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Sample records for bypass promotes expression

  1. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...... knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can...... be met by using promoter libraries. This approach generally consists of inserting a library of promoters in front of the gene to be studied, whereby the individual promoters might deviate either in their spacer sequences or bear slight deviations from the consensus sequence of a vegetative promoter...

  2. Hepatic phosphoenolpyruvate carboxykinase expression after gastric bypass surgery in rats with type 2 diabetes mellitus.

    Science.gov (United States)

    Wu, J; Hou, S S; Wang, W; Yin, M; Cheng, N; Ge, L L; Yin, J J; Xu, J

    2015-01-01

    The objective of this study was to investigate the mRNA expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) after gastric bypass surgery (GBS) in rats with type 2 diabetic mellitus (T2DM). Thirty-six male Goto-Kakizaki rats, aged 12 weeks, were randomly divided into the GBS, sham operation with diet restriction (SO), and sham operation alone (control) groups (N = 12 per group). Liver specimens from all rats were obtained during the operation and 8 weeks after operation. Blood lipid levels were measured before and 8 weeks after operation. Fasting blood glucose (FBG), food intake, and body weight were recorded at weekly time points after operation. The blood glucose area under the curve (AUC) was calculated, and insulin sensitivity indices (ISI) were assessed. The expression PEPCK mRNA and protein were measured by real-time polymerase chain reaction and western blot. Compared with those of the SO and control groups, the blood lipid levels and the FBG in the GBS group was significantly decreased (P rats while improving glucose tolerance and hyperglycemia, and the mechanism appears to be associated with a decrease of hepatic PEPCK mRNA and protein expression. PMID:26681041

  3. A promoter-level mammalian expression atlas

    KAUST Repository

    Forest, Alistair R R

    2014-03-26

    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

  4. Expression of Active Fluorophore Proteins in the Milk of Transgenic Pigs Bypassing the Secretory Pathway

    Science.gov (United States)

    Mukherjee, Ayan; Garrels, Wiebke; Talluri, Thirumala R.; Tiedemann, Daniela; Bősze, Zsuzsanna; Ivics, Zoltán; Kues, Wilfried A.

    2016-01-01

    We describe the expression of recombinant fluorescent proteins in the milk of two lines of transgenic pigs generated by Sleeping Beauty transposon-mediated genetic engineering. The Sleeping Beauty transposon consisted of an ubiquitously active CAGGS promoter driving a fluorophore cDNA, encoding either Venus or mCherry. Importantly, the fluorophore cDNAs did not encode for a signal peptide for the secretory pathway, and in previous studies of the transgenic animals a cytoplasmic localization of the fluorophore proteins was found. Unexpectedly, milk samples from lactating sows contained high levels of bioactive Venus or mCherry fluorophores. A detailed analysis suggested that exfoliated cells of the mammary epithelium carried the recombinant proteins passively into the milk. This is the first description of reporter fluorophore expression in the milk of livestock, and the findings may contribute to the development of an alternative concept for the production of bioactive recombinant proteins in the udder. PMID:27086548

  5. Methylation and Expression of Immune and Inflammatory Genes in the Offspring of Bariatric Bypass Surgery Patients

    Directory of Open Access Journals (Sweden)

    Frédéric Guénard

    2013-01-01

    Full Text Available Background. Maternal obesity, excess weight gain and overnutrition during pregnancy increase risks of obesity, type 2 diabetes mellitus, and cardiovascular disease in the offspring. Maternal biliopancreatic diversion is an effective treatment for severe obesity and is beneficial for offspring born after maternal surgery (AMS. These offspring exhibit lower severe obesity prevalence and improved cardiometabolic risk factors including inflammatory marker compared to siblings born before maternal surgery (BMS. Objective. To assess relationships between maternal bariatric surgery and the methylation/expression of genes involved in the immune and inflammatory pathways. Methods. A differential gene methylation analysis was conducted in a sibling cohort of 25 BMS and 25 AMS offspring from 20 mothers. Following differential gene expression analysis (23 BMS and 23 AMS, pathway analysis was conducted. Correlations between gene methylation/expression and circulating inflammatory markers were computed. Results. Five immune and inflammatory pathways with significant overrepresentation of both differential gene methylation and expression were identified. In the IL-8 pathway, gene methylation correlated with both gene expression and plasma C-reactive protein levels. Conclusion. These results suggest that improvements in cardiometabolic risk markers in AMS compared to BMS offspring may be mediated through differential methylation of genes involved in immune and inflammatory pathways.

  6. Pulmonary microRNA expression profiling in an immature piglet model of cardiopulmonary bypass-induced acute lung injury.

    Science.gov (United States)

    Li, Wenlei; Ma, Kai; Zhang, Sen; Zhang, Hao; Liu, Jinping; Wang, Xu; Li, Shoujun

    2015-04-01

    After surgery performed under cardiopulmonary bypass (CPB), severe lung injury often occurs in infants. MicroRNAs (miRNAs) are potentially involved in diverse pathophysiological processes via regulation of gene expression. The objective of this study was to investigate differentially expressed miRNAs and their potential target genes in immature piglet lungs in response to CPB. Fourteen piglets aged 18.6 ± 0.5 days were equally divided into two groups that underwent sham sternotomy or CPB. The duration of aortic cross-clamping was 2 h, followed by 2 h reperfusion. Lung injury was evaluated by lung function indices, levels of cytokines, and histological changes. We applied miRNA microarray and quantitative real-time polymerase chain reaction (qRT-PCR) analysis to determine miRNA expression. Meanwhile, qRT-PCR and enzyme-linked immunosorbent assay were used for validation of predicted mRNA targets. The deterioration of lung function and histopathological changes revealed the piglets' lungs were greatly impaired due to CPB. The levels of tumor necrosis factor alpha, interleukin 6, and interleukin 10 increased in the lung tissue after CPB. Using miRNA microarray, statistically significant differences were found in the levels of 16 miRNAs in the CPB group. Up-regulation of miR-21 was verified by PCR. We also observed down-regulation in the levels of miR-127, miR-145, and miR-204, which were correlated with increases in the expression of the products of their potential target genes PIK3CG, PTGS2, ACE, and IL6R in the CPB group, suggesting a potential role for miRNA in the regulation of inflammatory response. Our results show that CPB induces severe lung injury and dynamic changes in miRNA expression in piglet lungs. Moreover, the changes in miRNA levels and target gene expression may provide a basis for understanding the pathogenesis of CPB-induced injury to immature lungs. PMID:25347932

  7. MUS81 promotes common fragile site expression

    DEFF Research Database (Denmark)

    Ying, Songmin; Minocherhomji, Sheroy; Chan, Kok Lung;

    2013-01-01

    Fragile sites are chromosomal loci with a propensity to form gaps or breaks during early mitosis, and their instability is implicated as being causative in certain neurological disorders and cancers. Recent work has demonstrated that the so-called common fragile sites (CFSs) often impair the...... faithful disjunction of sister chromatids in mitosis. However, the mechanisms by which CFSs express their fragility, and the cellular factors required to suppress CFS instability, remain largely undefined. Here, we report that the DNA structure-specific nuclease MUS81-EME1 localizes to CFS loci in early...

  8. Precise regulation of gene expression dynamics favors complex promoter architectures.

    Directory of Open Access Journals (Sweden)

    Dirk Müller

    2009-01-01

    Full Text Available Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure.

  9. Promoter methylation regulates cyclooxygenase expression in breast cancer

    International Nuclear Information System (INIS)

    Overexpression of cyclooxygenase (COX-2) is commonly observed in human cancers. In a murine model of metastatic breast cancer, we observed that COX-2 expression and enzyme activity were associated with enhanced tumorigenic and metastatic potential. In contrast to the high COX-2 expression in metastatic tumors, transplantation of poorly tumorigenic tumor cell lines to syngeneic mice results in less COX-2 expression and less COX-2 activity in vivo. Aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in human cancer cell lines and in some human tumors of the gastrointestinal tract. Using bisulfite modification and a methylation-specific PCR, we examined the methylation status of the COX-2 promoter in a series of four closely-related murine mammary tumors differing in COX-2 expression and metastatic potential. We showed that line 410, which does not express COX-2 in vivo, exhibited evidence of promoter methylation. Interestingly, the metastatic counterpart of this cell (line 410.4) displayed only the unmethylated COX-2 promoter, as did two additional cell lines (lines 66.1 and 67). The methylation patterns observed in vitro were maintained when these murine mammary tumor lines were transplanted to syngeneic mice. Treatment with the DNA demethylating agent 5-aza-deoxycytidine increased COX-2 mRNA, increased protein and increased enzyme activity (prostaglandin synthesis). These results indicate that COX-2 promoter methylation may be one mechanism by which tumor cells regulate COX-2 expression. Upregulation of COX-2 expression in closely related metastatic lesions versus nonmetastatic lesions may represent a shift towards the unmethylated phenotype

  10. GSTP1 expression and promoter methylation in epithelial ovarian carcinoma

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    V Shilpa

    2014-01-01

    Full Text Available Context: GSTP1 is a subgroup of glutathione-S-transferase family, which provides cellular protection against free radical and carcinogenic compounds due to its detoxifying function. Altered GSTP1 activity due to down regulation of enzyme activity and DNA methylation has been reported in many tumors, although data for ovarian cancer are few. In this study, we aimed at determining the expression of GSTP1 in relation to the methylation of the GSTP1 promoter in epithelial ovarian cancer (EOC. Materials and Methods: GSTP1 mRNA expression and GSTP1 enzyme concentration were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR and enzyme-linked immunosorbent assay, respectively, in 88 EOCs, 14 low malignant potential (LMP tumors, and 20 benign tumors. The promoter methylation of GSTP1 gene was evaluated by methylation-specific PCR. Results: Reduced GSTP1 mRNA expression was observed in 49% EOCs, 21.4% LMP, and 45% benign tumors. Significantly lower levels of plasma GSTP1 were observed in all tumor samples compared to normal. GSTP1 promoter methylation was detected in 10 (11.4% EOCs and 1 (7.3% LMP tumors. No methylation was observed in benign tumors and normal ovaries. Conclusions: Our results show that there is a significant down regulation of GSTP1 expression while hypermethylation of the GSTP1 gene promoter is not very frequent in EOC. Further studies are needed to study underlying mechanisms leading to decreased expression.

  11. Platelets promote allergic asthma through the expression of CD154

    OpenAIRE

    Tian, Jun; ZHU, TIANYI; Liu, Juan; Guo, Zhenhong; Cao, Xuetao

    2014-01-01

    Platelet activation is associated with multiple immune responses and the pathogenesis of various immune-related diseases. However, the exact role and the underlying mechanism of platelets in the progression of allergic asthma remain largely unclear. In this study, we demonstrate that during antigen sensitization, platelets can be activated by ovalbumin (OVA) aerosol via the upregulation of CD154 (CD40L) expression. Platelet transfer promoted allergic asthma progression by inducing more severe...

  12. Promoter hypomethylation regulates CD133 expression in human gliomas

    Institute of Scientific and Technical Information of China (English)

    Kouichi Tabu; Ken Sasai; Taichi Kimura; Lei Wang; Eiko Aoyanagi; Shinji Kohsaka; Mishie Tanino; Hiroshi Nishihara; Shinya Tanaka

    2008-01-01

    Brain tumor-initiating cells (BTICs) have been enriched using antibodies against the cell surface protein CD133;however,the biological relevance and the regulatory mechanism of CD133 expression in human gliomas are not yet understood.In this study,we initially demonstrated that CD133 was overexpressed in high-grade human glioblastomas where CD133-positive cells were focally observed as a micro-cluster.In addition,CD133 transcripts with exon 1A,1B,or 1C were predominantly expressed in glioblastomas.To elucidate the mechanism regulating this aberrant expression of CD133,three proximal promoters (P1,P2,and P3) containing a CpG island were isolated.In U251MG and T98Gglioblastoma cells,the P1 region flanking exon 1A exhibited the highest activity among the three promoters,and this activity was significantly inactivated by in vitro methylation.After treatment with the demethylating agent 5-azacytidine and/or the histone deacetylase inhibitor valproic acid,the expression level of CD133 mRNA was significantly restored in glioma cells.Importantly,hypomethylation of CpG sites within the P1,P2,and P3 regions was observed by bisulfite sequencing in human glioblastoma tissues with abundant CD133 mRNA.Taken together,our results indicate that DNA hypomethylation is an important determinant of CD133 expression in glioblastomas,and this epigenetic event may be associated with the development of BTICs expressing CD133.

  13. Duodenal-jejunal bypass surgery on type 2 diabetic rats reduces the expression of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in the thoracic aorta

    Institute of Scientific and Technical Information of China (English)

    Maimaitiyusufu Wubulikasimu; Han Haifeng; Yan Zhibo; Zhang Xiang; Liu Shaozhuang; Zhang Guangyong; Kasimu Aimaiti

    2014-01-01

    Background Bariatric surgery offers a productive resolution of type 2 diabetes mellitus (T2DM).The development of T2DM vasculopathy is due to chronic inflammation,which increases matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression.This study sought to examine MMP-9 and TIMP-1 expression in the thoracic aorta after duodenal-jejunal bypass (DJB) surgery on a T2DM rat model induced by a high-fat diet and low dose streptozotocin (STZ).Methods Twenty-one T2DM Wistar rats induced by high-fat diet and low dose STZ were randomly divided into DJB and sham duodenal-jejunal bypass (S-DJB) groups.Ten Wistar rats were fed a normal diet as a control.Recovery of gastrointestinal function post-operation and resumption of a normal diet completed the experiment.Body weight,blood glucose,blood lipid levels,and MMP-9 and TIMP-1 expression levels in aortic endothelial cells were measured throughout.Results DJB rats showed significant weight loss 2 weeks post-operation compared with S-DJB rats.After surgery,DJB rats showed significant improvement and steady glycemic control with improved insulin sensitivity and glucose tolerance.They also exhibited improved lipid metabolism with a decrease in fasting free fatty acids (FFAs) and triglycerides (all P <0.05).Immunohistochemistry showed decreased MMP-9 and TIMP-1 expression 12 weeks after surgery (P < 0.01).Conclusions DJB surgery on an induced T2DM rat model improves blood glucose levels and lipids,following a high-fat diet and low dose STZ treatment.In addition,DJB decreased MMP-9 and TIMP-1 expression in vascular endothelial cells,which may play an important role in delaying the development of T2DM vascular disease.

  14. The mitochondrial plasmid of the true slime mold Physarum polycephalum bypasses uniparental inheritance by promoting mitochondrial fusion.

    Science.gov (United States)

    Sakurai, Rakusa; Nomura, Hideo; Moriyam, Yohsuke; Kawano, Shigeyuki

    2004-08-01

    Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF+ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF- strain KM88. Of the three mF- x mF+ crosses, only KM88 x JE8 displayed complete uniparental inheritance. However, in KM88 x TU111 and KM88 x NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion. PMID:15179521

  15. Platelets promote allergic asthma through the expression of CD154.

    Science.gov (United States)

    Tian, Jun; Zhu, Tianyi; Liu, Juan; Guo, Zhenhong; Cao, Xuetao

    2015-11-01

    Platelet activation is associated with multiple immune responses and the pathogenesis of various immune-related diseases. However, the exact role and the underlying mechanism of platelets in the progression of allergic asthma remain largely unclear. In this study, we demonstrate that during antigen sensitization, platelets can be activated by ovalbumin (OVA) aerosol via the upregulation of CD154 (CD40L) expression. Platelet transfer promoted allergic asthma progression by inducing more severe leukocyte infiltration and lung inflammation, elevated IgE production and strengthened T helper 2 (Th2) responses in asthma-induced mice. Accordingly, platelet depletion compromised allergic asthma progression. Cd154-deficient platelets failed to promote asthma development, indicating the requirement of CD154 for platelets to promote asthma progression. The mechanistic study showed that platelets inhibited the induction of Foxp3(+) regulatory T cells both in vivo and in vitro at least partially through CD154, providing an explanation for the increase of Th2 responses by platelet transfer. Our study reveals the previously unknown role of platelet CD154 in the promotion of asthma progression by polarizing Th2 responses and inhibiting regulatory T-cell generation and thus provides a potential clue for allergic disease interventions. PMID:25418472

  16. Analysis of promoter activity in transgenic plants by normalizing expression with a reference gene: anomalies due to the influence of the test promoter on the reference promoter

    Indian Academy of Sciences (India)

    Simran Bhullar; Suma Chakravarthy; Deepak Pental; Pradeep Kumar Burma

    2009-12-01

    Variations in transgene expression due to position effect and copy number are normalized when analysing and comparing the strengths of different promoters. In such experiments, the promoter to be tested is placed upstream to a reporter gene and a second expression cassette is introduced in a linked fashion in the same transfer DNA (T-DNA). Normalization in the activity of the test promoter is carried out by calculating the ratio of activities of the test and reference promoters. When an appropriate number of independent transgenic events are analysed, normalization facilitates assessment of the relative strengths of the test promoters being compared. In this study, using different modified versions of the Cauliflower Mosaic Virus (CaMV) 35S promoter expressing the reporter gene -glucuronidase (gus) (test cassette) linked to a chloramphenicol acetyl transferase (cat) gene under the wild-type 35S promoter (reference cassette) in transgenic tobacco lines, we observed that cat gene expression varied depending upon the strength of the modified 35S promoter expressing the gus gene. The 35S promoter in the reference cassette was found to have been upregulated in cases where the modified 35S promoter was weaker than the wild-type 35S promoter. Many studies have been carried out in different organisms to study the phenomenon of transcriptional interference, which refers to the reduced expression of the downstream promoter by a closely linked upstream promoter. However, we observed a positive interaction wherein the weakened activity of a promoter led to upregulation of a contiguous promoter. These observations suggest that, in situations where the promoters of the test and reference gene share the same transcription factors, the activity of the test promoter can influence the activity of the reference promoter in a way that the test promoter’s strength is underestimated when normalized by the reference promoter.

  17. Id1 expression promotes peripheral CD4+ T cell proliferation and survival upon TCR activation without co-stimulation

    International Nuclear Information System (INIS)

    Highlights: •Id1 expression enables naïve T cell proliferation without anti-CD28 co-stimulation. •Id1 expression facilitates T cells survival when stimulated with anti-CD3. •Elevation of IL-2 production by Id1 contributes increased proliferation and survival. •Id1 potentiates NF-κB activation by anti-CD3 stimulation. -- Abstract: Although the role of E proteins in the thymocyte development is well documented, much less is known about their function in peripheral T cells. Here we demonstrated that CD4 promoter-driven transgenic expression of Id1, a naturally occurring dominant-negative inhibitor of E proteins, can substitute for the co-stimulatory signal delivered by CD28 to facilitate the proliferation and survival of naïve CD4+ cells upon anti-CD3 stimulation. We next discovered that IL-2 production and NF-κB activity after anti-CD3 stimulation were significantly elevated in Id1-expressing cells, which may be, at least in part, responsible for the augmentation of their proliferation and survival. Taken together, results from this study suggest an important role of E and Id proteins in peripheral T cell activation. The ability of Id proteins to by-pass co-stimulatory signals to enable T cell activation has significant implications in regulating T cell immunity

  18. Specific expression of DR5 promoter in rice roots using a tCUP derived promoter-reporter system.

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    Jie Zhou

    Full Text Available Variation of transgene expression caused by either position effect at the insertion site or the promoter/enhancer elements employed for the expression of selectable marker genes has complicated phenotype characterization and caused misinterpretation. We have developed a reporter system in rice to analyze the influence of vector configuration, spacer and selectable marker gene promoter on the expression of the promoterless GUS reporter and DR5 promoter. Our results indicate that a spacer inserted between the reversed 35S promoter and the GUS reporter could reduce leaky expression of the reporter but was unable to block the nonspecific expression of DR5::GUS. Stacking the selectable marker unit in head to tail with the GUS reporter aided the gene specific expression of the GUS reporter under the DR5 promoter even when the 35S promoter is used for expression of the selectable marker. Compared to 35S under this configuration, a quick and distinctive expression of DR5::GUS was observed in the root cap, quiescent center and xylem cells in the root apical meristem by using the tCUP derived promoter (tCUP1 for selection, that is similar to the pattern obtained by a sensitive DR5 variant (DR5rev in Arabidopsis. These data suggest a conserved property of the tCUP promoter in preventing enhancer-promoter interactions in rice as it does in Arabidopsis, and also demonstrate that an analogous distal auxin maximum exists in roots of rice. Therefore, the tCUP promoter based selection system provides a new strategy for specific expression of transgenes in rice.

  19. Cooperative binding of transcription factors promotes bimodal gene expression response.

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    Pablo S Gutierrez

    Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

  20. Ring-Opening of the γ-OH-PdG Adduct Promotes Error-Free Bypass by the Sulfolobus solfataricus DNA Polymerase Dpo4

    OpenAIRE

    Shanmugam, Ganesh; Minko, Irina G.; Banerjee, Surajit; Christov, Plamen P.; Kozekov, Ivan D.; Rizzo, Carmelo J.; Lloyd, R. Stephen; Egli, Martin; Michael P. Stone

    2013-01-01

    Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one (γ-OH-PdG). When placed opposite deoxycytosine (dC) in DNA, γ-OH-PdG undergoes ring-opening to the N 2-(3-oxopropyl)-dG. Ring-opening of the adduct has been hypothesized to facilitate nonmutagenic bypass, particularly by DNA polymerases of the Y family. This study examined the bypass of γ-OH-PdG by Sulfolobus solfataricus ...

  1. STAT6 expression in glioblastoma promotes invasive growth

    International Nuclear Information System (INIS)

    different shRNA- silenced clones, but all had a reduction in 3H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1) and urokinase Plasminogen activator (uPA) as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt [1] gene expression microarray and clinical data, there was a significant difference in survival (P < 0.05) between glioma patients with up- and down-regulated STAT6. Decreased STAT6 expression correlated with longer survival times. In two subsets of patients with either grade IV tumors (GBM) or Grade II/III astrocytomas, there was a similar trend that however did not reach statistical significance. Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target

  2. STAT6 expression in glioblastoma promotes invasive growth

    Directory of Open Access Journals (Sweden)

    Silva Corinne M

    2011-05-01

    -type. There was some variation among the different shRNA- silenced clones, but all had a reduction in 3H-Thymidine uptake ranging from 35%- 70% in U-1242MG and 40- 50% in U-87MG cells. Additionally, STAT6- depleted cells were less invasive than controls in our in vitro transmembrane invasion assay. Invasiveness was decreased by 25-40% and 30-75% in U-1242MG and U-87MG cells, respectively. The microarray analysis identified matrix metalloproteinase 1 (MMP-1 and urokinase Plasminogen activator (uPA as potential STA6 target genes involved in the promotion of GBM cell invasion. In a Kaplan-Meier survival curve based on Rembrandt 1 gene expression microarray and clinical data, there was a significant difference in survival (P Conclusions Taken together, these findings suggest a role for STAT6 in enhancing cell proliferation and invasion in GBM, which may explain why up-regulation of STAT6 correlates with shorter survival times in glioma patients. This report thus identifies STAT6 as a new and potentially promising therapeutic target.

  3. Coronary Artery Bypass

    Science.gov (United States)

    ... to 3 days in the Intensive Care Unit (ICU). Life After Bypass After bypass surgery, your doctor will recommend that you join a cardiac rehabilitation program. These programs help you make lifestyle changes ...

  4. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

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    Jacob Daniela

    2005-06-01

    Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

  5. Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters

    OpenAIRE

    Li Junxin; Wang Juan; Wang Shaowen; Xing Miao; Yu Shaowen; Liu Gang

    2012-01-01

    Abstract Backgrounds The fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II. Results The transcriptional...

  6. A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa).

    Science.gov (United States)

    Samac, Deborah A; Tesfaye, Mesfin; Dornbusch, Melinda; Saruul, Purev; Temple, Stephen J

    2004-08-01

    The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa. PMID:15517994

  7. Cloning of a yeast alpha-amylase promoter and its regulated heterologous expression

    Science.gov (United States)

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR; Hooker, Brian S [Kennewick, WA; Anderson, Daniel B [Pasco, WA

    2003-04-01

    The present invention provides the promoter clone discovery of an alpha-amylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated alpha-amylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  8. Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization

    Institute of Scientific and Technical Information of China (English)

    Ti Liu; Tianhong Wang; Xian Li; Xuan Liu

    2008-01-01

    To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene (cbh1) promoters was obtained. The region from-677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from-620 to-820 of the modified cbh1 promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbh1 promoter, obtaining promoters with copy numbers 2, 4,and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase (gus) reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.

  9. Functional analysis of a reproductive organ predominant expressing promoter in cotton plants

    Institute of Scientific and Technical Information of China (English)

    REN Maozhi; CHEN Quanjia; LI Li; ZHANG Rui; GUO Sandui

    2005-01-01

    Transgenic Bt insect-resistant cotton plants have high insect resistance in the early stage of development, but relatively low resistance in the late stage. Substituting a reproductive organ-specific promoter for the CaMV35S promoter presently being used could be an ideal solution. For the first time, the promoter sequence of ADP-ribosylation factor 1 (arf1) gene was isolated from Gossypium hirsutumY18 by means of inverse PCR. The sequencing result discovered the unique structure of the arf1 promoter, including four promoter-specific elements, the initiator, TATA box, CAAT box and GC box, and also an intron in 5′-untranslation region. Four plant expression vectors were constructed for functional analysis of the promoter. Based on the pBI121 plant expression vector, four truncated arf1 promoters took the place of the CaMV35S promoter. These vectors were different only in their promoter regions. They were introduced into cotton plants via pollen tube pathway. Histochemical GUS staining and fluorescence quantitative analyses were performed to examine the expression patterns of the GUS gene driven by the 4 arf1 truncated promoters in transgenic cotton plants respectively. The results showed that the arf1 promoter was a typical reproductive organ-specific promoter. Hopefully, the arf1 promoter can be a regulatory element for designing cotton reproductive organs with desired characteristics.

  10. FANTOM4 EdgeExpressDB: an integrated database of promoters, genes, microRNAs, expression dynamics and regulatory interactions

    OpenAIRE

    Severin, J.; Waterhouse, A. M.; Kawaji, H.; Lassmann, T.; van Nimwegen, E; Balwierz, P. J.; de Hoon, M. J. L.; Hume, D A; Carninci, P.; Hayashizaki, Y.; Suzuki, H.; Daub, C.O.; Forrest, A. R. R.

    2009-01-01

    EdgeExpressDB is a novel database and set of interfaces for interpreting biological networks and comparing large high-throughput expression datasets that requires minimal development for new data types and search patterns. The FANTOM4 EdgeExpress database http://fantom.gsc.riken.jp/4/edgeexpress summarizes gene expression patterns in the context of alternative promoter structures and regulatory transcription factors and microRNAs using intuitive gene-centric and sub-network views. This is an ...

