in mouse via caspase 3 activation and glutathione (GSH decrease. Ex vivo experiments in 661W photoreceptor cells confirmed the low-glucose induction of death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. We evaluate herein retinal gene expression 4 h and 48 h after insulin-induced hypoglycemia. Microarray analysis demonstrated clusters of genes whose expression was modified by hypoglycemia and we discuss the potential implication of those genes in retinal cell death. In addition, we identify by gene set enrichment analysis, three important pathways, including lysosomal function, GSH metabolism and apoptotic pathways. Then we tested the effect of recurrent hypoglycemia (three successive 4h periods of hypoglycemia spaced by 48 h recovery on retinal cell death. Interestingly, exposure to multiple hypoglycemic events prevented GSH decrease and retinal cell death, or adapted the retina to external stress by restoring GSH level comparable to control situation. We hypothesize that scavenger GSH is a key compound in this apoptotic process, and maintaining "normal" GSH level, as well as a strict glycemic control, represents a therapeutic challenge in order to avoid side effects of diabetes, especially diabetic retinopathy.

  10. Gene expression microarray analysis of early oxygen-induced retinopathy in the rat.

    Science.gov (United States)

    Tea, Melinda; Fogarty, Rhys; Brereton, Helen M; Michael, Michael Z; Van der Hoek, Mark B; Tsykin, Anna; Coster, Douglas J; Williams, Keryn A

    2009-12-12

    Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague-Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague-Dawley list returned the term "response to hypoxia," absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1alpha were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9041-7) contains supplementary material, which is available to authorized users.

  11. Production of transgenic chickens expressing a tetracycline-inducible GFP gene.

    Science.gov (United States)

    Kwon, Mo Sun; Koo, Bon Chul; Roh, Ji Yeol; Kim, Minjee; Kim, Jin-Hoi; Kim, Teoan

    2011-07-15

    There is much interest in using farm animals as 'bioreactors' to produce large quantities of biopharmaceuticals. However, uncontrolled constitutive expression of foreign genes have been known to cause serious physiological disturbances in transgenic animals. The objective of this study was to test the feasibility of the controllable expression of an exogenous gene in the chicken. A retrovirus vector was designed to express GFP (green fluorescent protein) and rtTA (reverse tetracycline-controlled transactivator) under the control of the tetracycline-inducible promoter and the PGK (phosphoglycerate kinase) promoter, respectively. G0 founder chickens were produced by infecting the blastoderm of freshly laid eggs with concentrated retrovirus vector. Feeding the chickens obtained with doxycycline, a tetracycline derivative, resulted in emission of green body color under fluorescent light, and no apparent significant physiological dysfunctions. Successful germline transmission of the exogenous gene was also confirmed. Expression of the GFP gene reverted to the pre-induction levels when doxycycline was removed from the diet. The results showed that a tetracycline-inducible expression system in transgenic animals might be a promising solution to minimize physiological disturbances caused by the transgene. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts

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    Tui Neri

    2015-10-01

    Full Text Available Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41+ cells gradually gave rise to CD45+ cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.

  13. Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

    Institute of Scientific and Technical Information of China (English)

    Lu-yang YU; Bo LIN; Zhen-lin ZHANG; Li-he GUO

    2004-01-01

    AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS:PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and Westem blots. Urine amylase, NO generation, and histological examination were examined. RESULTS:Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen,duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

  14. Gene expression patterns underlying parasite-induced alterations in host behaviour and life history.

    Science.gov (United States)

    Feldmeyer, Barbara; Mazur, Johanna; Beros, Sara; Lerp, Hannes; Binder, Harald; Foitzik, Susanne

    2016-01-01

    Many parasites manipulate their hosts' phenotype. In particular, parasites with complex life cycles take control of their intermediate hosts' behaviour and life history to increase transmission to their definitive host. The proximate mechanisms underlying these parasite-induced alterations are poorly understood. The cestode Anomotaenia brevis affects the behaviour, life history and morphology of parasitized Temnothorax nylanderi ants and indirectly of their unparasitized nestmates. To gain insights on how parasites alter host phenotypes, we contrast brain gene expression patterns of T. nylanderi workers parasitized with the cestode, their unparasitized nestmates and unparasitized workers from unparasitized colonies. Over 400 differentially expressed genes between the three groups were identified, with most uniquely expressed genes detected in parasitized workers. Among these are genes that can be linked to the increased lifespan of parasitized workers. Furthermore, many muscle (functionality) genes are downregulated in these workers, potentially causing the observed muscular deformations and their inactive behaviour. Alterations in lifespan and activity could be adaptive for the parasite by increasing the likelihood that infected workers residing in acorns are eaten by their definitive host, a woodpecker. Our transcriptome analysis reveals numerous gene expression changes in parasitized workers and their uninfected nestmates and indicates possible routes of parasite manipulation. Although causality still needs to be established, parasite-induced alterations in lifespan and host behaviour appear to be partly explained by morphological muscle atrophy instead of central nervous system interference, which is often the core of behavioural regulation. Results of this study will shed light upon the molecular basis of antagonistic species interactions.

  15. Transgene-induced gene silencing is not affected by a change in ploidy level.

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    Daniela Pignatta

    Full Text Available BACKGROUND: Whole genome duplication, which results in polyploidy, is a common feature of plant populations and a recurring event in the evolution of flowering plants. Polyploidy can result in changes to gene expression and epigenetic instability. Several epigenetic phenomena, occurring at the transcriptional or post-transcriptional level, have been documented in allopolyploids (polyploids derived from species hybrids of Arabidopsis thaliana, yet findings in autopolyploids (polyploids derived from the duplication of the genome of a single species are limited. Here, we tested the hypothesis that an increase in ploidy enhances transgene-induced post-transcriptional gene silencing using autopolyploids of A. thaliana. METHODOLOGY/PRINCIPAL FINDINGS: Diploid and tetraploid individuals of four independent homozygous transgenic lines of A. thaliana transformed with chalcone synthase (CHS inverted repeat (hairpin constructs were generated. For each line diploids and tetraploids were compared for efficiency in post-transcriptional silencing of the endogenous CHS gene. The four lines differed substantially in their silencing efficiency. Yet, diploid and tetraploid plants derived from these plants and containing therefore identical transgene insertions showed no difference in the efficiency silencing CHS as assayed by visual scoring, anthocyanin assays and quantification of CHS mRNA. CONCLUSIONS/SIGNIFICANCE: Our results in A. thaliana indicated that there is no effect of ploidy level on transgene-induced post-transcriptional gene silencing. Our findings that post-transcriptional mechanisms were equally effective in diploids and tetraploids supports the use of transgene-driven post-transcriptional gene silencing as a useful mechanism to modify gene expression in polyploid species.

  16. Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.

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    Takashi Maejima

    Full Text Available Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3 and thrombomodulin (THBD, were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2 family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET, and real time chromosome conformation capture (3C assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.

  17. An efficient virus-induced gene silencing vector for maize functional genomics research.

    Science.gov (United States)

    Wang, Rong; Yang, Xinxin; Wang, Nian; Liu, Xuedong; Nelson, Richard S; Li, Weimin; Fan, Zaifeng; Zhou, Tao

    2016-04-01

    Maize is a major crop whose rich genetic diversity provides an advanced resource for genetic research. However, a tool for rapid transient gene function analysis in maize that may be utilized in most maize cultivars has been lacking, resulting in reliance on time-consuming stable transformation and mutation studies to obtain answers. We developed an efficient virus-induced gene silencing (VIGS) vector for maize based on a naturally maize-infecting cucumber mosaic virus (CMV) strain, ZMBJ-CMV. An infectious clone of ZMBJ-CMV was constructed, and a vascular puncture inoculation method utilizing Agrobacterium was optimized to improve its utility for CMV infection of maize. ZMBJ-CMV was then modified to function as a VIGS vector. The ZMBJ-CMV vector induced mild to moderate symptoms in many maize lines, making it useful for gene function studies in critically important maize cultivars, such as the sequenced reference inbred line B73. Using this CMV VIGS system, expression of two endogenous genes, ZmPDS and ZmIspH, was found to be decreased by 75% and 78%, respectively, compared with non-silenced tissue. Inserts with lengths of 100-300 bp produced the most complete transcriptional and visual silencing phenotypes. Moreover, genes related to autophagy, ZmATG3 and ZmATG8a, were also silenced, and it was found that they function in leaf starch degradation. These results indicate that our ZMBJ-CMV VIGS vector provides a tool for rapid and efficient gene function studies in maize. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  18. Inducible removal of UV-induced pyrimidine dimers from transcriptionally active and inactive genes of Saccharomyces cerevisiae.

    Science.gov (United States)

    Waters, R; Zhang, R; Jones, N J

    1993-05-01

    The prior UV irradiation of alpha haploid Saccharomyces cerevisiae with a UV dose of 25 J/m2 substantially increases the repairability of damage subsequently induced by a UV dose of 70 J/m2 given 1 h after the first irradiation. This enhancement of repair is seen at both the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells. The presence in the medium of the protein synthesis inhibitor, cycloheximide in the period between the two irradiations eliminated this effect. Enhanced repair still occurred if cycloheximide was present only after the final UV irradiation. This indicated that the first result is not due to cycloheximide merely blocking the synthesis of repair enzymes associated with a hypothetical rapid turnover of such molecules. The enhanced repairability is not the result of changes in chromatin accessibility without protein synthesis, merely caused by the repair of the damage induced by the prior irradiation. The data clearly show that a UV-inducible removal of pyrimidine dimers has occurred which involves the synthesis of new proteins. The genes known to possess inducible promoters, and which are involved in excision are RAD2, RAD7, RAD16 and RAD23. Studies with the rad7 and rad16 mutants which are defective in the ability to repair HML alpha and proficient in the repair of MAT alpha showed that in rad7, preirradiation enhanced the repair at MAT alpha, whereas in rad16 this increased repair of MAT alpha was absent. The preirradiation did not modify the inability to repair HML alpha in either strain. Thus RAD16 has a role in this inducible repair.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. In vivo gene therapy for pyridoxine-induced neuropathy by herpes simplex virus-mediated gene transfer of neurotrophin-3.

    Science.gov (United States)

    Chattopadhyay, Munmun; Wolfe, Darren; Huang, Shaohua; Goss, James; Glorioso, Joseph C; Mata, Marina; Fink, David J

    2002-01-01

    Neurotrophic factors have been demonstrated to prevent the development of peripheral neuropathy in animal models, but the therapeutic use of these factors in human disease has been limited by the short serum half-life and dose-limiting side effects of these potent peptides. We used peripheral subcutaneous inoculation with a replication-incompetent, genomic herpes simplex virus-based vector containing the coding sequence for neurotrophin-3 to transduce sensory neurons of the rat dorsal root ganglion in vivo, and found that expression of neurotrophin-3 from the vector protected peripheral sensory axons from neuropathy induced by intoxication with pyridoxine assessed by electrophysiological (foot sensory response amplitude, and conduction velocity, and H-wave), histological (nerve morphology and morphometry), and behavioral measures of proprioceptive function. In vivo gene transfer using herpes simplex virus vectors provides a unique option for treatment of diseases of the sensory peripheral nervous system.

  20. Interleukin-1-induced activation of the small GTPase Rac1 depends on receptor internalization and regulates gene expression.

    Science.gov (United States)

    Windheim, Mark; Hansen, Benjamin

    2014-01-01

    Interleukin 1 (IL-1) triggers the internalization of its cognate receptor from the plasma membrane. We recently demonstrated that this internalization is of critical importance for the IL-1-induced gene expression. In this study we report that the IL-1-induced activation of the small GTPase Rac1 requires receptor endocytosis. We further show that the depletion of Rac1 reduces the IL-1-dependent gene expression without affecting signaling events that are initiated at the plasma membrane. Collectively, we provide evidence for a key role of Rac1 in a pathway that regulates IL-1-induced gene expression depending on receptor endocytosis.

  1. Over-expressed Genes Detected by Suppression Subtractive Hybridization in Carcinoma Derived From Transformed 16HBE Cells Induced by BPDE

    Institute of Scientific and Technical Information of China (English)

    SHE-JUAN AN; JIA-KUN CHEN; LI-LI LIU; YAN-FENG ZHAO; XUE-MIN CHEN

    2005-01-01

    Objective To screen the over differentially expressed genes in carcinoma induced by BPDE-transformed 16HBE cells (16HBE-C cells). Methods The suppression subtractive hybridization (SSH) method was performed to profile differentially expressed genes between 16HBE-C cells and 16HBE cells. The cDNA fragments of differentially expressed genes were inserted into TA cloning vector and transformed competent E. coli strain. Positive clones were randomly picked up and identified by the colony PCR method. Dot blot was used to test the same source with the tester. The differentially expressed cDNA fragments were sequenced and compared with known genes and EST database in Genbank. Results Eight known genes were over-expressed in 16HBE-C cells including eukaryotic translation elongation factor 1 alpha 1, HIF-1 responsive RTP801, ribosomal protein L10 (RPL10), ribosomal protein S29 (RPS29), mitochondrion related genes, and laminin receptor 1. Three differentially expressed cDNA fragments could not be matched to the known genes but to the EST database. Conclusion The SSH method can detect differentially expressed genes between 16HBE-C and 16HBE cells. BPDE-induced carcinogenesis may be related to alteration of at least eight known genes and three unknown genes. These expression data provide a clue to further cloning novel genes and studying functions in BPDE-induced carcinoma.

  2. CLONING AND EXPRESSION OF A GENE MEDIATINGγ-RADIATION-INDUCED APOPTOSIS IN HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To identify the member of the caspase family proteases involved in γ-radiation-induced apoptosis in HL-60 cells and to study the expression of the caspase gene in normal, apoptotic cells and in immortal tu mor cells. Methods By using degenerate oligonucleotide primers encoding the highly conserved peptides that were pre sent in all known caspases, we performed RT-PCR on poly(A)RNA from γ-radiation-induced apoptotic HL-60 cells. Caspase-3 mRNA in apoptotic HL-60 cells and in human tumor cell lines was analyzed by Northern blot. Results The amplified DNA fragment was identified with caspase-3 cDNA by cloning and sequencing. The Northern blot analysis of caspase-3 mRNA of different human tumor cell lines showed that the caspase-3 gene transcript was more highly ex pressed in leukemia cell lines and the SH-SY5Y cell line than in HeLa and MCF-7 cells. It was more highly expressed in the radiation-induced apoptotic HL-60 cells than in control HL-60 cells. Conclusion These results indicated that caspase-3 was involved in γ-radiation-induced apoptosis in HL-60 cells. The high level of expression of caspase-3 may aid efforts to understand the insensitivity of some tumor cells to radiation, their inherent ability to survive, and apop tosis.

  3. Characterization of arecoline-induced effects on cytotoxicity in normal human gingival fibroblasts by global gene expression profiling.

    Science.gov (United States)

    Chiang, Shang-Lun; Jiang, Shih-Sheng; Wang, Yi-Jou; Chiang, Horn-Che; Chen, Ping-Ho; Tu, Hung-Pin; Ho, Kun-Yen; Tsai, Yu-Shan; Chang, I-Shou; Ko, Ying-Chin

    2007-11-01

    Areca nut is the most widely used psychoactive substance and an important environmental risk factor for development of oral premalignant lesions and cancer. Arecoline, the major alkaloid of areca nut, has been known to cause cytotoxicity and genotoxicity in mammalian cells in vivo and in vitro and even contributes to carcinogenicity. However, the susceptible genes accounting for arecoline-induced damage in normal human oral cells are still lacking, which possibly involves in initial molecular damage via alternation of gene expression level on biological pathways. The present study was undertaken to characterize the toxic effects of arecoline in gene expression profiling on normal human gingival fibroblasts (HGF) using cDNA microarray and quantitative real-time reverse transcription PCR. The cytotoxicity of arecoline on HGF-1 cell line was elevated in a dose-dependent manner (p arecoline determined from dose-response curve of the cytotoxicity, a large number of genes were significantly repressed than induced by arecoline in global gene expression profiling. Five induced- and seven repressed genes including glutathione synthetase were further validated, and their gene expression changes were increased in a dose-dependent manner in a concentration range of 50-150 microg/ml. In conclusion, we proposed a tentative model to explain arecoline-induced effects on contribution of oral pathogenesis. The findings identified that 12 susceptible genes can potentially serve as biomarkers of arecoline-induced damage in betel chewers.

  4. An elm EST database for identifying leaf beetle egg-induced defense genes

    Directory of Open Access Journals (Sweden)

    Büchel Kerstin

    2012-06-01

    Full Text Available Abstract Background Plants can defend themselves against herbivorous insects prior to the onset of larval feeding by responding to the eggs laid on their leaves. In the European field elm (Ulmus minor, egg laying by the elm leaf beetle ( Xanthogaleruca luteola activates the emission of volatiles that attract specialised egg parasitoids, which in turn kill the eggs. Little is known about the transcriptional changes that insect eggs trigger in plants and how such indirect defense mechanisms are orchestrated in the context of other biological processes. Results Here we present the first large scale study of egg-induced changes in the transcriptional profile of a tree. Five cDNA libraries were generated from leaves of (i untreated control elms, and elms treated with (ii egg laying and feeding by elm leaf beetles, (iii feeding, (iv artificial transfer of egg clutches, and (v methyl jasmonate. A total of 361,196 ESTs expressed sequence tags (ESTs were identified which clustered into 52,823 unique transcripts (Unitrans and were stored in a database with a public web interface. Among the analyzed Unitrans, 73% could be annotated by homology to known genes in the UniProt (Plant database, particularly to those from Vitis, Ricinus, Populus and Arabidopsis. Comparative in silico analysis among the different treatments revealed differences in Gene Ontology term abundances. Defense- and stress-related gene transcripts were present in high abundance in leaves after herbivore egg laying, but transcripts involved in photosynthesis showed decreased abundance. Many pathogen-related genes and genes involved in phytohormone signaling were expressed, indicative of jasmonic acid biosynthesis and activation of jasmonic acid responsive genes. Cross-comparisons between different libraries based on expression profiles allowed the identification of genes with a potential relevance in egg-induced defenses, as well as other biological processes, including signal transduction

  5. AMPKα is essential for acute exercise-induced gene responses but not for exercise training-induced adaptations in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Fentz, Joachim; Kjøbsted, Rasmus; Kristensen, Caroline Maag;

    2015-01-01

    -induced increases in exercise capacity and expression of metabolic proteins, as well as acute exercise-induced gene regulation, would be compromised in muscle-specific AMPKα1 and -α2 double-knockout (mdKO) mice. An acute bout of exercise increased skeletal muscle mRNA content of cytochrome c oxidase subunit I...

  6. Analysis of low temperature-induced genes (LTIG) in wine yeast during alcoholic fermentation.

    Science.gov (United States)

    Chiva, Rosana; López-Malo, Maria; Salvadó, Zoel; Mas, Albert; Guillamón, Jósé Manuel

    2012-11-01

    Fermentations carried out at low temperatures, that is, 10-15 °C, not only enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. In this study, we determined the transcriptional activity of 10 genes that were previously reported as induced by low temperatures and involved in cold adaptation, during fermentation with the commercial wine yeast strain QA23. Mutant and overexpressing strains of these genes were constructed in a haploid derivative of this strain to determine the importance of these genes in growth and fermentation at low temperature. In general, the deletion and overexpression of these genes did affect fermentation performance at low temperature. Most of the mutants were unable to complete fermentation, while overexpression of CSF1, HSP104, and TIR2 decreased the lag phase, increased the fermentation rate, and reached higher populations than that of the control strain. Another set of overexpressing strains were constructed by integrating copies of these genes in the delta regions of the commercial wine strain QA23. These new stable overexpressing strains again showed improved fermentation performance at low temperature, especially during the lag and exponential phases. Our results demonstrate the convenience of carrying out functional analysis in commercial strains and in an experimental set-up close to industrial conditions.