  11. Off-pump coronary artery bypass surgery is associated with fewer gene expression changes in the human myocardium in comparison with on-pump surgery

    OpenAIRE

    Ghorbel, Mohamed T.; Cherif, Myriam; Mokhtari, Amir; Bruno, Vito Domenico; Caputo, Massimo; Angelini, Gianni D

    2010-01-01

    Off-pump coronary artery bypass surgery reduces the myocardial injury associated with on pump surgery with cardiopulmonary bypass (CPB) and ischemic-cardioplegic arrest (CA). We sought to find a mechanistic explanation for this by comparing the transcriptomic changes in the myocardium of patients undergoing on- and off-pump surgery. Transcriptomic analyses were performed on left ventricular biopsies obtained from patients prior to (pre-op) and after completion of all coronary anastomoses (pos...

  12. Expression and aberrant promoter methylation of Wnt inhibitory factor-1 in human astrocytomas

    OpenAIRE

    Wu Jun; Liu Jinfang; Chen Fenghua; Fang Jiasheng; Wang Ying; Yang Zhuanyi; Wang Yanjin

    2010-01-01

    Abstract Background Wnt inhibitory factor-1(WIF-1) acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the expression and promoter methylation status of WIF-1 gene in human astrocytomas. Methods The tissue samples consisted of 53 astrocytomas and 6 normal brain tissues. The expression levels of WIF-1 were det...

  13. Expression and aberrant promoter methylation of Wnt inhibitory factor-1 in human astrocytomas

    Directory of Open Access Journals (Sweden)

    Wu Jun

    2010-03-01

    Full Text Available Abstract Background Wnt inhibitory factor-1(WIF-1 acts as a Wnt-antagonists and tumor suppressor, but hypermethylation of WIF-1 gene promoter and low expression activate Wnt signaling aberrantly and induce the development of various human tumors. With this work we intended to investigate the expression and promoter methylation status of WIF-1 gene in human astrocytomas. Methods The tissue samples consisted of 53 astrocytomas and 6 normal brain tissues. The expression levels of WIF-1 were determined by immunohistochemistry and semiquantitative RT-PCR. The results were analyzed in correlation with clinicopathological data. Methylation status of WIF-1 gene promoter was investigated using methylation specific PCR. The relationship between methylation and expression of the genes was analyzed. Results The average expression levels of WIF-1 protein and mRNA in astrocytomas were decreased significantly compared with normal control tissues. The protein and mRNA expression of WIF-1 gene in astrocytomas was decreased with the increase of pathological grade. Furthermore, WIF-1 promoter methylation was observed by MS-PCR in astrocytomas which showed significant reduction of WIF-1 expression. The WIF-1 promoter hypermethylation was associated with reduced expression of WIF-1 expression. Conclusion Our results demonstrate that the WIF-1 gene is frequently down-regulated or silenced in astrocytomas by aberrant promoter methylation. This may be an important mechanism in astrocytoma carcinogenesis.

  14. High level constitutive expression of luciferase reporter by lsd90 promoter in fission yeast.

    Directory of Open Access Journals (Sweden)

    Hemant Kumar Verma

    Full Text Available Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe.

  15. Twist expression promotes migration and invasion in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Twist, a transcription factor of the basic helix-loop-helix class, is reported to regulate cancer metastasis. It is known to induce epithelial-mesenchymal transition (EMT). In this study, we evaluated the expression of twist and its effect on cell migration in hepatocellular carcinoma (HCC). We examined twist expression using immunohistochemistry in 20 tissue samples of hepatocellular carcinoma, and assessed twist expression in HCC cell lines by RT-PCR and Western blot analysis. Ectopic twist expression was created by introducing a twist construct in the twist-negative HCC cell lines. Endogenous twist expression was blocked by twist siRNA in the twist-positive HCC cell lines. We studied EMT related markers, E-cadherin, Vimentin, and N-cadherin by Western blot analysis. Cell proliferation was measured by MTT assay, and cell migration was measured by in vitro wound healing assay. We used immunofluorescent vinculin staining to visualize focal adhesion. We detected strong and intermediate twist expression in 7 of 20 tumor samples, and no significant twist expression was found in the tumor-free resection margins. In addition, we detected twist expression in HLE, HLF, and SK-Hep1 cells, but not in PLC/RPF/5, HepG2, and Huh7 cells. Ectopic twist-expressing cells demonstrated enhanced cell motility, but twist expression did not affect cell proliferation. Twist expression induced epithelial-mesenchymal transition together with related morphologic changes. Focal adhesion contact was reduced significantly in ectopic twist-expressing cells. Twist-siRNA-treated HLE, HLF, and SK-Hep1 cells demonstrated a reduction in cell migration by 50, 40 and 18%, respectively. Twist induces migratory effect on hepatocellular carcinoma by causing epithelial-mesenchymal transition

  16. OCT4 increases BIRC5 and CCND1 expression and promotes cancer progression in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    OCT4 and BIRC5 are preferentially expressed in human cancer cells and mediate cancer cell survival and tumor maintenance. However, the molecular mechanism that regulates OCT4 and BIRC5 expression is not well characterized. By manipulating OCT4 and BIRC5 expression in hepatocellular carcinoma (HCC) cell lines, the regulatory mechanism of OCT4 on BIRC5 and CCND1 were investigated. Increasing or decreasing OCT4 expression could enhance or suppress BIRC5 expression, respectively, by regulating the activity of BIRC5 promoter. Because there is no binding site for OCT4 within BIRC5 promoter, the effect of OCT4 on BIRC5 promoter is indirect. An octamer motif for OCT4 in the CCND1 promoter has directly and partly participated in the regulation of CCND1 promoter activity, suggesting that OCT4 also could upregulated the expression of CCND1. Co-suppression of OCT4 and BIRC5 induced cancer cell apoptosis and cell cycle arrest, thereby efficiently inhibiting the proliferative activity of cancer cells and suppressing the growth of HCC xenogrfts in nude mice. OCT4 can upregulate BIRC5 and CCND1 expression by increasing their promoter activity. These factors collusively promotes HCC cell proliferation, and co-suppression of OCT4 and BIRC5 is potentially beneficial for HCC treatment

  17. Clogging the enrichment bypass

    International Nuclear Information System (INIS)

    The issue that has come to be known as the bypass is the latest dispute in the continuing saga of uranium trade between the United States and the Commonwealth of Indepenent States. Bypass deals now being conducted by US utilities have drawn heavy criticism from the US Department of Commerce but are perfectly legal under the terms of the Suspension Agreements. Urged on by the United States Enrichment Corporation, the US DOC intends to stop, or at least severely restrict, bypass deals. Using a recent agreement with Kazakhstan as a case study, this article reviews the implications of such bypass deals

  18. Analysis of promoter regions of co-expressed genes identified by microarray analysis

    Directory of Open Access Journals (Sweden)

    Höglund Mattias

    2006-08-01

    Full Text Available Abstract Background The use of global gene expression profiling to identify sets of genes with similar expression patterns is rapidly becoming a widespread approach for understanding biological processes. A logical and systematic approach to study co-expressed genes is to analyze their promoter sequences to identify transcription factors that may be involved in establishing specific profiles and that may be experimentally investigated. Results We introduce promoter clustering i.e. grouping of promoters with respect to their high scoring motif content, and show that this approach greatly enhances the identification of common and significant transcription factor binding sites (TFBS in co-expressed genes. We apply this method to two different dataset, one consisting of micro array data from 108 leukemias (AMLs and a second from a time series experiment, and show that biologically relevant promoter patterns may be obtained using phylogenetic foot-printing methodology. In addition, we also found that 15% of the analyzed promoter regions contained transcription factors start sites for additional genes transcribed in the opposite direction. Conclusion Promoter clustering based on global promoter features greatly improve the identification of shared TFBS in co-expressed genes. We believe that the outlined approach may be a useful first step to identify transcription factors that contribute to specific features of gene expression profiles.

  19. Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

    Directory of Open Access Journals (Sweden)

    Dalrymple Brian P

    2005-05-01

    Full Text Available Abstract Background The use of small interfering RNA (siRNA molecules in animals to achieve double-stranded RNA-mediated interference (RNAi has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III promoters of either mouse or human origin. Results To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene. Conclusion We have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.

  20. Modulation of gene expression by promoter mutagenesis in Corynebacterium glutamicum

    Czech Academy of Sciences Publication Activity Database

    Holátko, Jiří; Elišáková, Veronika; Prouza, Marek; Phensaijai, M.; Nešvera, Jan; Pátek, Miroslav

    Prague: Institute of Microbiology ASCR, Prague, Czech Republic, 2006, s. 132-132. [International Symposium on the Genetics of Industrial Microorganisms /10./. Praha (CZ), 24.06.2006-28.06.2006] Grant ostatní: EU(XE) ESF project No. CZ.04.3.07/4.2.01.1/0039 Institutional research plan: CEZ:AV0Z50200510 Keywords : c. glutamicum * promoters Subject RIV: EE - Microbiology, Virology

  1. Promoter DNA methylation regulates progranulin expression and is altered in FTLD

    OpenAIRE

    Banzhaf-Strathmann, Julia; Claus, Rainer; Mücke, Oliver; Rentzsch, Kristin; van der Zee, Julie; Engelborghs, Sebastiaan; De Deyn, Peter P.; Cruts, Marc; Van Broeckhoven, Christine; Plass, Christoph; Edbauer, Dieter

    2013-01-01

    Background Frontotemporal lobar degeneration (FTLD) is a heterogeneous group of neurodegenerative diseases associated with personality changes and progressive dementia. Loss-of-function mutations in the growth factor progranulin (GRN) cause autosomal dominant FTLD, but so far the pathomechanism of sporadic FTLD is unclear. Results We analyzed whether DNA methylation in the GRN core promoter restricts GRN expression and, thus, might promote FTLD in the absence of GRN mutations. GRN expression ...

  2. A promoter that drives gene expression preferentially in male transgenic rats

    OpenAIRE

    Li, Qiling; Ma, Yamin; Li, Wenzhi; Xu, Wei; Ma, Li; Fu, Guoxing; Tian, Xiaohua; Wang, Yueling; Li, Xu; Bythwood, Tameka; Richards, Jendai; Song, Qing

    2013-01-01

    Gender-preferential gene expression is a widespread phenomenon in humans. It is important to study how gender differences influence the pathogenesis of various diseases and response to specific drugs. The aim of this study is to determine if the mouse albumin enhancer/promoter may serve as the promoter to introduce gender-preferential gene expression in transgenic animals. We created four independent transgenic rat lines in which the human C-reactive protein (CRP) transgene was under the cont...

  3. Enhanced Expression of Endochitinase in Trichoderma harzianum with the cbh1 Promoter of Trichoderma reesei

    OpenAIRE

    Margolles-Clark, E.; Harman, G. E.; Penttila, M.

    1996-01-01

    Production of extracellular endochitinase could be increased 5-fold in the mycoparasite fungus Trichoderma harzianum by using the cellulase promoter cbh1 of Trichoderma reesei, whereas the total endochitinase activity increased 10-fold. The cbh1 promoter was not expressed on glucose and sucrose in T. harzianum and was induced by sophorose and on cellulase-inducing medium. The endogenous endochitinase gene was expressed at a low basal level on glucose and sucrose. No specific induction by crab...

  4. A regulatory toolbox of MiniPromoters to drive selective expression in the brain.

    Science.gov (United States)

    Portales-Casamar, Elodie; Swanson, Douglas J; Liu, Li; de Leeuw, Charles N; Banks, Kathleen G; Ho Sui, Shannan J; Fulton, Debra L; Ali, Johar; Amirabbasi, Mahsa; Arenillas, David J; Babyak, Nazar; Black, Sonia F; Bonaguro, Russell J; Brauer, Erich; Candido, Tara R; Castellarin, Mauro; Chen, Jing; Chen, Ying; Cheng, Jason C Y; Chopra, Vik; Docking, T Roderick; Dreolini, Lisa; D'Souza, Cletus A; Flynn, Erin K; Glenn, Randy; Hatakka, Kristi; Hearty, Taryn G; Imanian, Behzad; Jiang, Steven; Khorasan-zadeh, Shadi; Komljenovic, Ivana; Laprise, Stéphanie; Liao, Nancy Y; Lim, Jonathan S; Lithwick, Stuart; Liu, Flora; Liu, Jun; Lu, Meifen; McConechy, Melissa; McLeod, Andrea J; Milisavljevic, Marko; Mis, Jacek; O'Connor, Katie; Palma, Betty; Palmquist, Diana L; Schmouth, Jean-François; Swanson, Magdalena I; Tam, Bonny; Ticoll, Amy; Turner, Jenna L; Varhol, Richard; Vermeulen, Jenny; Watkins, Russell F; Wilson, Gary; Wong, Bibiana K Y; Wong, Siaw H; Wong, Tony Y T; Yang, George S; Ypsilanti, Athena R; Jones, Steven J M; Holt, Robert A; Goldowitz, Daniel; Wasserman, Wyeth W; Simpson, Elizabeth M

    2010-09-21

    The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies. PMID:20807748

  5. PPAR-β/δ activation promotes phospholipid transfer protein expression.

    Science.gov (United States)

    Chehaibi, Khouloud; Cedó, Lídia; Metso, Jari; Palomer, Xavier; Santos, David; Quesada, Helena; Naceur Slimane, Mohamed; Wahli, Walter; Julve, Josep; Vázquez-Carrera, Manuel; Jauhiainen, Matti; Blanco-Vaca, Francisco; Escolà-Gil, Joan Carles

    2015-03-15

    The peroxisome proliferator-activated receptor (PPAR)-β/δ has emerged as a promising therapeutic target for treating dyslipidemia, including beneficial effects on HDL cholesterol (HDL-C). In the current study, we determined the effects of the PPAR-β/δ agonist GW0742 on HDL composition and the expression of liver HDL-related genes in mice and cultured human cells. The experiments were carried out in C57BL/6 wild-type, LDL receptor (LDLR)-deficient mice and PPAR-β/δ-deficient mice treated with GW0742 (10mg/kg/day) or a vehicle solution for 14 days. GW0742 upregulated liver phospholipid transfer protein (Pltp) gene expression and increased serum PLTP activity in mice. When given to wild-type mice, GW0742 significantly increased serum HDL-C and HDL phospholipids; GW0742 also raised serum potential to generate preβ-HDL formation. The GW0742-mediated effects on liver Pltp expression and serum enzyme activity were completely abolished in PPAR-β/δ-deficient mice. GW0742 also stimulated PLTP mRNA expression in mouse J774 macrophages, differentiated human THP-1 macrophages and human hepatoma Huh7. Collectively, our findings demonstrate a common transcriptional upregulation by GW0742-activated PPAR-β/δ of Pltp expression in cultured cells and in mouse liver resulting in enhanced serum PLTP activity. Our results also indicate that PPAR-β/δ activation may modulate PLTP-mediated preβ-HDL formation and macrophage cholesterol efflux. PMID:25662586

  6. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    Directory of Open Access Journals (Sweden)

    Miyuki Uno

    2011-01-01

    Full Text Available OBJECTIVES: 1 To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT promoter to its gene and protein expression levels in glioblastoma and 2 to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001. However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297. The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing, and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.

  7. GenoCAD Plant Grammar to Design Plant Expression Vectors for Promoter Analysis.

    Science.gov (United States)

    Coll, Anna; Wilson, Mandy L; Gruden, Kristina; Peccoud, Jean

    2016-01-01

    With the rapid advances in prediction tools for discovery of new promoters and their cis-elements, there is a need to improve plant expression methodologies in order to facilitate a high-throughput functional validation of these promoters in planta. The promoter-reporter analysis is an indispensible approach for characterization of plant promoters. It requires the design of complex plant expression vectors, which can be challenging. Here, we describe the use of a plant grammar implemented in GenoCAD that will allow the users to quickly design constructs for promoter analysis experiments but also for other in planta functional studies. The GenoCAD plant grammar includes a library of plant biological parts organized in structural categories to facilitate their use and management and a set of rules that guides the process of assembling these biological parts into large constructs. PMID:27557770

  8. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

    Science.gov (United States)

    Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y

    2016-05-20

    Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality. PMID:26883397

  9. New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

    Science.gov (United States)

    Kanno, Alex I; Goulart, Cibelly; Rofatto, Henrique K; Oliveira, Sergio C; Leite, Luciana C C; McFadden, Johnjoe

    2016-04-15

    The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response. PMID:26850295

  10. Coordinated transcription factor and promoter engineering to establish strong expression elements in Saccharomyces cerevisiae.

    Science.gov (United States)

    Leavitt, John M; Tong, Alice; Tong, Joyce; Pattie, Jonathan; Alper, Hal S

    2016-07-01

    Gene expression requires the coordination of trans-acting factors and cis-DNA elements to initiate transcription. Here we present a coordinated approach that combines cis-acting element engineering with mutant trans-acting factors to engineer yeast promoters. Specifically, we first construct a hybrid promoter based on the ARO9 upstream region that exhibits high constitutive and inducible expression with respect to exogenous tryptophan. Next, we perform protein engineering to identify a mutant Aro80p that affords both high constitutive expression while retaining inducible traits. We then use this mutant trans-acting factor to drive expression and generate ultra-strong promoters with transcriptional output roughly 2 fold higher than TDH3 (GPD), one of the strongest promoters to-date. Finally, we used this element to construct a modular expression system capable of staged outputs resulting in a system with nearly 6-fold, 12-fold and 15-fold expression relative to the off-state. This work further highlights the potential of using endogenous transcription factors (including mutant factors) along with hybrid promoters to expand the yeast synthetic biology toolbox. PMID:27152757

  11. Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris

    OpenAIRE

    Prielhofer, Roland; Maurer, Michael; Klein, Joachim; Wenger, Jana; Kiziak, Christoph; Gasser, Brigitte; Mattanovich, Diethard

    2013-01-01

    Background Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. g...

  12. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs.

    Science.gov (United States)

    Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P; Komori, Hideyuki; Ohtani, Kiyoshi

    2014-07-18

    In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV

  13. Regional differences in gene expression and promoter usage in aged human brains

    KAUST Repository

    Pardo, Luba M.

    2013-02-19

    To characterize the promoterome of caudate and putamen regions (striatum), frontal and temporal cortices, and hippocampi from aged human brains, we used high-throughput cap analysis of gene expression to profile the transcription start sites and to quantify the differences in gene expression across the 5 brain regions. We also analyzed the extent to which methylation influenced the observed expression profiles. We sequenced more than 71 million cap analysis of gene expression tags corresponding to 70,202 promoter regions and 16,888 genes. More than 7000 transcripts were differentially expressed, mainly because of differential alternative promoter usage. Unexpectedly, 7% of differentially expressed genes were neurodevelopmental transcription factors. Functional pathway analysis on the differentially expressed genes revealed an overrepresentation of several signaling pathways (e.g., fibroblast growth factor and wnt signaling) in hippocampus and striatum. We also found that although 73% of methylation signals mapped within genes, the influence of methylation on the expression profile was small. Our study underscores alternative promoter usage as an important mechanism for determining the regional differences in gene expression at old age.

  14. Bypassing damaged nervous tissue

    CERN Document Server

    Shneider, M N

    2016-01-01

    We show the principal ability of bypassing damaged demyelinated portions of nervous tissue, thereby restoring its normal function for the passage of action potentials. We carry out a theoretical analysis on the basis of the synchronization mechanism of action potential propagation along a bundle of neurons, proposed recently in [1]. And we discuss the feasibility of implement a bypass to restore damaged nervous tissue and creating an artificial neuron network.

  15. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

    International Nuclear Information System (INIS)

    Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue

  16. Isolation and characterization of "GmScream" promoters that regulate highly expressing soybean (Glycine max Merr.) genes.

    Science.gov (United States)

    Zhang, Ning; McHale, Leah K; Finer, John J

    2015-12-01

    To increase our understanding of the regulatory components that control gene expression, it is important to identify, isolate and characterize new promoters. In this study, a group of highly expressed soybean (Glycine max Merr.) genes, which we have named "GmScream", were first identified from RNA-Seq data. The promoter regions were then identified, cloned and fused with the coding region of the green fluorescent protein (gfp) gene, for introduction and analysis in different tissues using 3 tools for validation. Approximately half of the GmScream promoters identified showed levels of GFP expression comparable to or higher than the Cauliflower Mosaic Virus 35S (35S) promoter. Using transient expression in lima bean cotyledonary tissues, the strongest GmScream promoters gave over 6-fold higher expression than the 35S promoter while several other GmScream promoters showed 2- to 3-fold higher expression. The two highest expressing promoters, GmScreamM4 and GmScreamM8, regulated two different elongation factor 1A genes in soybean. In stably transformed soybean tissues, GFP driven by the GmScreamM4 or GmScreamM8 promoter exhibited constitutive high expression in most tissues with preferentially higher expression in proliferative embryogenic tissues, procambium, vascular tissues, root tips and young embryos. Using deletion analysis of the promoter, two proximal regions of the GmScreamM8 promoter were identified as contributing significantly to high levels of gene expression. PMID:26706070

  17. Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver

    International Nuclear Information System (INIS)

    Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), α-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken β actin (ACT), nuclear factor κ B (NFκB), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promoter exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within First 5-7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein-Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy

  18. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

    DEFF Research Database (Denmark)

    Zhang, Qian; Wang, HongYi; Kantekure, Kanchan; Paterson, Jennifer C.; Liu, Xiaobin; Schaffer, Andreas; Paulos, Chrystal; Milone, Michael C.; Ødum, Niels; Turner, Suzanne; Marafioti, Teresa; Wasik, Mariusz A.

    2011-01-01

    protein. Stimulation of the ICOS receptor with anti-ICOS antibody or ICOS ligand-expressing B cells markedly enhanced proliferation of the ALK(+) TCL cells. These results demonstrate that NPM-ALK, acting through STAT3 as the gene transcriptional activator, induces the expression of ICOS, a cell growth......Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression via...... STAT3, which triggers the transcriptional activity of the ICOS gene promoter. In addition, STAT3 suppresses the expression of miR-219 that, in turn, selectively inhibits ICOS expression. ALK(+) TCL cell lines display extensive DNA methylation of the CpG island located within intron 1, the putative...

  19. Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters

    Directory of Open Access Journals (Sweden)

    Li Junxin

    2012-06-01

    Full Text Available Abstract Backgrounds The fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II. Results The transcriptional profiles of 13 key genes that participate in glucose metabolism in T. reesei were analyzed by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR. The results indicated that the mRNA levels of pdc (encoding pyruvate decarboxylase and eno (encoding enolase genes were much higher than other genes under high glucose conditions. Recombinant T. reesei strains that homologously expressed xylanase II were constructed by using the promoters of the pdc and eno genes, and they respectively produced 9266 IU/ml and 8866 IU/ml of xylanase activities in the cultivation supernatant in a medium with high glucose concentration. The productivities of xylanase II were 1.61 g/L (with the pdc promoter and 1.52 g/L (with the eno promoter, approximately accounted for 83% and 82% of the total protein secreted by T. reesei, respectively. Conclusions This work demonstrates the screening of constitutive promoters by using RT-qPCR in T. reesei, and has obtained the highest expression of recombinant xylanase II to date by using these promoters.

  20. Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

    Directory of Open Access Journals (Sweden)

    Barbara Arbeithuber

    2015-01-01

    Full Text Available Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5, a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type.

  1. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    International Nuclear Information System (INIS)

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  2. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    Energy Technology Data Exchange (ETDEWEB)

    Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: btm88939@kwansei.ac.jp [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

    2014-07-18

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  3. Adenovirus-mediated expression of an elastase-specific inhibitor (elafin): a comparison of different promoters.

    Science.gov (United States)

    Sallenave, J M; Xing, Z; Simpson, A J; Graham, F L; Gauldie, J

    1998-03-01

    This report describes the design and construction of three recombinant adenoviruses of serotype 5 (Ad5) expressing elafin (EL), also called elastase-specific inhibitor. Three promoters were chosen to drive the synthesis of elafin: the small (380 bp) human cytomegalovirus promoter (HCMV), the Ad2 major late promoter (MLP) and the mouse cytomegalovirus (MCMV) promoter. Human alveolar epithelial cells (A549), as well as rat and human primary pulmonary fibroblasts were infected with Ad5-HCMV-EL, Ad5-MLP-EL, Ad5-MCMV-EL and with the control Ad5-dl70/3. The MCMV promoter was the most efficient promoter in all cells studied. MLP was the least efficient promoter Intermediate between MCMV and MLP was HCMV which was able to induce significant amounts of elafin, particularly in human A549 cells. When compared in vivo in rat lungs, results were similar; MCMV was the only promoter which induced significant amounts of elafin as assessed by Northern blot analysis and ELISA, even with a low dose of virus (3 x 10(8) p.f.u.). Our data indicate that the MCMV promoter is the promoter of choice for the strong induction of adenovirus-mediated transgenes in the lung and suggest its suitability both in rodent experimental models and in humans for investigative and therapeutic purposes. PMID:9614555

  4. An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex

    Directory of Open Access Journals (Sweden)

    Bernd eKuhn

    2012-07-01

    Full Text Available Recording of identified neuronal network activity using genetically encoded calcium indicators (GECIs requires labeling that is cell type-specific and bright enough for the detection of functional signals. However, specificity and strong expression are often not achievable using the same promoter. Here we present a combinatorial approach for targeted expression and single-cell-level quantification in which a weak promoter is used to drive trans-amplification under a strong general promoter. We demonstrated this approach using recombinant adeno-associated viruses (rAAVs to deliver the sequence of the GECI D3cpv in the mouse cerebellar cortex. Direct expression under the human synapsin promoter (hSYN led to high levels of expression (50-100 µM in five interneuron types of the cerebellar cortex but not in Purkinje cells (PCs (≤10 μM, yielding sufficient contrast to allow functional signals to be recorded from somata and processes in awake animals using two-photon microscopy. When the hSYN promoter was used to drive expression of the tetracycline transactivator (tTA, a second rAAV containing the bidirectional TET promoter (Ptetbi could drive strong D3cpv expression in PCs (10-300 µM, enough to allow reliable complex spike detection in the dendritic arbor. An amplified approach should be of use in monitoring neural processing in selected cell types and boosting expression of optogenetic probes. Additionally, we overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by combining the virus injection with systemic pre-injection of hyperosmotic D-mannitol, and by this double the time window for functional imaging.

  5. High level transgenic expression of soybean (Glycine max GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline

    Directory of Open Access Journals (Sweden)

    Jones Michelle L

    2010-11-01

    Full Text Available Abstract Background Although numerous factors can influence gene expression, promoters are perhaps the most important component of the regulatory control process. Promoter regions are often defined as a region upstream of the transcriptional start. They contain regulatory elements that interact with regulatory proteins to modulate gene expression. Most genes possess their own unique promoter and large numbers of promoters are therefore available for study. Unfortunately, relatively few promoters have been isolated and characterized; particularly from soybean (Glycine max. Results In this research, a bioinformatics approach was first performed to identify members of the Gmubi (G.max ubiquitin and the GmERF (G. max Ethylene Response Factor gene families of soybean. Ten Gmubi and ten GmERF promoters from selected genes were cloned upstream of the gfp gene and successfully characterized using rapid validation tools developed for both transient and stable expression. Quantification of promoter strength using transient expression in lima bean (Phaseolus lunatus cotyledonary tissue and stable expression in soybean hairy roots showed that the intensity of gfp gene expression was mostly conserved across the two expression systems. Seven of the ten Gmubi promoters yielded from 2- to 7-fold higher expression than a standard CaMV35S promoter while four of the ten GmERF promoters showed from 1.5- to 2.2-times higher GFP levels compared to the CaMV35S promoter. Quantification of GFP expression in stably-transformed hairy roots of soybean was variable among roots derived from different transformation events but consistent among secondary roots, derived from the same primary transformation events. Molecular analysis of hairy root events revealed a direct relationship between copy number and expression intensity; higher copy number events displayed higher GFP expression. Conclusion In this study, we present expression intensity data on 20 novel soybean promoters

  6. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    OpenAIRE

    Miyuki Uno; Sueli Mieko Oba-Shinjo; Anamaria Aranha Camargo; Ricardo Pereira Moura; Paulo Henrique Aguiar; Hector Navarro Cabrera; Marcos Begnami; Sérgio Rosemberg; Manoel Jacobsen Teixeira; Suely Kazue Nagahashi Marie

    2011-01-01

    OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty...

  7. Promoter methylation of IGFBP-3 and p53 expression in ovarian endometrioid carcinoma

    OpenAIRE

    Huang Su-Cheng; Yang Hui-Wen; Chan Michael WY; Lin Ching-Wei; Torng Pao-Ling; Lin Chin-Tarng

    2009-01-01

    Abstract Background Insulin-like growth factor binding protein (IGFBP-3) is an antiproliferative, pro-apoptotic and invasion suppressor protein which is transcriptionally regulated by p53. Promoter methylation has been linked to gene silencing and cancer progression. We studied the correlation between IGFBP-3 and p53 expression as well as IGFBP-3 promoter methylation in ovarian endometrioid carcinoma (OEC) by immunohistochemical staining and quantitative methylation-specific PCR (qMSP). Addit...

  8. Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells

    International Nuclear Information System (INIS)

    Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription. The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter. We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals. BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC

  9. Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.

    OpenAIRE

    Varley, A.W.; Coulthard, M G; Meidell, R S; Gerard, R D; Munford, R S

    1995-01-01

    We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal lu...

  10. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    Directory of Open Access Journals (Sweden)

    V Shilpa

    2014-01-01

    Full Text Available Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6 -methyguanine-DNA methyltransferase (MGMT is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC tissue samples, 14 low malignant potential (LMP tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

  11. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    Science.gov (United States)

    Shilpa, V.; Bhagat, Rahul; Premalata, C.S.; Pallavi, V.R.; Ramesh, G.; Krishnamoorthy, Lakshmi

    2014-01-01

    Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O6-methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O6-position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC) tissue samples, 14 low malignant potential (LMP) tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP) after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression. PMID:25579142

  12. c-Myc activates BRCA1 gene expression through distal promoter elements in breast cancer cells

    International Nuclear Information System (INIS)

    The BRCA1 gene plays an important role in the maintenance of genomic stability. BRCA1 inactivation contributes to breast cancer tumorigenesis. An increasing number of transcription factors have been shown to regulate BRCA1 expression. c-Myc can act as a transcriptional activator, regulating up to 15% of all genes in the human genome and results from a high throughput screen suggest that BRCA1 is one of its targets. In this report, we used cultured breast cancer cells to examine the mechanisms of transcriptional activation of BRCA1 by c-Myc. c-Myc was depleted using c-Myc-specific siRNAs in cultured breast cancer cells. BRCA1 mRNA expression and BRCA1 protein expression were determined by quantitative RT-PCR and western blot, respectively and BRCA1 promoter activities were examined under these conditions. DNA sequence analysis was conducted to search for high similarity to E boxes in the BRCA1 promoter region. The association of c-Myc with the BRCA1 promoter in vivo was tested by a chromatin immunoprecipitation assay. We investigated the function of the c-Myc binding site in the BRCA1 promoter region by a promoter assay with nucleotide substitutions in the putative E boxes. BRCA1-dependent DNA repair activities were measured by a GFP-reporter assay. Depletion of c-Myc was found to be correlated with reduced expression levels of BRCA1 mRNA and BRCA1 protein. Depletion of c-Myc decreased BRCA1 promoter activity, while ectopically expressed c-Myc increased BRCA1 promoter activity. In the distal BRCA1 promoter, DNA sequence analysis revealed two tandem clusters with high similarity, and each cluster contained a possible c-Myc binding site. c-Myc bound to these regions in vivo. Nucleotide substitutions in the c-Myc binding sites in these regions abrogated c-Myc-dependent promoter activation. Furthermore, breast cancer cells with reduced BRCA1 expression due to depletion of c-Myc exhibited impaired DNA repair activity. The distal BRCA1 promoter region is associated with c

  13. Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter.

    Science.gov (United States)

    Zhang, Ai-Lian; Luo, Jin-Xian; Zhang, Tian-Yuan; Chen, Shou-Cai; Guan, Wen-Jun

    2004-06-01

    The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter (P(AOX1)) on pPIC9K resulting in plasmid pGAP9K. The recombinant expression vector pGAP9K-AS was constructed by inserting the angiostatin gene(AS) into pGAP9K. pGAP9K-AS was then transformed into P. pastoris GS115. The multi-copy integration transformant P. pastoris GS115 (pGAP9K-AS) was used to investigate the constitutive expression of angiostatin in P. pastoris. The expression of angiostatin reached its peak after 4 d of culture in P. pastoris GS115 (pGAP9K-AS) while the angiostatin expressed in P. pastoris GS115 (pPIC9K-AS) after 4 d of induction or 5 d of culture is only 70% of that expressed by P. pastoris GS115 (pGAP9K-AS). The AS expression in inducible system reached the peak after 6 d of induction but the expressed AS was only 86% of that from constitutive system. The results of anti-angiogenic and antitumor activity assay showed that AS expressed from both constitutive and inducible system inhibited the CAM angiogenesis and suppressed B16 melanoma in C57BL/6J mouse and that the tumor inhibition rates reached 90.63% and 90.54%, respectively. The above data indicates that the constitutive promoter P(GAP) can served as an effective alternative to the inductive promoter P(AOX1) to express AS and other proteins in P. pastoris. PMID:15490871

  14. Promoter-Specific Expression and Imprint Status of Marsupial IGF2

    OpenAIRE

    Stringer, Jessica M.; Suzuki, Shunsuke; Pask, Andrew J; Shaw, Geoff; Renfree, Marilyn B

    2012-01-01

    In mice and humans, IGF2 has multiple promoters to maintain its complex tissue- and developmental stage-specific imprinting and expression. IGF2 is also imprinted in marsupials, but little is known about its promoter region. In this study, three IGF2 transcripts were isolated from placental and liver samples of the tammar wallaby, Macropus eugenii. Each transcript contained a unique 5' untranslated region, orthologous to the non-coding exons derived from promoters P1–P3 in the human and mouse...

  15. c-Myc inhibits TP53INP1 expression via promoter methylation in esophageal carcinoma

    International Nuclear Information System (INIS)

    Research highlights: → TP53INP1 expression is down-regulated in esophageal carcinoma and is associated with CGI-131 methylation. → Inhibition of CGI-131 methylation upregulates TP53INP1 expression in ESCC cell lines. → Ectopic expression of TP53INP1 inhibits growth of ESCC cells by inducing apoptosis and inhibiting cell cycle progression. → c-Myc binds to the promoter of TP53INP1 in vivo and vitro and recruits DNMT3A to TP53INP1 promoter for CGI-131 methylation. -- Abstract: Tumor protein p53-induced nuclear protein 1 (TP53INP1) is a well known stress-induced protein that plays a role in both cell cycle arrest and p53-mediated apoptosis. Loss of TP53INP1 expression has been reported in human melanoma, breast carcinoma, and gastric cancer. However, TP53INP1 expression and its regulatory mechanism in esophageal squamous cell carcinoma (ESCC) remain unclear. Our findings are in agreement with previous reports in that the expression of TP53INP1 was downregulated in 28% (10/36 cases) of ESCC lesions, and this was accompanied by significant promoter methylation. Overexpression of TP53INP1 induced G1 cell cycle arrest and increased apoptosis in ESCC cell lines (EC-1, EC-109, EC-9706). Furthermore, our study showed that the oncoprotein c-Myc bound to the core promoter of TP53INP1 and recruited DNA methyltransferase 3A to methylate the local promoter region, leading to the inhibition of TP53INP1 expression. Our findings revealed that TP53INP1 is a tumor suppressor in ESCC and that c-Myc-mediated DNA methylation-associated silencing of TP53INP1 contributed to the pathogenesis of human ESCC.

  16. E4ORF3 Requirement for Achieving Long-Term Transgene Expression from the Cytomegalovirus Promoter in Adenovirus Vectors

    OpenAIRE

    Armentano, Donna; Smith, Michael P; Sookdeo, Cathleen C.; Zabner, Joseph; Perricone, Michael A; St. George, Judith A.; Wadsworth, Samuel C.; Gregory, Richard J.

    1999-01-01

    Analysis of transgene expression under the control of the cytomegalovirus (CMV) promoter from adenovirus vectors in which the E4 region was modified indicated that E4ORF3 is required for long-term expression in the murine lung. CMV promoter truncation led to the persistence of expression in the absence of E4, thus eliminating the ORF3 requirement.

  17. Efficient LEC2 activation of OLEOSIN expression requires two neighboring RY elements on its promoter

    Institute of Scientific and Technical Information of China (English)

    CHE NanYing; YANG Yang; LI YanDong; WANG LiLi; HUANG Ping; GAO Yin; An ChengCai

    2009-01-01

    As the main structural protein of oil body, OLEOSIN is highly expressed only during seed development. OLEOSIN promoter is a very useful tool for seed-specific gene engineering and seed bioreactor designing. The B3 domain transcription factor leafy cotyledon2 (LEC2) plays an important role in regulating seed development and seed-specific gene expression. Here, we first report how seed-specific B3 domain transcription factor leafy cotyledon2 (LEC2) efficiently activates OLEOSIN expression. The central promoter region of OLEOSIN, responsible for seed specificity and LEC2 activation, was determined by 5'-deletion analysis. Binding experiments in yeast cells and electrophoretic mobility shift assays showed that LEC2 specifically bound to two conserved RY elements in this region, in transient expression assays, mutation in either RY element dramatically reduced LEC2 activation of OLEOSIN promoter activity, while double mutation abolished it. Analysis of the distribution of RY elements in seed-specific genes activated by LEC2 also supported the idea that genes containing neighboring RY elements responded strongly to LEC2 activation. Therefore, we conclude that two neighboring RY elements are essential for efficient LEC2 activation of OLEOSIN expression. These findings will help us better utilize seed-specific promoter activity.

  18. Adenovirus E4-34kDa requires active proteasomes to promote late gene expression

    International Nuclear Information System (INIS)

    A complex of the Adenovirus (Ad) early region 1b 55-kDa protein (E1b-55kDa) and the early region 4 ORF6 34-kDa protein (E4-34kDa) promotes viral late RNA accumulation in the cytoplasm while inhibiting the transport of most newly synthesized cellular mRNA. The E4 ORF3 11-kDa protein (E4-11kDa) functionally compensates for at least some of the activities of this complex. We find that the same large central region of E4-34kDa that is required for proteasome-mediated degradation of p53 (J. Virol. 75, (2001) 699-709) is also required to promote viral late gene expression in a complementation assay. E4-34kDa does not promote late gene expression in complementation assays performed in the presence of proteasome inhibitors. A proteasome inhibitor also dramatically reduced late gene expression by a virus that lacks the E4-11kDa gene and therefore relies on E4-34kDa for late gene expression. Our results suggest that E4-34kDa activity in promoting late gene expression depends on the proteasome

  19. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

    OpenAIRE

    Zomerdijk, J C; Ouellette, M; ten Asbroek, A L; Kieft, R.; Bommer, A M; Clayton, C E; Borst, P

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region m...

  20. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish

    OpenAIRE

    Mosimann, Christian; Kaufman, Charles K.; Li, Pulin; Pugach, Emily K.; Tamplin, Owen J; Zon, Leonard I.

    2011-01-01

    Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopo...

  1. Motif discovery in promoters of genes co-localized and co-expressed during myeloid cells differentiation

    Science.gov (United States)

    Coppe, Alessandro; Ferrari, Francesco; Bisognin, Andrea; Danieli, Gian Antonio; Ferrari, Sergio; Bicciato, Silvio; Bortoluzzi, Stefania

    2009-01-01

    Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method for discovering putative regulatory elements, i.e. over-represented motifs, in a selected set of promoters, as compared with a background model. The combination of transcriptional, structural and functional data allowed the identification of sets of promoters pertaining to groups of genes co-expressed and co-localized in regions of the human genome. The application of motif discovery to 26 groups of genes co-expressed in myeloid cells differentiation and co-localized in the genome showed that there are more over-represented motifs in promoters of co-expressed and co-localized genes than in promoters of simply co-expressed genes (CEG). Motifs, which are similar to the binding sequences of known transcription factors, non-uniformly distributed along promoter sequences and/or occurring in highly co-expressed subset of genes were identified. Co-expressed and co-localized gene sets were grouped in two co-expressed genomic meta-regions, putatively representing functional domains of a high-level expression regulation. PMID:19059999

  2. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Science.gov (United States)

    Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

    2008-08-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

  3. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. Reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. Reinhardtii.

  4. Tissue specific promoters improve the localization of radiation-inducible gene expression

    International Nuclear Information System (INIS)

    Purpose: Site-specific activation of gene expression can be achieved by the use of a promoter that is induced by physical agents such as x-rays. The purpose of the present study was to determine whether site-specific activation of gene therapy can also be achieved within the vascular endothelium by use of radiation-inducible promoters. We studied induction of promoter-reporter gene constructs using previously identified radiation-promoters from c-jun, c-fos, Egr-1, ICAM-1, ELAM-1 after transfection into in the vascular endothelium. Methods: The following radiation-inducible genetic constructs were created: The ELAM-1 promoter fragment was cloned into pOGH to obtain the pE-sel(-587 +35)GH reporter construct. The ICAM-1 promoter fragment (-1162/+1) was cloned upstream of the CAT coding region of the pCAT-plasmid (Promega) after removal of the SV40 promoter by Bgl2/Stu1 digestion to create the pBS-CAT plasmid. The 132 to +170 bp segment of the 5' untranslated region of the c-jun promoter was cloned to the CAT reporter gene to create the -132/+170 cjun-CAT. The Egr-1 promoter fragment (-425/+75) was cloned upstream of the CAT coding region to create the pE425-CAT plasmid. Tandem repeats of the AP-1 binding site were cloned upstream of the CAT coding region (3 xTRE-CAT). Tandem repeats of the Egr binding site (EBS) were cloned upstream of the CAT coding region (EBS-CAT). Human vascular endothelial cells from both large vessel and small vessel origin (HUVEC and HMEC), as well as human tumor cell lines were transfected with plasmids -132/+170 cjun-CAT, pE425-CAT, 3 xTRE-CAT, EBS-CAT, pE-sel-GH and pBS-CAT by use of liposomes. Humor tumor cell lines included SQ20B (squamous), RIT3 (sarcoma), and HL525 (leukemia). Each plasmid was cotransfected with a plasmid containing a CMV promoter linked to the LacZ gene (1 μg). Transfected cells were treated with mock irradiation or x-rays. Cell extracts were assayed for reporter gene expression. Results: Radiation-induced gene

  5. Motif discovery in promoters of genes co-localized and co-expressed during myeloid cells differentiation

    OpenAIRE

    Coppe, Alessandro; Ferrari, Francesco; Bisognin, Andrea; Danieli, Gian Antonio; Ferrari, Sergio; Bicciato, Silvio; Bortoluzzi, Stefania

    2008-01-01

    Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method...

  6. GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line

    Institute of Scientific and Technical Information of China (English)

    Gunter Maubach; Michelle Chin Chia Lim; Chun-Yan Zhang; Lang Zhuo

    2006-01-01

    AIM: The GFAP was traditionally considered to be a biomarker for neural glia (mainly astrocytes and nonmyelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells (HSC). In this project,possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated.METHODS: The GFAP-lacZ transgene was transfected into various cell lines (HSC, hepatocyte, and other nonHSC cell types). The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression.RESULTS: The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a dose- and time-dependent manner, similar to the endogenous GFAP.CONCLUSION: In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

  7. Bypass Flow Study

    International Nuclear Information System (INIS)

    The purpose of the fluid dynamics experiments in the MIR (Matched Index of-Refraction) flow system at Idaho National Laboratory (INL) is to develop benchmark databases for the assessment of Computational Fluid Dynamics (CFD) solutions of the momentum equations, scalar mixing, and turbulence models for the flow ratios between coolant channels and bypass gaps in the interstitial regions of typical prismatic standard fuel element (SFE) or upper reflector block geometries of typical Modular High-temperature Gas-cooled Reactors (MHTGR) in the limiting case of negligible buoyancy and constant fluid properties. The experiments use Particle Image Velocimetry (PIV) to measure the velocity fields that will populate the bypass flow study database.

  8. Bypass Flow Study

    Energy Technology Data Exchange (ETDEWEB)

    Richard Schultz

    2011-09-01

    The purpose of the fluid dynamics experiments in the MIR (Matched Index of-Refraction) flow system at Idaho National Laboratory (INL) is to develop benchmark databases for the assessment of Computational Fluid Dynamics (CFD) solutions of the momentum equations, scalar mixing, and turbulence models for the flow ratios between coolant channels and bypass gaps in the interstitial regions of typical prismatic standard fuel element (SFE) or upper reflector block geometries of typical Modular High-temperature Gas-cooled Reactors (MHTGR) in the limiting case of negligible buoyancy and constant fluid properties. The experiments use Particle Image Velocimetry (PIV) to measure the velocity fields that will populate the bypass flow study database.

  9. Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering

    Directory of Open Access Journals (Sweden)

    Zou Gen

    2012-02-01

    Full Text Available Abstract Background Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes. In this study, the CREI binding sites within the cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid α-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus. Results The modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover

  10. Promotion

    OpenAIRE

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed.

  11. Aberrant promoter methylation and expression of UTF1 during cervical carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Samuel Guenin

    Full Text Available Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1 promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2'-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression.

  12. ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells.

    Science.gov (United States)

    Komaragiri, Shravan Kumar; Bostanthirige, Dhanushka H; Morton, Derrick J; Patel, Divya; Joshi, Jugal; Upadhyay, Sunil; Chaudhary, Jaideep

    2016-09-01

    Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function. PMID:27462022

  13. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters.

    Science.gov (United States)

    Song, Tianqiao; Ma, Zhenchuan; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

    2015-12-01

    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters. PMID:26714171

  14. Seladin-1 expression is regulated by promoter methylation in adrenal cancer

    International Nuclear Information System (INIS)

    Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC. A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands. The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 ± 686) than in normal adrenal glands (362 ± 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 ± 196) was significantly lower than in normal adrenal glands (3614 ± 949; p = 0.01). On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC

  15. The Best Bypass Surgery Trial

    DEFF Research Database (Denmark)

    Møller, Christian H; Jensen, Birte Østergaard; Gluud, Christian; Perko, Mario J; Lund, Jens T; Andersen, Lars Willy; Madsen, Jan Kyst; Hughes, Pia; Steinbrüchel, Daniel A

    2007-01-01

    Recent trials suggest that off-pump coronary artery bypass grafting (OPCAB) reduces the risk of mortality and morbidity compared with conventional coronary artery bypass grafting (CCAB) using cardiopulmonary bypass. Patients with a moderate- to high-risk of complications after CCAB may have addit...

  16. Electrical failure during cardiopulmonary bypass: a critical moment.

    Science.gov (United States)

    Durukan, Ahmet Baris; Gurbuz, Hasan Alper; Ozcelik, Gokhan; Yorgancioglu, Cem

    2016-06-01

    Electrical failure during cardiopulmonary bypass is a crisis situation for the cardiac surgical team. Fortunately, it has a low incidence with low morbidity and mortality rates. Notwithstanding, institutional preventative and management measures should be taken. Here, we report a case of electrical failure during cardiopulmonary bypass, which was successfully managed during the surgery, allowing the patient to recover uneventfully. These unwanted complications can only be managed by promoting awareness and putting in place strategies against them. PMID:27516788

  17. Cardiopulmonary bypass in pregnancy.

    Science.gov (United States)

    Pomini, F; Mercogliano, D; Cavalletti, C; Caruso, A; Pomini, P

    1996-01-01

    The cardiopathic patient can sustain acute heart failure during pregnancy. In such cases, if open heart operation is necessary to save the patient's life, the fetus could be seriously compromised after exposure to cardiopulmonary bypass. From 1958 to 1992, 69 reports of cardiac operations during pregnancy with the aid of cardiopulmonary bypass have been published. Maternal mortality was 2.9%. Embryofetal mortality was 20.2%. Examining only the last 40 patients, maternal and embryofetal mortality were 0.0% and 12.5%, respectively. Embryofetal mortality was 24.0% when hypothermia was used, compared with 0.0% while operating in normothermia. Maternal mortality did not change. The use of hypothermia during cardiopulmonary bypass provoked uterine contractions in several patients. Hypothermia decreases O2 exchange through the placenta. Pump flow and mean arterial pressure during cardiopulmonary bypass seem to be the most important parameters that influence fetal oxygenation. We speculate that cardiac operation is not a contraindication to pregnancy prolongation. PMID:8561577

  18. Aortic valve bypass

    DEFF Research Database (Denmark)

    Lund, Jens T; Jensen, Maiken Brit; Arendrup, Henrik; Ihlemann, Nikolaj

    2013-01-01

    In aortic valve bypass (AVB) a valve-containing conduit is connecting the apex of the left ventricle to the descending aorta. Candidates are patients with symptomatic aortic valve stenosis rejected for conventional aortic valve replacement (AVR) or transcatheter aortic valve implantation (TAVI...

  19. Bypassing AMPK Phosphorylation

    OpenAIRE

    Viollet, Benoit; Foretz, Marc; Schlattner, Uwe

    2014-01-01

    AMP-activated protein kinase (AMPK) functions as a signaling hub to balance energy supply with demand. Phosphorylation of activation loop Thr172 has been considered as an essential step in AMPK activation. In this issue of Chemistry & Biology, Scott and colleagues show that the small molecule direct AMPK activator, A-769662, bypasses this phosphorylation event, and acts synergistically with AMP on naive AMPK.

  20. Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes.

    Science.gov (United States)

    von Jonquieres, Georg; Fröhlich, Dominik; Klugmann, Claudia B; Wen, Xin; Harasta, Anne E; Ramkumar, Roshini; Spencer, Ziggy H T; Housley, Gary D; Klugmann, Matthias

    2016-01-01

    Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene

  1. Genetic variation in the proximal promoter of ABC and SLC superfamilies: liver and kidney specific expression and promoter activity predict variation.

    Directory of Open Access Journals (Sweden)

    Stephanie E Hesselson

    Full Text Available Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC and Solute Carrier (SLC superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

  2. Upregulation of HYAL1 expression in breast cancer promoted tumor cell proliferation, migration, invasion and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Jin-Xiang Tan

    Full Text Available Hyaluronic acid (HA is a component of the Extra-cellular matrix (ECM, it is closely correlated with tumor cell growth, proliferation, metastasis and angiogenesis, etc. Hyaluronidase (HAase is a HA-degrading endoglycosidase, levels of HAase are elevated in many cancers. Hyaluronidase-1 (HYAL1 is the major tumor-derived HAase. We previously demonstrated that HYAL1 were overexpression in human breast cancer. Breast cancer cells with higher HAase expression, exhibited significantly higher invasion ability through matrigel than those cells with lower HAase expression, and knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis. Here, to further elucidate the function of HYAL1 in breast cancer, we investigated the consequences of forcing HYAL1 expression in breast cancer cells by transfection of expression plasmid. Compared with control, HYAL1 up-regulated cells showed increased the HAase activity, and reduced the expression of HA in vitro. Meantime, upregulation of HYAL1 promoted the cell growth, migration, invasion and angiogenesis in vitro. Moreover, in nude mice model, forcing HYAL1 expression induced breast cancer cell xenograft tumor growth and angiogenesis. Interestingly, the HA expression was upregulated by forcing HYAL1 expression in vivo. These findings suggested that HYAL1-HA system is correlated with the malignant behavior of breast cancer.

  3. Induction of apolipoprotein E expression by TR4 orphan nuclear receptor via 5' proximal promoter region

    International Nuclear Information System (INIS)

    While other plasma lipoproteins are exclusively expressed in liver and intestine, apoliprotein E (apoE) is ubiquitously synthesized in many tissues. To understand the molecular mechanism of non-tissue-specific apoE expression, we tested the testicular orphan receptor 4 (TR4) effect on apoE expression in different cell lines, such as HepG2, COS-1, and H1299 cells. Gel shift assay and 5' promoter activity analyses identified one distinct hormone response element (TR4RE-DR0-apoE at -303 to -292 bp) that binds to TR4 and results in full induction of apoE gene transcription. TR4 also forms a complex with Sp1 to synergistically induce apoE expression via a region containing the TR4RE-DR0-apoE and the Sp1 binding site (-169 to -140 bp). Induction of apoE expression by TR4 was also confirmed at the mRNA and protein levels in H1299 cells. Together, our data demonstrate that TR4 can enhance apoE gene expression via binding to TR4RE-DR0 in apoE 5' promoter and this TR4 binding is essential for synergistic interaction with another transcription factor, Sp1

  4. Genome-wide patterns of promoter sharing and co-expression in bovine skeletal muscle

    Directory of Open Access Journals (Sweden)

    Dalrymple Brian P

    2011-01-01

    Full Text Available Abstract Background Gene regulation by transcription factors (TF is species, tissue and time specific. To better understand how the genetic code controls gene expression in bovine muscle we associated gene expression data from developing Longissimus thoracis et lumborum skeletal muscle with bovine promoter sequence information. Results We created a highly conserved genome-wide promoter landscape comprising 87,408 interactions relating 333 TFs with their 9,242 predicted target genes (TGs. We discovered that the complete set of predicted TGs share an average of 2.75 predicted TF binding sites (TFBSs and that the average co-expression between a TF and its predicted TGs is higher than the average co-expression between the same TF and all genes. Conversely, pairs of TFs sharing predicted TGs showed a co-expression correlation higher that pairs of TFs not sharing TGs. Finally, we exploited the co-occurrence of predicted TFBS in the context of muscle-derived functionally-coherent modules including cell cycle, mitochondria, immune system, fat metabolism, muscle/glycolysis, and ribosome. Our findings enabled us to reverse engineer a regulatory network of core processes, and correctly identified the involvement of E2F1, GATA2 and NFKB1 in the regulation of cell cycle, fat, and muscle/glycolysis, respectively. Conclusion The pivotal implication of our research is two-fold: (1 there exists a robust genome-wide expression signal between TFs and their predicted TGs in cattle muscle consistent with the extent of promoter sharing; and (2 this signal can be exploited to recover the cellular mechanisms underpinning transcription regulation of muscle structure and development in bovine. Our study represents the first genome-wide report linking tissue specific co-expression to co-regulation in a non-model vertebrate.

  5. Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

    Institute of Scientific and Technical Information of China (English)

    CHEN XiaoGuang; ZHANG YaJing; ZHENG XueLi; WANG ChunMei

    2007-01-01

    The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles stephensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I digestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively conserved in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.