  7. Icariin Is A PPARα Activator Inducing Lipid Metabolic Gene Expression in Mice

    Directory of Open Access Journals (Sweden)

    Yuan-Fu Lu

    2014-11-01

    Full Text Available Icariin is effective in the treatment of hyperlipidemia. To understand the effect of icariin on lipid metabolism, effects of icariin on PPARα and its target genes were investigated. Mice were treated orally with icariin at doses of 0, 100, 200, and 400 mg/kg, or clofibrate (500 mg/kg for five days. Liver total RNA was isolated and the expressions of PPARα and lipid metabolism genes were examined. PPARα and its marker genes Cyp4a10 and Cyp4a14 were induced 2-4 fold by icariin, and 4-8 fold by clofibrate. The fatty acid (FA binding and co-activator proteins Fabp1, Fabp4 and Acsl1 were increased 2-fold. The mRNAs of mitochondrial FA β-oxidation enzymes (Cpt1a, Acat1, Acad1 and Hmgcs2 were increased 2-3 fold. The mRNAs of proximal β-oxidation enzymes (Acox1, Ech1, and Ehhadh were also increased by icariin and clofibrate. The expression of mRNAs for sterol regulatory element-binding factor-1 (Srebf1 and FA synthetase (Fasn were unaltered by icariin. The lipid lysis genes Lipe and Pnpla2 were increased by icariin and clofibrate. These results indicate that icariin is a novel PPARα agonist, activates lipid metabolism gene expressions in liver, which could be a basis for its lipid-lowering effects and its beneficial effects against diabetes.

  8. Expression dynamics of secreted protease genes in Trichophyton rubrum induced by key host's proteinaceous components.

    Science.gov (United States)

    Leng, Wenchuan; Liu, Tao; Wang, Jin; Li, Ruoyu; Jin, Qi

    2009-11-01

    Trichophyton rubrum is the most common agent of dermatophytosis, a disease that affects millions of individuals worldwide. Its molecular pathogenicity mechanisms are still not completely elucidated. It has been widely recognized that proteases secreted by T. rubrum are the key virulence factors during host infection. However, our knowledge about the expression of its secreted proteases in host infection is still obscure. This investigation provides the expression patterns and dynamics of secreted protease genes belonging to the subtilisins (SUB) and metalloproteases (MEP) gene families in T. rubrum. The data was obtained under simulated host infection conditions through relative quantification of real time PCR. Keratin, collagen, and elastin induced the expression of similar protease genes, and the expression patterns and dynamics of these protease genes in media containing human skin sections were different from those in media containing individual protein substrates. According to the expression dynamics of these protease genes, we conclude that Sub3, Sub4, and Mep4 may be the dominant proteases secreted by T. rubrum during host infection, and that these proteases could be good targets for new antifungal chemotherapy and molecular diagnostic markers. This work presents useful molecular details to further our understanding of the pathogenesis of dermatophytosis.

  9. Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design.

    Science.gov (United States)

    Maruggi, Giulietta; Porcellini, Simona; Facchini, Giulia; Perna, Serena K; Cattoglio, Claudia; Sartori, Daniela; Ambrosi, Alessandro; Schambach, Axel; Baum, Christopher; Bonini, Chiara; Bovolenta, Chiara; Mavilio, Fulvio; Recchia, Alessandra

    2009-05-01

    The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.

  10. Ginsenoside Rg1-induced alterations in gene expression in TNF-α stimulated endothelial cells

    Institute of Scientific and Technical Information of China (English)

    吕俊萍; 马增春; 杨静; 黄坚; 王树人; 王升启

    2004-01-01

    Background In China the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The authors investigated the effect of ginsenoside Rg1 on the spectrum of gene expression in the endothelial cells stimulated by TNF-α and further explored the potential molecular mechanism of endothelial protection by ginsenoside Rg1.Methods Nitric oxide (NO) production in the cultured human umbilical vein endothelial cells(HUVECs) was measured by using an NO assay kit. A home-made oligonucleotide microarray containing approximately 400 cardiovascular disease-related genes was constructed. The alteration of the spectrum of gene expression induced by ginsenoside Rg1 in HUVECs which were activated by TNF-α were detected by oligonucleotide microarray analysis.Results NO production in HUVECs was decreased significantly after TNF-α treatment, while pretreatment with ginsenoside Rg1 enhanced NO production in TNF-αstimulated HUVECs. Ginsenoside Rg1 affected the expression levels of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-αstimulated HUVECs.Conclusions Ginsenoside Rg1 could enhance NO production and the expression of eNOS mRNA in TNF-α stimulated HUVECs. Ginsenoside Rg1 regulated sets of genes in endothelial cells and protected endothelial cells from TNF-αactivation. Microarray analysis provided us with valuable insights into the atheroprotective mechanism by gingsenoside Rg1.

  11. Selective serotonin reuptake inhibitor antidepressants potentiate methylphenidate (Ritalin)-induced gene regulation in the adolescent striatum.

    Science.gov (United States)

    Van Waes, Vincent; Beverley, Joel; Marinelli, Michela; Steiner, Heinz

    2010-08-01

    The psychostimulant methylphenidate (Ritalin) is used in conjunction with selective serotonin reuptake inhibitors (SSRIs) in the treatment of medical conditions such as attention-deficit hyperactivity disorder with anxiety/depression comorbidity and major depression. Co-exposure also occurs in patients on SSRIs who use psychostimulant 'cognitive enhancers'. Methylphenidate is a dopamine/norepinephrine reuptake inhibitor that produces altered gene expression in the forebrain; these effects partly mimic gene regulation by cocaine (dopamine/norepinephrine/serotonin reuptake inhibitor). We investigated whether the addition of SSRIs (fluoxetine or citalopram; 5 mg/kg) modified gene regulation by methylphenidate (2-5 mg/kg) in the striatum and cortex of adolescent rats. Our results show that SSRIs potentiate methylphenidate-induced expression of the transcription factor genes zif268 and c-fos in the striatum, rendering these molecular changes more cocaine-like. Present throughout most of the striatum, this potentiation was most robust in its sensorimotor parts. The methylphenidate + SSRI combination also enhanced behavioral stereotypies, consistent with dysfunction in sensorimotor striatal circuits. In so far as such gene regulation is implicated in psychostimulant addiction, our findings suggest that SSRIs may enhance the addiction potential of methylphenidate.

  12. Exogenous PTEN Gene Induces Apoptosis in Breast Carcinoma Cell Line MDA468

    Institute of Scientific and Technical Information of China (English)

    CHEN Qingyong; WANG Chunyou; JIANG Chunfang; CHEN Daoda

    2007-01-01

    The effects and mechanisms of exogenous phosphatase and tensin homolog deleted from chromosome ten (PTEN) gene on phosphatase activity-dependent apoptosis of breast cancer cell line MDA468 were investigated. PTEN gene packaged with lipofectin was transferred into breast cancer cell line MDA468 and parental MDA468 cells served as controls. RT-PCR and Western blot were done to detect the expression of target genes. The expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by epidermal growth factor (EGF) was also detected. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. MDA468 cells transfected with PTEN gene could express PTEN mRNA and protein. PTEN decreased the phosphorylation level of AKT protein and down-regulated FAK protein expression in MDA468 stimulated by EGF. The apoptosis rate was 21.68%. PTEN induced breast cancer apoptosis phosphatase activity-dependently. The mechanism is possibly relatedwith phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB)/AKT signaling pathway. Those results may provide new clues on the gene therapy in breast cancer.

  13. MUC1 induces drug resistance in pancreatic cancer cells via upregulation of multidrug resistance genes.

    Science.gov (United States)

    Nath, S; Daneshvar, K; Roy, L D; Grover, P; Kidiyoor, A; Mosley, L; Sahraei, M; Mukherjee, P

    2013-06-17

    MUC1 (CD227), a membrane tethered mucin glycoprotein, is overexpressed in >60% of human pancreatic cancers (PCs), and is associated with poor prognosis, enhanced metastasis and chemoresistance. The objective of this study was to delineate the mechanism by which MUC1 induces drug resistance in human (BxPC3 and Capan-1) and mouse (KCKO, KCM) PC cells. We report that PC cells that express high levels of MUC1 exhibit increased resistance to chemotherapeutic drugs (gemcitabine and etoposide) in comparison with cells that express low levels of MUC1. This chemo resistance was attributed to the enhanced expression of multidrug resistance (MDR) genes including ABCC1, ABCC3, ABCC5 and ABCB1. In particular, levels of MRP1 protein encoded by the ABCC1 gene were significantly higher in the MUC1-high PC cells. In BxPC3 and Capan-1 cells MUC1 upregulates MRP1 via an Akt-dependent pathway, whereas in KCM cells MUC1-mediated MRP1 upregulation is via an Akt-independent mechanism. In KCM, BxPC3 and Capan-1 cells, the cytoplasmic tail motif of MUC1 associates directly with the promoter region of the Abcc1/ABCC1 gene, indicating a possible role of MUC1 acting as a transcriptional regulator of this gene. This is the first report to show that MUC1 can directly regulate the expression of MDR genes in PC cells, and thus confer drug resistance.

  14. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus

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    Sang-Ho Park

    2016-08-01

    Full Text Available Grapevine Algerian latent virus (GALV is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP, but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

  15. Development of Virus-Induced Gene Expression and Silencing Vector Derived from Grapevine Algerian Latent Virus.

    Science.gov (United States)

    Park, Sang-Ho; Choi, Hoseong; Kim, Semin; Cho, Won Kyong; Kim, Kook-Hyung

    2016-08-01

    Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

  16. Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Sheng-Jun Zhang; Hong-Ying Chen; Zhi-Xin Chen; Xiao-Zhong Wang

    2005-01-01

    AIM: To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells.METHODS: HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 μg/mLG418 and named HL-7702/HBV-encoded X protein (HBx)cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively.RESULTS: RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semiquantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than those in control.CONCLUSION: HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL,Bax/Bcl-2, and c-myc gene in a dose-dependent manner.

  17. Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge.

    Science.gov (United States)

    van Wees, S C; Luijendijk, M; Smoorenburg, I; van Loon, L C; Pieterse, C M

    1999-11-01

    Selected strains of nonpathogenic rhizobacteria from the genus Pseudomonas are capable of eliciting broad-spectrum induced systemic resistance (ISR) in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). In Arabidopsis, the ISR pathway functions independently of salicylic acid (SA) but requires responsiveness to jasmonate and ethylene. Here, we demonstrate that known defense-related genes, i.e. the SA-responsive genes PR-1, PR-2, and PR-5, the ethylene-inducible gene Hel, the ethylene- and jasmonate-responsive genes ChiB and Pdf1.2, and the jasmonate-inducible genes Atvsp, Lox1, Lox2, Pall, and Pin2, are neither induced locally in the roots nor systemically in the leaves upon induction of ISR by Pseudomonas fluorescens WCS417r. In contrast, plants infected with the virulent leaf pathogen Pseudomonas syringae pv. tomato (Pst) or expressing SAR induced by preinfecting lower leaves with the avirulent pathogen Pst(avrRpt2) exhibit elevated expression levels of most of the defense-related genes studied. Upon challenge inoculation with Pst, PR gene transcripts accumulated to a higher level in SAR-expressing plants than in control-treated and ISR-expressing plants, indicating that SAR involves potentiation of SA-responsive PR gene expression. In contrast, pathogen challenge of ISR-expressing plants led to an enhanced level of Atvsp transcript accumulation. The otherjasmonate-responsive defense-related genes studied were not potentiated during ISR, indicating that ISR is associated with the potentiation of specific jasmonate-responsive genes.

  18. Selective serotonin re-uptake inhibitors potentiate gene blunting induced by repeated methylphenidate treatment: Zif268 versus Homer1a.

    Science.gov (United States)

    Van Waes, Vincent; Vandrevala, Malcolm; Beverley, Joel; Steiner, Heinz

    2014-11-01

    There is a growing use of psychostimulants, such as methylphenidate (Ritalin; dopamine re-uptake inhibitor), for medical treatments and as cognitive enhancers in the healthy. Methylphenidate is known to produce some addiction-related gene regulation. Recent findings in animal models show that selective serotonin re-uptake inhibitors (SSRIs), including fluoxetine, can potentiate acute induction of gene expression by methylphenidate, thus indicating an acute facilitatory role for serotonin in dopamine-induced gene regulation. We investigated whether repeated exposure to fluoxetine, in conjunction with methylphenidate, in adolescent rats facilitated a gene regulation effect well established for repeated exposure to illicit psychostimulants such as cocaine-blunting (repression) of gene inducibility. We measured, by in situ hybridization histochemistry, the effects of a 5-day repeated treatment with methylphenidate (5 mg/kg), fluoxetine (5 mg/kg) or a combination on the inducibility (by cocaine) of neuroplasticity-related genes (Zif268, Homer1a) in the striatum. Repeated methylphenidate treatment alone produced minimal gene blunting, while fluoxetine alone had no effect. In contrast, fluoxetine added to methylphenidate robustly potentiated methylphenidate-induced blunting for both genes. This potentiation was widespread throughout the striatum, but was most robust in the lateral, sensorimotor striatum, thus mimicking cocaine effects. For illicit psychostimulants, blunting of gene expression is considered part of the molecular basis of addiction. Our results thus suggest that SSRIs, such as fluoxetine, may increase the addiction liability of methylphenidate.

  19. Changes in gene expression induced by aromatic amine drugs: testing the danger hypothesis.

    Science.gov (United States)

    Ng, Winnie; Uetrecht, Jack

    2013-01-01

    Virtually all drugs that contain a primary aromatic amine are associated with a high incidence of idiosyncratic drug reactions (IDRs), suggesting that this functional group has biological effects that may be used as biomarkers to predict IDR risk. Most IDRs exhibit evidence of immune involvement and the ability of aromatic amines to form reactive metabolites and redox cycle may be responsible for initiation of an immune response through induction of cell stress, as postulated by the Danger Hypothesis. If true, danger signals could be biomarkers of IDR risk. A previous attempt to test the Danger Hypothesis found that sulfamethoxazole (SMX), the only aromatic amine tested, was also the only drug not associated with an increase of cell stress genes in mice. To ensure that these observations were not species-specific, and to determine biomarkers of IDR risk common to aromatic amines, rats were treated with SMX and two other aromatic amine drugs, dapsone (DDS) and aminoglutethimide (AMG), and hepatic gene expression was determined using microarrays. As in mice, SMX induced minimal gene changes in the rat, and none indicated cell stress, whereas DDS and AMG induced several changes including up-regulation of enzymes such as aldo-keto reductase, glutathione-S-transferase, and aldehyde dehydrogenase, which may represent danger signals. Early insulin-induced hepatic gene (Eiih) was up-regulated by all three drugs. Some mRNA changes were observed in the Keap-1-Nrf2-ARE pathway; however, the pattern was significantly different for each drug. Overall, the most salient finding was that the changes in the liver were minimal, even though aromatic amines cause a high incidence of IDRs. The liver generates a large number of reactive species; however, the ability of aromatic amines to be bioactivated by cells of the immune system may be why they cause a high incidence of IDRs.

  20. Acute overactive endocannabinoid signaling induces glucose intolerance, hepatic steatosis, and novel cannabinoid receptor 1 responsive genes.

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    Maxwell A Ruby

    Full Text Available Endocannabinoids regulate energy balance and lipid metabolism by stimulating the cannabinoid receptor type 1 (CB1. Genetic deletion and pharmacological antagonism have shown that CB1 signaling is necessary for the development of obesity and related metabolic disturbances. However, the sufficiency of endogenously produced endocannabinoids to cause hepatic lipid accumulation and insulin resistance, independent of food intake, has not been demonstrated. Here, we show that a single administration of isopropyl dodecylfluorophosphonate (IDFP, perhaps the most potent pharmacological inhibitor of endocannabinoid degradation, increases hepatic triglycerides (TG and induces insulin resistance in mice. These effects involve increased CB1 signaling, as they are mitigated by pre-administration of a CB1 antagonist (AM251 and in CB1 knockout mice. Despite the strong physiological effects of CB1 on hepatic lipid and glucose metabolism, little is known about the downstream targets responsible for these effects. To elucidate transcriptional targets of CB1 signaling, we performed microarrays on hepatic RNA isolated from DMSO (control, IDFP and AM251/IDFP-treated mice. The gene for the secreted glycoprotein lipocalin 2 (lcn2, which has been implicated in obesity and insulin resistance, was among those most responsive to alterations in CB1 signaling. The expression pattern of IDFP mice segregated from DMSO mice in hierarchal cluster analysis and AM251 pre-administration reduced (>50% the majority (303 of 533 of the IDFP induced alterations. Pathway analysis revealed that IDFP altered expression of genes involved in lipid, fatty acid and steroid metabolism, the acute phase response, and amino acid metabolism in a CB1-dependent manner. PCR confirmed array results of key target genes in multiple independent experiments. Overall, we show that acute IDFP treatment induces hepatic TG accumulation and insulin resistance, at least in part through the CB1 receptor, and

  1. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.

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    Robert A Koza

    2006-05-01

    Full Text Available High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity.

  2. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.

    Science.gov (United States)

    Koza, Robert A; Nikonova, Larissa; Hogan, Jessica; Rim, Jong-Seop; Mendoza, Tamra; Faulk, Christopher; Skaf, Jihad; Kozak, Leslie P

    2006-05-01

    High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity.

  3. Hepatitis B virus x gene and cyanobacterial toxins promote aflatoxin B1-induced hepatotumorigenesis in mice

    Institute of Scientific and Technical Information of China (English)

    Min Lian; Ying Liu; Shun-Zhang Yu; Geng-Sun Qian; Shu-Guang Wan; Kenneth R Dixon

    2006-01-01

    AIM: To assess the combinative role of aflatoxin B1 (AFB1),cyanobacterial toxins (cyanotoxins), and hepatitis B virus (HBV) x gene in hepatotumorigenicity.METHODS: One-week-old animals carrying HBV x gene and their wild-type littermates were intraperitoneally (ip) injected with either single-dose AFB1 [6 mg/kg body weight (bw)], repeated-dose cyanotoxins (microcystinLR or nodularin, 10 μg/kg bw oncea week for 15 wk),DMSO (vehicle control) alone, or AFB1 followed by cyanotoxins a week later, and were sacrificed at 24 and 52 wk post-treatment.RESULTS: AFB1 induced liver tumors in 13 of 29 (44.8%) transgenic mice at 52 wk post-treatment, significantly more frequent than in wild-type mice (13.3%). This significant difference was not shown in the 24-wk study. Compared with AFB1 exposure alone, MC-LR and nodularin yielded approximately 3-fold and 6-fold increases in the incidence of AFB1-induced liver tumors in wild-type animals at 24 wk, respectively. HBV x gene did not further elevate the risk associated with coexposure to AFB1 and cyanotoxins. With the exception of an MC-LR-dosed wild-type mouse, no liver tumor was observed in mice treated with cyanotoxins alone at 24 wk. Neither DMSO-treated transgenic mice nor their wild-type littermates had pathologic alterations relevant to hepatotumorigenesis in even up to 52 wk.CONCLUSION: HBV x gene and nodularin promote the development of AFB1-induced liver tumors. Co-exposure to AFB1 and MC-LR tends to elevate the risk of liver tumors at 24 wk relative to exposure to one of them.The combinative effect of AFB1, cyanotoxins and HBVx on hepatotumorigenesis is weak at 24 wk.