  6. The E2 transactivator of bovine papillomavirus type 1 is expressed from multiple promoters.

    OpenAIRE

    Vaillancourt, P; Nottoli, T; Choe, J; Botchan, M R

    1990-01-01

    The E2 proteins of bovine papillomavirus type 1 (BPV-1) are a family of site-specific DNA-binding proteins which regulate viral transcription by repression and activation. Repressors E2-TR and E8/E2 are expressed from promoters P5 (P3080) and P3 (P890), respectively. Previous reports have provided evidence that the transcript for the 48-kilodalton transactivator is initiated from a promoter proximal to the open reading frame encoding this protein (P2440 or P4). Our studies extend these findin...

  7. Nuclear trafficking of EGFR by Vps34 represses Arf expression to promote lung tumor cell survival.

    Science.gov (United States)

    Dayde, D; Guerard, M; Perron, P; Hatat, A-S; Barrial, C; Eymin, B; Gazzeri, S

    2016-07-28

    Epidermal growth factor receptor (EGFR) is a cell surface receptor that has an essential role in cell proliferation and survival, and overexpression of EGFR is a common feature of human cancers. In Non-small-cell lung cancer (NSCLC), activating mutations of EGFR have also been described. We recently showed that mutant EGFR-L858R inhibits the expression of the p14ARF tumor-suppressor protein to promote cell survival. In this study, we defined the molecular bases by which EGFR controls Arf expression. Using various lung tumor models, we showed that EGF stimulation inhibits Arf transcription by a mechanism involving the nuclear transport and recruitment of EGFR to the Arf promoter. We unraveled the vesicular trafficking protein Vps34 as a mediator of EGFR nuclear trafficking and showed that its neutralization prevents the accumulation of EGFR to the Arf promoter in response to ligand activation. Finally, in lung tumor cells that carry mutant EGFR-L858R, we demonstrated that inhibition of Vps34 using small interfering RNA restrains nuclear EGFR location and restores Arf expression leading to apoptosis. These findings identify the Arf tumor suppressor as a new transcriptional target of nuclear EGFR and highlight Vps34 as an important regulator of the nuclear EGFR/Arf survival pathway. As a whole, they provide a mechanistic explanation to the inverse correlation between nuclear expression of EGFR and overall survival in NSCLC patients. PMID:26686095

  8. A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants.

    Science.gov (United States)

    Schenk, P M; Sagi, L; Remans, T; Dietzgen, R G; Bernard, M J; Graham, M W; Manners, J M

    1999-04-01

    A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the beta-glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter. PMID:10380808

  9. Novel strong tissue specific promoter for gene expression in human germ cells

    Directory of Open Access Journals (Sweden)

    Kuzmin Denis

    2010-08-01

    Full Text Available Abstract Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102, where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter. To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1, whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293. In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X. The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12, and an important role - in the rest two cell lines.

  10. Maximal Expression of the Evolutionarily Conserved Slit2 Gene Promoter Requires Sp1.

    Science.gov (United States)

    Saunders, Jacquelyn; Wisidagama, D Roonalika; Morford, Travis; Malone, Cindy S

    2016-08-01

    Slit2 is a neural axon guidance and chemorepellent protein that stimulates motility in a variety of cell types. The role of Slit2 in neural development and neoplastic growth and migration has been well established, while the genetic mechanisms underlying regulation of the Slit2 gene have not. We identified the core and proximal promoter of Slit2 by mapping multiple transcriptional start sites, analyzing transcriptional activity, and confirming sequence homology for the Slit2 proximal promoter among a number of species. Deletion series and transient transfection identified the Slit2 proximal promoter as within 399 base pairs upstream of the start of transcription. A crucial region for full expression of the Slit2 proximal promoter lies between 399 base pairs and 296 base pairs upstream of the start of transcription. Computer modeling identified three transcription factor-binding consensus sites within this region, of which only site-directed mutagenesis of one of the two identified Sp1 consensus sites inhibited transcriptional activity of the Slit2 proximal promoter (-399 to +253). Bioinformatics analysis of the Slit2 proximal promoter -399 base pair to -296 base pair region shows high sequence conservation over twenty-two species, and that this region follows an expected pattern of sequence divergence through evolution. PMID:26456684

  11. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter.

    Science.gov (United States)

    Vassileva, A; Chugh, D A; Swaminathan, S; Khanna, N

    2001-06-01

    High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in

  12. Cloning of the chrysanthemum UEP1 promoter and comparative expression in florets and leaves of Dendranthema grandiflora.

    Science.gov (United States)

    Annadana, S; Beekwilder, M J; Kuipers, G; Visser, P B; Outchkourov, N; Pereira, A; Udayakumar, M; De Jong, J; Jongsma, M A

    2002-08-01

    To attain high transgene expression in petal tissue of ray florets of chrysanthemum an endogenous ubiquitin extension protein (UEP1) promoter was cloned and tested with the beta-glucuronidase (GUS) reporter gene. Expression levels were compared with four heterologous promoters: chalcone synthase (chs-A) and zinc finger transcription factor (EPF2-5) from petunia, eceriferum (CER6) from Arabidopsis and multicystatin (PMC) from potato. The comparison of the expression levels of the different constructs in ray florets, disc florets, and leaves is presented. The highest mean expression in petal tissue of ray and disc florets was conferred by the UEP1 promoter, followed by CER6 and EPF2-5. The UEP1 promoter in ray florets confers over 50-fold enhancement in expression as compared to CaMV 35S-based promoters. PMID:12212845

  13. Characterization of constitutive promoters for piggyBac transposon-mediated stable transgene expression in mesenchymal stem cells (MSCs.

    Directory of Open Access Journals (Sweden)

    Sheng Wen

    Full Text Available Multipotent mesenchymal stem cells (MSCs can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40, one housekeeping gene promoter (UbC, and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH. CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.

  14. MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms

    Directory of Open Access Journals (Sweden)

    Print Cristin G

    2011-01-01

    Full Text Available Abstract Background Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs in the human genome is a difficult challenge. Therefore, a reverse strategy, which identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the SNP-related mRNAs will be useful. Those mRNA transcripts that are expressed at two distinct abundances in proportion to SNP allele frequency may warrant further study. Matrix metalloproteinase 1 (MMP1 is important in both normal development and in numerous pathologies. Although much research has been conducted to investigate the expression of MMP1 in many different cell types and conditions, the regulation of its expression is still not fully understood. Results In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC microarray data from 15 individuals. We found that MMP1 mRNA abundance was bimodally distributed in un-treated HUVECs and showed a bimodal response to inflammatory mediator treatment. RT-PCR and MMP1 activity assays confirmed the bimodal regulation and DNA sequencing of 69 individuals identified an MMP1 gene promoter polymorphism that segregated precisely with the MMP1 bimodal expression. Chromatin immunoprecipation (ChIP experiments indicated that the transcription factors (TFs ETS1, ETS2 and GATA3, bind to the MMP1 promoter in the region of this polymorphism and may contribute to the bimodal expression. Conclusions We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticans to understand and use. This method identified bimodal endothelial cell expression of MMP1, which appears to be biologically significant with implications for inflammatory disease. (271 Words

  15. MYC Expression Promotes the Proliferation of Neural Progenitor Cells in Culture and In Vivo

    Directory of Open Access Journals (Sweden)

    Dan Fults

    2002-01-01

    Full Text Available Primitive neuroectodermal tumors. (20PNETs are pediatric brain tumors that result from defects in signaling molecules governing the growth and differentiation of neural progenitor cells. We used the RCAS-TVA system to study the growth effects of three genetic alterations implicated in human PNETs on a subset of neural progenitor cells that express the intermediate filament protein, nestin. The genetic alterations tested were: 1 overexpression of the cellular oncoprotein, MYC; 2 activation of transcription factor, β-catenin; and 3 haploinsufficiency of Ptc, the hedgehog receptor gene. The RCAS-TVA system uses an avian retroviral vector, RCAS, to target gene expression to specific cell types in transgenic mice. To express exogenous genes in neural progenitor cells, we used Ntv-a mice. In these mice, the Nestin gene promoter drives expression of TVA, the cell surface receptor for the virus. Ectopic expression of MYC, but not activated β-catenin, promoted the proliferation of neural progenitor cells in culture and in the cerebral leptomeninges in vivo. These effects were equally penetrant in mice with Ptc+/− and Ptc+/+ genetic backgrounds. Although overexpression of MYC is not sufficient to cause intraparenchymal tumors, it may facilitate PNET formation by sustaining the growth of undifferentiated progenitor cells.

  16. Reduced CTGF expression promotes cell growth, migration, and invasion in nasopharyngeal carcinoma.

    Directory of Open Access Journals (Sweden)

    Yan Zhen

    Full Text Available BACKGROUND: The role of CTGF varies in different types of cancer. The purpose of this study is to investigate the involvement of CTGF in tumor progression and prognosis of human nasopharyngeal carcinoma (NPC. EXPERIMENTAL DESIGN: CTGF expression levels were examined in NPC tissues and cells, nasopharynx (NP tissues, and NP69 cells. The effects and molecular mechanisms of CTGF expression on cell proliferation, migration, invasion, and cell cycle were also explored. RESULTS: NPC cells exhibited decreased mRNA expression of CTGF compared to immortalized human nasopharyngeal epithelial cell line NP69. Similarly, CTGF was observed to be downregulated in NPC compared to normal tissues at mRNA and protein levels. Furthermore, reduced CTGF was negatively associated with the progression of NPC. Knocking down CTGF expression enhanced the colony formation, cell migration, invasion, and G1/S cell cycle transition. Mechanistic analysis revealed that CTGF suppression activated FAK/PI3K/AKT and its downstream signals regulating the cell cycle, epithelial-mesenchymal transition (EMT and MMPs. Finally, DNA methylation microarray revealed a lack of hypermethylation at the CTGF promoter, suggesting other mechanisms are associated with suppression of CTGF in NPC. CONCLUSION: Our study demonstrates that reduced expression of CTGF promoted cell proliferation, migration, invasion and cell cycle progression through FAK/PI3K/AKT, EMT and MMP pathways in NPC.

  17. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    International Nuclear Information System (INIS)

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have

  18. Characterization of Promoter Elements Regulating the Expression of the Human Neurotensin/Neuromedin N Gene*

    OpenAIRE

    Wang, Xiaofu; Gulhati, Pat; Li, Jing; Dobner, Paul R.; Weiss, Heidi; Townsend, Courtney M.; Evers, B. Mark

    2010-01-01

    Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine N cells in the distal small intestine. We have identified key regulatory elements in the promoter region that are involved in human NT/N (hNT/N) gene expression in the novel human endocrine cell line, BON, which resembles intestinal N cells in several important aspects including NT/N precursor protein processing, ratios of different NT/N mRNA isoforms, and high levels...

  19. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Wei [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yang, Guifang [Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Cai, Xiaojun [Department of Ophthalmology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Falck, John R. [Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390 (United States); Yang, Jing, E-mail: yangjingliu@yahoo.com.cn [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2012-10-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have

  20. Construction of an effective protein expression system using the tpl promoter in Escherichia coli.

    Science.gov (United States)

    Koyanagi, Takashi; Katayama, Takane; Hirao, Ai; Suzuki, Hideyuki; Kumagai, Hidehiko

    2005-09-01

    An effective protein expression system was constructed in Escherichia coli using the promoter of the tyrosine phenol-lyase (tpl) gene of Erwinia herbicola. This system involves a mutant form of the TyrR protein with an enhanced ability to activate tpl and the TutB protein with an ability to transport L-tyrosine (an inducer of Tpl). The highest expression level obtained for this system was more than twice that obtained for the tac system, although it was lower than the level obtained for the T7 system, as revealed with the lac-reporter assay and SDS-polyacrylamide gel electrophoresis. PMID:16215823

  1. Expression characteristics of GFP driven by NAC1 promoter and its responses to auxin and gibberellin

    Institute of Scientific and Technical Information of China (English)

    WANG Youhua; DUAN Liusheng; LU Mengzhu; LI Zhaohu; WANG Minjie; ZHAI Zhixi

    2006-01-01

    A 1050 bp fragment upstream transcription start site of a transcription factor gene NAC1 in Arabidopsis thaliana was amplified and cloned into plasmid pRD420 to construct a green fluorescent protein(GFP) fusion system under the control of NAC1 promoter. Plasmids were introduced into tobacco by Agrobacterium mediated method to regenerate plants with NAC1-GFP gene, and expression pattern of NAC1-GFP and its responses to auxin and gibberellin (GA) were observed. GFP was found to accumulate specifically in root, and was detected after treatment of auxin, N-1-Naphthylphthalamic acid (NPA, an auxin antagonist) or GA3. It was indicated that the expression of GFP driven by NAC1 promoter was induced not only by auxin but also by GAs, suggesting that NAC1 mediated both the auxin signaling and the GAs signaling involved in lateral roots development.

  2. Mutation of the TERT promoter, switch to active chromatin, and monoallelic TERT expression in multiple cancers.

    Science.gov (United States)

    Stern, Josh Lewis; Theodorescu, Dan; Vogelstein, Bert; Papadopoulos, Nickolas; Cech, Thomas R

    2015-11-01

    Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression. PMID:26515115

  3. The pea END1 promoter drives anther-specific gene expression in different plant species.

    Science.gov (United States)

    Gómez, María D; Beltrán, José-Pío; Cañas, Luis A

    2004-10-01

    END1 was isolated by an immunosubtractive approach intended to identify specific proteins present in the different pea (Pisum sativum L.) floral organs and the genes encoding them. Following this strategy we obtained a monoclonal antibody (mAbA1) that specifically recognized a 26-kDa protein (END1) only detected in anther tissues. Northern blot assays showed that END1 is expressed specifically in the anther. In situ hybridization and immunolocalization assays corroborated the specific expression of END1 in the epidermis, connective, endothecium and middle layer cells during the different stages of anther development. END1 is the first anther-specific gene isolated from pea. The absence of a practicable pea transformation method together with the fact that no END1 homologue gene exists in Arabidopsis prevented us from carrying out END1 functional studies. However, we designed functional studies with the END1 promoter in different dicot species, as the specific spatial and temporal expression pattern of END1 suggested, among other things, the possibility of using its promoter region for biotechnological applications. Using different constructs to drive the uidA (beta-glucuronidase) gene controlled by the 2.7-kb isolated promoter sequence we have proven that the END1 promoter is fully functional in the anthers of transgenic Arabidopsis thaliana (L.) Heynh., Nicotiana tabacum L. (tobacco) and Lycopersicon esculentum Mill. (tomato) plants. The presence in the -330-bp region of the promoter sequence of three putative CArG boxes also suggests that END1 could be a target gene of MADS-box proteins and that, subsequently, it would be activated by genes controlling floral organ identity. PMID:15221384

  4. Sex differences in estrogen receptor promoter expression in the area postrema

    Institute of Scientific and Technical Information of China (English)

    Chunxiao Zhang; Tomohiro Hamada

    2013-01-01

    Estrogen receptor α is widely distributed in the rat brain, but the tissue- or target-specificity of the estrogen receptor α gene promoters remains unknown. In the present study, we used transgenic rats expressing enhanced green fluorescent protein under the control of the estrogen receptor α 0/B promoter to examine expression driven by this promoter in two significant nuclei that regulate cardiovascular activity, the area postrema and the nucleus tractus solitarius. Immunohistochemistry showed that enhanced green fluorescent protein-labeled cells were distributed in the area postrema and the nucleus tractus solitarius of both female and male transgenic rats, and a neural network of enhanced green fluorescent protein-positive fibers was seen between the area postrema and the nucleus tractus solitarius. The number of enhanced green fluorescent protein-labeled cells in the area postrema of female rats was significantly higher than in the males, but no significant difference was found in the number of enhanced green fluorescent protein-labeled cells in the nucleus tractus solitarius. The sex differences in the number of enhanced green fluorescent protein-labeled cells in the area postrema was not affected after ovariectomy or 17β-estradiol benzoate treatment in adult rats. Our results suggest that the effects of estrogen in the area postrema are related to the expression of estrogen receptor α under the control of the 0/B promoter, and changes in the sex hormone environment in the adult period do not affect estrogen receptor α expression in the area postrema or the nucleus tractus solitarius.

  5. Loss of expression and promoter methylation of SLIT2 are associated with sessile serrated adenoma formation.

    Directory of Open Access Journals (Sweden)

    Andrew D Beggs

    2013-05-01

    Full Text Available Serrated adenomas form a distinct subtype of colorectal pre-malignant lesions that may progress to malignancy along a different molecular pathway than the conventional adenoma-carcinoma pathway. Previous studies have hypothesised that BRAF mutation and promoter hypermethylation plays a role, but the evidence for this is not robust. We aimed to carry out a whole-genome loss of heterozygosity analysis, followed by targeted promoter methylation and expression analysis to identify potential pathways in serrated adenomas. An initial panel of 9 sessile serrated adenomas (SSA and one TSA were analysed using Illumina Goldengate HumanLinkage panel arrays to ascertain regions of loss of heterozygosity. This was verified via molecular inversion probe analysis and microsatellite analysis of a further 32 samples. Methylation analysis of genes of interest was carried out using methylation specific PCR (verified by pyrosequencing and immunohistochemistry used to correlate loss of expression of genes of interest. All experiments used adenoma samples and normal tissue samples as control. SSA samples were found on whole-genome analysis to have consistent loss of heterozygosity at 4p15.1-4p15.31, which was not found in the sole TSA, adenomas, or normal tissues. Genes of interest in this region were PDCH7 and SLIT2, and combined MSP/IHC analysis of these genes revealed significant loss of SLIT2 expression associated with promoter methylation of SLIT2. Loss of expression of SLIT2 by promoter hypermethylation and loss of heterozygosity events is significantly associated with serrated adenoma development, and SLIT2 may represent a epimutated tumour suppressor gene according to the Knudson "two hit" hypothesis.

  6. The expression of foreign gene under the control of cauliflower mosaic virus 35s RNA promoter

    Institute of Scientific and Technical Information of China (English)

    WangHao; BaiYongyan

    1990-01-01

    The promoter region of cauliflower mosaic virus (CaMV) 35s RNA was employed to construct an intermediate expression vector which can be used in Ti plasmid system of Agrobacterium iumefaciens.The original plasmid,which contains a polylinker between CaMV 35s RNA and its 3' termination signal in pUC18 was modified to have another antibiotic resistance marker (kanamycin resistance gene Kmr) to facilitate the selection of recombinant with Ti plasmid.Octopine synthase (ocs) structural gene was inserted into this vector downstream of CaMV 35s RNA promoter.This chimaeric gene was introduced into integrative Ti plasmid vector pGV 3850,and then transformed into Nicotiana tobaccum the chimaeric gene into tobacco cells.In both cases,the expression of ocs gene was demonstrated.The amount of octopine was much more than the nopaline synthesized by nopaline synthase (nos) gene transferred at the same time with Ti plasmid vector.This demonstrated that CaMV 35s RNA promoter is stronger in transcriptional function than the promoter of nos in tobacco cells.

  7. Analysis of the sericin1 promoter and assisted detection of exogenous gene expression efficiency in the silkworm Bombyx mori L.

    OpenAIRE

    Ye, Lupeng; Qian, Qiujie; Zhang, Yuyu; You, Zhengying; Che, Jiaqian; Song, Jia; Zhong, Boxiong

    2015-01-01

    In genetics, the promoter is one of the most important regulatory elements controlling the spatiotemporal expression of a target gene. However, most studies have focused on core or proximal promoter regions, and information on regions that are more distant from the 5′-flanking region of the proximal promoter is often lacking. Here, approximately 4-kb of the sericin1 (Ser1) promoter was predicted to contain many potential transcriptional factor binding sites (TFBSs). Transgenic experiments hav...

  8. Axillobifemoral bypass grafting

    Directory of Open Access Journals (Sweden)

    Davidović Lazar B.

    2004-01-01

    Full Text Available INTRODUCTION Axillo-femoral bypass (AxF means connecting the axillar and femoral artery with the graft that is placed subcutaneously [1]. Usually, this graft is connected with contralateral femoral artery via one accessory subcutaneous graft, and this connection is known as axillobifemoral bypass (AxFF. This extra-anatomic procedure is an alternative method to the standard reconstruction of aortoiliac region when there are contraindications for general or local reasons. OBJECTIVE The objective of this paper is to show early and late results of AxFF bypass grafting as well as to show the indications for AxFF bypass. METHODS The sample consisted of 37 patients. The procedure was performed in 28 patients who suffered from aortoiliac occlusive disease and who were at high risk due to the comorbidity- in one patient with the rupture of juxtarenal aneurysm of abdominal aorta; in five patients with aortoenteric fistula, in two patients with iatrogenic lesion of abdominal aorta and in one female patient with anus preternaturalis definitivus who was treated for rectovaginal fistula. Donor's right axillary artery was used in 26 cases (70.3%, and donor's left axillary artery was used in 9 cases (29.7%. Dacron graft was used in 34 patients and Polytetrafluo-roethlylene graft was used in three patients. Simultaneously, profundo-plastic was done in four patients and femoro-popliteal bypass was performed in three patients. In five patients who suffered from aortoenteric fistula, simultaneous intervention of gastrointerstinal system has been done, x2 test was used for statistical evaluation and life table method was used for verification of late graft patency. RESULTS The rate of early postoperative mortality was 13.5%. The causes of death were: sepsis -1, MOFS - 3, and infarct myocardium -1. The mean follow up period was 40.1 months, ranging from six months to 17 years. During the follow up period, an early graft thrombosis was identified in two and late graft

  9. Expression in Arabidopsis of a nucellus-specific promoter from watermelon (Citrullus lanatus).

    Science.gov (United States)

    Dwivedi, Krishna K; Roche, Dominique; Carman, John G

    2010-11-01

    Though many tissue-specific promoters have been identified, few have been associated specifically with the angiospermous megasporangium (nucellus). In the present study the 2000-bp regulatory region upstream to the watermelon, Citrullus lanatus (Thunb.) Matsum & Nakai, gene WM403 (GenBank accession no. AF008925), which shows nucellus-specific expression, was cloned from watermelon gDNA and fused to the β-glucuronidase reporter gene (GUS). The resulting plasmid, WM403 Prom::GUS(+), which also contained NPTII, was transformed into Arabidopsis thaliana ecotype Co1-0. Seedlings were selected on kanamycin-containing medium, and transformants were confirmed by PCR. GUS assays of T(3) transformants revealed weak promoter activation in epidermal layers of the placenta and locule septum during premeiotic ovule development but strong activation in the nucellus, embryo sac and early embryo, from early embryo sac formation to early globular embryo formation. Expression in seeds was absent thereafter. These results indicate that the WM403 promoter may be useful in driving nucellus-specific gene expression in plants including candidate genes for important nucellus-specific traits such as apospory or adventitious embryony. PMID:21802614

  10. Cloning and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene and the use of its promoter for expression in Myrothecium gramineum, a novel expression host.

    Science.gov (United States)

    De Maeseneire, Sofie L; Dauvrin, Thierry; Jonniaux, Jean-Luc; Van Bogaert, Inge N A; Soetaert, Wim K; Vandamme, Erick J M C

    2008-04-01

    At our laboratory, research has focused on the development of Myrothecium gramineum as a novel expression host. The glyceraldehyde-3-phosphate dehydrogenase (gpd)-promoter of M. gramineum was isolated and characterized (Genbank accession number EF486690). In order to prove its functionality and to explore the potential of M. gramineum as a novel fungal expression host, use of this gpd-promoter for the expression of a fungal alpha-amylase was investigated. Myrothecium gramineum was transformed with pGPDlpAmyAO, containing the gpd-promoter followed by the amy3 encoding sequence of Aspergillus oryzae. Study of the amylase production indicated that the promoter can be successfully used for the expression of heterologous proteins in M. gramineum. To the best of our knowledge, this is the first time a homologous expression system has been described for M. gramineum. PMID:18294194

  11. Derepression of hTERT gene expression promotes escape from oncogene-induced cellular senescence.

    Science.gov (United States)

    Patel, Priyanka L; Suram, Anitha; Mirani, Neena; Bischof, Oliver; Herbig, Utz

    2016-08-23

    Oncogene-induced senescence (OIS) is a critical tumor-suppressing mechanism that restrains cancer progression at premalignant stages, in part by causing telomere dysfunction. Currently it is unknown whether this proliferative arrest presents a stable and therefore irreversible barrier to cancer progression. Here we demonstrate that cells frequently escape OIS induced by oncogenic H-Ras and B-Raf, after a prolonged period in the senescence arrested state. Cells that had escaped senescence displayed high oncogene expression levels, retained functional DNA damage responses, and acquired chromatin changes that promoted c-Myc-dependent expression of the human telomerase reverse transcriptase gene (hTERT). Telomerase was able to resolve existing telomeric DNA damage response foci and suppressed formation of new ones that were generated as a consequence of DNA replication stress and oncogenic signals. Inhibition of MAP kinase signaling, suppressing c-Myc expression, or inhibiting telomerase activity, caused telomere dysfunction and proliferative defects in cells that had escaped senescence, whereas ectopic expression of hTERT facilitated OIS escape. In human early neoplastic skin and breast tissue, hTERT expression was detected in cells that displayed features of senescence, suggesting that reactivation of telomerase expression in senescent cells is an early event during cancer progression in humans. Together, our data demonstrate that cells arrested in OIS retain the potential to escape senescence by mechanisms that involve derepression of hTERT expression. PMID:27503890

  12. Transgenic mice expressing yellow fluorescent protein under control of the human tyrosine hydroxylase promoter.

    Science.gov (United States)

    Choi, Eun Yang; Yang, Jae Won; Park, Myung Sun; Sun, Woong; Kim, Hyun; Kim, Seung U; Lee, Myung Ae

    2012-10-01

    Pathogenesis of Parkinson's disease and related catecholaminergic neurological disorders is closely associated with changes in the levels of tyrosine hydroxylase (TH). Therefore, investigation of the regulation of the TH gene system should assist in understanding the pathomechanisms involved in these neurological disorders. To identify regulatory domains that direct human TH expression in the central nervous system (CNS), we generated two transgenic mouse lines in which enhanced yellow fluorescent protein (EYFP) is expressed under the control of either 3.2-kb (hTHP-EYFP construct) human TH promoter or 3.2-kb promoter with 2-kb 3'-flanking regions (hTHP-ex3-EYFP construct) of the TH gene. In the adult transgenic mouse brain, the hTHP-EYFP construct directs neuron-specific EYFP expression in various CNS areas, such as olfactory bulb, striatum, interpeduncular nucleus, cerebral cortex, hippocampus, and particularly dentate gyrus. Although these EYFP-positive cells were identified as mature neurons, few EYFP-positive cells were TH-positive neurons. On the other hand, we could detect the EYFP mRNA expression in a subset of neurons in the olfactory bulb, midbrain, and cerebellum, in which expression of endogenous TH is enriched, with hTHP-ex3-EYFP transgenic mice. These results indicate that the 3.2-kb sequence upstream of the TH gene is not sufficient for proper expression and that the 2-kb sequence from the translation start site to exon 3 is necessary for expression of EYFP in a subset of catecholaminergic neurons. PMID:22714400

  13. Aryl hydrocarbon receptor downregulates MYCN expression and promotes cell differentiation of neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Pei-Yi Wu

    Full Text Available Neuroblastoma (NB is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR, a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, AHR expression in NB tumors was found to correlate highly with histological grade of differentiation. In vitro studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulating MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation.

  14. Stepping back to move forward: Expressive writing promotes self-distancing.

    Science.gov (United States)

    Park, Jiyoung; Ayduk, Özlem; Kross, Ethan

    2016-04-01

    Prior research indicates that expressive writing enhances well-being by leading people to construct meaningful narratives that explain distressing life experiences. But how does expressive writing facilitate meaning-making? We addressed this issue in 2 longitudinal studies by examining whether and how expressive writing promotes self-distancing, a process that facilitates meaning-making. At baseline in both studies, participants reflected on a distressing life experience. In Study 1 participants were then randomly assigned to write about their distressing experience or a non-emotional topic for 15 min on 3 consecutive days; in Study 2 participants were randomly assigned to write or think about their distressing experience or write about a non-emotional topic for the same amount of time. One day following the intervention, expressive writing participants in both studies self-distanced more when they reflected over their distressing experience compared with participants in the other conditions, which in turn led them to experience less emotional reactivity 1 month (Studies 1 and 2) and 6 months (Study 2) after the intervention. Analyses using data from both studies indicated that expressive writing reduced physical symptoms indirectly through its effects on self-distancing and emotional reactivity [that is, expressive writing group (vs. comparison groups) → greater self-distancing → less emotional reactivity → fewer physical symptoms]. Finally, linguistic analyses using essays from both studies indicated that increased use of causation words and decreased use of negative emotion words and first-person singular pronouns predicted increases in self-distancing over time. These findings demonstrate that expressive writing promotes self-distancing and illustrate how it does so. PMID:26461252

  15. Targeting gene expression to endothelium in transgenic animals: a comparison of the human ICAM-2, PECAM-1 and endoglin promoters.

    Science.gov (United States)

    Cowan, Peter J; Shinkel, Trixie A; Fisicaro, Nella; Godwin, James W; Bernabéu, Carmelo; Almendro, Nuria; Rius, Carlos; Lonie, Andrew J; Nottle, Mark B; Wigley, Peter L; Paizis, Kathy; Pearse, Martin J; d'Apice, Anthony J F

    2003-05-01

    It is highly likely that successful pig-to-human xenotransplantation of vascularized organs will require genetic modification of the donor pig, and in particular of donor vascular endothelium. Promoters are generally tested in transgenic mice before generating transgenic pigs. Several promoters have been used to drive endothelial cell-specific expression in mice but none have yet been tested in pigs. We compared the promoters of three human genes that are predominantly expressed in vascular endothelium: intercellular adhesion molecule 2 (ICAM-2), platelet endothelial cell adhesion molecule 1 (PECAM-1) and endoglin. Expression of human complement regulatory proteins (hCRPs), directed by each of the promoters in mice, was largely restricted to vascular endothelium and leukocyte subpopulations. However, expression from the PECAM-1 promoter was weak in liver and non-uniform in the small vessels of heart, kidney, and lung. Conversely, expression from the endoglin promoter was consistently strong in the small vessels of these organs but was absent in larger vessels. The ICAM-2 promoter, which produced strong and uniform endothelial expression in all organs examined, was therefore used to generate hCRP transgenic pigs. Leukocytes from 57 pigs containing at least one intact transgene were tested for transgene expression by flow cytometry. Forty-seven of these transgenic pigs were further analyzed by immunohistochemical staining of liver biopsies, and 18 by staining of heart and kidney sections. Only two of the pigs showed expression, which appeared to be restricted to vascular endothelium in heart and kidney but was markedly weaker than in transgenic mice produced with the same batch of DNA. Thus, in this case, promoter performance in mice and pigs was not equivalent. The weak expression driven by the human ICAM-2 promoter in pigs relative to mice suggests the need for additional regulatory elements to achieve species-specific gene expression in pigs. PMID:12694542

  16. Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM.

    Directory of Open Access Journals (Sweden)

    Nameeta Shah

    Full Text Available O⁶-methylguanine DNA-methyltransferase (MGMT promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089-13.097], p<0.0001. To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301-7.27], p = 0.007. We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical

  17. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    International Nuclear Information System (INIS)

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study

  18. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  19. Prognostic impact of MGMT promoter methylation and MGMT and CD133 expression in colorectal adenocarcinoma

    Science.gov (United States)

    2014-01-01

    Background New biomarkers are needed for the prognosis of advanced colorectal cancer, which remains incurable by conventional treatments. O6-methylguanine DNA methyltransferase (MGMT) methylation and protein expression have been related to colorectal cancer treatment failure and tumor progression. Moreover, the presence in these tumors of cancer stem cells, which are characterized by CD133 expression, has been associated with chemoresistance, radioresistance, metastasis, and local recurrence. The objective of this study was to determine the prognostic value of CD133 and MGMT and their possible interaction in colorectal cancer patients. Methods MGMT and CD133 expression was analyzed by immunohistochemistry in 123 paraffin-embedded colorectal adenocarcinoma samples, obtaining the percentage staining and intensity. MGMT promoter methylation status was obtained by using bisulfite modification and methylation-specific PCR (MSP). These values were correlated with clinical data, including overall survival (OS), disease-free survival (DFS), tumor stage, and differentiation grade. Results Low MGMT expression intensity was significantly correlated with shorter OS and was a prognostic factor independently of treatment and histopathological variables. High percentage of CD133 expression was significantly correlated with shorter DFS but was not an independent factor. Patients with low-intensity MGMT expression and ≥50% CD133 expression had the poorest DFS and OS outcomes. Conclusions Our results support the hypothesis that MGMT expression may be an OS biomarker as useful as tumor stage or differentiation grade and that CD133 expression may be a predictive biomarker of DFS. Thus, MGMT and CD133 may both be useful for determining the prognosis of colorectal cancer patients and to identify those requiring more aggressive adjuvant therapies. Future studies will be necessary to determine its clinical utility. PMID:25015560

  20. NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

    International Nuclear Information System (INIS)

    NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2

  1. An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila

    Science.gov (United States)

    Larsen, Camilla; Franch-Marro, Xavier; Hartenstein, Volker; Alexandre, Cyrille; Vincent, Jean-Paul

    2006-01-01

    Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic males. Many animals expressing GFP in distinct patterns can be recovered with relatively little effort. As expected, many insertions cause loss of function. After insertion at a genomic location, specific parts of the transposon can be excised by FLP recombinase, thus allowing it to induce conditional misexpression of the tagged gene. Therefore, both gain- and loss-of-function studies can be carried out with a single insertion in a gene identified by virtue of its expression pattern. Using this promoter trap approach, we have identified a group of cells that innervate the calyx of the mushroom body and could thus define a previously unrecognized memory circuit. PMID:17093046

  2. Importance of the bovine papillomavirus P2443 promoter in the regulation of E2 and E5 expression.

    OpenAIRE

    Spalholz, B A

    1993-01-01

    The full-length bovine papillomavirus E2 gene product (E2TA), which has a direct role in DNA replication and functions as a transcriptional activator, can be expressed from an unspliced mRNA transcribed from the P2443 promoter or from spliced mRNAs transcribed from other upstream promoters. The regulation of E2 expression from these promoters is still in question. In the background of wild-type protein coding sequences, this study identified the P2443 promoter as the major source of E2TA as w...

  3. Kalrn promoter usage and isoform expression respond to chronic cocaine exposure

    Directory of Open Access Journals (Sweden)

    Ma Xin-Ming

    2011-02-01

    Full Text Available Abstract Background The long-term effects of cocaine on behavior are accompanied by structural changes in excitatory glutamatergic synapses onto the medium spiny neurons of the striatum. The Kalrn gene encodes several functionally distinct isoforms; these multidomain guanine nucleotide exchange factors (GEFs contain additional domains known to interact with phosphatidylinositides as well as with a number of different proteins. Through their activation of Rho proteins and their interactions with other proteins, the different Kalirin isoforms affect cytoskeletal organization. Chronic exposure of adult male rodents to cocaine increases levels of Kalirin 7 in the striatum. When exposed chronically to cocaine, mice lacking Kalirin 7, the major adult isoform, fail to show an increase in dendritic spine density in the nucleus accumbens, show diminished place preference for cocaine, and exhibit increased locomotor activity in response to cocaine. Results The use of alternate promoters and 3'-terminal exons of the mouse Kalrn gene were investigated using real-time quantitative polymerase chain reaction. While the two most distal full-length Kalrn promoters are used equally in the prefrontal cortex, the more proximal of these promoters accounts for most of the transcripts expressed in the nucleus accumbens. The 3'-terminal exon unique to the Kalirin 7 isoform accounts for a greater percentage of the Kalrn transcripts in prefrontal cortex than in nucleus accumbens. Western blot analyses confirmed these differences. Chronic cocaine treatment increases usage of the promoter encoding the Δ-Kalirin isoforms but does not alter full-length Kalirin promoter usage. Usage of the 3'-terminal exon unique to Kalirin 7 increases following chronic cocaine exposure. Conclusions Kalrn promoter and 3'-terminal exon utilization are region-specific. In the nucleus accumbens, cocaine-mediated alterations in promoter usage and 3'-terminal exon usage favor expression of

  4. Tumour necrosis factor alpha downregulates human hemojuvelin expression via a novel response element within its promoter

    Directory of Open Access Journals (Sweden)

    Salama Mohamed

    2012-09-01

    Full Text Available Abstract Background Iron homeostasis is chiefly regulated by hepcidin whose expression is tightly controlled by inflammation, iron stores, and hypoxia. Hemojuvelin (HJV is a bone morphogenetic protein co-receptor that has been identified as a main upstream regulator of hepcidin expression; HJV mutations are associated with a severe form of iron overload (Juvenile haemochromatosis. Currently however, there is no information on how HJV is regulated by inflammation. Methods To study the regulation of Hjv expression by inflammation and whether Hfe has a role in that regulation, control and LPS-injected wild type and Hfe KO mice were used. Moreover, human hepatoma cells (HuH7 were used to study the effect of IL-6 and TNF-α on HJV mRNA expression. Results Here we show that LPS repressed hepatic Hjv and BMPs, while it induced hepcidin 1 expression in wild-type and Hfe KO mice with no effect on hepatic pSMAD 1, 5, 8 protein levels. In addition, exogenous TNF-α (20 ng/mL decreased HJV mRNA and protein expression to 40% of control with no effect on hepcidin mRNA expression in 24 hours. On the other hand, IL-6 induced hepcidin mRNA and protein expression with no effect on HJV mRNA expression levels. Moreover, using the HJV promoter-luciferase reporter fusion construct (HJVP1.2-luc, we showed that the basal luciferase activity of HJVP1.2-luc was inhibited by 33% following TNF-α treatment of HuH7 transfected cells suggesting that the TNF-α down-regulation is exerted at the transcriptional level. Additionally, mutation of a canonical TNF- alpha responsive element (TNFRE within HJVP1.2-luc abolished TNF-α response suggesting that this TNFRE is functional. Conclusions From these results, we conclude that TNF-α suppresses HJV transcription possibly via a novel TNFRE within the HJV promoter. In addition, the results suggest that the proposed link between inflammation and BMP-SMAD signalling is independent of HJV and BMP ligands.

  5. Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

    International Nuclear Information System (INIS)

    The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erbβΔE in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erbβΔE expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erbβ siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erbβ expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erbβ was recruited to the Srebp-1c promoter. Moreover, Rev-erbβ trans-activated the Srebp-1c promoter, in contrast, Rev-erbβ efficiently repressed the Rev-erbα promoter, a previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erbβ; and (ii) increased Rev-erbβ and Srebp-1c mRNA expression. These data suggest that Rev-erbβ has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.

  6. Distinct promoter activation mechanisms modulate noise-driven HIV gene expression

    Science.gov (United States)

    Chavali, Arvind K.; Wong, Victor C.; Miller-Jensen, Kathryn

    2015-12-01

    Latent human immunodeficiency virus (HIV) infections occur when the virus occupies a transcriptionally silent but reversible state, presenting a major obstacle to cure. There is experimental evidence that random fluctuations in gene expression, when coupled to the strong positive feedback encoded by the HIV genetic circuit, act as a ‘molecular switch’ controlling cell fate, i.e., viral replication versus latency. Here, we implemented a stochastic computational modeling approach to explore how different promoter activation mechanisms in the presence of positive feedback would affect noise-driven activation from latency. We modeled the HIV promoter as existing in one, two, or three states that are representative of increasingly complex mechanisms of promoter repression underlying latency. We demonstrate that two-state and three-state models are associated with greater variability in noisy activation behaviors, and we find that Fano factor (defined as variance over mean) proves to be a useful noise metric to compare variability across model structures and parameter values. Finally, we show how three-state promoter models can be used to qualitatively describe complex reactivation phenotypes in response to therapeutic perturbations that we observe experimentally. Ultimately, our analysis suggests that multi-state models more accurately reflect observed heterogeneous reactivation and may be better suited to evaluate how noise affects viral clearance.

  7. A novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses.

    Science.gov (United States)

    Wennier, Sonia T; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the identification of the MVA13.5L promoter, a unique and novel naturally occurring tandem promoter in MVA composed of two 44 nucleotide long repeated motifs, each containing an early promoter element. The MVA13.5L gene is highly conserved across orthopoxviruses, yet its function is unknown. The unique structure of its promoter is not found for any other gene in the MVA genome and is also conserved in other orthopoxviruses. Comparison of the MVA13.5L promoter activity with synthetic poxviral promoters revealed that the MVA13.5L promoter produced higher levels of protein early during infection in HeLa cells and particularly in MDBK cells, a cell line in which MVA replication stops at an early stage before the expression of late genes. Finally, a recombinant antigen expressed under the control of this novel promoter induced high antibody titers and increased CD8 T cell responses in homologous prime-boost immunization compared to commonly used promoters. In particular, the recombinant antigen specific CD8 T cell responses dominated over the immunodominant B8R vector-specific responses after three vaccinations and even more during the memory phase. These results have identified the native MVA13.5L promoter as a new potent promoter for use in MVA vectored preventive and therapeutic vaccines. PMID:23951355

  8. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

    Science.gov (United States)

    Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

  9. Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons

    DEFF Research Database (Denmark)

    Durham, Paul L; Dong, Penny X; Belasco, Kevin T;

    2004-01-01

    -expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV-lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT(1)) agonists......We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures....... Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the beta-galactosidase reporter gene (AdCGRP-lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP-lacZ reporter gene was preferentially expressed in neurons, with 91% co...

  10. Skp2 is over-expressed in breast cancer and promotes breast cancer cell proliferation.

    Science.gov (United States)

    Zhang, Wenwen; Cao, Lulu; Sun, Zijia; Xu, Jing; Tang, Lin; Chen, Weiwei; Luo, Jiayan; Yang, Fang; Wang, Yucai; Guan, Xiaoxiang

    2016-05-18

    The F box protein Skp2 is oncogenic. Skp2 and Skp2B, an isoform of Skp2 are overexpressed in breast cancer. However, little is known regarding the mechanism by which Skp2B promotes the occurrence and development of breast cancer. Here, we determined the expression and clinical outcomes of Skp2 in breast cancer samples and cell lines using breast cancer database, and investigated the role of Skp2 and Skp2B in breast cancer cell growth, apoptosis and cell cycle arrest. We obtained Skp2 is significantly overexpressed in breast cancer samples and cell lines, and high Skp2 expression positively correlated with poor prognosis of breast cancer. Both Skp2 and Skp2B could promote breast cancer cell proliferation, inhibit cell apoptosis, change the cell cycle distribution and induce the increased S phase cells and therefore induce cell proliferation in breast cancer cells. Moreover, the 2 isoforms could both suppress PIG3 expression via independent pathways in the breast cancer cells. Skp2 suppressed p53 and inhibited PIG3-induced apoptosis, while Skp2B attenuated the function of PIG3 by inhibiting PHB. Our results indicate that Skp2 and Skp2B induce breast cancer cell development and progression, making Skp2 and Skp2B potential molecular targets for breast cancer therapy. PMID:27111245

  11. Specific-sized hyaluronan fragments promote expression of human β-defensin 2 in intestinal epithelium.

    Science.gov (United States)

    Hill, David R; Kessler, Sean P; Rho, Hyunjin K; Cowman, Mary K; de la Motte, Carol A

    2012-08-31

    Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human β-defensin 2 (HβD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HβD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HβD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HβD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HβD2 protein. PMID:22761444

  12. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Dhananjay M Nawandar

    2015-10-01

    Full Text Available Epstein-Barr virus (EBV is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL in immunosuppressed patients. However, the cellular mechanism(s that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1 promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

  13. In Vitro Cultured Rat Islets Express Genes That both Prevent and Promote Angiogenesis

    Directory of Open Access Journals (Sweden)

    Tillmar L

    2004-03-01

    Full Text Available CONTEXT: Endogenous pancreatic islets are supported by a dense sinusoidal capillary system which is disrupted following isolation and culture in vitro. A rapid and accurate revascularization is therefore crucial for the survival and functioning of the transplanted islet. Although a blood flow is established in islet grafts within 1-2 weeks, these islets show poor development of intra-islet capillaries. To improve the revascularization process and the arrangement of the new blood vessels, islet production of the factors governing these processes needs to be further characterized. OBJECTIVE: To study the expression of factors which regulate angiogenesis in cultured rat islets. DESIGN AND MAIN OUTCOME MEASURES: Rat islets were isolated and cultured for one week. After 6 hours of exposure to normoxic (21% O2 or hypoxic (1% O2 conditions, mRNA expression was evaluated by the GEArray Angiogenesis 1 and 2 systems. The expression of the vascular endothelial growth factor (VEGF, the tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 1 (Tie1 and acidic fibroblast growth factor (aFGF, was further evaluated by semi-quantitative RT-PCR. RESULTS: We found the expression of 19 genes that code for factors either promoting or preventing angiogenesis. Only VEGF and Tie1 were upregulated in response to hypoxia. CONCLUSION: Hypoxia-induced islet vascularization may involve VEGF and Tie-induced signaling events. The results also show that cultured islets express genes which prevent angiogenesis concurrently with genes coding for factors stimulating angiogenesis. The balance between these factors is probably of vital importance for the revascularization process in transplanted islets. Thus, pharmacologic or genetic attenuation of islet-derived angiostatic factors may prove beneficial in promoting islet revascularization in future transplantation trials.

  14. TGF-β promotes glioma cell growth via activating Nodal expression through Smad and ERK1/2 pathways

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jing [Department of Neurology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang (China); Liu, Su-zhi [Department of Neurology, The Affiliated Taizhou Hospital, Wenzhou Medical University, Taizhou 317000, Zhejiang (China); Lin, Yan; Cao, Xiao-pan [Department of Neurology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang (China); Liu, Jia-ming, E-mail: wzljm@126.com [School of Environmental Science and Public Health, Wenzhou Medical University, Wenzhou 325035, Zhejiang (China)

    2014-01-17

    Highlights: •TGF-β promoted Nodal expression in glioma cells. •TGF-β promoted Nodal expression via activating Smad and ERK1/2 pathways. •TGF-β promotes glioma cell growth via activating Nodal expression. -- Abstract: While there were certain studies focusing on the mechanism of TGF-β promoting the growth of glioma cells, the present work revealed another novel mechanism that TGF-β may promote glioma cell growth via enhancing Nodal expression. Our results showed that Nodal expression was significantly upregulated in glioma cells when TGF-β was added, whereas the TGF-β-induced Nodal expression was evidently inhibited by transfection Smad2 or Smad3 siRNAs, and the suppression was especially significant when the Smad3 was downregulated. Another, the attenuation of TGF-β-induced Nodal expression was observed with blockade of the ERK1/2 pathway also. Further detection of the proliferation, apoptosis, and invasion of glioma cells indicated that Nodal overexpression promoted the proliferation and invasion of tumor cells and inhibited their apoptosis, resembling the effect of TGF-β addition. Downregulation of Nodal expression via transfection Nodal-specific siRNA in the presence of TGF-β weakened the promoting effect of the latter on glioma cells growth, and transfecting Nodal siRNA alone in the absence of exogenous TGF-β more profoundly inhibited the growth of glioma cells. These results demonstrated that while both TGF-β and Nodal promoted glioma cells growth, the former might exert such effect by enhancing Nodal expression, which may form a new target for glioma therapy.

  15. Expression of Fbxo7 in haematopoietic progenitor cells cooperates with p53 loss to promote lymphomagenesis.

    Directory of Open Access Journals (Sweden)

    Mikhail Lomonosov

    Full Text Available Fbxo7 is an unusual F box protein that augments D-type cyclin complex formation with Cdk6, but not Cdk4 or Cdk2, and its over-expression has been demonstrated to transform immortalised fibroblasts in a Cdk6-dependent manner. Here we present new evidence in vitro and in vivo on the oncogenic potential of this regulatory protein in primary haematopoietic stem and progenitor cells (HSPCs. Increasing Fbxo7 expression in HSPCs suppressed their colony forming ability in vitro, specifically decreasing CD11b (Mac1 expression, and these effects were dependent on an intact p53 pathway. Furthermore, increased Fbxo7 levels enhanced the proliferative capacity of p53 null HSPCs when they were grown in reduced concentrations of stem cell factor. Finally, irradiated mice reconstituted with p53 null, but not wild-type, HSPCs expressing Fbxo7 showed a statistically significant increase in the incidence of T cell lymphoma in vivo. These data argue that Fbxo7 negatively regulates the proliferation and differentiation of HSPCs in a p53-dependent manner, and that in the absence of p53, Fbxo7 expression can promote T cell lymphomagenesis.

  16. Coronary Artery Bypass

    Directory of Open Access Journals (Sweden)

    Kadri Ceberut

    2011-01-01

    Full Text Available Ancient schwannoma is a rare variant of neural tumors though rarely seen in the thorax. The combination with coronary artery diseases is also rare. Here we describe a 66 year-old male who had undergone one-stage combined surgery for thoracic ancient schwannomas removal and coronary artery disease. The masses were, respectively, 13 cm in the middle mediastinum and 5 cm in diameter originating from the intercostal nerve. The tumors were successfully removed using sternotomy, and then a coronary artery bypass grafting was performed. Here we discuss this rare tumor in relation to the relevant literature.

  17. Expression System Based on an MTIIa Promoter to Produce hPSA in Mammalian Cell Cultures.

    Science.gov (United States)

    Santos, Anderson K; Parreira, Ricardo C; Resende, Rodrigo R

    2016-01-01

    Because of the limitations of standard culture techniques, the development of new recombinant protein expression systems with biotechnological potential is a key challenge. Ideally, such systems should be able to effectively and accurately synthesize a protein of interest with intrinsic metabolic capacity. Here, we describe such a system that was designed based on a plasmid vector containing promoter elements derived from the metallothionein MTIIa promoter, as well as processing and purification elements. This promoter can be induced by heavy metals in a culture medium to induce the synthesis of human prostate-specific antigen (hPSA), which has been modified to insert elements for purification, proteolysis, and secretion. We optimized hPSA production in this system by comparing the effects and contributions of ZnCl2, CdCl2, and CuSO4 in HEK293FT, HeLa, BHK-21, and CHO-K1 cells. We also compared the effectiveness of three different transfection agents: multi-walled carbon nanotubes, Lipofectamine 2000, and X-tremeGENE HP Reagent. hPSA production was confirmed via the detection of enhanced green fluorescent protein fluorescence, and cell viability was determined. The expression of hPSA was compared with that of the native protein produced by LNCaP cells, using enzyme-linked immunosorbent assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis. X-tremeGENE reagent, the BHK-21 cell line, and CuSO4 showed the highest hPSA production rates. Furthermore, BHK-21 cells were more resistant to the oxidative stress caused by 100 μM CuSO4. These results suggest that the proposed optimized inducible expression system can effectively produce recombinant proteins with desired characteristics for a wide range of applications in molecular biology. PMID:27582737

  18. Effect of 5 '-deletion of Arabi dopsis profilin2 promoter on its vascular specific expression

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Based on the published sequence of profilin2promoter of Arabidopsis thaliana, a full-length promoter ments with length of 1380, 1153, 969 and 597 bp were then fused with gus (uidA) gene respectively. Constructed plant expression vectors were individually transferred into Kalan choe laciniata and transgenic plants regenerated. GUS his tochemical assay confirmed that the full-length promoter Pfnl.7 was vascular-specific. Deletion assays showed that profilin2 promoter could be divided into three parts. Dele tion of fragment 1 ( -1667--1380 bp) resulted in constitu tive expression, suggesting that element(s) responsible for vascular-specific expression might exist in this region. Frag ment 2 located at -1153 - -597 bp strongly inhibited gus gene expression. Fragment 3 ( -597 - -1 bp) is considered as a basic domain of profilin2.

  19. PP2A mediated AMPK inhibition promotes HSP70 expression in heat shock response.

    Directory of Open Access Journals (Sweden)

    Ting Wang

    Full Text Available BACKGROUND: Under stress, AMP-activated protein kinase (AMPK plays a central role in energy balance, and the heat shock response is a protective mechanism for cell survival. The relationship between AMPK activity and heat shock protein (HSP expression under stress is unclear. METHODOLOGY/PRINCIPAL FINDINGS: We found that heat stress induced dephosphorylation of AMPKα subunit (AMPKα in various cell types from human and rodent. In HepG2 cells, the dephosphorylation of AMPKα under heat stress in turn caused dephosphorylation of acetyl-CoA carboxylase and upregulation of phosphoenolpyruvate carboxykinase, two downstream targets of AMPK, confirming the inhibition of AMPK activity by heat stress. Treatment of HepG2 cells with phosphatase 2A (PP2A inhibitor okadaic acid or inhibition of PP2A expression by RNA interference efficiently reversed heat stress-induced AMPKα dephosphorylation, suggesting that heat stress inhibited AMPK through activation of PP2A. Heat stress- and other HSP inducer (CdCl(2, celastrol, MG132-induced HSP70 expression could be inhibited by AICAR, an AMPK specific activator. Inhibition of AMPKα expression by RNA interference reversed the inhibitory effect of AICAR on HSP70 expression under heat stress. These results indicate that AMPK inhibition under stress contribute to HSP70 expression. Mechanistic studies showed that activation of AMPK by AICAR had no effect on heat stress-induced HSF1 nuclear translocation, phosphorylation and binding with heat response element in the promoter region of HSP70 gene, but significantly decreased HSP70 mRNA stability. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that during heat shock response, PP2A mediated AMPK inhibition upregulates HSP70 expression at least partially through stabilizing its mRNA, which suggests a novel mechanism for HSP induction under stress.

  20. An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters

    OpenAIRE

    Song, Tianqiao; MA, ZHENCHUAN; Shen, Danyu; Li, Qi; Li, Wanlin; Su, Liming; Ye, Tingyue; Zhang, Meixiang; Wang, Yuanchao; Dou, Daolong

    2015-01-01

    Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana en...