  4. Anchoring ethinylestradiol induced gene expression changes with testicular morphology and reproductive function in the medaka.

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    Hilary D Miller

    Full Text Available Environmental estrogens are ubiquitous in the environment and can cause detrimental effects on male reproduction. In fish, a multitude of effects from environmental estrogens have been observed including altered courting behavior and fertility, sex reversal, and gonadal histopathology. However, few studies in fish assess the impacts of estrogenic exposure on a physiological endpoint, such as reproduction, as well as the associated morphologic response and underlying global gene expression changes. This study assessed the implications of a 14 day sub-chronic exposure of ethinylestradiol (EE2; 1.0 or 10.0 µg/L EE2 on male medaka fertility, testicular histology and testicular gene expression. The findings demonstrate that a 14 day exposure to EE2 induced impaired male reproductive capacity and time- and dose-dependent alterations in testicular morphology and gene expression. The average fertilization rate/day following the exposure for control, 1.0 and 10.0 µg/L EE2 was 91.3% (±4.4, 62.8% (±8.3 and 28.8% (±5.8, respectively. The testicular morphologic alterations included increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. These changes were highly associated with testicular gene expression changes using a medaka-specific microarray. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell cycle and proliferation, collagen production/extracellular matrix organization, hormone signaling, male reproduction and protein ubiquitination among others. These findings highlight the importance of anchoring global gonadal gene expression changes with morphology and ultimately with tissue/organ function.

  5. Gene expression analysis of induced pluripotent stem cells from aneuploid chromosomal syndromes

    Science.gov (United States)

    2013-01-01

    Background Human aneuploidy is the leading cause of early pregnancy loss, mental retardation, and multiple congenital anomalies. Due to the high mortality associated with aneuploidy, the pathophysiological mechanisms of aneuploidy syndrome remain largely unknown. Previous studies focused mostly on whether dosage compensation occurs, and the next generation transcriptomics sequencing technology RNA-seq is expected to eventually uncover the mechanisms of gene expression regulation and the related pathological phenotypes in human aneuploidy. Results Using next generation transcriptomics sequencing technology RNA-seq, we profiled the transcriptomes of four human aneuploid induced pluripotent stem cell (iPSC) lines generated from monosomy × (Turner syndrome), trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome), and partial trisomy 11:22 (Emanuel syndrome) as well as two umbilical cord matrix iPSC lines as euploid controls to examine how phenotypic abnormalities develop with aberrant karyotype. A total of 466 M (50-bp) reads were obtained from the six iPSC lines, and over 13,000 mRNAs were identified by gene annotation. Global analysis of gene expression profiles and functional analysis of differentially expressed (DE) genes were implemented. Over 5000 DE genes are determined between aneuploidy and euploid iPSCs respectively while 9 KEGG pathways are overlapped enriched in four aneuploidy samples. Conclusions Our results demonstrate that the extra or missing chromosome has extensive effects on the whole transcriptome. Functional analysis of differentially expressed genes reveals that the genes most affected in aneuploid individuals are related to central nervous system development and tumorigenesis. PMID:24564826

  6. Maternal diet during pregnancy induces gene expression and DNA methylation changes in fetal tissues in sheep

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    Xianyong eLan

    2013-04-01

    Full Text Available Studies in rats and mice have established that maternal nutrition induces epigenetic modifications, sometimes permanently, that alter gene expression in the fetus, which in turn leads to phenotypic changes. However, limited data is available on the influence of maternal diet on epigenetic modifications and gene expression in sheep. Therefore, the objectives of this study were to investigate the impact of different maternal dietary energy sources on the expression of imprinted genes in fetuses in sheep. Ewes were naturally bred to a single sire and from d 67 ± 3 of gestation until necropsy (d 130 ± 1, they were fed one of three diets of alfalfa haylage (HY; fiber, corn (CN; starch, or dried corn distiller’s grains (DG; fiber plus protein plus fat. A total of 26 fetuses were removed from the dams and longissimus dorsi, semitendinosus, perirenal adipose depot, and subcutaneous adipose depot tissues were collected for expression and DNA methylation analyses. Expression analysis of nine imprinted genes and three DNA methylatransferase (DNMTs genes showed significant effects of the different maternal diets on the expression of these genes. The methylation levels of CpG islands of both IGF2R and H19 were higher in HY and DG than CN fetuses in both males and females. This result is consistent with the low amino acid content of the CN diet, a source of methyl group donors, compared to HY and DG diets. Thus, results of this study provide evidence of association between maternal nutrition during pregnancy and transcriptomic and epigenomic alterations of imprinted genes and DNMTs in the fetal tissues.

  7. A role for FACT in repopulation of nucleosomes at inducible genes.

    Science.gov (United States)

    Voth, Warren P; Takahata, Shinya; Nishikawa, Joy L; Metcalfe, Benjamin M; Näär, Anders M; Stillman, David J

    2014-01-01

    Xenobiotic drugs induce Pleiotropic Drug Resistance (PDR) genes via the orthologous Pdr1/Pdr3 transcription activators. We previously identified the Mediator transcription co-activator complex as a key target of Pdr1 orthologs and demonstrated that Pdr1 interacts directly with the Gal11/Med15 subunit of the Mediator complex. Based on an interaction between Pdr1 and the FACT complex, we show that strains with spt16 or pob3 mutations are sensitive to xenobiotic drugs and display diminished PDR gene induction. Although FACT acts during the activation of some genes by assisting in the nucleosomes eviction at promoters, PDR promoters already contain nucleosome-depleted regions (NDRs) before induction. To determine the function of FACT at PDR genes, we examined the kinetics of RNA accumulation and changes in nucleosome occupancy following exposure to a xenobiotic drug in wild type and FACT mutant yeast strains. In the presence of normal FACT, PDR genes are transcribed within 5 minutes of xenobiotic stimulation and transcription returns to basal levels by 30-40 min. Nucleosomes are constitutively depleted in the promoter regions, are lost from the open reading frames during transcription, and the ORFs are wholly repopulated with nucleosomes as transcription ceases. While FACT mutations cause minor delays in activation of PDR genes, much more pronounced and significant defects in nucleosome repopulation in the ORFs are observed in FACT mutants upon transcription termination. FACT therefore has a major role in nucleosome redeposition following cessation of transcription at the PDR genes, the opposite of its better-known function in nucleosome disassembly.

  8. A role for FACT in repopulation of nucleosomes at inducible genes.

    Directory of Open Access Journals (Sweden)

    Warren P Voth

    Full Text Available Xenobiotic drugs induce Pleiotropic Drug Resistance (PDR genes via the orthologous Pdr1/Pdr3 transcription activators. We previously identified the Mediator transcription co-activator complex as a key target of Pdr1 orthologs and demonstrated that Pdr1 interacts directly with the Gal11/Med15 subunit of the Mediator complex. Based on an interaction between Pdr1 and the FACT complex, we show that strains with spt16 or pob3 mutations are sensitive to xenobiotic drugs and display diminished PDR gene induction. Although FACT acts during the activation of some genes by assisting in the nucleosomes eviction at promoters, PDR promoters already contain nucleosome-depleted regions (NDRs before induction. To determine the function of FACT at PDR genes, we examined the kinetics of RNA accumulation and changes in nucleosome occupancy following exposure to a xenobiotic drug in wild type and FACT mutant yeast strains. In the presence of normal FACT, PDR genes are transcribed within 5 minutes of xenobiotic stimulation and transcription returns to basal levels by 30-40 min. Nucleosomes are constitutively depleted in the promoter regions, are lost from the open reading frames during transcription, and the ORFs are wholly repopulated with nucleosomes as transcription ceases. While FACT mutations cause minor delays in activation of PDR genes, much more pronounced and significant defects in nucleosome repopulation in the ORFs are observed in FACT mutants upon transcription termination. FACT therefore has a major role in nucleosome redeposition following cessation of transcription at the PDR genes, the opposite of its better-known function in nucleosome disassembly.

  9. Distinct lithium-induced gene expression effects in lymphoblastoid cell lines from patients with bipolar disorder.

    Science.gov (United States)

    Fries, Gabriel R; Colpo, Gabriela D; Monroy-Jaramillo, Nancy; Zhao, Junfei; Zhao, Zhongming; Arnold, Jodi G; Bowden, Charles L; Walss-Bass, Consuelo

    2017-09-19

    Lithium is the most commonly prescribed medication for the treatment of bipolar disorder (BD), yet the mechanisms underlying its beneficial effects are still unclear. We aimed to compare the effects of lithium treatment in lymphoblastoid cell lines (LCLs) from BD patients and controls. LCLs were generated from sixty-two BD patients (based on DSM-IV) and seventeen healthy controls matched for age, sex, and ethnicity. Patients were recruited from outpatient clinics from February 2012 to October 2014. LCLs were treated with 1mM lithium for 7 days followed by microarray gene expression assay and validation by real-time quantitative PCR. Baseline differences between groups, as well as differences between vehicle- and lithium-treated cells within each group were analyzed. The biological significance of differentially expressed genes was examined by pathway enrichment analysis. No significant differences in baseline gene expression (adjusted p-value Lithium treatment of LCLs from controls did not lead to any significant differences. However, lithium altered the expression of 236 genes in LCLs from patients; those genes were enriched for signaling pathways related to apoptosis. Among those genes, the alterations in the expression of PIK3CG, SERP1 and UPP1 were validated by real-time PCR. A significant correlation was also found between circadian functioning and CEBPG and FGF2 expression levels. In summary, our results suggest that lithium treatment induces expression changes in genes associated with the apoptosis pathway in BD LCLs. The more pronounced effects of lithium in patients compared to controls suggest a disease-specific effect of this drug. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

  10. Exercise induces transient transcriptional activation of the PGC-1α gene in human skeletal muscle

    Science.gov (United States)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1α (PGC-1α) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC-1α transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl-CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P 40-fold) and mRNA content (7- to 10-fold), peaking within 2 h after exercise. Activation of PGC-1α was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF-1) mRNA, a gene induced by PGC-1α in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor α, and calcineurin Aα and Aβ mRNA were elevated (≈2- to 6-fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1α transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC-1α may coordinate the activation of metabolic genes in human muscle in response to exercise. PMID:12563009

  11. Exercise induces transient transcriptional activation of the PGC-1alpha gene in human skeletal muscle.

    Science.gov (United States)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P Darrell

    2003-02-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1alpha (PGC-1alpha) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC-1alpha transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl-CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P 40-fold) and mRNA content (7- to 10-fold), peaking within 2 h after exercise. Activation of PGC-1alpha was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF-1) mRNA, a gene induced by PGC-1alpha in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor alpha, and calcineurin Aalpha and Abeta mRNA were elevated (approximately 2- to 6-fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC-1alpha transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC-1alpha may coordinate the activation of metabolic genes in human muscle in response to exercise.

  12. Ketamine and Imipramine Reverse Transcriptional Signatures of Susceptibility and Induce Resilience-Specific Gene Expression Profiles.

    Science.gov (United States)

    Bagot, Rosemary C; Cates, Hannah M; Purushothaman, Immanuel; Vialou, Vincent; Heller, Elizabeth A; Yieh, Lynn; LaBonté, Benoit; Peña, Catherine J; Shen, Li; Wittenberg, Gayle M; Nestler, Eric J

    2017-02-15

    Examining transcriptional regulation by antidepressants in key neural circuits implicated in depression and understanding the relation to transcriptional mechanisms of susceptibility and natural resilience may help in the search for new therapeutic agents. Given the heterogeneity of treatment response in human populations, examining both treatment response and nonresponse is critical. We compared the effects of a conventional monoamine-based tricyclic antidepressant, imipramine, and a rapidly acting, non-monoamine-based antidepressant, ketamine, in mice subjected to chronic social defeat stress, a validated depression model, and used RNA sequencing to analyze transcriptional profiles associated with susceptibility, resilience, and antidepressant response and nonresponse in the prefrontal cortex (PFC), nucleus accumbens, hippocampus, and amygdala. We identified similar numbers of responders and nonresponders after ketamine or imipramine treatment. Ketamine induced more expression changes in the hippocampus; imipramine induced more expression changes in the nucleus accumbens and amygdala. Transcriptional profiles in treatment responders were most similar in the PFC. Nonresponse reflected both the lack of response-associated gene expression changes and unique gene regulation. In responders, both drugs reversed susceptibility-associated transcriptional changes and induced resilience-associated transcription in the PFC. We generated a uniquely large resource of gene expression data in four interconnected limbic brain regions implicated in depression and its treatment with imipramine or ketamine. Our analyses highlight the PFC as a key site of common transcriptional regulation by antidepressant drugs and in both reversing susceptibility- and inducing resilience-associated molecular adaptations. In addition, we found region-specific effects of each drug, suggesting both common and unique effects of imipramine versus ketamine. Copyright © 2016 Society of Biological

  13. Function of DNA methyltransferase 3a in lead (Pb(2+) )-Induced Cyclooxygenase-2 gene.

    Science.gov (United States)

    Tsai, Yao-Ting; Chang, Che-Mai; Wang, Jaw-Yuan; Hou, Ming-Feng; Wang, Ju-Ming; Shiurba, Robert; Chang, Wen-Chang; Chang, Wei-Chiao

    2015-09-01

    Lead ions (Pb(2+) ) are toxic industrial pollutants associated with chronic inflammatory diseases in humans and animals. Previously, we found that Pb(2+) ions induce COX-2 gene expression via the EGF receptor/nuclear factor-κB signal transduction pathway in epidermoid carcinoma cell line A431. In this study, to see whether Pb(2+) ions affect COX-2 expression by epigenetic mechanisms, we looked at the mRNAs of DNA methyltransferases (DNMTs) using real-time PCR of total RNA from these cells. Cells exposed to Pb(2+) had low levels of DNMT3a mRNA, whereas the levels of DNMT1 and DNMT3b mRNAs remained unchanged. Pretreatment of cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5 μM) followed by Pb(2+) (1 μM) significantly increased levels of COX-2 mRNA compared with cells treated with Pb(2+) alone. Overexpression of tumor suppressor gene Rb correlated with an increase in COX-2 mRNA and a decrease in DNMT3a mRNA. Conversely, overexpression of transcription factor E2F1 correlated with a decrease in COX-2 mRNA and an increase in DMNT3a mRNA. Pretreatment with EGFR inhibitors AG1478 and PD153035 significantly limited Pb(2+) -induced reduction in DNMT3a mRNA. In addition, gene knockdown of DNMT3a with short hairpin RNA correlated with increased COX-2 mRNA induced by Pb(2+) . Our findings suggest Pb(2+) ions induce COX-2 expression indirectly by reducing DNMT3a methylation of the COX-2 promoter via transcription factors Rb and E2F1. © 2014 Wiley Periodicals, Inc.

  14. Pharmacological Inhibition of DNA Methylation Induces Proinvasive and Prometastatic Genes In Vitro and In Vivo

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    Bushra Ateeq

    2008-03-01

    Full Text Available The mechanism of action of DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR, a potential anticancer agent is believed to be activated by the demethylation of tumor suppressor genes. We tested here the hypothesis that demethylating agents also demethylate and activate genes involved in invasion and metastasis and therefore might increase the risk of developing tumor metastasis. The effect of 5-aza-CdR on noninvasive human breast cancer cells MCF-7 and ZR-75-1 was evaluated by cell proliferation, invasion, and migration assay. The ability of 5-aza-CdR to activate a panel of silenced prometastatic and tumor suppressor genes was evaluated using reverse transcription-polymerase chain reaction and bisulfite DNA sequence analysis in vitro and for change in tumor growth and gene expression in vivo. Treatment of MCF-7 and ZR-75-1 with 5-aza-CdR diminished cell proliferation, induced tumor suppressor RASSF1A, and altered cell cycle kinetics' G2/M-phase cell cycle arrest. While these effects of 5-aza-CdR slowed the growth of tumors in nude mice, it also induced a battery of prometastatic genes, namely, uPA, CXCR4, HEPARANASE, SYNUCLEIN γ, and transforming growth factor-beta (TGF-β, by demethylation of their promoters. These results draw attention to the critical role of demethylation as a potential mechanism that can promote the development and progression of tumor metastasis after demethylation therapy as an anticancer treatment.

  15. Phosphate Modulates Transcription of Soybean VspB and Other Sugar-Inducible Genes.

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    Sadka, A.; DeWald, D. B.; May, G. D.; Park, W. D.; Mullet, J. E.

    1994-05-01

    The soybean vegetative storage protein genes VspA and VspB encode vacuolar glycoprotein acid phosphatases. Transcription of the Vsp is synergistically activated by jasmonic acid or methyl jasmonate (MeJA) and soluble sugars. The action of these modulators is mediated by two different DNA domains in the VspB promoter. In this study, we present new data regarding VspB regulation by sucrose and inorganic phosphate, which suggest a common mechanism of transcriptional control for Vsp and other sugar-inducible genes. We found that the sugar-mediated activation of VspB expression was inhibited by phosphate. Deletion analysis and transient assays in tobacco protoplasts identified a 130-bp DNA domain in the VspB promoter that mediates both sucrose induction and phosphate inhibition. Transcription mediated by this DNA domain was induced by phosphate elimination from the protoplast incubation medium, even in the absence of sucrose. The effect of sucrose and phosphate on VspB expression was studied in vivo in several ways. Depletion of phosphate from soybean cell cultures by the addition of mannose stimulated VspB expression, even in the absence of sucrose or MeJA. In illuminated soybean leaves treated with MeJA, inhibition of photosynthetic electron transport by DCMU decreased VspB expression. In contrast, VspB expression in soybean leaves stimulated by phosphate depletion was not influenced by DCMU. Moreover, sucrose-stimulated expression of the sugar-responsive genes lipoxygenase A and chalcone synthase of soybean and proteinase inhibitor II and class I patatin of potato was inhibited by phosphate. Like VspB, these genes were stimulated by phosphate depletion in the absence of exogenous sucrose. We propose that sugar-responsive genes are activated, in part, by accumulation of sugar-phosphates and concomitant reduction of cellular phosphate levels. These data may help explain recruitment of the Vsp, which encode acid phosphatases, as vegetative storage proteins.

  16. Inducible defenses stay up late: temporal patterns of immune gene expression in Tenebrio molitor.

    Science.gov (United States)

    Johnston, Paul R; Makarova, Olga; Rolff, Jens

    2013-12-06

    The course of microbial infection in insects is shaped by a two-stage process of immune defense. Constitutive defenses, such as engulfment and melanization, act immediately and are followed by inducible defenses, archetypically the production of antimicrobial peptides, which eliminate or suppress the remaining microbes. By applying RNAseq across a 7-day time course, we sought to characterize the long-lasting immune response to bacterial challenge in the mealworm beetle Tenebrio molitor, a model for the biochemistry of insect immunity and persistent bacterial infection. By annotating a hybrid de novo assembly of RNAseq data, we were able to identify putative orthologs for the majority of components of the conserved insect immune system. Compared with Tribolium castaneum, the most closely related species with a reference genome sequence and a manually curated immune system annotation, the T. molitor immune gene count was lower, with lineage-specific expansions of genes encoding serine proteases and their countervailing inhibitors accounting for the majority of the deficit. Quantitative mapping of RNAseq reads to the reference assembly showed that expression of genes with predicted functions in cellular immunity, wound healing, melanization, and the production of reactive oxygen species was transiently induced immediately after immune challenge. In contrast, expression of genes encoding antimicrobial peptides or components of the Toll signaling pathway and iron sequestration response remained elevated for at least 7 days. Numerous genes involved in metabolism and nutrient storage were repressed, indicating a possible cost of immune induction. Strikingly, the expression of almost all antibacterial peptides followed the same pattern of long-lasting induction, regardless of their spectra of activity, signaling possible interactive roles in vivo.

  17. [Cadmium induces p53-dependent apoptosis through the inhibition of Ube2d family gene expression].

    Science.gov (United States)

    Tokumoto, Maki; Satoh, Masahiko

    2012-01-01

    Cadmium (Cd), a harmful metal, exerts severe toxic effects on various tissues such as those in the kidney, liver, lung, and bone. In particular, renal toxicity with damage to proximal tubule cells is caused by chronic exposure to Cd. However, the molecular mechanism underlying chronic Cd renal toxicity remains to be understood. In this review, we present our recent findings since we examined to search for the target molecules involved in the renal toxicity of Cd using toxicogenomics. In NRK-52E rat renal tubular epithelial cells, we found using DNA microarrays that Cd suppressed the expression of the gene encoding Ube2d4, a member of the Ube2d family. The Ube2d family consists of selective ubiquitin-conjugating enzymes associated with p53 degradation. Moreover, Cd suppressed the expressions of genes encoding all Ube2d family members (Ube2d1/2/3/4) prior to the appearance of cytotoxicity in NRK-52E cells. Cd markedly increased p53 protein level and induced p53 phosphorylation and apoptosis in the cells. In vivo studies showed that chronic Cd exposure also suppressed Ube2d family gene expression and induced p53 accumulation and apoptosis in the renal tubules of the mouse kidney. These findings suggest that Cd causes p53-dependent apoptosis due to the inhibition of p53 degradation through the down-regulation of Ube2d family genes in NRK-52E cells and mouse kidney. Thus, the Ube2d family genes may be one of the key targets of renal toxicity caused by Cd.

  18. Salicylic Acid Induced Self-excision of the Marker Gene npt Ⅱ Using CreloxP System from Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    B.G. Ma; X.Y. Duan; L.X. Zhang; H.P. Zhang; L.Z. Pan; J.X. Niu

    2007-01-01

    @@ Transgenic plants, despite a great deal of scientific evidences, have induced a number of environmental and consumer concerns for long time because various antibiotic resistance genes, according to common transformation strategies, will accompany engineered crops at all times.

  19. Effect of TAK1 gene silencing on the apoptosis of Kasumi-1 cells induced by arsenic trioxide

    Institute of Scientific and Technical Information of China (English)

    许锦霞

    2013-01-01

    Objective To study the effect of transforming growth factor-βactivated kinase-1 (TAK1) gene silencing on the proliferation and apoptosis of Kasumi-1 cells induced by arsenic trioxide (As2O3) .Methods Acute myeloid

  20. Temporal gene expression profiling of the rat knee joint capsule during immobilization-induced joint contractures.

    Science.gov (United States)

    Wong, Kayleigh; Sun, Fangui; Trudel, Guy; Sebastiani, Paola; Laneuville, Odette

    2015-05-26

    Contractures of the knee joint cause disability and handicap. Recovering range of motion is recognized by arthritic patients as their preference for improved health outcome secondary only to pain management. Clinical and experimental studies provide evidence that the posterior knee capsule prevents the knee from achieving full extension. This study was undertaken to investigate the dynamic changes of the joint capsule transcriptome during the progression of knee joint contractures induced by immobilization. We performed a microarray analysis of genes expressed in the posterior knee joint capsule following induction of a flexion contracture by rigidly immobilizing the rat knee joint over a time-course of 16 weeks. Fold changes of expression values were measured and co-expressed genes were identified by clustering based on time-series analysis. Genes associated with immobilization were further analyzed to reveal pathways and biological significance and validated by immunohistochemistry on sagittal sections of knee joints. Changes in expression with a minimum of 1.5 fold changes were dominated by a decrease in expression for 7732 probe sets occurring at week 8 while the expression of 2251 probe sets increased. Clusters of genes with similar profiles of expression included a total of 162 genes displaying at least a 2 fold change compared to week 1. Functional analysis revealed ontology categories corresponding to triglyceride metabolism, extracellular matrix and muscle contraction. The altered expression of selected genes involved in the triglyceride biosynthesis pathway; AGPAT-9, and of the genes P4HB and HSP47, both involved in collagen synthesis, was confirmed by immunohistochemistry. Gene expression in the knee joint capsule was sensitive to joint immobility and provided insights into molecular mechanisms relevant to the pathophysiology of knee flexion contractures. Capsule responses to immobilization was dynamic and characterized by modulation of at least three

  1. ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

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    Hafner Mathias

    2006-04-01

    Full Text Available Abstract Background TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT. Methods The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. Results After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. Conclusion Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling.

  2. Analyzing gene function in adult long-term hematopoietic stem cells using the interferon inducible Mx1-Cre mouse system.

    Science.gov (United States)

    Gudmundsson, Kristbjorn Orri; Oakley, Kevin; Han, Yufen; Du, Yang

    2014-01-01

    Long-term hematopoietic stem cells (LT-HSCs) have the ability to self-renew and differentiate into all blood cell lineages. Understanding the genetic networks that regulate LT-HSC function in the adult bone marrow requires inducible gene targeting and bone marrow transplantations. In this chapter we describe the use of the inducible Mx1-Cre mouse model to delete genes in LT-HSCs and methodologies for examining the function of LT-HSCs following deletion.

  3. Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

    Science.gov (United States)

    Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

    2016-08-05

    Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation.

  4. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    Science.gov (United States)

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  5. The tobacco smoke component acrolein induces glucocorticoid resistant gene expression via inhibition of histone deacetylase.

    Science.gov (United States)

    Randall, Matthew J; Haenen, Guido R M M; Bouwman, Freek G; van der Vliet, Albert; Bast, Aalt

    2016-01-05

    Chronic obstructive pulmonary disease (COPD) is the leading cause of cigarette smoke-related death worldwide. Acrolein, a crucial reactive electrophile found in cigarette smoke mimics many of the toxic effects of cigarette smoke-exposure in the lung. In macrophages, cigarette smoke is known to hinder histone deacetylases (HDACs), glucocorticoid-regulated enzymes that play an important role in the pathogenesis of glucocorticoid resistant inflammation, a common feature of COPD. Thus, we hypothesize that acrolein plays a role in COPD-associated glucocorticoid resistance. To examine the role of acrolein on glucocorticoid resistance, U937 monocytes, differentiated with PMA to macrophage-like cells were treated with acrolein for 0.5h followed by stimulation with hydrocortisone for 8h, or treated simultaneously with LPS and hydrocortisone for 8h without acrolein. GSH and nuclear HDAC activity were measured, or gene expression was analyzed by qPCR. Acrolein-mediated TNFα gene expression was not suppressed by hydrocortisone whereas LPS-induced TNFα expression was suppressed. Acrolein also significantly inhibited nuclear HDAC activity in macrophage-like cells. Incubation of recombinant HDAC2 with acrolein led to the formation of an HDAC2-acrolein adduct identified by mass spectrometry. Therefore, these results suggest that acrolein-induced inflammatory gene expression is resistant to suppression by the endogenous glucocorticoid, hydrocortisone.

  6. PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance.

    Science.gov (United States)

    de Leeuw, R; Flach, K; Bentin Toaldo, C; Alexi, X; Canisius, S; Neefjes, J; Michalides, R; Zwart, W

    2013-07-25

    Protein kinase A (PKA)-induced estrogen receptor alpha (ERα) phosphorylation at serine residue 305 (ERαS305-P) can induce tamoxifen (TAM) resistance in breast cancer. How this phospho-modification affects ERα specificity and translates into TAM resistance is unclear. Here, we show that S305-P modification of ERα reprograms the receptor, redirecting it to new transcriptional start sites, thus modulating the transcriptome. By altering the chromatin-binding pattern, Ser305 phosphorylation of ERα translates into a 26-gene expression classifier that identifies breast cancer patients with a poor disease outcome after TAM treatment. MYC-target genes and networks were significantly enriched in this gene classifier that includes a number of selective targets for ERαS305-P. The enhanced expression of MYC increased cell proliferation in the presence of TAM. We demonstrate that activation of the PKA signaling pathway alters the transcriptome by redirecting ERα to new transcriptional start sites, resulting in altered transcription and TAM resistance.

  7. The Arabidopsis AN3-YDA Gene Cascade Induces Anthocyanin Accumulation by Regulating Sucrose Levels

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    Lai-Sheng Meng

    2016-11-01

    Full Text Available Anthocyanin accumulation specifically depends on sucrose (Suc signalling/levels. However, the gene cascades specifically involved in the Suc signalling/level-mediated anthocyanin biosynthetic pathway are still unknown. Arabidopsis ANGUSTIFOLIA3 (AN3, a transcription coactivator, is involved in the regulation of leaf shape and drought tolerance. Recently, an AN3-CONSTITUTIVE PHOTOMORPHOGENIC 1 gene cascade has been reported to regulate the light signalling-mediated anthocyanin accumulation. Target gene analysis indicates that AN3 is associated with the YODA (YDA promoter, a mitogen-activated protein kinase kinase kinase, in vivo for inducing anthocyanin accumulation. Indeed, loss-of-function mutants of YDA showed significantly increased anthocyanin accumulation. YDA mutation can also suppress the decrease in an3-4 anthocyanin accumulation. Further analysis indicates that the mutations of AN3 and YDA disrupt the normal Suc levels because of the changes of invertase activity in mutants of an3 or yda, which in turn induces the alterations of anthocyanin accumulation in mutants of an3 or yda via unknown regulatory mechanisms.

  8. Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation.

    Science.gov (United States)

    Zhang, Hai; Zhang, Guo; Gonzalez, Frank J; Park, Sung-Min; Cai, Dongsheng

    2011-07-01

    Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.

  9. Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation.

    Directory of Open Access Journals (Sweden)

    Hai Zhang

    2011-07-01

    Full Text Available Hypoxia-inducible factor (HIF is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.

  10. Homeobox gene Dlx-2 is implicated in metabolic stress-induced necrosis

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    Lim Sung-Chul

    2011-09-01

    Full Text Available Abstract Background In contrast to tumor-suppressive apoptosis and autophagic cell death, necrosis promotes tumor progression by releasing the pro-inflammatory and tumor-promoting cytokine high mobility group box 1 (HMGB1, and its presence in tumor patients is associated with poor prognosis. Thus, necrosis has important clinical implications in tumor development; however, its molecular mechanism remains poorly understood. Results In the present study, we show that Distal-less 2 (Dlx-2, a homeobox gene of the Dlx family that is involved in embryonic development, is induced in cancer cell lines dependently of reactive oxygen species (ROS in response to glucose deprivation (GD, one of the metabolic stresses occurring in solid tumors. Increased Dlx-2 expression was also detected in the inner regions, which experience metabolic stress, of human tumors and of a multicellular tumor spheroid, an in vitro model of solid tumors. Dlx-2 short hairpin RNA (shRNA inhibited metabolic stress-induced increase in propidium iodide-positive cell population and HMGB1 and lactate dehydrogenase (LDH release, indicating the important role(s of Dlx-2 in metabolic stress-induced necrosis. Dlx-2 shRNA appeared to exert its anti-necrotic effects by preventing metabolic stress-induced increases in mitochondrial ROS, which are responsible for triggering necrosis. Conclusions These results suggest that Dlx-2 may be involved in tumor progression via the regulation of metabolic stress-induced necrosis.

  11. Applicability of gene expression and systems biology to develop pharmacogenetic predictors; antipsychotic-induced extrapyramidal symptoms as an example.

    Science.gov (United States)

    Mas, Sergi; Gassó, Patricia; Lafuente, Amelia

    2015-11-01

    Pharmacogenetics has been driven by a candidate gene approach. The disadvantage of this approach is that is limited by our current understanding of the mechanisms by which drugs act. Gene expression could help to elucidate the molecular signatures of antipsychotic treatments searching for dysregulated molecular pathways and the relationships between gene products, especially protein-protein interactions. To embrace the complexity of drug response, machine learning methods could help to identify gene-gene interactions and develop pharmacogenetic predictors of drug response. The present review summarizes the applicability of the topics presented here (gene expression, network analysis and gene-gene interactions) in pharmacogenetics. In order to achieve this, we present an example of identifying genetic predictors of extrapyramidal symptoms induced by antipsychotic.

  12. A novel PRD I and TG binding activity involved in virus-induced transcription of IFN-A genes.

    Science.gov (United States)

    Génin, P; Bragança, J; Darracq, N; Doly, J; Civas, A

    1995-01-01

    Comparative analysis of the inducible elements of the mouse interferon A4 and A11 gene promoters (IE-A4 and IE-A11) by transient transfection experiments, DNase 1 footprinting and electrophoretic mobility shift assays resulted in identification of a virus-induced binding activity suggested to be involved in NDV-induced activation of transcription of these genes. The virus-induced factor, termed VIF, is activated early by contact of virions with cells. It specifically recognizes the PRD I-like domain shared by both inducible elements, as well as the TG-like domain of IE-A4. This factor, distinct from the IRF-1, IRF-2 and the alpha F1 binding proteins and presenting a different affinity pattern from that of the TG protein, is proposed as a candidate for IFN-type I gene regulation. Images PMID:8559665

  13. Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries

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    Fletcher Sue

    2007-07-01

    Full Text Available Abstract Background Antisense oligonucleotides (AOs can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting. Results Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers. Conclusion The combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino

  14. TGFβ-dependent gene expression shows that senescence correlates with abortive differentiation along several lineages in Myc-induced lymphomas.

    Science.gov (United States)

    Müller, Judith; Samans, Birgit; van Riggelen, Jan; Fagà, Giovanni; Peh K N, Raquel; Wei, Chia-Lin; Müller, Heiko; Amati, Bruno; Felsher, Dean; Eilers, Martin

    2010-12-01

    Deregulated expression of Myc under the control of an immunoglobulin enhancer induces lymphoma formation in mice. The development of lymphomas is limited by TGFβ-dependent senescence and high levels of Myc expression are continuously required to antagonize senescence. The biological processes underlying senescence are not fully resolved. We report here a comprehensive analysis of TGFβ-dependent alterations in gene expression when the Myc transgene is switched off. Our data show that Myc-induced target genes are downregulated in a TGFβ-independent manner. In contrast, TGFβ is required to upregulate a broad spectrum of genes that are characteristic of different T-cell lineages when Myc is turned off. The analysis reveals a significant overlap between these Myc-repressed genes with genes that are targets of polycomb repressive complexes in embryonic stem cells. Therefore, TGFβ-dependent senescence is associated with gene expression patterns indicative of abortive cellular differentiation along several lineages.

  15. Ethanol induced astaxanthin accumulation and transcriptional expression of carotenogenic genes in Haematococcus pluvialis.

    Science.gov (United States)

    Wen, Zewen; Liu, Zhiyong; Hou, Yuyong; Liu, Chenfeng; Gao, Feng; Zheng, Yubin; Chen, Fangjian

    2015-10-01

    Haematococcus pluvialis is one of the most promising natural sources of astaxanthin. However, inducing the accumulation process has become one of the primary obstacles in astaxanthin production. In this study, the effect of ethanol on astaxanthin accumulation was investigated. The results demonstrated that astaxanthin accumulation occurred with ethanol addition even under low-light conditions. The astaxanthin productivity could reach 11.26 mg L(-1) d(-1) at 3% (v/v) ethanol, which was 2.03 times of that of the control. The transcriptional expression patterns of eight carotenogenic genes were evaluated using real-time PCR. The results showed that ethanol greatly enhanced transcription of the isopentenyl diphosphate (IPP) isomerase genes (ipi-1 and ipi-2), which were responsible for isomerization reaction of IPP and dimethylallyl diphosphate (DMAPP). This finding suggests that ethanol induced astaxanthin biosynthesis was up-regulated mainly by ipi-1 and ipi-2 at transcriptional level, promoting isoprenoid synthesis and substrate supply to carotenoid formation. Thus ethanol has the potential to be used as an effective reagent to induce astaxanthin accumulation in H. pluvialis.

  16. BDE-209 inhibits pluripotent genes expression and induces apoptosis in human embryonic stem cells.

    Science.gov (United States)

    Du, Lili; Sun, Wen; Zhang, Huili; Chen, Dunjin

    2016-05-01

    Decabromodiphenyl ether (BDE-209) has been detected in human serum, semen, placenta, cord blood and milk worldwide. However, little is known regarding the potential effects on the early human embryonic development of BDE-209. In this study, human embryonic stem cell lines FY-hES-10 and FY-hES-26 were used to evaluate the potential effects and explore the toxification mechanisms using low-level BDE-209 exposure. Our data showed that BDE-209 exposure (1, 10 and 100 nM) reduced the expression of pluripotent genes such as OCT4, SOX2 and NANOG and induced human embryonic stem cells (hESCs) apoptosis. The downregulation of BIRC5/BCL2 and upregulation of BAX were related to apoptosis of hESCs induced by BDE-209 exposure. A mechanism study showed that OCT4 down-regulation accompanied by OCT4 promoter hypermethylation and increasing miR-145/miR-335 levels, OCT4 inhibitors. Moreover, BDE-209 could increase the generation of intracellular reactive oxygen species (ROS) and decrease SOD2 expression. The ROS increase and OCT4 downregulation after BDE-209 exposure could be reversed partly by antioxidant N-acetylcysteine supplement. These findings showed that BDE-209 exposure could decrease pluripotent genes expression via epigenetic regulation and induce apoptosis through ROS generation in human embryonic stem cells in vitro.

  17. Methylation-Associated Gene Silencing of RARB in Areca Carcinogens Induced Mouse Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Zi-Lun Lai

    2014-01-01

    Full Text Available Regarding oral squamous cell carcinoma (OSCC development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO, or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis.

  18. Methylation-Associated Gene Silencing of RARB in Areca Carcinogens Induced Mouse Oral Squamous Cell Carcinoma

    Science.gov (United States)

    Tsou, Yung-An; Fan, Shin-Ru; Tsai, Ming-Hsui; Chen, Hsiao-Ling; Chang, Nai-Wen; Cheng, Ju-Chien

    2014-01-01

    Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis. PMID:25197641

  19. Linkage of the human inducible nitric oxide synthase gene to type 1 diabetes.

    Science.gov (United States)

    Johannesen, J; Pie, A; Pociot, F; Kristiansen, O P; Karlsen, A E; Nerup, J

    2001-06-01

    Exposure of human pancreatic islets to a mixture of cytokines induces expression of the inducible nitric oxide synthase (iNOS), impairs beta-cell function, and induces apoptosis. We performed a mutational scanning of all 27 exons of the human NOS2 gene and linkage transmission disequilibrium testing of identified NOS2 polymorphisms in a Danish nationwide type 1 diabetes mellitus (IDDM) family collection. Mutational screening was performed using PCR-amplified exons, followed by single stranded conformation polymorphism and verification of potential polymorphisms by sequencing. The transmission disequilibrium test was performed in an IDDM family material comprising 257 Danish families; 154 families were affected sibling pair families, and 103 families were simplex families. In total, 10 polymorphisms were identified in 8 exons, of which 4 were tested in the family material. A C/T single nucleotide polymorphism in exon 16 resulting in an amino acid substitution, Ser(608)Leu, showed linkage to IDDM in human leukocyte antigen DR3/4-positive affected offspring (P = 0.008; corrected P = 0.024). No other distorted transmission patterns were found for any other tested single nucleotide polymorphism or constructed haplotypes with the exception of those including data from exon 16. In conclusion, linkage of the human NOS2 gene to IDDM in a subset of patients supports a pathogenic role of nitric oxide in human IDDM.