  1. Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans

    OpenAIRE

    Conine, Colin C.; Moresco, James J.; Gu, Weifeng; Shirayama, Masaki; Conte, Darryl; Yates, John R.; Mello, Craig C

    2013-01-01

    During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expre...

  2. Identification of novel motif patterns to decipher the promoter architecture of co-expressed genes in Arabidopsis thaliana

    OpenAIRE

    López, Yosvany; Patil, Ashwini; Nakai, Kenta

    2013-01-01

    Background The understanding of the mechanisms of transcriptional regulation remains a challenge for molecular biologists in the post-genome era. It is hypothesized that the regulatory regions of genes expressed in the same tissue or cell type share a similar structure. Though several studies have analyzed the promoters of genes expressed in specific metazoan tissues or cells, little research has been done in plants. Hence finding specific patterns of motifs to explain the promoter architectu...

  3. Spiritual Bypass: A Preliminary Investigation

    Science.gov (United States)

    Cashwell, Craig S.; Glosoff, Harriet L.; Hammond, Cheree

    2010-01-01

    The phenomenon of spiritual bypass has received limited attention in the transpersonal psychology and counseling literature and has not been subjected to empirical inquiry. This study examines the phenomenon of spiritual bypass by considering how spirituality, mindfulness, alexithymia (emotional restrictiveness), and narcissism work together to…

  4. Leptin promotes breast cancer cell migration and invasion via IL-18 expression and secretion.

    Science.gov (United States)

    Li, Kuangfa; Wei, Lan; Huang, Yunxiu; Wu, Yang; Su, Min; Pang, Xueli; Wang, Nian; Ji, Feihu; Zhong, Changli; Chen, Tingmei

    2016-06-01

    In recent years, crosstalk between tumor microenvironment and cancer cells have received increasing attention. Accumulating research data suggests that leptin, a key adipokine secreted from adipocytes, plays important roles in breast cancer development. In our study, the effects of leptin on polarization of tumor-associated macrophages (TAMs) and promotion of the invasiveness of tumor cells were investigated. THP1 cells were used to differentiate M2 polarization macrophages. After stimulated by leptin, we established a co-culture system of tumor cells and macrophages to evaluate the function of leptin-induced macrophages in the migration and invasion of breast cancer cells. The gene and protein expressions were analyzed and the underlying mechanisms were evaluated. Moreover, pathological human specimens, and xenografts in nude mice, were detected to strengthen the in vitro results. Leptin elevated the expression of an array of cytokines in TAMs, IL-18 was the most increased, with an activation of the NF-κB/NF-κB1 signalling pathway. Additionally, after treated with leptin, TAMs significantly promoted the migration and invasion of breast cancer cells. However, these effects of leptin were abolished by the co-incubation of Bay11‑7082, a pharmacological NF-κB inhibitor. Leptin also directly stimulated IL-18 expression in breast cancer cells, which, differently, was via the PI3K/AKT-ATF-2 signaling pathway. In vivo studies showed that malignant breast carcinoma exhibited strong higher expression of Leptin, IL-8, and TAMs markers. Xenograft tumor-bearing mouse models showed that leptin significantly increased tumor volume, enhanced lung metastases, and increased expression of IL-8 and TAM markers, which were abolished by depletion of macrophages by clophosome-clodronate liposomes (CCL). Leptin could induce IL-18 expression both in TAMs and breast cancer cells. Leptin-induced IL-18 expression was regulated via NF-κB/NF-κB1 signaling in TAMs, while via PI3K

  5. Extracellular expression of alkaline phytase in Pichia pastoris: Influence of signal peptides, promoters and growth medium

    Directory of Open Access Journals (Sweden)

    Mimi Yang

    2015-06-01

    Full Text Available Alkaline phytase isolated from pollen grains of Lilium longiflorum (LlALP possesses unique catalytic and thermal stability properties that suggest it has the potential to be used as a feed supplement. However, substantial amounts of active enzymes are needed for animal feed studies and endogenous levels of LlALP in lily pollen are too low to provide the required amounts. Active rLlALP2 (coded by LlAlp2, one of two isoforms of alkaline phytase cDNA identified in lily pollen has been successfully expressed in intracellular compartments of Pichia pastoris, however enzyme yields have been modest (25–30 mg/L and purification of the enzyme has been challenging. Expression of foreign proteins to the extracellular medium of P. pastoris greatly simplifies protein purification because low levels of endogenous proteins are secreted by the yeast. In this paper, we first describe the generation of P. pastoris strains that will secrete rLlALP2 to the extracellular medium. Data presented here indicates that deletion of native signal peptides at the N- and C-termini of rLlALP2 enhanced α-mating factor (α-MF-driven secretion by four-fold; chicken egg white lysozyme signal peptide was ineffective in the extracellular secretion of rLlALP2. Second, we describe our efforts to increase expression levels by employing a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP in place of the strong, tightly controlled promoter of alcohol oxidase 1 gene (PAOX1. PGAP enhanced the extracellular expression levels of rLlALP2 compared to PAOX1. Finally, we report on the optimization of the culture medium to enhance yields of rLlALP2. The strength of PGAP varies depending on the carbon source available for cell growth; secreted expression of rLlALP2 was highest when glycerol was the carbon source. The addition of histidine and Triton X-100 also enhanced extracellular expression. Taken together, the employment of PGAP under optimized culture

  6. Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

    Directory of Open Access Journals (Sweden)

    Wen-Qin Bai

    Full Text Available Bioactive gibberellins (GAs comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

  7. The stress-regulated transcription factor CHOP promotes hepatic inflammatory gene expression, fibrosis, and oncogenesis.

    Directory of Open Access Journals (Sweden)

    Diane DeZwaan-McCabe

    Full Text Available Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC. Although these conditions also lead to integrated stress response (ISR or unfolded protein response (UPR activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. Chop-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. Chop-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases.

  8. Human papillomavirus type 16 E7 perturbs DREAM to promote cellular proliferation and mitotic gene expression.

    Science.gov (United States)

    DeCaprio, J A

    2014-07-31

    The study of the small DNA tumor viruses continues to provide valuable new insights into oncogenesis and fundamental biological processes. Although much has already been revealed about how the human papillomaviruses (HPVs) can transform cells and contribute to cervical and oropharyngeal cancer, there clearly is much more to learn. In this issue of Oncogene, Pang et al., doi:10.1038/onc.2013.426, demonstrate that the high-risk HPV16 E7 oncogene can promote cellular proliferation by interacting with the DREAM (DP, RB-like, E2F and MuvB) complex at two distinct phases of the cell cycle. Consistent with earlier work, HPV16 E7 can bind to the retinoblastoma tumor suppressor (RB) family member p130 (RBL2) protein and promote its proteasome-mediated destruction thereby disrupting the DREAM complex and can prevent exit from the cell cycle into quiescence. In addition, they demonstrate that HPV16 E7 can bind to MuvB core complex in association with BMYB and FOXM1 and activate gene expression during the G2 and M phase of the cell cycle. Thus, HPV16 E7 acts to prevent exit from the cell cycle entry and promotes mitotic proliferation and may account for the high levels of FOXM1 often observed in poor-risk cervical cancers. PMID:24166507

  9. Human papillomavirus type 16 E7 perturbs DREAM to promote cellular proliferation and mitotic gene expression

    Science.gov (United States)

    DeCaprio, James A.

    2014-01-01

    Study of the small DNA tumor viruses continues to provide valuable new insights into oncogenesis and fundamental biological processes. While much has already been revealed about how the human papillomaviruses (HPVs) can transform cells and contribute to cervical and oropharyngeal cancer, there clearly is much more to learn. In this issue of Oncogene, Pang et al. demonstrate that the high-risk HPV16 E7 oncogene can promote cellular proliferation by interacting with the DREAM (DP, RB-like, E2F and MuvB) complex at two distinct phases of the cell cycle (1). Consistent with earlier work, HPV16 E7 can bind to the retinoblastoma tumor suppressor (RB) family member p130 (RBL2) protein and promote its proteasome-mediated destruction thereby disrupting the DREAM complex and prevent exit from the cell cycle into quiescence. In addition, they demonstrate that HPV16 E7 can bind to MuvB core complex in association with BMYB and FOXM1 and activate gene expression during the G2 and M phase of the cell cycle. Thus, HPV16 E7 acts to prevent exit from the cell cycle entry and promotes mitotic proliferation and may account for the high levels of FOXM1 often observed in poor risk cervical cancers. PMID:24166507

  10. Flow characteristics in narrowed coronary bypass graft

    Science.gov (United States)

    Bernad, S. I.; Bosioc, A.; Bernad, E. S.; Petre, I.; Totorean, A. F.

    2016-06-01

    Tortuous saphenous vein graft (SVG) hemodynamics was investigated using computational fluid dynamics (CFD) techniques. Computed tomography (CT) technology is used for non-invasive bypass graft assessment 7 days after surgery. CT investigation shown two regions with severe shape remodelling first is an elbow type contortion and second is a severe curvature with tortuous area reduction. In conclusion, the helical flow induced by vessel torsion may stabilize the blood flow in the distal part of the SVG, reducing the flow disturbance and suppressing the flow separation, but in the distal end of the graft, promote the inflammatory processes in the vessels.

  11. The murine biglycan: Complete cDNA cloning, genomic organization, promoter function, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Wegrowski, Y.; Pillarisetti, J.; Danielson, K.G.; Iozzo, R.V. [Thomas Jefferson Univ., Philadelphia, PA (United States); Suzuki, S. [Univ. of Southern California, Los Angeles, CA (United States)

    1995-11-01

    Biglycan is a ubiquitous chondroitin/dermatan sulfate proteoglycan that belongs to a growing family of proteins harboring leucine-rich repeats. We have cloned and sequenced the cDNA containing the complete murine biglycan, elucidated its genomic organization, and demonstrated functional promoter activity of its 5{prime} flanking region. The deduced biglycan protein core was highly conserved across species. However, the mouse biglycan (Bgn) gene was significantly larger than the human counterpart, primarily because of a large > 4.5-kb intron between exons 1 and 2. The mouse Bgn gene spanned over 9.5 kb of continuous DNA and comprised 8 exons, with a perfectly conserved intron/exon organization vis-a-vis the human counterpart. The promoter region was enriched in GC dinucleotide and contained numerous cis-acting elements including binding sites for SP-1, AP-1, and AP-2 factors. It lacked TATA and CAAT boxes typical of housekeeping genes. In support of this, primer extension analysis showed the existence of multiple transcription start sites. Transient cell transfection assays with a construct comprising the 548 hp upstream of the major transcription start site fused to the chloramphenicol acetyl transferase reporter gene showed functional promoter activity. Internal and 5{prime} deletion constructs showed that the distal promoter of the Bgn gene was required for full transcriptional activity. In contrast to the homologous proteoglycan decorin, the highest expression of biglycan mRNA was observed in lung, liver, and spleen of adult mouse and the lowest in skin, heart, and kidney. These results will be useful for the study of biglycan gene regulation and for the generation of mice with targeted null mutation of the Bgn gene. 56 refs., 7 figs., 1 tab.

  12. Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

    Science.gov (United States)

    Zhang, Jian

    2009-01-01

    Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

  13. Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.

  14. Cardiopulmonary bypass in pregnancy

    Directory of Open Access Journals (Sweden)

    Mukul Chandra Kapoor

    2014-01-01

    Full Text Available Cardiac surgery carried out on cardiopulmonary bypass (CPB in a pregnant woman is associated with poor neonatal outcomes although maternal outcomes are similar to cardiac surgery in non-pregnant women. Most adverse maternal and fetal outcomes from cardiac surgery during pregnancy are attributed to effects of CPB. The CPB is associated with utero-placental hypoperfusion due to a number of factors, which may translate into low fetal cardiac output, hypoxia and even death. Better maternal and fetal outcomes may be achieved by early pre-operative optimization of maternal cardiovascular status, use of perioperative fetal monitoring, optimization of CPB, delivery of a viable fetus before the operation and scheduling cardiac surgery on an elective basis during the second trimester.

  15. Construction of chimeric inducible promoters by elicitors of rice fungal blast pathogen and their expression in transgenic rice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The promoter fragments of wheat GstA1 and potato Gst1 have been amplified by PCR, cloned and fused respectively to the minimal promoter sequence of rice actin gene (Act1)) and its 5′ untranslated leader sequence together with GUS. The constructs with 2 chimeric promoters (WGA and PGA) have been transferred into rice in order to analyze their inducibility patterns in transgenic rice plants. The results show that: WGA and PGA are both inducible by elicitors of Pyricularia oryzae in transgenic rice cells; the intronⅠ of rice Act1 gene is important for the heterogenic expression of monocot and dicot promoter elements in rice; and the Act1 minimal promoter and its 5′untranslated leader sequence produced low level background expression in rice.

  16. Transgenic expression of the human growth hormone minigene promotes pancreatic β-cell proliferation.

    Science.gov (United States)

    Baan, Mieke; Kibbe, Carly R; Bushkofsky, Justin R; Harris, Ted W; Sherman, Dawn S; Davis, Dawn Belt

    2015-10-01

    Transgenic mouse models are designed to study the role of specific proteins. To increase transgene expression the human growth hormone (hGH) minigene, including introns, has been included in many transgenic constructs. Until recently, it was thought that the hGH gene was not spliced, transcribed, and translated to produce functional hGH protein. We generated a transgenic mouse with the transcription factor Forkhead box M1 (FoxM1) followed by the hGH minigene, under control of the mouse insulin promoter (MIP) to target expression specifically in the pancreatic β-cell. Expression of FoxM1 in isolated pancreatic islets in vitro stimulates β-cell proliferation. We aimed to investigate the effect of FoxM1 on β-cell mass in a mouse model for diabetes mellitus. However, we found inadvertent coexpression of hGH protein from a spliced, bicistronic mRNA. MIP-FoxM1-hGH mice had lower blood glucose and higher pancreatic insulin content, due to increased β-cell proliferation. hGH signals through the murine prolactin receptor, and expression of its downstream targets tryptophan hydroxylase-1 (Tph1), tryptophan hydroxylase-2 (Tph2), and cytokine-inducible SH2 containing protein (Cish) was increased. Conversely, transcriptional targets of FoxM1 were not upregulated. Our data suggest that the phenotype of MIP-FoxM1-hGH mice is due primarily to hGH activity and that the FoxM1 protein remains largely inactive. Over the past decades, multiple transgenic mouse strains were generated that make use of the hGH minigene to increase transgene expression. Our work suggests that each will need to be carefully screened for inadvertent hGH production and critically evaluated for the use of proper controls. PMID:26202070

  17. MicroRNA genes preferentially expressed in dendritic cells contain sites for conserved transcription factor binding motifs in their promoters

    Directory of Open Access Journals (Sweden)

    Huynen Martijn A

    2011-06-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play a fundamental role in the regulation of gene expression by translational repression or target mRNA degradation. Regulatory elements in miRNA promoters are less well studied, but may reveal a link between their expression and a specific cell type. Results To explore this link in myeloid cells, miRNA expression profiles were generated from monocytes and dendritic cells (DCs. Differences in miRNA expression among monocytes, DCs and their stimulated progeny were observed. Furthermore, putative promoter regions of miRNAs that are significantly up-regulated in DCs were screened for Transcription Factor Binding Sites (TFBSs based on TFBS motif matching score, the degree to which those TFBSs are over-represented in the promoters of the up-regulated miRNAs, and the extent of conservation of the TFBSs in mammals. Conclusions Analysis of evolutionarily conserved TFBSs in DC promoters revealed preferential clustering of sites within 500 bp upstream of the precursor miRNAs and that many mRNAs of cognate TFs of the conserved TFBSs were indeed expressed in the DCs. Taken together, our data provide evidence that selected miRNAs expressed in DCs have evolutionarily conserved TFBSs relevant to DC biology in their promoters.

  18. Temperature influences on the expression of GFP promoted by the upstream sequence of cpcB from Arthrospira platensis

    Science.gov (United States)

    Lu, Yongzhong; Zhang, Xuecheng

    2007-07-01

    In order to investigate the regulation mechanism of the phycocyanin gene, a series of functional analyses of the upstream sequence of cpcB gene from Arthrospira platensis were conducted in E. coli with green fluorescent protein encoding gene (gfp) as the reporter. Results showed that the gfp gene could express at a high level under the promotion of the upstream sequence, suggesting the existence of some strong promoter elements in it. The expression of GFP was influenced by temperature. Higher temperature led to higher expression level. The bioinformatics analyses followed by mutation analyses on the secondary structure of translation initiation region (TIR) revealed that RNA thermosensor might account for the temperature regulation.

  19. Intramuscular Delivery of Adenovirus Serotype 5 Vector Expressing Humanized Protective Antigen Induces Rapid Protection against Anthrax That May Bypass Intranasally Originated Preexisting Adenovirus Immunity

    OpenAIRE

    Wu, Shipo; Zhang, Zhe; Yu, Rui; Zhang, Jun; Liu, Ying; Song, Xiaohong; YI, SHAOQIONG; Liu, Ju; Chen, Jianqin; Yin, Ying; Xu, Junjie; Hou, Lihua; Chen, Wei

    2014-01-01

    Developing an effective anthrax vaccine that can induce a rapid and sustained immune response is a priority for the prevention of bioterrorism-associated anthrax infection. Here, we developed a recombinant replication-deficient adenovirus serotype 5-based vaccine expressing the humanized protective antigen (Ad5-PAopt). A single intramuscular injection of Ad5-PAopt resulted in rapid and robust humoral and cellular immune responses in Fisher 344 rats. Animals intramuscularly inoculated with a s...

  20. Forced expression of Hnf1b/Foxa3 promotes hepatic fate of embryonic stem cells.

    Science.gov (United States)

    Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Hakhamaneshi, Mohammad Saeed; Ebadifar, Asghar; Fathi, Fardin; Baharvand, Hossein

    2016-05-20

    Embryonic stem (ES) cell-derived hepatocytes have the potential to be used for basic research, regenerative medicine, and drug discovery. Recent reports demonstrated that in addition to conventional differentiation inducers such as chemical compounds and cytokines, overexpression of lineage-specific transcription factors could induce ES cells to differentiate to a hepatic fate. Here, we hypothesized that lentivirus-mediated inducible expression of hepatic lineage transcription factors could enhance mouse ES cells to hepatocyte-like cells. We screened the effects of candidate transcription factors Hnf1b, Hnf1a, Hnf4a, Foxa1, Foxa3 and Hex, and determined that the combination of Hnf1b/Foxa3 promoted expression of several hepatic lineage-specific markers and proteins, in addition to glycogen storage, ICG uptake, and secretion of albumin and urea. The differentiated cells were engraftable and expressed albumin when transplanted into a carbon tetrachloride-injured mouse model. These results demonstrated the crucial role of Hnf1b and Foxa3 in hepatogenesis in vitro and provided a valuable tool for the efficient differentiation of HLCs from ES cells. PMID:27107701

  1. MeCP2 enforces Foxp3 expression to promote regulatory T cells' resilience to inflammation.

    Science.gov (United States)

    Li, Chaoran; Jiang, Shan; Liu, Si-Qi; Lykken, Erik; Zhao, Lin-tao; Sevilla, Jose; Zhu, Bo; Li, Qi-Jing

    2014-07-01

    Forkhead box P3(+) (Foxp3(+)) regulatory T cells (Tregs) are crucial for peripheral tolerance. During inflammation, steady Foxp3 expression in Tregs is essential for maintaining their lineage identity and suppressive function. However, the molecular machinery governing Tregs' resilience to inflammation-induced Foxp3 destabilization remains elusive. Here, we demonstrate that methyl-CpG binding protein 2 (MeCP2), an eminent epigenetic regulator known primarily as the etiological factor of Rett syndrome, is critical to sustain Foxp3 expression in Tregs during inflammation. In response to inflammatory stimuli, MeCP2 is specifically recruited to the Conserved Non-Coding sequence 2 region of the foxp3 locus, where it collaborates with cAMP responsive element binding protein 1 to promote local histone H3 acetylation, thereby counteracting inflammation-induced epigenetic silencing of foxp3. Consequently, Treg-specific deletion of MeCP2 causes spontaneous immune activation in mice and failure in protection against autoimmunity. Furthermore, we demonstrate that Foxp3 expression in MeCP2-deficient Tregs diminishes with time, resulting in their failure to suppress effector T-cell-mediated colitis. Thus, MeCP2 serves as a critical safeguard that confers Tregs with resilience against inflammation. PMID:24958888

  2. MeCP2 enforces Foxp3 expression to promote regulatory T cells’ resilience to inflammation

    Science.gov (United States)

    Li, Chaoran; Jiang, Shan; Liu, Si-Qi; Lykken, Erik; Zhao, Lin-tao; Sevilla, Jose; Zhu, Bo; Li, Qi-Jing

    2014-01-01

    Forkhead box P3+ (Foxp3+) regulatory T cells (Tregs) are crucial for peripheral tolerance. During inflammation, steady Foxp3 expression in Tregs is essential for maintaining their lineage identity and suppressive function. However, the molecular machinery governing Tregs’ resilience to inflammation-induced Foxp3 destabilization remains elusive. Here, we demonstrate that methyl-CpG binding protein 2 (MeCP2), an eminent epigenetic regulator known primarily as the etiological factor of Rett syndrome, is critical to sustain Foxp3 expression in Tregs during inflammation. In response to inflammatory stimuli, MeCP2 is specifically recruited to the Conserved Non-Coding sequence 2 region of the foxp3 locus, where it collaborates with cAMP responsive element binding protein 1 to promote local histone H3 acetylation, thereby counteracting inflammation-induced epigenetic silencing of foxp3. Consequently, Treg-specific deletion of MeCP2 causes spontaneous immune activation in mice and failure in protection against autoimmunity. Furthermore, we demonstrate that Foxp3 expression in MeCP2-deficient Tregs diminishes with time, resulting in their failure to suppress effector T-cell–mediated colitis. Thus, MeCP2 serves as a critical safeguard that confers Tregs with resilience against inflammation. PMID:24958888

  3. A Novel Naturally Occurring Tandem Promoter in Modified Vaccinia Virus Ankara Drives Very Early Gene Expression and Potent Immune Responses

    OpenAIRE

    Wennier, Sonia T.; Brinkmann, Kay; Steinhäußer, Charlotte; Mayländer, Nicole; Mnich, Claudia; Wielert, Ursula; Dirmeier, Ulrike; Hausmann, Jürgen; Chaplin, Paul; Steigerwald, Robin

    2013-01-01

    Modified vaccinia virus Ankara (MVA) has been shown to be suitable for the generation of experimental vaccines against cancer and infectious diseases, eliciting strong humoral and cellular immune responses. In viral vectored vaccines, strong recombinant antigen expression and timing of expression influence the quantity and quality of the immune response. Screening of synthetic and native poxvirus promoters for strong protein expression in vitro and potent immune responses in vivo led to the i...

  4. IL-17 expression by breast-cancer-associated macrophages: IL-17 promotes invasiveness of breast cancer cell lines

    OpenAIRE

    ZHU, XINGWU; Mulcahy, Lori A; Mohammed, Rabab AA; Lee, Andrew HS; Franks, Hester A; Kilpatrick, Laura; Yilmazer, Acelya; Paish, E. Claire; Ellis, Ian O; Patel, Poulam M; Jackson, Andrew M

    2008-01-01

    Introduction IL-17 plays an important role in autoimmunity, promoting autoimmunity, inflammation and invasion in multiple sclerosis, rheumatoid arthritis and type I diabetes. The role of IL-17 in cancer is unclear, however, as there are few studies examining IL-17 protein expression in cancer. We therefore examined IL-17 protein expression in human breast cancer and modelled its potential biological significance in vitro. Methods Immunohistochemistry was used to determine IL-17 expression in ...

  5. Wheat chloroplast targeted sHSP26 promoter confers heat and abiotic stress inducible expression in transgenic Arabidopsis Plants.

    Directory of Open Access Journals (Sweden)

    Neetika Khurana

    Full Text Available The small heat shock proteins (sHSPs have been found to play a critical role in physiological stress conditions in protecting proteins from irreversible aggregation. To characterize the hloroplast targeted sHSP26 promoter in detail, deletion analysis of the promoter is carried out and analysed via transgenics in Arabidopsis. In the present study, complete assessment of the importance of CCAAT-box elements along with Heat shock elements (HSEs in the promoter of sHSP26 was performed. Moreover, the importance of 5' untranslated region (UTR has also been established in the promoter via Arabidopsis transgenics. An intense GUS expression was observed after heat stress in the transgenics harbouring a full-length promoter, confirming the heat-stress inducibility of the promoter. Transgenic plants without UTR showed reduced GUS expression when compared to transgenic plants with UTR as was confirmed at the RNA and protein levels by qRT-PCR and GUS histochemical assays, thus suggesting the possible involvement of some regulatory elements present in the UTR in heat-stress inducibility of the promoter. Promoter activity was also checked under different abiotic stresses and revealed differential expression in different deletion constructs. Promoter analysis based on histochemical assay, real-time qPCR and fluorimetric analysis revealed that HSEs alone could not transcribe GUS gene significantly in sHSP26 promoter and CCAAT box elements contribute synergistically to the transcription. Our results also provide insight into the importance of 5`UTR of sHsp26 promoter thus emphasizing the probable role of imperfect CCAAT-box element or some novel cis-element with respect to heat stress.

  6. Characterization of FeDREB1 promoter involved in cold- and drought-inducible expression from common buckwheat (Fagopyrum esculentum).

    Science.gov (United States)

    Fang, Z W; Xu, X Y; Gao, J F; Wang, P K; Liu, Z X; Feng, B L

    2015-01-01

    C-repeat-binding factor (CBF)/dehydration-responsive element (DREB) transcription factors play key roles in plant stress responses. However, little information is available on the regulation of CBF/DREB expression. In this study, we isolated and characterized the FeDREB1 promoter sequence from the common buckwheat accession Xinong 9976. To identify the upstream region of the FeDREB1 gene required for promoter activity, we constructed a series of FeDREB1 promoter deletion derivatives. Each deletion construct was analyzed through Agrobacterium-mediated transient transformation in tobacco leaves treated with 4°C cold or drought stress. Promoter-beta-glucuronidase fusion assays revealed that the pCD1 (-270 bp) deletion in the upstream region of FeDREB1 could activate expression of the GUS gene at 4°C. The pCD1 (-270 bp), pCD2 (-530 bp), and pCD3 (-904 bp) deletion induced low-level GUS expression under drought stress. However, the pCD4 (-1278 bp) deletion clearly activated GUS gene expression. Our results suggest that sections pCD1 (-270 bp) and pCD4 (-1278 bp) in the FeDREB1 gene promoter are new sources of induced promoters for adversity-resistance breeding in plant genetic engineering. PMID:26214481

  7. Lysine-specific histone demethylase 1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch.

    Science.gov (United States)

    Sun, Hao; Liang, Lining; Li, Yuan; Feng, Chengqian; Li, Lingyu; Zhang, Yixin; He, Songwei; Pei, Duanqing; Guo, Yunqian; Zheng, Hui

    2016-01-01

    Lysine-specific histone demethylase 1 (LSD1) regulates histone methylation and influences the epigenetic state of cells during the generation of induced pluripotent stem cells (iPSCs). Here we reported that LSD1 inhibition via shRNA or specific inhibitor, tranylcypromine, promoted reprogramming at early stage via two mechanisms. At early stage of reprogramming, LSD1 inhibition increased the retrovirus-mediated exogenous expression of Oct4, Klf4, and Sox2 by blocking related H3K4 demethylation. Since LSD1 inhibition still promoted reprogramming even when iPSCs were induced with small-molecule compounds in a virus-free system, additional mechanisms should be involved. When RNA-seq was used for analysis, it was found that LSD1 inhibition reversed some gene expression changes induced by OKS, which subsequently promoted reprogramming. For example, by partially rescuing the decreased expression of Hif1α, LSD1 inhibition reversed the up-regulation of genes in oxidative phosphorylation pathway and the down-regulation of genes in glycolysis pathway. Such effects facilitated the metabolic switch from oxidative phosphorylation to glycolysis and subsequently promoted iPSCs induction. In addition, LSD1 inhibition also promoted the conversion from pre-iPSCs to iPSCs by facilitating the similar metabolic switch. Therefore, LSD1 inhibition promotes reprogramming by facilitating the expression of exogenous transcriptional factors and metabolic switch. PMID:27481483

  8. Studying Gene Expression: Database Searches and Promoter Fusions to Investigate Transcriptional Regulation in Bacteria

    Directory of Open Access Journals (Sweden)

    Betsy M. Martinez- Vaz

    2010-04-01

    Full Text Available A laboratory project was designed to illustrate how to search biological databases and utilize the information provided by these resources to investigate transcriptional regulation in Escherichia coli. The students searched several databases (NCBI Genomes, RegulonDB and EcoCyc to learn about gene function, regulation, and the organization of transcriptional units. A fluorometer and GFP promoter fusions were used to obtain fluorescence data and measure changes in transcriptional activity. The class designed and performed experiments to investigate the regulation of genes necessary for biosynthesis of amino acids and how expression is affected by environmental signals and transcriptional regulators. Assessment data showed that this activity enhanced students’ knowledge of databases, reporter genes and transcriptional regulation.

  9. Cloning and functional analysis of the promoters that upregulate carotenogenic gene expression during flower development in Gentiana lutea.

    Science.gov (United States)

    Zhu, Changfu; Yang, Qingjie; Ni, Xiuzhen; Bai, Chao; Sheng, Yanmin; Shi, Lianxuan; Capell, Teresa; Sandmann, Gerhard; Christou, Paul

    2014-04-01

    Over the last two decades, many carotenogenic genes have been cloned and used to generate metabolically engineered plants producing higher levels of carotenoids. However, comparatively little is known about the regulation of endogenous carotenogenic genes in higher plants, and this restricts our ability to predict how engineered plants will perform in terms of carotenoid content and composition. During petal development in the Great Yellow Gentian (Gentiana lutea), carotenoid accumulation, the formation of chromoplasts and the upregulation of several carotenogenic genes are temporally coordinated. We investigated the regulatory mechanisms responsible for this coordinated expression by isolating five G. lutea carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH and GlLYCE) promoters by inverse polymerase chain reaction (PCR). Each promoter was sufficient for developmentally regulated expression of the gusA reporter gene following transient expression in tomato (Solanum lycopersicum cv. Micro-Tom). Interestingly, the GlLYCB and GlBCH promoters drove high levels of gusA expression in chromoplast-containing mature green fruits, but low levels in chloroplast-containing immature green fruits, indicating a strict correlation between promoter activity, tomato fruit development and chromoplast differentiation. As well as core promoter elements such as TATA and CAAT boxes, all five promoters together with previously characterized GlZEP promoter contained three common cis-regulatory motifs involved in the response to methyl jasmonate (CGTCA) and ethylene (ATCTA), and required for endosperm expression (Skn-1_motif, GTCAT). These shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation. Our data provide insight into the regulatory basis of the coordinated upregulation of carotenogenic gene expression during flower development in G. lutea. PMID:24256196

  10. Photon-induced cell migration and integrin expression promoted by DNA integration of HPV16 genome

    International Nuclear Information System (INIS)

    Persistent human papilloma virus 16 (HPV16) infections are a major cause of cervical cancer. The integration of the viral DNA into the host genome causes E2 gene disruption which prevents apoptosis and increases host cell motility. In cervical cancer patients, survival is limited by local infiltration and systemic dissemination. Surgical control rates are poor in cases of parametrial infiltration. In these patients, radiotherapy (RT) is administered to enhance local control. However, photon irradiation itself has been reported to increase cell motility. In cases of E2-disrupted cervical cancers, this phenomenon would impose an additional risk of enhanced tumor cell motility. Here, we analyze mechanisms underlying photon-increased migration in keratinocytes with differential E2 gene status. Isogenic W12 (intact E2 gene status) and S12 (disrupted E2 gene status) keratinocytes were analyzed in fibronectin-based and serum-stimulated migration experiments following single photon doses of 0, 2, and 10 Gy. Quantitative FACS analyses of integrin expression were performed. Migration and adhesion are increased in E2 gene-disrupted keratinocytes. E2 gene disruption promotes attractability by serum components, therefore, effectuating the risk of local infiltration and systemic dissemination. In S12 cells, migration is further increased by photon RT which leads to enhanced expression of fibronectin receptor integrins. HPV16-associated E2 gene disruption is a main predictor of treatment-refractory cancer virulence. E2 gene disruption promotes cell motility. Following photon RT, E2-disrupted tumors bear the risk of integrin-related infiltration and dissemination. (orig.)

  11. Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Ilchung [Ames Laboratory; Ray, Judhajeet [Ames Laboratory; Gupta, Vinayak [Iowa State University; Ilgu, Muslum [Ames Laboratory; Beasley, Jonathan [Iowa State University; Bendickson, Lee [Ames Laboratory; Mehanovic, Samir [Molecular Express; Kraus, George A. [Iowa State University; Nilsen-Hamilton, Marit [Ames Laboratory

    2014-04-20

    We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from the GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.

  12. Methylation of the claudin 1 promoter is associated with loss of expression in estrogen receptor positive breast cancer.

    Directory of Open Access Journals (Sweden)

    Francescopaolo Di Cello

    Full Text Available Downregulation of the tight junction protein claudin 1 is a frequent event in breast cancer and is associated with recurrence, metastasis, and reduced survival, suggesting a tumor suppressor role for this protein. Tumor suppressor genes are often epigenetically silenced in cancer. Downregulation of claudin 1 via DNA promoter methylation may thus be an important determinant in breast cancer development and progression. To investigate if silencing of claudin 1 has an epigenetic etiology in breast cancer we compared gene expression and methylation data from 217 breast cancer samples and 40 matched normal samples available through the Cancer Genome Atlas (TCGA. Moreover, we analyzed claudin 1 expression and methylation in 26 breast cancer cell lines. We found that methylation of the claudin 1 promoter CpG island is relatively frequent in estrogen receptor positive (ER+ breast cancer and is associated with low claudin 1 expression. In contrast, the claudin 1 promoter was not methylated in most of the ER-breast cancers samples and some of these tumors overexpress claudin 1. In addition, we observed that the demethylating agents, azacitidine and decitabine can upregulate claudin 1 expression in breast cancer cell lines that have a methylated claudin 1 promoter. Taken together, our results indicate that DNA promoter methylation is causally associated with downregulation of claudin 1 in a subgroup of breast cancer that includes mostly ER+ tumors, and suggest that epigenetic therapy to restore claudin 1 expression might represent a viable therapeutic strategy in this subtype of breast cancer.

  13. EphA4 expression promotes network activity and spine maturation in cortical neuronal cultures

    Directory of Open Access Journals (Sweden)

    Clifford Meredith A

    2011-05-01

    Full Text Available Abstract Background Neurons form specific connections with targets via synapses and patterns of synaptic connectivity dictate neural function. During development, intrinsic neuronal specification and environmental factors guide both initial formation of synapses and strength of resulting connections. Once synapses form, non-evoked, spontaneous activity serves to modulate connections, strengthening some and eliminating others. Molecules that mediate intercellular communication are particularly important in synaptic refinement. Here, we characterize the influences of EphA4, a transmembrane signaling molecule, on neural connectivity. Results Using multi-electrode array analysis on in vitro cultures, we confirmed that cortical neurons mature and generate spontaneous circuit activity as cells differentiate, with activity growing both stronger and more patterned over time. When EphA4 was over-expressed in a subset of neurons in these cultures, network activity was enhanced: bursts were longer and were composed of more spikes than in control-transfected cultures. To characterize the cellular basis of this effect, dendritic spines, the major excitatory input site on neurons, were examined on transfected neurons in vitro. Strikingly, while spine number and density were similar between conditions, cortical neurons with elevated levels of EphA4 had significantly more mature spines, fewer immature spines, and elevated colocalization with a mature synaptic marker. Conclusions These results demonstrate that experimental elevation of EphA4 promotes network activity in vitro, supporting spine maturation, producing more functional synaptic pairings, and promoting more active circuitry.

  14. Bee Venom Promotes Hair Growth in Association with Inhibiting 5α-Reductase Expression.

    Science.gov (United States)

    Park, Seeun; Erdogan, Sedef; Hwang, Dahyun; Hwang, Seonwook; Han, Eun Hye; Lim, Young-Hee

    2016-06-01

    Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 d. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and Western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5α-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)2 and 7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5α-reductase inhibitor and hair growth promoter. PMID:27040904

  15. Structure of the gene for human β2-adrenergic receptor: expression and promoter characterization

    International Nuclear Information System (INIS)

    The genomic gene coding for the human β2-adrenergic receptor (β2AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with β2AR properties. Southern blot analyses with β2AR-specific probes show that a single β2AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the β2AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins

  16. Inflammation Promotes Expression of Stemness-Related Properties in HBV-Related Hepatocellular Carcinoma.

    Directory of Open Access Journals (Sweden)

    Te-Sheng Chang

    Full Text Available The expression of cancer stemness is believed to reduce the efficacy of current therapies against hepatocellular carcinoma (HCC. Understanding of the stemness-regulating signaling pathways incurred by a specific etiology can facilitate the development of novel targets for individualized therapy against HCC. Niche environments, such as virus-induced inflammation, may play a crucial role. However, the mechanisms linking inflammation and stemness expression in HCC remain unclear. Here we demonstrated the distinct role of inflammatory mediators in expressions of stemness-related properties involving the pluripotent octamer-binding transcription factor 4 (OCT4 in cell migration and drug resistance of hepatitis B virus-related HCC (HBV-HCC. We observed positive immunorecognition for macrophage chemoattractant protein 1 (MCP-1/CD68 and OCT4/NANOG in HBV-HCC tissues. The inflammation-conditioned medium (inflamed-CM generated by lipopolysaccharide-stimulated U937 human leukemia cells significantly increased the mRNA and protein levels of OCT4/NANOG preferentially in HBV-active (HBV+HBsAg+ HCC cells. The inflamed-CM also increased the side population (SP cell percentage, green fluorescent protein (GFP-positive cell population, and luciferase activity of OCT4 promoter-GFP/luciferase in HBV-active HCC cells. Furthermore, the inflamed-CM upregulated the expressions of insulin-like growth factor-I (IGF-I/IGF-I receptor (IGF-IR and activated IGF-IR/Akt signaling in HBV-HCC. The IGF-IR phosphorylation inhibitor picropodophyllin (PPP suppressed inflamed-CM-induced OCT4 and NANOG levels in HBV+HBsAg+ Hep3B cells. Forced expression of OCT4 significantly increased the secondary sphere formation and cell migration, and reduced susceptibility of HBV-HCC cells to cisplatin, bleomycin, and doxorubicin. Taking together, our results show that niche inflammatory mediators play critical roles in inducing the expression of stemness-related properties involving IGF

  17. Heart bypass surgery - minimally invasive

    Science.gov (United States)

    ... MIDCAB; Robot assisted coronary artery bypass; RACAB; Keyhole heart surgery ... To perform this surgery: The heart surgeon will make a 3- to 5-inch-long surgical cut in the left part of your chest between your ribs ...

  18. The elicitor-inducible alfalfa isoflavone reductase promoter confers different patterns of developmental expression in homologous and heterologous transgenic plants.

    Science.gov (United States)

    Oommen, A; Dixon, R A; Paiva, N L

    1994-01-01

    In legumes, the synthesis of infection- and elicitor-inducible antimicrobial phytoalexins occurs via the isoflavonoid branch of the phenylpropanoid pathway. To study transcriptional regulation of isoflavonoid pathway-specific genes, we have isolated the gene encoding isoflavone reductase (IFR), which is the enzyme that catalyzes the penultimate step in the synthesis of the phytoalexin medicarpin in alfalfa. Chimeric gene fusions were constructed between 765- and 436-bp promoter fragments of the IFR gene and the beta-glucuronidase reporter gene and transferred to alfalfa and tobacco by Agrobacterium-mediated transformation. Both promoter fragments conferred elicitor-mediated expression in cell suspension cultures derived from transgenic plants of both species and fungal infection-mediated expression in leaves of transgenic alfalfa. Developmental expression directed by both promoter fragments in transgenic alfalfa was observed only in the root meristem, cortex, and nodules, which is consistent with the accumulation of endogenous IFR transcripts. However, in transgenic tobacco, expression from the 765-bp promoter was observed in vegetative tissues (root meristem and cortex, inner vascular tissue of stems and petioles, leaf tips, and stem peripheries adjacent to petioles) and in reproductive tissues (stigma, placenta, base of the ovary, receptacle, seed, tapetal layer, and pollen grains), whereas the 436-bp promoter was expressed only in fruits, seed, and pollen. These data indicate that infection/elicitor inducibility of the IFR promoter in both species and developmental expression in alfalfa are determined by sequences downstream of position -436, whereas sequences between -436 and -765 confer a complex pattern of strong ectopic developmental expression in the heterologous species that lacks the isoflavonoid pathway. PMID:7866024

  19. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression.

    Science.gov (United States)

    Sadasivam, Subhashini; Duan, Shenghua; DeCaprio, James A

    2012-03-01

    Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex-comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4-serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis. PMID:22391450

  20. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression

    Science.gov (United States)

    Sadasivam, Subhashini; Duan, Shenghua; DeCaprio, James A.

    2012-01-01

    Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex—comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4—serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis. PMID:22391450

  1. Development of useful recombinant promoter and its expression analysis in different plant cells using confocal laser scanning microscopy.

    Directory of Open Access Journals (Sweden)

    Deepak Kumar

    Full Text Available BACKGROUND: Designing functionally efficient recombinant promoters having reduced sequence homology and enhanced promoter activity will be an important step toward successful stacking or pyramiding of genes in a plant cell for developing transgenic plants expressing desired traits(s. Also basic knowledge regarding plant cell specific expression of a transgene under control of a promoter is crucial to assess the promoter's efficacy. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed a set of 10 recombinant promoters incorporating different up-stream activation sequences (UAS of Mirabilis mosaic virus sub-genomic transcript (MS8, -306 to +27 and TATA containing core domains of Figwort mosaic virus sub-genomic transcript promoter (FS3, -271 to +31. Efficacies of recombinant promoters coupled to GUS and GFP reporter genes were tested in tobacco protoplasts. Among these, a 369-bp long hybrid sub-genomic transcript promoter (MSgt-FSgt showed the highest activity in both transient and transgenic systems. In a transient system, MSgt-FSgt was 10.31, 2.86 and 2.18 times more active compared to the CaMV35S, MS8 and FS3 promoters, respectively. In transgenic tobacco (Nicotiana tabaccum, var. Samsun NN and Arabidopsis plants, the MSgt-FSgt hybrid promoter showed 14.22 and 7.16 times stronger activity compared to CaMV35S promoter respectively. The correlation between GUS activity and uidA-mRNA levels in transgenic tobacco plants were identified by qRT-PCR. Both CaMV35S and MSgt-FSgt promoters caused gene silencing but the degree of silencing are less in the case of the MSgt-FSgt promoter compared to CaMV35S. Quantification of GUS activity in individual plant cells driven by the MSgt-FSgt and the CaMV35S promoter were estimated using confocal laser scanning microscopy and compared. CONCLUSION AND SIGNIFICANCE: We propose strong recombinant promoter MSgt-FSgt, developed in this study, could be very useful for high-level constitutive expression of transgenes in

  2. HBx induced AFP receptor expressed to activate PI3K/AKT signal to promote expression of Src in liver cells and hepatoma cells

    International Nuclear Information System (INIS)

    Hepatitis B virus (HBV)-X protein(HBx) is a transactivator of host several cellular genes including alpha-fetoprotein(AFP) and AFP receptor(AFPR) which contributes to HBV-associated tumor development. The expression of AFP/AFPR are correlated with hepatocellular carcinoma(HCC)-initial cells. But the role of AFP and AFPR in promoting occurrence of HBV-related HCC were still unclear. A total of 71 clinical patients’ liver specimens, normal human liver cells L-02 and HCC cell lines, PLC/PRF/5 were selected for analyzing the effects of HBx on expression of AFP, AFPR and Src. The expression of goal proteins were detected by Immunohistochemical stained and Western blotting; HBx-expressed vectors were constructed and transfected into L-02 cells, laser confocal microscopy was applied to observe expression and location of AFP, AFPR and Src in the normal liver cells and HCC cells, soft agar colony formation assay was used to observe colonies formed of the cells. We confirmed HBx gives preference to promote the expression of AFP and AFPR; HBx priors to up-regulate the expression of AFPR and AFP in L-02 cells and in normal liver specimens; AFPR signal been able to stimulate Src expression. The results also indicated that phosphatidylinositol 3-kinase(PI3K) inhibitors Ly294002 and GDC0941 effectively suppress AFPR mediated up-regulation expression of Src in AFPR positive HCC lines. HBx priors to drive the expression of AFP and AFPR to promote expression of Src in normal liver cells and hepatoma cells; AFP and AFPR maybe play pivotal role in HBV-related hepatocarcinogenesis; Targeting AFPR is an available therapeutic strategy of HCC. The online version of this article (doi:10.1186/s12885-015-1384-9) contains supplementary material, which is available to authorized users

  3. Myristic Acid (MA) Promotes Adipogenic Gene Expression and the Differentiation of Porcine Intramuscular Adipocyte Precursor Cells

    Institute of Scientific and Technical Information of China (English)

    LU Nai-sheng; ZHANG Yong-liang; JIANG Qing-yan; SHU Gang; XIE Qiu-ping; ZHU Xiao-tong; GAO Ping; ZHOU Gui-xuan; WANG Song-bo; WANG Li-na; XI Qian-yun

    2014-01-01

    Intramuscular fat (IMF) content is considered to be a key factor that affects the marbling, tenderness, juiciness and lfavor of pork. To investigate the effects of myristic acid (MA) on the differentiation of porcine intramuscular adipocytes, cells were isolated from longissimus dorsi muscle (LDM) and treated with 0, 10, 50 or 100μmol L-1 MA. The results showed that MA signiifcantly promotes the differentiation of intramuscular adipocytes in a dose-dependent manner. MA also led to a parallel increase in the expression of peroxisome proliferator activated receptor-γ(PPARγ) and adipose-related genes, such as glucose transporter 1 (GLUT1), lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4/aP2), fatty acid translocase (FAT), acetyl-CoA carboxylaseα(ACCα), adipose triglyceride lipase (ATGL) and fatty acid synthase (FASN). However, no signiifcant effects of MA were observed on the expression of CAAT enhancer binding protein-α(C/EBPα) or hormone sensitive lipase (HSL). The expression of pyruvate dehydrogenase kinase 4 (PDK4) was increased by MA during the early stages of differentiation (day 1-3). In addition, MA also increased the absolute content of C14 (P<0.001) and saturated fatty acids (SFA) (P<0.05) to varying degrees, but no effects were observed on other fatty acids. These results suggest that MA might be able to enhance the IMF content of pork and increase the accumulation of myristic and myristoleic acid in muscle, which might have beneifcial implications for human health.

  4. Modified GFAP promoter auto-regulates tet-activator expression for increased transactivation and reduced tTA-associated toxicity.

    Science.gov (United States)

    Barton, Michael D; Dunlop, J W; Psaltis, G; Kulik, J; DeGennaro, L; Kwak, Seung P

    2002-05-30

    Transactivator tTA is a necessary component of the tetracycline-regulated inducible gene system. While several transgenic animals have been described that express tTA in the central nervous system (CNS), their tTA levels are often limited, presumably due to toxic effects. We evaluated methods for auto-regulating tTA levels in astrocytes by modifying the transgenic promoter human GFAP (hGFAP). The hGFAP promoter carrying a single copy of the tet-operon in place of a native enhancer element (GFAPtetO1) drove expression of tTA at low levels during un-stimulated, basal condition. However the same promoter auto-induced expression of tTA to significant levels after tetracycline withdrawal. Glial cell-specificity of the promoter remained uncompromised during both basal and induced conditions. Transgenic rats were developed using the auto-inducible GFAPtetO1 promoter that expressed tTA mRNA to high levels in the brain. Expression was widespread within the CNS but enriched in astrocyte-rich regions including the cerebellum. Primary cerebellar astrocytes from GFAPtetO1 rats transfected with 07LacZ produced substantially greater inducibility of reporter gene compared to GFAP-tTA transgenic rats. Finally, GFAPtetO1 rats exhibited severe motor/gait deficit when bred to homozygosity. This phenotype was attributable to developmental abnormalities of the cerebellum and was completely abrogated by doxycycline administration. These results suggest that developmental toxicity resulting from tTA expression can be circumvented and tTA transgenics with high transactivation potential can be developed using the auto-activation strategy. Promoter modification presented here may be useful in developing highly inducible transgenic strategies without loss in tissue-specificity. PMID:12007834

  5. Statins Activate Human PPAR Promoter and Increase PPAR mRNA Expression and Activation in HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Makoto Seo

    2008-01-01

    Full Text Available Statins increase peroxisome proliferator-activated receptor (PPAR mRNA expression, but the mechanism of this increased PPAR production remains elusive. To examine the regulation of PPAR production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin on human PPAR promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1 Majority of statins enhanced PPAR promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPAR promoter. This enhancement may be mediated by statin-induced HNF-4. (2 PPAR mRNA expression was increased by statin treatment. (3 The PPAR levels in nuclear fractions were increased by statin treatment. (4 Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPAR/RXR expression vectors. In summary, these data demonstrate that PPAR production and activation are upregulated through the PPAR promoter activity by statin treatment.

  6. Reduced Ets Domain-containing Protein Elk1 Promotes Pulmonary Fibrosis via Increased Integrin αvβ6 Expression.

    Science.gov (United States)

    Tatler, Amanda L; Habgood, Anthony; Porte, Joanne; John, Alison E; Stavrou, Anastasios; Hodge, Emily; Kerama-Likoko, Cheryl; Violette, Shelia M; Weinreb, Paul H; Knox, Alan J; Laurent, Geoffrey; Parfrey, Helen; Wolters, Paul John; Wallace, William; Alberti, Siegfried; Nordheim, Alfred; Jenkins, Gisli

    2016-04-29

    Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFβ1 is considered central to the pathogenesis of IPF. A major mechanism of TGFβ1 activation in the lung involves the epithelially restricted αvβ6 integrin. Expression of the αvβ6 integrin is dramatically increased in IPF. How αvβ6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the β6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvβ6 integrin expression in vitro We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvβ6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease. PMID:26861876

  7. Expression pattern and core region analysis of AtMPK3 promoter in response to environmental stresses

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The protein kinase AtMPK3,a component of the MAP kinase cascade,plays an important role in stress signal transduction in plant cells. To clarify how AtMPK3 is regulated at the transcriptional level in response to various environmental factors, the 1016-bp promoter sequence upstream of the transcription start site of the AtMPK3 gene was isolated. Analyses of the promoter sequence using plant promoter databases revealed that the AtMPK3 promoter contains many potential cis-acting elements involved in environmental stress responses. We constructed four deletion mutants of the AtMPK3 promoter, and introduced the intact and truncated promoter sequences fused to the β-glucuronidase (GUS) gene into Arabidopsis. GUS histochemical staining and quantitative fluorometric GUS assays were performed to visualize and compare the expression patterns in response to different environmental stimuli. The region between-188 and-62 upstream of the transcription start site was identified as the essential DNA sequence of the AtMPK3 promoter for responses to drought, high salinity, low temperature, and wounding. These results advance our understanding of the molecular mechanisms controlling AtMPK3 expression in response to different environmental stimuli.

  8. Relationship between Expression of the Human Alpha-Fetoprotein Gene and DNA Methylation Status of the Promoter Region

    Institute of Scientific and Technical Information of China (English)

    Lijun Chen; Wei Wang; Qiuyue Jin; Ruimin Wang; Wenliang Hu

    2006-01-01

    OBJECTIVE DNA methylation has been regarded as an important epigenetic signature reflecting the transcription state of DNA in cells. This study was to conducted to assess the relationship between human alpha-fetoprotein (AFP) gene expression and the DNA methylation status of the promoter region in three different cells, namely two human hepatocellular carcinoma (HCC) cell lines and normal human fibroblasts.METHODS Transcription of the AFP gene was verified by RT-PCR. After bisulphate treatment of DNA, the methods of MSP and BSP were used to analyze the methylation density and status within single DNA strands of two closely spaced CpG dinucleotides of the promoter region in the different cells.RESULTS RT-PCR analysis indicated that the expression of the AFP gene in HepG2 cells was significantly higher than in SMMC-7721 cells,and that the AFP gene was not expressed in normal human fibroblasts.By MSP and BSP we observed that the promoter region was demethylated in the AFP-high-expressing cell lines, and that the sites of -2,494 bp and -2,431 bp in the AFP genomic sequence can be used as detection sites for early tumorous diagnosis.CONCLUSION These results indicate that the DNA methylation state of the promoter region has a negative correlation with AFP gene expression.

  9. Multiple Promoters in the WNK1 Gene: One Controls Expression of a Kidney-Specific Kinase-Defective Isoform

    Science.gov (United States)

    Delaloy, Celine; Lu, Jingyu; Houot, Anne-Marie; Disse-Nicodeme, Sandra; Gasc, Jean-Marie; Corvol, Pierre; Jeunemaitre, Xavier

    2003-01-01

    WNK1 is a serine-threonine kinase, the expression of which is affected in pseudohypoaldosteronism type II, a Mendelian form of arterial hypertension. We characterized human WNK1 transcripts to determine the molecular mechanisms governing WNK1 expression. We report the presence of two promoters generating two WNK1 isoforms with a complete kinase domain. Further variations are achieved by the use of two polyadenylation sites and tissue-specific splicing. We also determined the structure of a kidney-specific isoform regulated by a third promoter and starting at a novel exon. This transcript is kinase defective and has a predominant expression in the kidney compared to the other WNK1 isoforms, with, furthermore, a highly restricted expression profile in the distal convoluted tubule. We confirmed that the ubiquitous and kidney-specific promoters are functional in several cells lines and identified core promoters and regulatory elements. In particular, a strong enhancer element upstream from the kidney-specific exon seems specific to renal epithelial cells. Thus, control of human WNK1 gene expression of kinase-active or -deficient isoforms is mediated predominantly through the use of multiple transcription initiation sites and tissue-specific regulatory elements. PMID:14645531

  10. Bypass Rewiring and Robustness of Complex Networks

    CERN Document Server

    Park, Junsang

    2016-01-01

    A concept of bypass rewiring is introduced and random bypass rewiring is analytically and numerically investigated with simulations. Our results show that bypass rewiring makes networks robust against removal of nodes including random failures and attacks. Especially, random bypass rewiring connects all nodes except the removed nodes on an even degree infinite network and makes the percolation threshold $0$ for arbitrary occupation probabilities. In our example, the even degree network is more robust than the original network with random bypass rewiring while the original network is more robust than the even degree networks without random bypass. We propose a greedy bypass rewiring algorithm which guarantees the maximum size of the largest component at each step, assuming which node will be removed next is unknown. The simulation result shows that the greedy bypass rewiring algorithm improves the robustness of the autonomous system of the Internet under attacks more than random bypass rewiring.