  20. DNMT3B inhibits the re-expression of genes associated with induced pluripotency.

    Science.gov (United States)

    Wongtrakoongate, Patompon; Li, Jianliang; Andrews, Peter W

    2014-02-15

    DNMT3B is a de novo DNA methyltransferase that is highly expressed in mouse and human embryonic stem (ES) cells and has been shown to be essential for differentiation of mouse ES cells toward different lineages. In the present study, we found that DNMT3B is rapidly down-regulated in human ES cells during retinoic acid (RA)-induced differentiation compared with DNMT3A2, which is also highly expressed in ES cells. Silencing of DNMT3B in human ES cells by an inducible shRNAi system leads to a reduction of clonal ability of the stem cells, while expression of OCT4 and NANOG is unchanged. By contrast, the germline-specific genes VASA and SCP3 and the surface antigen BE12 are down regulated following DNMT3B knockdown. Upon retinoic acid-induced differentiation, we found that depletion of DNMT3B leads to a decrease in expression of the surface antigen A2B5 and of neural tube-associated genes PAX7 and BRN3A. Consistent with its importance in stem cell differentiation, we observed that silencing of DNMT3B facilitates the generation of cells that bear the hallmarks of pluripotency. Our findings suggest a role of DNMT3B in controlling the differentiation of human ES cells and in the generation of iPS cells.

  1. Deletion of the Men1 Gene Prevents Streptozotocin-Induced Hyperglycemia in Mice

    Directory of Open Access Journals (Sweden)

    Yuqing Yang

    2010-01-01

    Full Text Available Diabetes ultimately results from an inadequate number of functional beta cells in the islets of Langerhans. Enhancing proliferation of functional endogenous beta cells to treat diabetes remains underexplored. Here, we report that excision of the Men1 gene, whose loss-of-function mutation leads to inherited multiple endocrine neoplasia type 1 (MEN1, rendered resistant to streptozotocin-induced hyperglycemia in a tamoxifen-inducible and temporally controlled Men1 excision mouse model as well as in a tissue-specific Men1 excision mouse model. Men1 excision prevented mice from streptozotocin-induced hyperglycemia mainly through increasing the number of functional beta cells. BrdU incorporation by beta cells, islet size, and circulating insulin levels were significantly increased in Men1-excised mice. Membrane localization of glucose transporter 2 was largely preserved in Men1-excised beta cells, but not in Men1-expressing beta cells. Our findings suggest that repression of menin, a protein encoded by the Men1 gene, might be a valuable means to maintain or increase the number of functional endogenous beta cells to prevent or ameliorate diabetes.

  2. Molecular cloning and characterization of two hypersensitive induced reaction genes from wheat infected by stripe rust pathogen

    Science.gov (United States)

    A novel gene induced during hypersensitive reaction (HIR) in wheat was identified using in silico cloning and designated as TaHIR2. The TaHIR2 gene was deduced to encode a 284-amino acid protein, whose molecular mass and isoelectric point (pI) were 31.05 kD and 5.18, respectively. Amino acid sequenc...

  3. Myostatin propeptide gene delivery by gene gun ameliorates muscle atrophy in a rat model of botulinum toxin-induced nerve denervation.

    Science.gov (United States)

    Tsai, Sen-Wei; Tung, Yu-Tang; Chen, Hsiao-Ling; Yang, Shang-Hsun; Liu, Chia-Yi; Lu, Michelle; Pai, Hui-Jing; Lin, Chi-Chen; Chen, Chuan-Mu

    2016-02-01

    Muscle atrophy is a common symptom after nerve denervation. Myostatin propeptide, a precursor of myostatin, has been documented to improve muscle growth. However, the mechanism underlying the muscle atrophy attenuation effects of myostatin propeptide in muscles and the changes in gene expression are not well established. We investigated the possible underlying mechanisms associated with myostatin propeptide gene delivery by gene gun in a rat denervation muscle atrophy model, and evaluated gene expression patterns. In a rat botulinum toxin-induced nerve denervation muscle atrophy model, we evaluated the effects of wild-type (MSPP) and mutant-type (MSPPD75A) of myostatin propeptide gene delivery, and observed changes in gene activation associated with the neuromuscular junction, muscle and nerve. Muscle mass and muscle fiber size was moderately increased in myostatin propeptide treated muscles (pmuscle regulatory factors, neurite outgrowth factors (IGF-1, GAP43) and acetylcholine receptors was observed. Our results demonstrate that myostatin propeptide gene delivery, especially the mutant-type of MSPPD75A, attenuates muscle atrophy through myogenic regulatory factors and acetylcholine receptor regulation. Our data concluded that myostatin propeptide gene therapy may be a promising treatment for nerve denervation induced muscle atrophy. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Social defeat during adolescence and adulthood differentially induce BDNF-regulated immediate early genes

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    Caroline M. Coppens

    2011-11-01

    Full Text Available Stressful life events generally enhance the vulnerability for the development of human psychopathologies such as anxiety disorders and depression. The incidence rates of adult mental disorders steeply rises during adolescence in parallel with a structural and functional reorganization of the neural circuitry underlying stress reactivity. However, the mechanisms underlying susceptibility to stress and manifestation of mental disorders during adolescence are little understood. We hypothesized that heightened sensitivity to stress during adolescence reflects age-dependent differences in the expression of activity-dependent genes involved in synaptic plasticity. Therefore, we compared the effect of social stress during adolescence with social stress in adulthood on the expression of a panel of genes linked to induction of long-term potentiation (LTP and brain-derived neurotrophic factor (BDNF signaling. We show that social defeat during adolescence and adulthood differentially regulates expression of the immediate early genes BDNF, Arc, Carp, and Tieg1, as measured by qPCR in tissue lysates from prefrontal cortex, nucleus accumbens, and hippocampus. In the hippocampus, mRNA levels for all four genes were robustly elevated following social defeat in adolescence, whereas none were induced by defeat in adulthood. The relationship to coping style was also examined using adult reactive and proactive coping rats. Gene expression levels of reactive and proactive animals were similar in the prefrontal cortex and hippocampus. However, a trend toward a differential expression of BDNF and Arc mRNA in the nucleus accumbens was detected. BDNF mRNA was increased in the nucleus accumbens of proactive defeated animals, whereas the expression level in reactive defeated animals was comparable to control animals. The results demonstrate striking differences in immediate early gene expression in response to social defeat in adolescent and adult rats.

  5. Pregnancy-Induced Changes in Systemic Gene Expression among Healthy Women and Women with Rheumatoid Arthritis.

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    Anuradha Mittal

    Full Text Available Pregnancy induces drastic biological changes systemically, and has a beneficial effect on some autoimmune conditions such as rheumatoid arthritis (RA. However, specific systemic changes that occur as a result of pregnancy have not been thoroughly examined in healthy women or women with RA. The goal of this study was to identify genes with expression patterns associated with pregnancy, compared to pre-pregnancy as baseline and determine whether those associations are modified by presence of RA.In our RNA sequencing (RNA-seq dataset from 5 healthy women and 20 women with RA, normalized expression levels of 4,710 genes were significantly associated with pregnancy status (pre-pregnancy, first, second and third trimesters over time, irrespective of presence of RA (False Discovery Rate (FDR-adjusted p value<0.05. These genes were enriched in pathways spanning multiple systems, as would be expected during pregnancy. A subset of these genes (n = 256 showed greater than two-fold change in expression during pregnancy compared to baseline levels, with distinct temporal trends through pregnancy. Another 98 genes involved in various biological processes including immune regulation exhibited expression patterns that were differentially associated with pregnancy in the presence or absence of RA.Our findings support the hypothesis that the maternal immune system plays an active role during pregnancy, and also provide insight into other systemic changes that occur in the maternal transcriptome during pregnancy compared to the pre-pregnancy state. Only a small proportion of genes modulated by pregnancy were influenced by presence of RA in our data.

  6. Isolation and Expression Analysis of a Novel Abiotic Stress-Induced Gene W89 from Wheat

    Institute of Scientific and Technical Information of China (English)

    ZHANG Rui-yue; XU Zhao-shi; LI Lian-cheng; CHEN Ming; MA You-zhi

    2007-01-01

    Water stress and cold stress are important factors restricting plant growth. However, there is little knowledge on the function of stress-responsive genes in plants. Therefore, it is necessary to clone some important genes to study the mechanism of plant adaptation to abiotic stress for improvement of plant resistance. A putative water stress-induced gene, W89, was cloned from the cDNA library of drought-treated wheat seedlings by phage hybridization in situ, and its entire length was obtained using 5'-rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR). The full-length cDNA of W89 consists of 2 392 bp and contains a 1 896 bp open reading frame (ORF) encoding a 631 amino acid protein. Southern blot analysis indicated that W89 was a single-copy gene. RT-PCR analysis revealed that the expression of W89 was upregulated by drought, cold, and abscisic acid (ABA). Amino acid sequence analysis discovered that W89 had a conserved region of DUF248 (pfam03141), which contained a methyltransferase domain with a sterile alpha motif (SAM)-binding motif. Phylogenetic analysis showed that W89 was 66% identical to Oryza sativa dehydration-responsive protein (BAD67956). It was supposed that W89 was a novel dehydration-responsive protein encoding gene. On the basis of the functions of methyltransferase and the SAM-binding motif, the SAM-binding motif of W89 was supposed to be connected with other proteins or transcription factors to transduce stress signals and finally regulate the expression of stress-responsive genes on the early stage of drought stress.

  7. Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters.

    Science.gov (United States)

    Kovalchuk, Nataliya; Jia, Wei; Eini, Omid; Morran, Sarah; Pyvovarenko, Tatiana; Fletcher, Stephen; Bazanova, Natalia; Harris, John; Beck-Oldach, Kontanze; Shavrukov, Yuri; Langridge, Peter; Lopato, Sergiy

    2013-08-01

    Constitutive over-expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3- to 6-week delays in flowering compared to control plants. In this work, two cold-inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low-to-moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up-regulation of cold-responsive target genes. The OsWRKY71 promoter-driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild-type plants. The WRKY71-TaDREB3 promoter-transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.

  8. Nutrition-induced ketosis alters metabolic and signaling gene networks in liver of periparturient dairy cows.

    Science.gov (United States)

    Loor, Juan J; Everts, Robin E; Bionaz, Massimo; Dann, Heather M; Morin, Dawn E; Oliveira, Rosane; Rodriguez-Zas, Sandra L; Drackley, James K; Lewin, Harris A

    2007-12-19

    Dairy cows are highly susceptible after parturition to developing liver lipidosis and ketosis, which are costly diseases to farmers. A bovine microarray platform consisting of 13,257-annotated oligonucleotides was used to study hepatic gene networks underlying nutrition-induced ketosis. On day 5 postpartum, 14 Holstein cows were randomly assigned to ketosis-induction (n = 7) or control (n = 7) groups. Cows in the ketosis-induction group were fed at 50% of day 4 intake until they developed signs of clinical ketosis, and cows in the control group were fed ad libitum throughout the treatment period. Liver was biopsied at 10-14 (ketosis) or 14 days postpartum (controls). Feed restriction increased blood concentrations of nonesterified fatty acids and beta-hydroxybutyrate, but decreased glucose. Liver triacylglycerol concentration also increased. A total of 2,415 genes were altered by ketosis (false discovery rate = 0.05). Ingenuity Pathway Analysis revealed downregulation of genes associated with oxidative phosphorylation, protein ubiquitination, and ubiquinone biosynthesis with ketosis. Other molecular adaptations included upregulation of genes and nuclear receptors associated with cytokine signaling, fatty acid uptake/transport, and fatty acid oxidation. Genes downregulated during ketosis included several associated with cholesterol metabolism, growth hormone signaling, proton transport, and fatty acid desaturation. Feed restriction and ketosis resulted in previously unrecognized alterations in gene network expression underlying key cellular functions and discrete metabolic events. These responses might help explain well-documented physiological adaptations to reduced feed intake in early postpartum cows and, thus, provide molecular targets that might be useful in prevention and treatment of liver lipidosis and ketosis.

  9. Adult Drosophila melanogaster evolved for antibacterial defense invest in infection-induced expression of both humoral and cellular immunity genes

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    McGraw Elizabeth A

    2011-08-01

    Full Text Available Abstract Background While the transcription of innate immunity genes in response to bacterial infection has been well-characterised in the Drosophila model, we recently demonstrated the capacity for such transcription to evolve in flies selected for improved antibacterial defense. Here we use this experimental system to examine how insects invest in constitutive versus infection-induced transcription of immunity genes. These two strategies carry with them different consequences with respect to energetic and pleiotropic costs and may be more or less effective in improving defense depending on whether the genes contribute to humoral or cellular aspects of immunity. Findings Contrary to expectation we show that selection preferentially increased the infection-induced expression of both cellular and humoral immunity genes. Given their functional roles, infection induced increases in expression were expected for the humoral genes, while increases in constitutive expression were expected for the cellular genes. We also report a restricted ability to improve transcription of immunity genes that is on the order of 2-3 fold regardless of total transcription level of the gene. Conclusions The evolved increases in infection-induced expression of the cellular genes may result from specific cross talk with humoral pathways or from generalised strategies for enhancing immunity gene transcription. A failure to see improvements in constitutive expression of the cellular genes suggests either that increases might come at too great a cost or that patterns of expression in adults are decoupled from the larval phase where increases would be most effective. The similarity in fold change increase across all immunity genes may suggest a shared mechanism for the evolution of increased transcription in small, discrete units such as duplication of cis-regulatory elements.

  10. Use of Substrate-Induced Gene Expression in Metagenomic Analysis of an Aromatic Hydrocarbon-Contaminated Soil

    OpenAIRE

    2016-01-01

    Metagenomics allows the study of genes related to xenobiotic degradation in a culture-independent manner, but many of these studies are limited by the lack of genomic context for metagenomic sequences. This study combined a phenotypic screen known as substrate-induced gene expression (SIGEX) with whole-metagenome shotgun sequencing. SIGEX is a high-throughput promoter-trap method that relies on transcriptional activation of a green fluorescent protein (GFP) reporter gene in response to an ind...

  11. Predator-induced defences in Daphnia pulex: Selection and evaluation of internal reference genes for gene expression studies with real-time PCR

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    Gilbert Don

    2010-06-01

    Full Text Available Abstract Background The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data. Results In this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, TATA box binding protein (Tbp syntaxin 16 (Stx16, X-box binding protein 1 (Xbp1 and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP, was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are

  12. Induced Pluripotency and Gene Editing in Disease Modelling: Perspectives and Challenges

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    Yu Fen Samantha Seah

    2015-12-01

    Full Text Available Embryonic stem cells (ESCs are chiefly characterized by their ability to self-renew and to differentiate into any cell type derived from the three main germ layers. It was demonstrated that somatic cells could be reprogrammed to form induced pluripotent stem cells (iPSCs via various strategies. Gene editing is a technique that can be used to make targeted changes in the genome, and the efficiency of this process has been significantly enhanced by recent advancements. The use of engineered endonucleases, such as homing endonucleases, zinc finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs and Cas9 of the CRISPR system, has significantly enhanced the efficiency of gene editing. The combination of somatic cell reprogramming with gene editing enables us to model human diseases in vitro, in a manner considered superior to animal disease models. In this review, we discuss the various strategies of reprogramming and gene targeting with an emphasis on the current advancements and challenges of using these techniques to model human diseases.

  13. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.

    Science.gov (United States)

    Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.

  14. Clozapine induces oxidative stress and proapoptotic gene expression in neutrophils of schizophrenic patients.

    Science.gov (United States)

    Fehsel, Karin; Loeffler, Stefan; Krieger, Klaus; Henning, Uwe; Agelink, Markus; Kolb-Bachofen, Victoria; Klimke, Ansgar

    2005-10-01

    The present study examined cellular effects of the atypical antipsychotic drug clozapine on blood cells of treated patients with and without clozapine-induced agranulocytosis (CA). Blood from one patient who commenced clozapine treatment was examined at weekly intervals for 128 days. Olanzapine-treated (n = 5) and polymedicated (n = 14) schizophrenic patients, as well as healthy subjects (n = 19) and septic shock patients (n = 8), were studied for comparison. We observed dramatically increased numbers of native neutrophils stained for superoxide anion production (P genes p53 (P genes did not correlate to the percentage of apoptotic neutrophils (2.0% +/- 1.3%), but in CA patients about 37% of the neutrophils show morphologic signs of apoptosis (P genes decreased significantly. In conclusion, high production of reactive oxygen species in neutrophils of clozapine-treated patients, together with increased expression of proapoptotic genes, suggests that neutrophils are predisposed to apoptosis in schizophrenic patients under clozapine therapy. The correlation between drug and proapoptotic markers was highest for clozapine and bax alpha as well as superoxide anion radicals. This indicates oxidative mitochondrial stress in neutrophils of clozapine-treated patients which probably contributes to the induction of apoptosis and sudden loss of neutrophils and their precursors in CA patients.

  15. The ubiquitin hydrolase USP22 contributes to 3'-end processing of JAK-STAT-inducible genes.

    Science.gov (United States)

    Chipumuro, Edmond; Henriksen, Melissa A

    2012-02-01

    The JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway drives cellular growth, differentiation, and the immune response. STAT-activated gene expression is both rapid and transient and requires dynamic post-translational modification of the chromatin template. We previously showed that monoubiquitination of histone H2B (ubH2B) is highly dynamic at the STAT1 target gene, interferon regulatory factor 1 (IRF1), suggesting that a deubiquitinase is recruited during gene activation. Here, we report that RNAi-mediated knockdown of the ubiquitin hydrolase, USP22, results in 2-fold higher ubH2B, and 2-fold lower transcriptional elongation at IRF1. We also demonstrate that USP22 depletion diminishes 3'-end cleavage/polyadenylation by 2- to 3-fold. Furthermore, the polyadenylation factor CPSF73 is not effectively recruited, and serine 2 phosphorylation (Ser2P) of the C-terminal domain of RNA polymerase II is also disrupted. The transcriptional and processing defects observed in the USP22-knockdown cells are reversed by transient USP22 overexpression. Together, these results suggest that ubH2B helps recruit polyadenylation factors to STAT1-activated genes. We propose a working model, wherein a cycle of H2B ubiquitination/deubiquitination specifies Ser2P to regulate elongation and 3'-end processing of JAK-STAT-inducible mRNAs. These results further elaborate USP22 function and its role as a putative cancer stem cell marker.

  16. Inducers and autoinducers on Salmonella enterica serovar Typhimurium motility, growth and gene expression

    Directory of Open Access Journals (Sweden)

    Rita de Cássia dos Santos da Conceição

    2015-12-01

    Full Text Available Genus Salmonella bacteria are among the major pathogenic microorganisms in food. This bacterium pathogenicity is related to a number of virulence factors, among which its flagella. Flagellum expression is one of the virulence factors modulated by Quorum Sensing. Epinephrine produced by mammals uses the same signaling pathway of the 3 bacteria autoinducer. This study evaluated the effect of molecules inducer (epinephrine and autoinducers (autoinducer 2 and autoinducer 3 and their association with the motility, growth and expression genes flhC, fliA, fliY, motA, motB e fliC of Salmonella Typhimurium (ST. Initially, ST was inoculated in BHI. Then, motility assays, growth curves and gene expression were performed by testing different concentrations of epinephrine (50, 125, 250, 500µM, conditioned medium (10 and 50% and a combination of these. ST was exposed to different concentrations of epinephrine, conditioned medium and an association of both. Following, motility assays, bacterial growth and gene expression were performed. The results obtained showed that the combination of 500uM epinephrine with 50% conditioned medium increased ST bacterial motility by increasing the expression of genes involved in flagellum assembly.

  17. Identification of cadmium-induced Agaricus blazei genes through suppression subtractive hybridization.

    Science.gov (United States)

    Wang, Liling; Li, Haibo; Wei, Hailong; Wu, Xueqian; Ke, Leqin

    2014-01-01

    Cadmium (Cd) is one of the most serious environmental pollutants. Filamentous fungi are very promising organisms for controlling and reducing the amount of heavy metals released by human and industrial activities. However, the molecular mechanisms involved in Cd accumulation and tolerance of filamentous fungi are not fully understood. Agaricus blazei Murrill, an edible mushroom with medicinal properties, demonstrates high tolerance for heavy metals, especially Cd. To investigate the molecular mechanisms underlying the response of A. blazei after Cd exposure, we constructed a forward subtractive library that represents cadmium-induced genes in A. blazei under 4 ppm Cd stress for 14 days using suppression subtractive hybridization combined with mirror orientation selection. Differential screening allowed us to identify 39 upregulated genes, 26 of which are involved in metabolism, protein fate, cellular transport, transport facilitation and transport routes, cell rescue, defense and virulence, transcription, and the action of proteins with a binding function, and 13 are encoding hypothetical proteins with unknown functions. Induction of six A. blazei genes after Cd exposure was further confirmed by RT-qPCR. The cDNAs isolated in this study contribute to our understanding of genes involved in the biochemical pathways that participate in the response of filamentous fungi to Cd exposure.

  18. Hypoxia-inducible genes encoding small EF-hand proteins in rice and tomato.

    Science.gov (United States)

    Otsuka, Chie; Minami, Ikuko; Oda, Kenji

    2010-01-01

    Rice has evolved metabolic and morphological adaptations to low-oxygen stress to grow in submerged paddy fields. To characterize the molecular components that mediate the response to hypoxia in rice, we identified low-oxygen stress early response genes by microarray analysis. Among the highly responsive genes, five genes, OsHREF1 to OsHREF5, shared strong homology. They encoded small proteins harboring two EF-hands, typical Ca(2+)-binding motifs. Homologous genes were found in many land plants, including SlHREF in tomato, which is also strongly induced by hypoxia. SlHREF induction was detected in both roots and shoots of tomato plants under hypoxia. With the exception of OsHREF5, OsHREF expression was unaffected by drought, salinity, cold, or osmotic stress. Fluorescent signals of green fluorescent protein-fused OsHREFs were detected in the cytosol and nucleus. Ruthenium red, an inhibitor of intracellular Ca(2+) release, repressed induction of OsHREF1-4 under hypoxia. The HREFs may be related to the Ca(2+) response to hypoxia.

  19. Expression of ACC Oxidase Gene from Sugarcane Induced by Hormones and Environmental Force

    Institute of Scientific and Technical Information of China (English)

    WANG Ai-qin; YANG Li-tao; WANG Zi-zhang; WEI Yu-tuo; HE Long-fei; LI Yang-rui

    2005-01-01

    In the present study, a full-length cDNA encoding 1-aminocyclopropane-1-carboxylic acid oxidase gene has been cloned from sugarcane (named GZ-ACO). Two primers were designed for coding the ORF in the full-length cDNA of GZ-ACO gene from sugarcane. PCR amplification was performed with sugarcane DNA template, and a fragment of 1 104 bp (GZ34)was obtained. GZ34 was labeled with [α-32p] dCTP as the probe and used for hybridization after cloning and sequencing.Southern blotting analysis indicated that there were at least three other sequences, which weakly hybridized with the GZ34. Northern analysis showed that GZ34 was strongly induced by treatment with IAA, BA, ethephon, LiC1 and cold stress, respectively. As a contrast, the mRNA for ACO gene was at lower levels for both the light-grown and dark-grown plants without additional treatment. There were two transcripts in the dark-grown plants and three transcripts in the treatments with IAA, BA and cold stress, but there was only one transcript in ethephon treatment. It showed that GZ-ACO might be a gene connected with ethylene formation and take part in response to the induction of plant hormone and environmental stress.

  20. Myeloid translocation gene-16 co-repressor promotes degradation of hypoxia-inducible factor 1.

    Directory of Open Access Journals (Sweden)

    Parveen Kumar

    Full Text Available The myeloid translocation gene 16 (MTG16 co-repressor down regulates expression of multiple glycolytic genes, which are targets of the hypoxia-inducible factor 1 (HIF1 heterodimer transcription factor that is composed of oxygen-regulated labile HIF1α and stable HIF1β subunits. For this reason, we investigated whether MTG16 might regulate HIF1 negatively contributing to inhibition of glycolysis and stimulation of mitochondrial respiration. A doxycycline Tet-On system was used to control levels of MTG16 in B-lymphoblastic Raji cells. Results from co-association studies revealed MTG16 to interact with HIF1α. The co-association required intact N-terminal MTG16 residues including Nervy Homology Region 1 (NHR1. Furthermore, electrophoretic mobility shift assays demonstrated an association of MTG16 with hypoxia response elements (HREs in PFKFB3, PFKFB4 and PDK1 promoters in-vitro. Results from chromatin immunoprecipitation assays revealed co-occupancy of these and other glycolytic gene promoters by HIF1α, HIF1β and MTG16 in agreement with possible involvement of these proteins in regulation of glycolytic target genes. In addition, MTG16 interacted with prolyl hydroxylase D2 and promoted ubiquitination and proteasomal degradation of HIF1α. Our findings broaden the area of MTG co-repressor functions and reveal MTG16 to be part of a protein complex that controls the levels of HIF1α.

  1. Influence of suppressor gene p16 on retinoic acid inducing cancer cell A549 differentiation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the role of suppressor gene p16 in the process of differential regulation of retinoic acid (RA) on the A549 lung cancer cells.Methods Tumor suppressor gene p16 was transferred into A549 cells and the cells were treated with all-trans retinoic acid (ATR) at the dosage of 5×10-6 mol/L for 4 d. After that, the proliferation and differentiation of A549 cells were examined by growth curve and cytometry analysis, the change of lung lineage-specific marker MUC1 was tested by immunohistochemical staining. Meanwhile, Western blot was used to observe the change of p16 protein expression in A549 cells treated with ATRA.Results ATRA could obviously inhibit the growth and induce the differentiation of A549 Cells that were transferred with p16 gene. There were more cells arrested in G1/G0 phase and the expression of MUG1 was markedly down-regulated than in control cells. The expression of p16 protein was up-regulated in A549 cells treated with ATRA.Conclusion Suppressor gene p16 could enhance the effects of RA and proliferated suppression and differential induction of A549 cells.

  2. Interactome of Radiation-Induced microRNA-Predicted Target Genes

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    Tenzin W. Lhakhang

    2012-01-01

    Full Text Available The microRNAs (miRNAs function as global negative regulators of gene expression and have been associated with a multitude of biological processes. The dysfunction of the microRNAome has been linked to various diseases including cancer. Our laboratory recently reported modulation in the expression of miRNA in a variety of cell types exposed to ionizing radiation (IR. To further understand miRNA role in IR-induced stress pathways, we catalogued a set of common miRNAs modulated in various irradiated cell lines and generated a list of predicted target genes. Using advanced bioinformatics tools we identified cellular pathways where miRNA predicted target genes function. The miRNA-targeted genes were found to play key roles in previously identified IR stress pathways such as cell cycle, p53 pathway, TGF-beta pathway, ubiquitin-mediated proteolysis, focal adhesion pathway, MAPK signaling, thyroid cancer pathway, adherens junction, insulin signaling pathway, oocyte meiosis, regulation of actin cytoskeleton, and renal cell carcinoma pathway. Interestingly, we were able to identify novel targeted pathways that have not been identified in cellular radiation response, such as aldosterone-regulated sodium reabsorption, long-term potentiation, and neutrotrophin signaling pathways. Our analysis indicates that the miRNA interactome in irradiated cells provides a platform for comprehensive modeling of the cellular stress response to IR exposure.

  3. Fitness Effects of Network Non-Linearity Induced by Gene Expression Noise

    Science.gov (United States)

    Ray, Christian; Cooper, Tim; Balazsi, Gabor

    2012-02-01

    In the non-equilibrium dynamics of growing microbial cells, metabolic enzymes can create non-linearities in metabolite concentration because of non-linear degradation (utilization): an enzyme can saturate in the process of metabolite utilization. Increasing metabolite production past the saturation point then results in an ultrasensitive metabolite response. If the production rate of a metabolite depends on a second enzyme or other protein-mediated process, uncorrelated gene expression noise can thus cause transient metabolite concentration bursts. Such bursts are physiologically unnecessary and may represent a source of selection against the ultrasensitive switch, especially if the fluctuating metabolic intermediate is toxic. Selection may therefore favor correlated gene expression fluctuations for enzymes in the same pathway, such as by same-operon membership in bacteria. Using a modified experimental lac operon system, we are undertaking a combined theoretical-experimental approach to demonstrate that (i) the lac operon has an implicit ultrasensitive switch that we predict is avoided by gene expression correlations induced by same-operon membership; (ii) bacterial growth rates are sensitive to crossing the ultrasensitive threshold. Our results suggest that correlations in intrinsic gene expression noise are exploited by evolution to ameliorate the detrimental effects of nonlinearities in metabolite concentrations.

  4. Human paraoxonase gene cluster overexpression alleviates angiotensin II-induced cardiac hypertrophy in mice.

    Science.gov (United States)

    Pei, Jian-Fei; Yan, Yun-Fei; Tang, Xiaoqiang; Zhang, Yang; Cui, Shen-Shen; Zhang, Zhu-Qin; Chen, Hou-Zao; Liu, De-Pei

    2016-11-01

    Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure. The paraoxonase (PON) gene cluster (PC) protects against atherosclerosis and coronary artery diseases. However, the role of PC in the heart is largely unknown. To evaluate the roles of PC in cardiac hypertrophy, transgenic mice carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences were studied. We demonstrated that the PC transgene (PC-Tg) protected mice from cardiac hypertrophy induced by Ang II; these mice had reduced heart weight/body weight ratios, decreased left ventricular wall thicknesses and increased fractional shortening compared with wild-type (WT) control. The same protective tendency was also observed with an Apoe (-/-) background. Mechanically, PC-Tg normalized the disequilibrium of matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) in hypertrophic hearts, which might contribute to the protective role of PC-Tg in cardiac fibrosis and, thus, protect against cardiac remodeling. Taken together, our results identify a novel anti-hypertrophic role for the PON gene cluster, suggesting a possible strategy for the treatment of cardiac hypertrophy through elevating the levels of the PON gene family.

  5. Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer.

    Science.gov (United States)

    Raynal, Noël J-M; Lee, Justin T; Wang, Youjun; Beaudry, Annie; Madireddi, Priyanka; Garriga, Judith; Malouf, Gabriel G; Dumont, Sarah; Dettman, Elisha J; Gharibyan, Vazganush; Ahmed, Saira; Chung, Woonbok; Childers, Wayne E; Abou-Gharbia, Magid; Henry, Ryan A; Andrews, Andrew J; Jelinek, Jaroslav; Cui, Ying; Baylin, Stephen B; Gill, Donald L; Issa, Jean-Pierre J

    2016-03-15

    Targeting epigenetic pathways is a promising approach for cancer therapy. Here, we report on the unexpected finding that targeting calcium signaling can reverse epigenetic silencing of tumor suppressor genes (TSG). In a screen for drugs that reactivate silenced gene expression in colon cancer cells, we found three classical epigenetic targeted drugs (DNA methylation and histone deacetylase inhibitors) and 11 other drugs that induced methylated and silenced CpG island promoters driving a reporter gene (GFP) as well as endogenous TSGs in multiple cancer cell lines. These newly identified drugs, most prominently cardiac glycosides, did not change DNA methylation locally or histone modifications globally. Instead, all 11 drugs altered calcium signaling and triggered calcium-calmodulin kinase (CamK) activity, leading to MeCP2 nuclear exclusion. Blocking CamK activity abolished gene reactivation and cancer cell killing by these drugs, showing that triggering calcium fluxes is an essential component of their epigenetic mechanism of action. Our data identify calcium signaling as a new pathway that can be targeted to reactivate TSGs in cancer.

  6. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Mizoshiri, N. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kishida, T. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, K. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Shirai, T.; Terauchi, R.; Tsuchida, S. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mori, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Ejima, A. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Sato, Y. [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Arai, Y.; Fujiwara, H. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan); Yamamoto, T.; Kanamura, N. [Department of Dental Medicine, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, O., E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kubo, T. [Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2015-11-27

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genes and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.

  7. CHD1 regulates cell fate determination by activation of differentiation-induced genes

    DEFF Research Database (Denmark)

    Baumgart, Simon J; Najafova, Zeynab; Hossan, Tareq

    2017-01-01

    . Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close......The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start...... to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination....

  8. Pregnancy-Induced Changes in Systemic Gene Expression among Healthy Women and Women with Rheumatoid Arthritis

    DEFF Research Database (Denmark)

    Mittal, Anuradha; Pachter, Lior; Nelson, J Lee

    2015-01-01

    Background Pregnancy induces drastic biological changes systemically, and has a beneficial effect on some autoimmune conditions such as rheumatoid arthritis (RA). However, specific systemic changes that occur as a result of pregnancy have not been thoroughly examined in healthy women or women...... with RA. The goal of this study was to identify genes with expression patterns associated with pregnancy, compared to pre-pregnancy as baseline and determine whether those associations are modified by presence of RA. Results In our RNA sequencing (RNA-seq) dataset from 5 healthy women and 20 women with RA......, normalized expression levels of 4,710 genes were significantly associated with pregnancy status (pre-pregnancy, first, second and third trimesters) over time, irrespective of presence of RA (False Discovery Rate (FDR)-adjusted p value

  9. Zscan4 transiently reactivates early embryonic genes during the generation of induced pluripotent stem cells.

    Science.gov (United States)

    Hirata, Tetsuya; Amano, Tomokazu; Nakatake, Yuhki; Amano, Misa; Piao, Yulan; Hoang, Hien G; Ko, Minoru S H

    2012-01-01

    The generation of induced pluripotent stem cells (iPSCs) by the forced expression of defined transcription factors in somatic cells holds great promise for the future of regenerative medicine. However, the initial reprogramming mechanism is still poorly understood. Here we show that Zscan4, expressed transiently in2-cell embryos and embryonic stem cells (ESCs), efficiently produces iPSCs from mouse embryo fibroblasts when coexpressed with Klf4, Oct4, and Sox2. Interestingly, the forced expression of Zscan4 is required onlyfor the first few days of iPSC formation. Microarray analysis revealed transient and early induction of preimplantation-specific genes in a Zscan4-dependent manner. Our work indicates that Zscan4 is a previously unidentified potent natural factor that facilitates the reprogramming process and reactivates early embryonic genes.

  10. Phosphoribulokinase mediates nitrogenase-induced carbon dioxide fixation gene repression in Rhodobacter sphaeroides

    Science.gov (United States)

    Farmer, Ryan M.

    2015-01-01

    In many organisms there is a balance between carbon and nitrogen metabolism. These observations extend to the nitrogen-fixing, nonsulfur purple bacteria, which have the classic family of P(II) regulators that coordinate signals of carbon and nitrogen status to regulate nitrogen metabolism. Curiously, these organisms also possess a reverse mechanism to regulate carbon metabolism based on cellular nitrogen status. In this work, studies in Rhodobacter sphaeroides firmly established that the activity of the enzyme that catalyses nitrogen fixation, nitrogenase, induces a signal that leads to repression of genes encoding enzymes of the Calvin–Benson–Bassham (CBB) CO2 fixation pathway. Additionally, genetic and metabolomic experiments revealed that NADH-activated phosphoribulokinase is an intermediate in the signalling pathway. Thus, nitrogenase activity appears to be linked to cbb gene repression through phosphoribulokinase. PMID:26306848

  11. CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells

    Science.gov (United States)

    Kim, Eun Ji; Kang, Ki Ho; Ju, Ji Hyeon

    2017-01-01

    Recent advances in genome editing with programmable nucleases have opened up new avenues for multiple applications, from basic research to clinical therapy. The ease of use of the technology—and particularly clustered regularly interspaced short palindromic repeats (CRISPR)—will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. Here, we highlight the progress made in correcting gene mutations in monogenic hereditary disorders and discuss various CRISPR-associated applications, such as cancer research, synthetic biology, and gene therapy using induced pluripotent stem cells. The challenges, ethical issues, and future prospects of CRISPR-based systems for human research are also discussed. PMID:28049282

  12. Optimization of a Virus-Induced Gene Silencing System with Soybean yellow common mosaic virus for Gene Function Studies in Soybeans

    OpenAIRE

    Kil Hyun Kim; Seungmo Lim; Yang Jae Kang; Min Young Yoon; Moon Nam; Tae Hwan Jun; Min-Jung Seo; Seong-Bum Baek; Jeom-Ho Lee; Jung-Kyung Moon; Suk-Ha Lee; Su-Heon Lee; Hyoun-Sub Lim; Jae Sun Moon; Chang-Hwan Park

    2016-01-01

    Virus-induced gene silencing (VIGS) is an effective tool for the study of soybean gene function. Successful VIGS depends on the interaction between virus spread and plant growth, which can be influenced by environmental conditions. Recently, we developed a new VIGS system derived from the Soybean yellow common mosaic virus (SYCMV). Here, we investigated several environmental and developmental factors to improve the efficiency of a SYCMV-based VIGS system to optimize the functional analysis of...

  13. Bioinformatics analysis of the factors controlling type I IFN gene expression in autoimmune disease and virus-induced immunity

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    Di eFeng

    2013-09-01

    Full Text Available Patients with systemic lupus erythematosus (SLE and Sjögren's syndrome (SS display increased levels of type I IFN-induced genes. Plasmacytoid dendritic cells (PDCs are natural interferon producing cells and considered to be a primary source of IFN-α in these two diseases. Differential expression patterns of type I IFN inducible transcripts can be found in different immune cell subsets and in patients with both active and inactive autoimmune disease. A type I IFN gene signature generally consists of three groups of IFN-induced genes - those regulated in response to virus-induced type I IFN, those regulated by the IFN-induced mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK pathway, and those by the IFN-induced phosphoinositide-3 kinase (PI-3K pathway. These three groups of type I IFN-regulated genes control important cellular processes such as apoptosis, survival, adhesion, and chemotaxis, that when dysregulated, contribute to autoimmunity. With the recent generation of large datasets in the public domain from next-generation sequencing and DNA microarray experiments, one can perform detailed analyses of cell type-specific gene signatures as well as identify distinct transcription factors that differentially regulate these gene signatures. We have performed bioinformatics analysis of data in the public domain and experimental data from our lab to gain insight into the regulation of type I IFN gene expression. We have found that the genetic landscape of the IFNA and IFNB genes are occupied by transcription factors, such as insulators CTCF and cohesin, that negatively regulate transcription, as well as IRF5 and IRF7, that positively and distinctly regulate IFNA subtypes. A detailed understanding of the factors controlling type I IFN gene transcription will significantly aid in the identification and development of new therapeutic strategies targeting the IFN pathway in autoimmune disease.

  14. IGF-I induced genes in stromal fibroblasts predict the clinical outcome of breast and lung cancer patients

    Directory of Open Access Journals (Sweden)

    Herrmann Richard

    2010-01-01

    Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-I signalling is important for cancer initiation and progression. Given the emerging evidence for the role of the stroma in these processes, we aimed to characterize the effects of IGF-I on cancer cells and stromal cells separately. Methods We used an ex vivo culture model and measured gene expression changes after IGF-I stimulation with cDNA microarrays. In vitro data were correlated with in vivo findings by comparing the results with published expression datasets on human cancer biopsies. Results Upon stimulation with IGF-I, breast cancer cells and stromal fibroblasts show some common and other distinct response patterns. Among the up-regulated genes in the stromal fibroblasts we observed a significant enrichment in proliferation associated genes. The expression of the IGF-I induced genes was coherent and it provided a basis for the segregation of the patients into two groups. Patients with tumours with highly expressed IGF-I induced genes had a significantly lower survival rate than patients whose tumours showed lower levels of IGF-I induced gene expression (P = 0.029 - Norway/Stanford and P = 7.96e-09 - NKI dataset. Furthermore, based on an IGF-I induced gene expression signature derived from primary lung fibroblasts, a separation of prognostically different lung cancers was possible (P = 0.007 - Bhattacharjee and P = 0.008 - Garber dataset. Conclusion Expression patterns of genes induced by IGF-I in primary breast and lung fibroblasts accurately predict outcomes in breast and lung cancer patients. Furthermore, these IGF-I induced gene signatures derived from stromal fibroblasts might be promising predictors for the response to IGF-I targeted therapies. See the related commentary by Werner and Bruchim: http://www.biomedcentral.com/1741-7015/8/2

  15. Embryonic stem cell-derived microvesicles induce gene expression changes in Muller cells of the retina.

    Directory of Open Access Journals (Sweden)

    Diana Katsman

    Full Text Available Cell-derived microvesicles (MVs, recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  16. Transcriptional inhibition of the Catalase gene in phosphine-induced oxidative stress in Drosophila melanogaster.

    Science.gov (United States)

    Liu, Tao; Li, Li; Zhang, Fanhua; Wang, Yuejin

    2015-10-01

    Phosphine (PH3) is a toxic substance to pest insects and is therefore commonly used in pest control. The oxidative damage induced by PH3 is considered to be one of the primary mechanisms of its toxicity in pest insects; however, the precise mode of PH3 action in this process is still unclear. In this study, we evaluated the responses of several oxidative biomarkers and two of the main antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD), after fumigation treatment with PH3 in Drosophila melanogaster as a model system. The results showed that larvae exposed to sub-lethal levels of PH3 (0.028 mg/L) exhibited lower aerobic respiration rates and higher levels of hydrogen peroxide (H2O2) and lipid peroxidation (LPO). Furthermore, unlike SOD, the activity and expression of CAT and its encoding gene were downregulated by PH3 in a time- and dose-dependent manner. Finally, the responses of six potential transcription factors of PH3 were determined by real-time polymerase chain reaction to explore the regulation mechanism of DmCAT by PH3. There were no significant effects of PH3 on three nuclear factor-kappa B homologs (DORSAL, DIF, and RELISH) or two activator protein-1 genes (JUN and FOS), while dramatic inhibition of DNA replication-related element factor (DREF) expression was observed after fumigation with PH3, suggesting that PH3 could inhibit the expression of DmCAT via the DRE/DREF system. These results confirmed that PH3 induces oxidative stress and targets CAT by downregulating its encoding gene in Drosophila. Our results provide new insight into the signal transduction mechanism between PH3 and its target genes.

  17. Hydrogen peroxide induces adaptive response and differential gene expression in human embryo lung fibroblast cells.

    Science.gov (United States)

    Wei, Qinzhi; Huang, Haiyan; Yang, Linqing; Yuan, Jianhui; Yang, Xiaohua; Liu, Yungang; Zhuang, Zhixiong

    2014-04-01

    Hydrogen peroxide (H2 O2 ), a substance involved in cellular oxidative stress, has been observed to induce an adaptive response, which is characterized by a protection against the toxic effect of H2 O2 at higher concentrations. However, the molecular mechanism for the adaptive response remains unclear. In particular, the existing reports on H2 O2 -induced adaptive response are limited to animal cells and human tumor cells, and relatively normal human cells have never been observed for an adaptive response to H2 O2 . In this study, a human embryo lung fibroblast (MRC-5) cell line was used to model an adaptive response to H2 O2 , and the relevant differential gene expressions by using fluoro mRNA differential display RT-PCR. The results showed significant suppression of cytotoxicity of H2 O2 (1100 μM, 1 h) after pretreatment of the cells with H2 O2 at lower concentrations (0.088-8.8 μM, 24 h), as indicated by cell survival, lactate dehydrogenase release, and the rate of apoptotic cells. Totally 60 mRNA components were differentially expressed compared to untreated cells, and five of them (sizing 400-600 bp) which demonstrated the greatest increase in expression were cloned and sequenced. They showed identity with known genes, such as BCL-2, eIF3S5, NDUFS4, and RPS10. Real time RT-PCR analysis of the five genes displayed a pattern of differential expression consistent with that by the last method. These five genes may be involved in the induction of adaptive response by H2 O2 in human cells, at least in this particular cell type. Copyright © 2012 Wiley Periodicals, Inc.

  18. Obesity induces upregulation of genes involved in myocardial Ca2+ handling

    Directory of Open Access Journals (Sweden)

    A.P. Lima-Leopoldo

    2008-07-01

    Full Text Available Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+ handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c, sarcolemmal Na+/Ca2+ exchanger (NCX, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a, ryanodine receptor (RyR2, and phospholamban (PLB mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control or high-fat diet (obese for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05 but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.

  19. Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy

    Science.gov (United States)

    Nakhaei-Rad, Saeideh; Montenegro-Venegas, Carolina; Pina-Fernández, Eneko; Marini, Claudia; Santos, Monica; Ahmadian, Mohammad R.; Stork, Oliver; Zenker, Martin

    2017-01-01

    Noonan syndrome (NS) is characterized by reduced growth, craniofacial abnormalities, congenital heart defects, and variable cognitive deficits. NS belongs to the RASopathies, genetic conditions linked to mutations in components and regulators of the Ras signaling pathway. Approximately 50% of NS cases are caused by mutations in PTPN11. However, the molecular mechanisms underlying cognitive impairments in NS patients are still poorly understood. Here, we report the generation and characterization of a new conditional mouse strain that expresses the overactive Ptpn11D61Y allele only in the forebrain. Unlike mice with a global expression of this mutation, this strain is viable and without severe systemic phenotype, but shows lower exploratory activity and reduced memory specificity, which is in line with a causal role of disturbed neuronal Ptpn11 signaling in the development of NS-linked cognitive deficits. To explore the underlying mechanisms we investigated the neuronal activity-regulated Ras signaling in brains and neuronal cultures derived from this model. We observed an altered surface expression and trafficking of synaptic glutamate receptors, which are crucial for hippocampal neuronal plasticity. Furthermore, we show that the neuronal activity-induced ERK signaling, as well as the consecutive regulation of gene expression are strongly perturbed. Microarray-based hippocampal gene expression profiling revealed profound differences in the basal state and upon stimulation of neuronal activity. The neuronal activity-dependent gene regulation was strongly attenuated in Ptpn11D61Y neurons. In silico analysis of functional networks revealed changes in the cellular signaling beyond the dysregulation of Ras/MAPK signaling that is nearly exclusively discussed in the context of NS at present. Importantly, changes in PI3K/AKT/mTOR and JAK/STAT signaling were experimentally confirmed. In summary, this study uncovers aberrant neuronal activity-induced signaling and regulation

  20. Robust TLR4-induced gene expression patterns are not an accurate indicator of human immunity

    Science.gov (United States)

    2010-01-01

    Background Activation of Toll-like receptors (TLRs) is widely accepted as an essential event for defence against infection. Many TLRs utilize a common signalling pathway that relies on activation of the kinase IRAK4 and the transcription factor NFκB for the rapid expression of immunity genes. Methods 21 K DNA microarray technology was used to evaluate LPS-induced (TLR4) gene responses in blood monocytes from a child with an IRAK4-deficiency. In vitro responsiveness to LPS was confirmed by real-time PCR and ELISA and compared to the clinical predisposition of the child and IRAK4-deficient mice to Gram negative infection. Results We demonstrated that the vast majority of LPS-responsive genes in IRAK4-deficient monocytes were greatly suppressed, an observation that is consistent with the described role for IRAK4 as an essential component of TLR4 signalling. The severely impaired response to LPS, however, is inconsistent with a remarkably low incidence of Gram negative infections observed in this child and other children with IRAK4-deficiency. This unpredicted clinical phenotype was validated by demonstrating that IRAK4-deficient mice had a similar resistance to infection with Gram negative S. typhimurium as wildtype mice. A number of immunity genes, such as chemokines, were expressed at normal levels in human IRAK4-deficient monocytes, indicating that particular IRAK4-independent elements within the repertoire of TLR4-induced responses are expressed. Conclusions Sufficient defence to Gram negative immunity does not require IRAK4 or a robust, 'classic' inflammatory and immune response. PMID:20105294

  1. On the mechanism of gene amplification induced under stress in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available Gene amplification is a collection of processes whereby a DNA segment is reiterated to multiple copies per genome. It is important in carcinogenesis and resistance to chemotherapeutic agents, and can underlie adaptive evolution via increased expression of an amplified gene, evolution of new gene functions, and genome evolution. Though first described in the model organism Escherichia coli in the early 1960s, only scant information on the mechanism(s of amplification in this system has been obtained, and many models for mechanism(s were possible. More recently, some gene amplifications in E. coli were shown to be stress-inducible and to confer a selective advantage to cells under stress (adaptive amplifications, potentially accelerating evolution specifically when cells are poorly adapted to their environment. We focus on stress-induced amplification in E. coli and report several findings that indicate a novel molecular mechanism, and we suggest that most amplifications might be stress-induced, not spontaneous. First, as often hypothesized, but not shown previously, certain proteins used for DNA double-strand-break repair and homologous recombination are required for amplification. Second, in contrast with previous models in which homologous recombination between repeated sequences caused duplications that lead to amplification, the amplified DNAs are present in situ as tandem, direct repeats of 7-32 kilobases bordered by only 4 to 15 base pairs of G-rich homology, indicating an initial non-homologous recombination event. Sequences at the rearrangement junctions suggest nonhomologous recombination mechanisms that occur via template switching during DNA replication, but unlike previously described template switching events, these must occur over long distances. Third, we provide evidence that 3'-single-strand DNA ends are intermediates in the process, supporting a template-switching mechanism. Fourth, we provide evidence that lagging

  2. Protective effect of the thioltransferase gene on in vivo UVR-300 nm-induced cataract.

    Science.gov (United States)

    Kronschläger, Martin; Galichanin, Konstantin; Ekström, Joakim; Lou, Marjorie F; Söderberg, Per G

    2012-01-25

    To determine the protection factor (PF) for glutaredoxin-1 (Grx1) with regard to UVR-induced cataract by comparison of in vivo ultraviolet radiation (UVR) lens toxicity between double knockout Grx1⁻/⁻ and Grx1⁺/⁺ mice. Twenty Grx1⁺/⁺ mice and 20 Grx1⁻/⁻ mice were unilaterally exposed in vivo to UVR for 15 minutes. Groups of four animals each received 0.0, 2.1, 2.9, 3.6, and 4.1 kJ/m(2) UVR-300 nm. At 48 hours after UVR exposure, light-scattering in the exposed and contralateral nonexposed lenses was measured quantitatively. Macroscopic lens changes were documented with dark-field illumination photography. UVR-300 nm induced subcapsular and cortical cataract in Grx1⁻/⁻ and Grx1⁺/⁺ mice. In both Grx1⁻/⁻ and Grx1⁺/⁺, the light-scattering intensified with increased in vivo exposure doses of UVR-300 nm. The intensity of forward light-scattering was higher in the lenses of Grx1⁻/⁻ mice than in the lenses of Grx1⁺/⁺ mice. The threshold dose for in vivo UVR-300 nm-induced cataract, expressed as MTD(2.3:16), was 3.8 in the Grx1⁺/⁺ group and 3.0 in the Grx1⁻/⁻ group, resulting in a PF of 1.3. The PF is an objective relative measure of protective properties. The Grx1 gene is associated with an in vivo PF of 1.3. This result signifies that the presence of the gene allows a 1.3 times longer in vivo exposure to UVR, at equivalent irradiance, than the absence of the gene before early-onset, UVR-induced cataract occurs. This finding indicates the important role of the Grx1 gene in the oxidation defense system of the lens.

  3. Hypoxia attenuates purinergic P2X receptor-induced inflammatory gene expression in brainstem microglia

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    Smith SMC

    2013-08-01

    Full Text Available Stephanie MC Smith,1,2 Gordon S Mitchell,1,2 Scott A Friedle,3 Christine M Sibigtroth,1 Stéphane Vinit,1 Jyoti J Watters1–31Department of Comparative Biosciences, 2Comparative Biomedical Sciences Training Program, 3Program in Cellular and Molecular Biology, University of Wisconsin, Madison, WI, USAAbstract: Hypoxia and increased extracellular nucleotides are frequently coincident in the brainstem. Extracellular nucleotides are potent modulators of microglial inflammatory gene expression via P2X purinergic receptor activation. Although hypoxia is also known to modulate inflammatory gene expression, little is known about how hypoxia or P2X receptor activation alone affects inflammatory molecule production in brainstem microglia, nor how hypoxia and P2X receptor signaling interact when they occur together. In the study reported here, we investigated the ability of a brief episode of hypoxia (2 hours in the presence and absence of the nonselective P2X receptor agonist 2′(3′-O-(4-benzoylbenzoyladenosine-5′-triphosphate (BzATP to promote inflammatory gene expression in brainstem microglia in adult rats. We evaluated inducible nitric oxide synthase (iNOS, tumor necrosis factor alpha (TNFα, and interleukin (IL-6 messenger RNA levels in immunomagnetically isolated brainstem microglia. While iNOS and IL-6 gene expression increased with hypoxia and BzATP alone, TNFα expression was unaffected. Surprisingly, BzATP-induced inflammatory effects were lost after hypoxia, suggesting that hypoxia impairs proinflammatory P2X-receptor signaling. We also evaluated the expression of key P2X receptors activated by BzATP, namely P2X1, P2X4, and P2X7. While hypoxia did not alter their expression, BzATP upregulated P2X4 and P2X7 mRNAs; these effects were ablated in hypoxia. Although both P2X4 and P2X7 receptor expression correlated with increased microglial iNOS and IL-6 levels in microglia from normoxic rats, in hypoxia, P2X7 only correlated with IL-6, and P2X

  4. Expression patterns and action analysis of genes associated with drug-induced liver diseases during rat liver regeneration

    Institute of Scientific and Technical Information of China (English)

    Qian-Ji Ning; Shao-Wei Qin; Cun-Shuan Xu

    2006-01-01

    AIM: To study the action of the genes associated with drug-induced liver diseases at the gene transcriptional level during liver regeneration (LR) in rats.METHODS: The genes associated with drug-induced liver diseases were obtained by collecting the data from databases and literature, and the gene expression changes in the regenerating liver were checked by the Rat Genome 230 2.0 array.RESULTS: The initial and total expression numbers of genes occurring in phases of 0.5-4 h after partial hepatectomy (PH), 4-6 h after PH (G0/G1 transition),6-66 h after PH (cell proliferation), 66-168 h after PH (cell differentiation and structure-function reconstruction) were 21, 3, 9, 2 and 21, 9, 19, 18, respectively. It is illustrated that the associated genes were mainly triggered at the initial stage of LR and worked at different phases. According to their expression similarity,these genes were classified into 5 types: only upregulated (12 genes), predominantly up-regulated (4genes), only down-regulated (11 genes), predominantly down-regulated (3 genes), and approximately up-/down-regulated (2 genes). The total times of their upand down-expression were 130 and 79, respectively,demonstrating that expression of most of the genes was increased during LR, while a few decreased. The cell physiological and biochemical activities during LR were staggered according to the time relevance and were diverse and complicated in gene expression patterns.CONCLUSION: Drug metabolic capacity in regenerating liver was enhanced. Thirty-two genes play important roles during liver regeneration in rats.

  5. The microRNA machinery regulates fasting-induced changes in gene expression and longevity in Caenorhabditis elegans.

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    Kogure, Akiko; Uno, Masaharu; Ikeda, Takako; Nishida, Eisuke

    2017-07-07

    Intermittent fasting (IF) is a dietary restriction regimen that extends the lifespans of Caenorhabditis elegans and mammals by inducing changes in gene expression. However, how IF induces these changes and promotes longevity remains unclear. One proposed mechanism involves gene regulation by microRNAs (miRNAs), small non-coding RNAs (∼22 nucleotides) that repress gene expression and whose expression can be altered by fasting. To test this proposition, we examined the role of the miRNA machinery in fasting-induced transcriptional changes and longevity in C. elegans We revealed that fasting up-regulated the expression of the miRNA-induced silencing complex (miRISC) components, including Argonaute and GW182, and the miRNA-processing enzyme DRSH-1 (the ortholog of the Drosophila Drosha enzyme). Our lifespan measurements demonstrated that IF-induced longevity was suppressed by knock-out or knockdown of miRISC components and was completely inhibited by drsh-1 ablation. Remarkably, drsh-1 ablation inhibited the fasting-induced changes in the expression of the target genes of DAF-16, the insulin/IGF-1 signaling effector in C. elegans Fasting-induced transcriptome alterations were substantially and modestly suppressed in the drsh-1 null mutant and the null mutant of ain-1, a gene encoding GW182, respectively. Moreover, miRNA array analyses revealed that the expression levels of numerous miRNAs changed after 2 days of fasting. These results indicate that components of the miRNA machinery, especially the miRNA-processing enzyme DRSH-1, play an important role in mediating IF-induced longevity via the regulation of fasting-induced changes in gene expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Development of a Heat-Shock Inducible Gene Expression System in the Red Alga Cyanidioschyzon merolae

    Science.gov (United States)

    Sumiya, Nobuko; Fujiwara, Takayuki; Kobayashi, Yusuke; Misumi, Osami; Miyagishima, Shin-ya

    2014-01-01

    The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage. PMID:25337786

  7. Cannabidivarin (CBDV suppresses pentylenetetrazole (PTZ-induced increases in epilepsy-related gene expression

    Directory of Open Access Journals (Sweden)

    Naoki Amada

    2013-11-01

    Full Text Available To date, anticonvulsant effects of the plant cannabinoid, cannabidivarin (CBDV, have been reported in several animal models of seizure. However, these behaviourally observed anticonvulsant effects have not been confirmed at the molecular level. To examine changes to epilepsy-related gene expression following chemical convulsant treatment and their subsequent control by phytocannabinoid administration, we behaviourally evaluated effects of CBDV (400 mg/kg, p.o. on acute, pentylenetetrazole (PTZ: 95 mg/kg, i.p.-induced seizures, quantified expression levels of several epilepsy-related genes (Fos, Casp 3, Ccl3, Ccl4, Npy, Arc, Penk, Camk2a, Bdnf and Egr1 by qPCR using hippocampal, neocortical and prefrontal cortical tissue samples before examining correlations between expression changes and seizure severity. PTZ treatment alone produced generalised seizures (median: 5.00 and significantly increased expression of Fos, Egr1, Arc, Ccl4 and Bdnf. Consistent with previous findings, CBDV significantly decreased PTZ-induced seizure severity (median: 3.25 and increased latency to the first sign of seizure. Furthermore, there were correlations between reductions of seizure severity and mRNA expression of Fos, Egr1, Arc, Ccl4 and Bdnf in the majority of brain regions in the CBDV+PTZ treated group. When CBDV treated animals were grouped into CBDV responders (criterion: seizure severity ≤3.25 and non-responders (criterion: seizure severity >3.25, PTZ-induced increases of Fos, Egr1, Arc, Ccl4 and Bdnf expression were suppressed in CBDV responders. These results provide the first molecular confirmation of behaviourally observed effects of the non-psychoactive, anticonvulsant cannabinoid, CBDV, upon chemically-induced seizures and serve to underscore its suitability for clinical development.

  8. Heart failure-inducible gene therapy targeting protein phosphatase 1 prevents progressive left ventricular remodeling.

    Directory of Open Access Journals (Sweden)

    Yosuke Miyazaki

    Full Text Available BACKGROUND: The targeting of Ca(2+ cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR Ca(2+ ATPase, or ablation of phospholamban (PLN and associated protein phosphatase 1 (PP1 protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca(2+ uptake in the SR among the three PP1 isoforms, thereby contributing to Ca(2+ downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9 vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice. METHODS: We created an adeno-associated virus 9 (AAV9 vector encoding PP1β short-hairpin RNA (shRNA or negative control (NC shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP promoter conjugated to emerald-green fluorescence protein (EmGFP and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA were injected into the tail vein (2×10(11 GC/mouse of muscle LIM protein deficient mice (MLPKO, followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months. RESULTS: In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months. CONCLUSION: Heart failure-inducible

  9. Role of 5-HT2C receptor gene variants in antipsychotic-induced weight gain

    Directory of Open Access Journals (Sweden)

    Brandl EJ

    2011-08-01

    Full Text Available Tessa JM Wallace, Clement C Zai, Eva J Brandl, Daniel J MüllerNeurogenetics Section, Center for Addiction and Mental Health, Department of Psychiatry, University of Toronto, Toronto, ON, CanadaAbstract: Antipsychotic-induced weight gain is a serious side effect of antipsychotic medication that can lead to increased morbidity, mortality, and non-compliance in patients. Numerous single nucleotide polymorphisms have been studied for association with antipsychotic-induced weight gain in an attempt to find genetic predictors of this side effect. An ability to predict this side effect could lead to personalized treatment plans for predisposed individuals, which could significantly decrease the prevalence and severity of weight gain. Variations in the serotonin receptor 2c gene (HTR2C have emerged as promising candidates for prediction of antipsychotic-induced weight gain. Specifically, the well-studied -759C/T promoter polymorphism has been associated with weight gain in diverse populations, although some studies have reported no association. This discrepancy is likely due to heterogeneity in study design with respect to ethnicity, treatment duration, and other variables. Notably, the association between HTR2C and antipsychotic-induced weight gain appears strongest in short-term studies on patients with limited or no previous antipsychotic treatment. Other, less extensively studied promoter polymorphisms (-697C/G, -997G/A, and -1165A/G have also emerged as potential predictors of antipsychotic-induced weight gain. Conversely, the well-studied intronic polymorphism Cys23Ser does not appear to be associated. With further research on both HTR2C and other genetic and environmental predictors of antipsychotic-induced weight gain, a predictive test could one day be created to screen patients and provide preventative or alternative treatment for those who are predisposed to this serious side effect.Keywords: HTR2C, pharmacogenomics, promoter polymorphism

  10. Early estrogen-induced gene 1, a novel RANK signaling component, is essential for osteoclastogenesis

    Institute of Scientific and Technical Information of China (English)

    Han Kyoung Choi; Hye Ri Kang; Eutteum Jung; Tae Eon Kim; Jing Jing Lin; Soo Young Lee

    2013-01-01

    The receptor activator of NF-kB (RANK) and immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptors are essential factors involved in regulating osteoclast formation and bone remodeling.Here,we identify early estrogen-induced gene 1 (EEIG1) as a novel RANK ligand (RANKL)-inducible protein that physically interacts with RANK and further associates with Gab2,PLCγ2 and Tec/Btk kinases upon RANKL stimulation.EEIG1 positively regulates RANKL-induced osteoclast formation,likely due to its ability to facilitate RANKL-stimulated PLCγ2 phosphorylation and NFATc1 induction.In addition,an inhibitory peptide designed to block RANK-EEIG1 interaction inhibited RANKL-induced bone destruction by reducing osteoclast formation.Together,our results identify EEIG1 as a novel RANK signaling component controlling RANK-mediated osteoclast formation,and suggest that targeting EEIG1 might represent a new therapeutic strategy for the treatment of pathological bone resorption.

  11. Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I

    Directory of Open Access Journals (Sweden)

    Qin Lan

    2011-12-01

    Full Text Available Abstract Background The nonstructural protein 1 (NSP1 of rotavirus has been reported to block interferon (IFN signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs and (or the β-transducin repeat containing protein (β-TrCP. However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms. Results The NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s other than IRFs. Overexpression of NSP1 can block IFN-β promoter activation induced by the retinoic acid inducible gene I (RIG-I, but does not inhibit IFN-β activation induced by the mitochondrial antiviral-signaling protein (MAVS, indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way. Conclusions Our findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus.

  12. RNAseq analysis reveals pathways and candidate genes associated with salinity tolerance in a spaceflight-induced wheat mutant.

    Science.gov (United States)

    Xiong, Hongchun; Guo, Huijun; Xie, Yongdun; Zhao, Linshu; Gu, Jiayu; Zhao, Shirong; Li, Junhui; Liu, Luxiang

    2017-06-02

    Salinity stress has become an increasing threat to food security worldwide and elucidation of the mechanism for salinity tolerance is of great significance. Induced mutation, especially spaceflight mutagenesis, is one important method for crop breeding. In this study, we show that a spaceflight-induced wheat mutant, named salinity tolerance 1 (st1), is a salinity-tolerant line. We report the characteristics of transcriptomic sequence variation induced by spaceflight, and show that mutations in genes associated with sodium ion transport may directly contribute to salinity tolerance in st1. Furthermore, GO and KEGG enrichment analysis of differentially expressed genes (DEGs) between salinity-treated st1 and wild type suggested that the homeostasis of oxidation-reduction process is important for salt tolerance in st1. Through KEGG pathway analysis, "Butanoate metabolism" was identified as a new pathway for salinity responses. Additionally, key genes for salinity tolerance, such as genes encoding arginine decarboxylase, polyamine oxidase, hormones-related, were not only salt-induced in st1 but also showed higher expression in salt-treated st1 compared with salt-treated WT, indicating that these genes may play important roles in salinity tolerance in st1. This study presents valuable genetic resources for studies on transcriptome variation caused by induced mutation and the identification of salt tolerance genes in crops.

  13. Molecular characterization of hap complex components responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii.

    Science.gov (United States)

    Oda, Saori; Yurimoto, Hiroya; Nitta, Nobuhisa; Sasano, Yu; Sakai, Yasuyoshi

    2015-03-01

    We identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2Δ, Cbhap3Δ, and Cbhap5Δ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

  14. The Gene Expression Profile of D-galactose Induced Aging Model Rat Using cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Li Min(李珉); Wang Gang; Zhang Wei; Wang Miqu; Zhang Yizheng

    2004-01-01

    In order to study the molecular mechanism of D-galactose induced aging model, cDNA microarray is used to analyze gene expression profiles of both normal and D-galactose induced aging model rats. D-galactose induced aging model rats are injected with D-galactose, while normal rats are injected with physiological saline as control. After 7 weeks, the two groups of rats are killed simultaneously. Their livers are harvested for genome-wide expression analysis. D-galactose treated rats showed changes in gene expression associated with increase or decrease in xenobiotic metabolism, protein metabolism and energy metabolism.

  15. [Advances in molecular mechanisms of bacterial resistance caused by stress-induced transfer of resistance genes--a review].

    Science.gov (United States)

    Sun, Dongchang; Wang, Bing; Zhu, Lihong

    2013-07-04

    The transfer of resistance gene is one of the most important causes of bacterial resistance. Recent studies reveal that stresses induce the transfer of antibiotic resistance gene through multiple mechanisms. DNA damage stresses trigger bacterial SOS response and induce the transfer of resistance gene mediated by conjugative DNA. Antibiotic stresses induce natural bacterial competence for transformation in some bacteria which lack the SOS system. In addition, our latest studies show that the general stress response regulator RpoS regulates a novel type of resistance gene transfer which is mediated by double-stranded plasmid DNA and occurs exclusively on the solid surface. In this review, we summarized recent advances in SOS dependent and independent stress-induced DNA transfer which is mediated by conjugation and transformation respectively, and the transfer of double-stranded plasmid DNA on the solid surface which is regulated by RpoS. We propose that future work should address how stresses activate the key regulators and how these regulators control the expression of gene transfer related genes. Answers to the above questions would pave the way for searching for candidate targets for controlling bacterial resistance resulted from the transfer of antibiotic genes.

  16. Overexpression of superoxide dismutase 3 gene blocks high-fat diet-induced obesity, fatty liver and insulin resistance.

    Science.gov (United States)

    Cui, R; Gao, M; Qu, S; Liu, D

    2014-09-01

    Oxidative stress has an important role in the development of obesity and obesity-associated metabolic disorders. As an endogenous antioxidant enzyme, superoxide dismutase 3 (SOD3) has the potential to affect diet-induced obesity and obesity-associated complications. In the current work, we overexpressed SOD3 in C57BL/6 mice fed a high-fat diet (HFD) to study its effect on HFD-induced obesity, fatty liver and insulin resistance. We demonstrated that the Sod3 gene transfer blocked HFD-induced obesity, fatty liver and insulin resistance. Real-time PCR analysis of adipose and liver tissues revealed that overexpression of the Sod3 gene suppressed expression of pro-inflammatory genes in adipose tissue including F4/80, Tnfα, Cd11c, Mcp1 and Il6, and increased expression of anti-inflammatory genes such as adiponectin. In the liver, high levels of SOD3 activity in animals enhanced expression of the genes responsible for energy expenditure including Cpt1α, Cpt1β, Pgc1α, Pgc1β and Ucp2. These results suggest that overexpression of the Sod3 gene through gene transfer is an effective approach in preventing diet-induced obesity and obesity-associated complications.

  17. Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

    Science.gov (United States)

    Makuch, Edyta; Kuropatwa, Marianna; Kurowska, Ewa; Ciekot, Jaroslaw; Klopotowska, Dagmara; Matuszyk, Janusz

    2014-07-05

    Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

  18. The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

    Science.gov (United States)

    Kim, Jong-Hyun; Lee, Sang-Hee; Sohn, Hae-Jin; Lee, Jinyoung; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

    2012-12-01

    The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

  19. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  20. Methylmercury-induced changes in gene transcription associated with neuroendocrine disruption in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Richter, Catherine A.; Martyniuk, Christopher J.; Annis, Mandy L.; Brumbaugh, William G.; Chasar, Lia C.; Denslow, Nancy D.; Tillitt, Donald E.

    2014-01-01

    Methyl-mercury (MeHg) is a potent neuroendocrine disruptor that impairs reproductive processes in fish. The objectives of this study were to (1) characterize transcriptomic changes induced by MeHg exposure in the female largemouth bass (LMB) hypothalamus under controlled laboratory conditions, (2) investigate the health and reproductive impacts of MeHg exposure on male and female largemouth bass (LMB) in the natural environment, and (3) identify MeHg-associated gene expression patterns in whole brain of female LMB from MeHg-contaminated habitats. The laboratory experiment was a single injection of 2.5 μg MeHg/g body weight for 96 h exposure. The field survey compared river systems in Florida, USA with comparably lower concentrations of MeHg (Wekiva, Santa Fe, and St. Johns Rivers) in fish and one river system with LMB that contained elevated concentrations of MeHg (St. Marys River). Microarray analysis was used to quantify transcriptomic responses to MeHg exposure. Although fish at the high-MeHg site did not show overt health or reproductive impairment, there were MeHg-responsive genes and pathways identified in the laboratory study that were also altered in fish from the high-MeHg site relative to fish at the low-MeHg sites. Gene network analysis suggested that MeHg regulated the expression targets of neuropeptide receptor and steroid signaling, as well as structural components of the cell. Disease-associated gene networks related to MeHg exposure, based upon expression data, included cerebellum ataxia, movement disorders, and hypercalcemia. Gene responses in the CNS are consistent with the documented neurotoxicological and neuroendocrine disrupting effects of MeHg in vertebrates.

  1. Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum.

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    Verónica S Di Stilio

    Full Text Available Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS of a PHYTOENE DESATURASE ortholog (TdPDS can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05, as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.

  2. Analysis of tumor heterogeneity and cancer gene networks using deep sequencing of MMTV-induced mouse mammary tumors.

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    Christiaan Klijn

    Full Text Available Cancer develops through a multistep process in which normal cells progress to malignant tumors via the evolution of their genomes as a result of the acquisition of mutations in cancer driver genes. The number, identity and mode of action of cancer driver genes, and how they contribute to tumor evolution is largely unknown. This study deployed the Mouse Mammary Tumor Virus (MMTV as an insertional mutagen to find both the driver genes and the networks in which they function. Using deep insertion site sequencing we identified around 31000 retroviral integration sites in 604 MMTV-induced mammary tumors from mice with mammary gland-specific deletion of Trp53, Pten heterozygous knockout mice, or wildtype strains. We identified 18 known common integration sites (CISs and 12 previously unknown CISs marking new candidate cancer genes. Members of the Wnt, Fgf, Fgfr, Rspo and Pdgfr gene families were commonly mutated in a mutually exclusive fashion. The sequence data we generated yielded also information on the clonality of insertions in individual tumors, allowing us to develop a data-driven model of MMTV-induced tumor development. Insertional mutations near Wnt and Fgf genes mark the earliest "initiating" events in MMTV induced tumorigenesis, whereas Fgfr genes are targeted later during tumor progression. Our data shows that insertional mutagenesis can be used to discover the mutational networks, the timing of mutations, and the genes that initiate and drive tumor evolution.

  3. Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes.

    Science.gov (United States)

    Waring, R B; May, G S; Morris, N R

    1989-06-30

    Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.

  4. Identification of target genes of transcription factor CEBPB in acute promyelocytic leukemia cells induced by all-trans retinoic acid

    Institute of Scientific and Technical Information of China (English)

    Lei Yu; Yang-De Zhang; Jun Zhou; De-Ming Yao; Xiang Li

    2013-01-01

    Objective: To indentify target genes of transcription factor CCAAT enhancer-binding proteinβ (CEBPB) in acute promyelocytic leukemia cells induced by all-trans retinoic acid. Methods:A new strategy for high-throughput identification of direct target genes was established by combining chromatin immunoprecipitation (ChIP) with in vitro selection. Then, 106 potential CEBPB binding fragments from the genome of the all-trans retinoic acid (ATRA)-treated NB4 cells were identified. Results: Of them, 82 were mapped in proximity to known or previously predicted genes; 7 were randomly picked up for further confirmation by ChIP-PCR and 3 genes (GALM, ITPR2 and ORM2) were found to be specifically up-regulated in the ATRA-treated NB4 cells, indicating that they might be the down-stream target genes of ATRA. Conclusions: Our results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.

  5. Adenovirus-mediated hypoxia-inducible factor-1 alpha gene transfer induces angiogenesis and neurogenesis following cerebral ischemia in rats

    Institute of Scientific and Technical Information of China (English)

    Wanfu Wu; Xiu Chen; Zhen Yu; Changlin Hu; Wenqin Cai

    2008-01-01

    BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) accumulates under conditions of hypoxia. HIF-1α target genes have pleiotropic effects on neurogenesis, neuroprotection and angiogenesis in the brain.OBJECTIVE: To investigate whether a recombinant adenovirus carrying HIF-1α can increase the expression of HIF-1α in vivo and thus promote angiogenesis and neurogenesis in a rat model of focal cerebral ischemia.DESIGN, TIME AND SETTING: The randomized, controlled experiment was performed at the Department of Neurobiology, Third Military Medical University of Chinese PLA from September 2006 to October 2007.MATERIALS: 68 healthy adult male Sprague-Dawley (SD) rats, weighing 230-250 g, were used. HIF-1α antibody was purchased from Wuhan Boster Company. Vascular endothelial growth factor (VEGF) antibody was purchased from Santa Cruz Biotech Company.METHODS: All 68 rats were induced with a transient middle cerebral artery occlusion (MCAO), according to the method of intra-luminal vascular occlusion. 54 rats, in which MCAO was successfully induced, were randomly divided into adenovirus (Ad) group and recombinant adenovirus with HIF-1αgene (Ad-HIF-1α) group (27 rats for each group). Rats were injected with 10 μL Ad (Ad group) or Ad-HIF-1α (Ad-HIF-1α group) into the lateral ventricle, 1 day after MCAO induction. MAIN OUTCOME MEASURES: Reverse transcription polymerase chain reaction was used to measure the expression of HIF-1α and of VEGF. Immunohistochemistry was used to detect the localization of HIF-1α, VEGF and factor Ⅷ in ischemic penumbra. Rat newborn nerve cells were labeled with 5-bromodeoxyuridine (BrdU) after ischemia. BrdU/neurofilament 200 (NF200) and BrdU/glial fibrillary acidic protein (GFAP) double labeled immunofluorescent histochemistry was used to identify the differentiation of newborn cells. Neurological function was evaluated using the modified neurological severity score (NSS).RESULTS: Compared with Ad, Ad-HIF-1αenhanced the expression of HIF-1

  6. Utilization of Alternative Polyadenylation Signals in the Novel Human Apoptosis-Inducing Gene hap

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    hap, a novel human apoptosis-inducing gene which can interact with another newly discovered apoptosis-inducing gene ASY, was identified by cloning its cDNAs from human lung cell line (WI-38) cDNA library. Two major mRNA species (1.8 and 2. 7 kb in length, respectively) were previously identified by Northern blot analysis of poly(A)+ RNA from human multiple tissues using partial hap cDNA as a probe. In the present work, the molecular mechanism accounting for the generation of the two hap transcripts were investigated. The rapid amplification of cDNA 3'-ends (3'-RACE) technique and the sequential Southern blot analysis, in conjunction with the sequencing analysis demonstrated that the two hap transcripts derive from the alternative polyadenylation site selection: a AATAAA signal at position 1 528 -1 533 nt for the 1.8 kb hap mRNA; and a AATAAA signal at position 2 375 -2 380 nt for the 2. 7 kb hap mRNA. Furthermore, a number of regulatory elements within hap 3'-untranslated region (3'-UTR) were also examined.

  7. Cohesin protects genes against γH2AX Induced by DNA double-strand breaks.

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    Pierre Caron

    2012-01-01

    Full Text Available Chromatin undergoes major remodeling around DNA double-strand breaks (DSB to promote repair and DNA damage response (DDR activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome.

  8. Water-deficit inducible expression of a cytokinin biosynthetic gene IPT improves drought tolerance in cotton.

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    Sundaram Kuppu

    Full Text Available Water-deficit stress is a major environmental factor that limits agricultural productivity worldwide. Recent episodes of extreme drought have severely affected cotton production in the Southwestern USA. There is a pressing need to develop cotton varieties with improved tolerance to water-deficit stress for sustainable production in wat