  11. O-methylguanine-DNA methyltransferase (MGMT mRNA expression predicts outcome in malignant glioma independent of MGMT promoter methylation.

    Directory of Open Access Journals (Sweden)

    Simone Kreth

    Full Text Available BACKGROUND: We analyzed prospectively whether MGMT (O(6-methylguanine-DNA methyltransferase mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. METHODOLOGY/PRINCIPAL FINDINGS: ADULT PATIENTS WITH A HISTOLOGICALLY PROVEN MALIGNANT ASTROCYTOMA (GLIOBLASTOMA: N = 53, anaplastic astrocytoma: N = 10 were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA database (N = 229 glioblastomas. Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001; the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001; however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6 did significantly worse than those with low transcriptional activity (p<0.01. Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6 did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001. Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT m

  12. Decreased chicken ovalbumin upstream promoter transcription factor II expression in tamoxifen-resistant breast cancer cells.

    Science.gov (United States)

    Riggs, Krista A; Wickramasinghe, Nalinie S; Cochrum, Renate K; Watts, Mary Beth; Klinge, Carolyn M

    2006-10-15

    Tamoxifen (TAM) is successfully used for the treatment and prevention of breast cancer. However, many patients that are initially TAM responsive develop tumors that are antiestrogen/TAM resistant (TAM-R). The mechanism behind TAM resistance in estrogen receptor alpha (ERalpha)-positive tumors is not understood. The orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF)-I interacts directly with 4-hydroxytamoxifen (4-OHT)- and estradiol (E(2))-occupied ERalpha, corepressors NCoR and SMRT, and inhibit E(2)-induced gene transcription in breast cancer cells. Here we tested the hypothesis that reduced COUP-TFI and COUP-TFII correlate with TAM resistance. We report for the first time that COUP-TFII, but not COUP-TFI, is reduced in three antiestrogen/TAM-R cell lines derived from TAM-sensitive (TAM-S) MCF-7 human breast cancer cells and in MDA-MB-231 cells compared with MCF-7. ERalpha and ERbeta protein expression was not different between TAM-S and TAM-R cells, but progesterone receptor (PR) was decreased in TAM-R cells. Further, E(2) increased COUP-TFII transcription in MCF-7, but not TAM-R, cells. Importantly, reexpression of COUP-TFII in TAM-S cells to levels comparable to those in MCF-7 was shown to increase 4-OHT-mediated growth inhibition and increased apoptosis. Conversely, knockdown of COUP-TFII in TAM-S MCF-7 cells blocked growth inhibitory activity and increased 4-OHT agonist activity. 4-OHT increased COUP-TFII-ERalpha interaction approximately 2-fold in MCF-7 cells. COUP-TFII expression in TAM-R cells also inhibited 4-OHT-induced endogenous PR and pS2 mRNA expression. These data indicate that reduced COUP-TFII expression correlates with acquired TAM resistance in human breast cancer cell lines and that COUP-TFII plays a role in regulating the growth inhibitory activity of TAM in breast cancer cells. PMID:17047084

  13. Expression and Cellular Immunogenicity of a Transgenic Antigen Driven by Endogenous Poxviral Early Promoters at Their Authentic Loci in MVA

    Science.gov (United States)

    Orubu, Toritse; Alharbi, Naif Khalaf; Lambe, Teresa; Gilbert, Sarah C.; Cottingham, Matthew G.

    2012-01-01

    CD8+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens. PMID:22761956

  14. Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA.

    Directory of Open Access Journals (Sweden)

    Toritse Orubu

    Full Text Available CD8(+ T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8(+ T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens.

  15. Treatment of canine leukocyte adhesion deficiency by foamy virus vectors expressing CD18 from a PGK promoter

    OpenAIRE

    Bauer, Thomas R; Olson, Erik M.; Huo, Yunwen; Tuschong, Laura M; Allen, James M; Li, Yi; Burkholder, Tanya H; Russell, David W.

    2011-01-01

    Proto-oncogene activation caused by retroviral vector integration can cause malignancies in gene therapy trials. This has led investigators to search for less genotoxic vectors with minimal enhancer activity and a decreased risk of influencing neighboring chromosomal gene expression after integration. We previously showed that foamy virus vectors expressing the canine CD18 gene from an internal murine stem cell virus promoter could cure canine leukocyte adhesion deficiency. Here we have repea...

  16. Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2

    OpenAIRE

    Meng, Anming; Tang, Hong; Ong, Bruce A.; Farrell, Michael J.; Lin, Shuo

    1997-01-01

    We have used zebrafish embryos to dissect the promoter activity of a gene with a complex expression pattern during embryogenesis. GATA-2 is a transcription factor required for hematopoiesis and is dynamically expressed in hematopoietic tissues and in the central nervous system. Using constructs containing zebrafish GATA-2 genomic flanking sequences and the green fluorescent protein (GFP) reporter gene, we demonstrate that distinct regulatory domains are required for hematopoietic, enveloping ...

  17. A study of tumor suppressor gene expression and promoter methylation for the identification of prognostic markers in glioblastoma

    OpenAIRE

    Vaitkienė Grigaitė, Paulina

    2013-01-01

    Glioblastoma (GBM) is the most common primary brain tumor in adults. This study attempted to identify the genes potentially regulated by promoter methylation in GBM. Therefore, we analyzed the expression of COX7A1, SPINT1, AREG, NPTX2, and KRT81 in glioblastomas and human brain tissue and investigated if there were any associations between the expression and methylation of these genes. Moreover, we aimed to determine the methylation frequency of 11 genes (AREG, CASP8, CD81, DcR...

  18. Multiple Promoters in the WNK1 Gene: One Controls Expression of a Kidney-Specific Kinase-Defective Isoform

    OpenAIRE

    Delaloy, Celine; Lu, Jingyu; Houot, Anne-Marie; Disse-Nicodeme, Sandra; Gasc, Jean-Marie; Corvol, Pierre; Jeunemaitre, Xavier

    2003-01-01

    WNK1 is a serine-threonine kinase, the expression of which is affected in pseudohypoaldosteronism type II, a Mendelian form of arterial hypertension. We characterized human WNK1 transcripts to determine the molecular mechanisms governing WNK1 expression. We report the presence of two promoters generating two WNK1 isoforms with a complete kinase domain. Further variations are achieved by the use of two polyadenylation sites and tissue-specific splicing. We also determined the structure of a ki...

  19. Reduced syncytin-1 expression levels in placental syndromes correlates with epigenetic hypermethylation of the ERVW-1 promoter region.

    Directory of Open Access Journals (Sweden)

    Matthias Ruebner

    Full Text Available Terminal differentiation of villous cytotrophoblasts (CT ends in formation of the multinucleated syncytiotrophoblast representing the fetal-maternal interface. Aberrations during this cell-fusion process are associated with Intrauterine Growth Restriction (IUGR, Preeclampsia (PE and High Elevated Liver and Low Platelets (HELLP Syndrome. Syncytin-1, the envelope gene of the human Endogenous Retrovirus ERVW-1, is one of the most important genes involved in cell-fusion and showed decreased gene expression during these pathological pregnancies. The aim of this study was to determine the methylation pattern of the entire promoter of ERVW-1 and to correlate these findings with the expression profile of Syncytin-1 in the placental syndromes. 14 isolated villous cytotrophoblasts from control (n = 3, IUGR (n = 3, PE (n = 3, PE/IUGR (n = 3 and HELLP/IUGR (n = 2 placentae were used to determine the mean methylation level (ML for the ERVW-1 promoter region. ML rose significantly from 29% in control CTs to 49% in IUGR, 53% in PE, 47% in PE/IUGR and 64% in HELLP/IUGR indicating an epigenetic down-regulation of Syncytin-1 by promoter hypermethylation. DNA demethylation of the trophoblast like cell lines BeWo, JEG-3 and JAR with 5-AZA-2'desoxycytidine (AZA showed an increased Syncytin-1 expression and fusion ability in all cell lines. Promoter activity of the 5'LTR could be inhibited by hypermethylation 42-fold using a luciferase based reporter-gene assay. Finally overexpression of the methyltransferases DNMT3a and LSH could be responsible for a decreased Syncytin-1 expression by promoter hypermethylation of ERVW-1. Our study linked decreased Syncytin-1 expression to an epigenetic hypermethylation of the entire promoter of ERVW-1. Based on our findings we are predicting a broad aberrant epigenetic DNA-methylation pattern in pathological placentae affecting placentogenesis, but also the development of the fetus and the mother during pregnancy.

  20. PDGF induces SphK1 expression via Egr-1 to promote pulmonary artery smooth muscle cell proliferation.

    Science.gov (United States)

    Sysol, Justin R; Natarajan, Viswanathan; Machado, Roberto F

    2016-06-01

    Pulmonary arterial hypertension (PAH) is a progressive, life-threatening disease for which there is currently no curative treatment available. Pathologic changes in this disease involve remodeling of the pulmonary vasculature, including marked proliferation of pulmonary artery smooth muscle cells (PASMCs). Recently, the bioactive lipid sphingosine-1-phosphate (S1P) and its activating kinase, sphingosine kinase 1 (SphK1), have been shown to be upregulated in PAH and promote PASMC proliferation. The mechanisms regulating the transcriptional upregulation of SphK1 in PASMCs are unknown. In this study, we investigated the role of platelet-derived growth factor (PDGF), a PAH-relevant stimuli associated with enhanced PASMC proliferation, on SphK1 expression regulation. In human PASMCs (hPASMCs), PDGF significantly increased SphK1 mRNA and protein expression and induced cell proliferation. Selective inhibition of SphK1 attenuated PDGF-induced hPASMC proliferation. In silico promoter analysis for SphK1 identified several binding sites for early growth response protein 1 (Egr-1), a PDGF-associated transcription factor. Luciferase assays demonstrated that PDGF activates the SphK1 promoter in hPASMCs, and truncation of the 5'-promoter reduced PDGF-induced SphK1 expression. Stimulation of hPASMCs with PDGF induced Egr-1 protein expression, and direct binding of Egr-1 to the SphK1 promoter was confirmed by chromatin immunoprecipitation analysis. Inhibition of ERK signaling prevented induction of Egr-1 by PDGF. Silencing of Egr-1 attenuated PDGF-induced SphK1 expression and hPASMC proliferation. These studies demonstrate that SphK1 is regulated by PDGF in hPASMCs via the transcription factor Egr-1, promoting cell proliferation. This novel mechanism of SphK1 regulation may be a therapeutic target in pulmonary vascular remodeling in PAH. PMID:27099350

  1. Promoter methylation-associated loss of ID4 expression is a marker of tumour recurrence in human breast cancer

    International Nuclear Information System (INIS)

    Inhibitor of DNA binding/Inhibitor of differentiation 4 (ID4) is a critical factor for cell proliferation and differentiation in normal vertebrate development. ID4 has regulative functions for differentiation and growth of the developing brain. The role of ID1, ID2 and ID3 are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of ID3 expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of ID4 in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. ID4 promoter methylation, ID4 mRNA expression and ID4 protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of ID4 promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of ID4 promoter methylation with ID4 mRNA and ID4 protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between ID4 promoter methylation and clinicopathological parameters. Frequent ID4 promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between ID4 promoter methylation and loss of ID4 expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both transcription and protein level

  2. SERPINB5 and AKAP12 -- Expression and promoter methylation of metastasis suppressor genes in pancreatic ductal adenocarcinoma

    International Nuclear Information System (INIS)

    Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool. Metastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot. In general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes' promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05). AKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly

  3. Construction of Synthetic Promoter-Based Expression Cassettes for the Production of Cadaverine in Recombinant Corynebacterium glutamicum.

    Science.gov (United States)

    Oh, Young Hoon; Choi, Jae Woo; Kim, Eun Young; Song, Bong Keun; Jeong, Ki Jun; Park, Kyungmoon; Kim, Il-Kwon; Woo, Han Min; Lee, Seung Hwan; Park, Si Jae

    2015-08-01

    Corynebacterium glutamicum is an important microorganism in the biochemical industry for the production of various platform chemicals. However, despite its importance, a limited number of studies have been conducted on how to constitute gene expression cassettes in engineered C. glutamicum to obtain desired amounts of the target products. Therefore, in this study, six expression cassettes for the expression of the second lysine decarboxylase of Escherichia coli, LdcC, were constructed using six synthetic promoters with different strengths and were examined in C. glutamicum for the production of cadaverine. Among six expression cassettes, the expression of the E. coli ldcC gene under the PH30 promoter supported the highest production of cadaverine in flask and fed-batch cultivations. A fed-batch fermentation of recombinant C. glutamicum expressing E. coli ldcC gene under the PH30 promoter resulted in the production of 40.91 g/L of cadaverine in 64 h. This report is expected to contribute toward developing engineered C. glutamicum strains to have desired features. PMID:26047931

  4. The p38 SAPK pathway regulates the expression of the MMP-9 collagenase via AP-1-dependent promoter activation.

    Science.gov (United States)

    Simon, C; Simon, M; Vucelic, G; Hicks, M J; Plinkert, P K; Koitschev, A; Zenner, H P

    2001-12-10

    The invasive phenotype of cancers critically depends on the expression of proteases such as the M(R) 92,000 type IV collagenase (MMP-9). Several growth factors and oncogenes were found to increase promoter activity and as a consequence protease expression. This frequently requires the activation of the transcription factor AP-1 by signal transduction cascades such as the ERK and JNK pathways. We have previously demonstrated that the tumor promoter TPA can induce MMP-9 expression via a third signaling cascade, the p38 pathway. Considering that TPA is a potent activator of AP-1, we hypothesized that this transcription factor might also be required for p38 pathway-dependent MMP-9 regulation. While dominant negative p38 and MKK-6 mutants reduced MMP-9 promoter activity in CAT assays, a construct encoding an activating mutation in the MKK-6 protein potently stimulated it. This was mediated via 144 bp of the 5'flanking region of the wild-type promoter, which contains an AP-1 site at -79. Both point mutations in this motif and the expression of a c-jun protein lacking its transactivation domain and therefore acting as a dominant negative AP-1 mutant abrogated MKK-6-dependent promoter stimulation. Finally SB 203580, a specific p38 pathway inhibitor, reduced MMP-9 expression/secretion and in vitro invasion of cancer cells. Thus, our results provide evidence that also the third SAPK/MAPK signaling cascade, the p38 signal transduction pathway, stimulates MMP-9 expression in an AP-1-dependent fashion. PMID:11716547

  5. Epigenetic and Proteomic Expression Changes Promoted by Eating Addictive-Like Behavior.

    Science.gov (United States)

    Mancino, Samantha; Burokas, Aurelijus; Gutiérrez-Cuesta, Javier; Gutiérrez-Martos, Miriam; Martín-García, Elena; Pucci, Mariangela; Falconi, Anastasia; D'Addario, Claudio; Maccarrone, Mauro; Maldonado, Rafael

    2015-11-01

    An increasing perspective conceptualizes obesity and overeating as disorders related to addictive-like processes that could share common neurobiological mechanisms. In the present study, we aimed at validating an animal model of eating addictive-like behavior in mice, based on the DSM-5 substance use disorder criteria, using operant conditioning maintained by highly palatable chocolate-flavored pellets. For this purpose, we evaluated persistence of food-seeking during a period of non-availability of food, motivation for food, and perseverance of responding when the reward was associated with a punishment. This model has allowed identifying extreme subpopulations of mice related to addictive-like behavior. We investigated in these subpopulations the epigenetic and proteomic changes. A significant decrease in DNA methylation of CNR1 gene promoter was revealed in the prefrontal cortex of addict-like mice, which was associated with an upregulation of CB1 protein expression in the same brain area. The pharmacological blockade (rimonabant 3 mg/kg; i.p.) of CB1 receptor during the late training period reduced the percentage of mice that accomplished addiction criteria, which is in agreement with the reduced performance of CB1 knockout mice in this operant training. Proteomic studies have identified proteins differentially expressed in mice vulnerable or not to addictive-like behavior in the hippocampus, striatum, and prefrontal cortex. These changes included proteins involved in impulsivity-like behavior, synaptic plasticity, and cannabinoid signaling modulation, such as alpha-synuclein, phosphatase 1-alpha, doublecortin-like kinase 2, and diacylglycerol kinase zeta, and were validated by immunoblotting. This model provides an excellent tool to investigate the neurobiological substrate underlying the vulnerability to develop eating addictive-like behavior. PMID:25944409

  6. Thymic stromal lymphopoietin gene promoter polymorphisms and expression levels in Graves’ disease and Graves’ ophthalmopathy

    Directory of Open Access Journals (Sweden)

    Tsai Kun-Hsi

    2012-11-01

    Full Text Available Abstract Background Graves disease (GD is an organ-specific autoimmune disease characterized by hyperthyroidism, diffuse goiter, autoantibodies against thyroid-specific antigens, and dermopathy. Studies of GD have demonstrated the importance of the Th2 and Th17 immune responses in mediating disease progression. In the present study, we investigated the role of a Th2 cytokine, thymic stromal lymphopoietin (TSLP, in GD and Th17 differentiation. Methods In this study, we genotyped 470 patients with GD at 3 single nucleotide polymorphisms (SNPs in TSLP. In addition, the serum concentrations of TSLP were determined in 432 patients and 272 controls. Ten patients and controls each were further screened using in vitro Th17 differentiation assays. The SNPs were genotyped using ABI TaqMan® SNP genotyping assays. For the Th17 differentiation assays, peripheral blood mononuclear cells (PBMCs isolated from the patients and controls were placed into Th17 differentiation media, and interleukin 17 expression levels were determined. Results Haplotype analysis indicated that patients with the Ht3 (TCC haplotype have a 3.28-fold higher risk of developing GD (p = 0.007, whereas those with the Ht5 (TCG haplotype had a 0.03-fold, reduced risk of developing GD (p = 1 × 10−14. SNP rs3806933 (p = 0.007 was associated with female Graves ophthalmopathy (GO. TSLP expression levels were higher in GD patients than in control subjects, and TLSP was also shown to promote the differentiation of Th17 cells in GD patients. Conclusions These results suggest that polymorphisms in TSLP may be used as genetic markers for the diagnosis and prognosis of GD. Furthermore, TLSP may be a target for treating GD.

  7. Design and characterization of a dual-mode promoter with activation and repression capability for tuning gene expression in yeast.

    Science.gov (United States)

    Mazumder, Mostafizur; McMillen, David R

    2014-08-01

    Modularity in controlling gene expression artificially is becoming an essential aspect of synthetic biology. Artificial transcriptional control of gene expression is one of the most well-developed methods for the design of novel synthetic regulatory networks. Such networks are intended to help understand natural cellular phenomena and to enable new biotechnological applications. Promoter sequence manipulation with cis-regulatory elements is a key approach to control gene expression transcriptionally. Here, we have designed a promoter that can be both activated and repressed, as a contribution to the library of synthetic biological 'parts'. Starting with the minimal cytochrome C (minCYC) promoter in yeast, we incorporated five steroid hormone responsive elements (SHREs) and one lac operator site, respectively, upstream and downstream of the TATA box. This allows activation through the testosterone-responsive androgen receptor, and repression through the LacI repressor. Exposure to varying concentrations of testosterone (to vary activation) and IPTG (to vary repression) demonstrated the ability to tune the promoter's output curve over a wide range. By integrating activating and repressing signals, the promoter permits a useful form of signal integration, and we are optimistic that it will serve as a component in future regulatory networks, including feedback controllers. PMID:25056312

  8. Study on the polymorphisms and promoter methylation and expression of the glutathione Stransferases P1 gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    张友才

    2006-01-01

    Objective To study the relationship between hepatocellular carcinoma (HCC) and the polymorphisms, promoter methylation, and expression of glutathione S-transferases P1 gene (GST)P1 gene. Methods Using methylation -special PCR (MSP), the methylated status of CpG islands of GSTP1 gene in tumor tissues of 53 HCC and its adjacent nontumor tissues were studied. The en-

  9. A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs.

    Directory of Open Access Journals (Sweden)

    Peter J Romanienko

    Full Text Available The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6. However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice.

  10. A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs

    Science.gov (United States)

    Romanienko, Peter J.; Giacalone, Joseph; Ingenito, Joanne; Wang, Yijie; Isaka, Mayumi; Johnson, Thomas; You, Yun; Mark, Willie H.

    2016-01-01

    The genomes of more than 50 organisms have now been manipulated due to rapid advancement of gene editing technology. One way to perform gene editing in the mouse using the CRISPR/CAS system, guide RNA (gRNA) and CAS9 mRNA transcribed in vitro are microinjected into fertilized eggs that are then allowed to develop to term. As a rule, gRNAs are tested first in tissue culture cells and the one with the highest locus-specific cleavage activity is chosen for microinjection. For cell transfections, gRNAs are typically expressed using the human U6 promoter (hU6). However, gRNAs for microinjection into zygotes are obtained by in vitro transcription from a T7 bacteriophage promoter in a separate plasmid vector. Here, we describe the design and construction of a combined U6T7 hybrid promoter from which the same gRNA sequence can be expressed. An expression vector containing such a hybrid promoter can now be used to generate gRNA for testing in mammalian cells as well as for microinjection purposes. The gRNAs expressed and transcribed from this vector are found to be functional in cells as well as in mice. PMID:26849369

  11. Structure of two solanum tuberosum steroidal glycoalkaloid glycosyltransferase genes and expression of their promoters in transgenic potatoes

    Science.gov (United States)

    The Sgt2 gene in potato encodes a solanidine glucosyltransferase and is present as two distinct alleles expressed in cultivated potatoes. Promoter regions upstream from both steroidal glycoalkaloid biosynthetic gene alleles, Sgt2.1 and Sgt2.2, were isolated from Solanum tuberosum cv. Russet Burbank ...

  12. Repression of Pumilio Protein Expression by Rbfox1 Promotes Germ Cell Differentiation.

    Science.gov (United States)

    Carreira-Rosario, Arnaldo; Bhargava, Varsha; Hillebrand, Jens; Kollipara, Rahul K; Ramaswami, Mani; Buszczak, Michael

    2016-03-01

    RNA-binding Fox (Rbfox) proteins have well-established roles in regulating alternative splicing, but specific Rbfox isoforms lack nuclear localization signals and accumulate in the cytoplasm. The potential splicing-independent functions of these proteins remain unknown. Here we demonstrate that cytoplasmic Drosophila Rbfox1 regulates germ cell development and represses the translation of mRNAs containing (U)GCAUG elements within their 3'UTRs. During germline cyst differentiation, Rbfox1 targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development. Mis-expression of pumilio results in the formation of germline tumors, which contain cysts that break down and dedifferentiate back to single, mitotically active cells. Together, these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. The finding that Rbfox proteins regulate mRNA translation has implications for Rbfox-related diseases. PMID:26954550

  13. MiR-595 targeting regulation of SOX7 expression promoted cell proliferation of human glioblastoma.

    Science.gov (United States)

    Hao, Yu; Zhang, Shubao; Sun, Shaojun; Zhu, Jianxin; Xiao, Yilei

    2016-05-01

    Increasing evidence indicated that dysregulation of microRNAs (miRNAs) were involved with human disease including cancer. Recently, miR-595 was reported as a tumor promoter in malignant mesothelioma. However, the underlying mechanism of miR-595 in human glioblastoma (GBM) cells have not been well elucidated. Therefore, in this study, we investigated the biological functions and molecular mechanisms of miR-595 in human GBM. MiR-595 expression was significantly upregulated in GBM tissues and cells. We modified miR-595 levels in GBM cells and investigated their effects on the cell proliferation by MTT, colony formation and anchorage-independent growth assays. We found that miR-595 significantly increased GBM cell proliferation. Bioinformatic analysis predicted that miR-595 may target the 3'-UTR of SOX7and suppressed its translation, and further confirmed by luciferase assay. In sum, these observations together indicated that miR-595 played a critical role in carcinogenesis by suppression of SOX7, and may serve as a therapeutic target for the treatment of GBM. PMID:27133048

  14. Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression.

    Science.gov (United States)

    Lee, Mon-Juan; Chen, Yuhsin; Huang, Yuan-Pin; Hsu, Yi-Chiang; Chiang, Lan-Hsin; Chen, Tzu-Yu; Wang, Gwo-Jaw

    2013-12-01

    Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines. PMID:23794266

  15. Surface Expression of Precursor N-cadherin Promotes Tumor Cell Invasion

    Directory of Open Access Journals (Sweden)

    Deborah Maret

    2010-12-01

    Full Text Available The expression of N-cadherin (NCAD has been shown to correlate with increased tumor cell motility and metastasis. However, NCAD-mediated adhesion is a robust phenomenon and therefore seems to be inconsistent with the “release” from intercellular adhesion required for invasion. We show that in the most invasive melanoma and brain tumor cells, altered posttranslational processing results in abundant nonadhesive precursor N-cadherin (proNCAD at the cell surface, although total NCAD levels remain constant. We demonstrate that aberrantly processed proNCAD promotes cell migration and invasion in vitro. Furthermore, in human tumor specimens, we find high levels of proNCAD as well, supporting an overall conclusion that proNCAD and mature NCAD coexist on these tumor cell surfaces and that it is the ratio between these functionally antagonistic moieties that directly correlates with invasion potential. Our work provides insight into what may be a widespread mechanism for invasion and metastasis and challenges the current dogma of the functional roles played by classic cadherins in tumor progression.

  16. Co-regulated expression of HAND2 and DEIN by a bidirectional promoter with asymmetrical activity in neuroblastoma

    Directory of Open Access Journals (Sweden)

    Berthold Frank

    2009-04-01

    Full Text Available Abstract Background HAND2, a key regulator for the development of the sympathetic nervous system, is located on chromosome 4q33 in a head-to-head orientation with DEIN, a recently identified novel gene with stage specific expression in primary neuroblastoma (NB. Both genes are expressed in primary NB as well as most NB cell lines and are separated by a genomic sequence of 228 bp. The similar expression profile of both genes suggests a common transcriptional regulation mediated by a bidirectional promoter. Results Northern Blot analysis of DEIN and HAND2 in 20 primary NBs indicated concurrent expression levels of the two genes, which was confirmed by microarray analysis of 236 primary NBs (Pearson's correlation coefficient r = 0.65. While DEIN expression in the latter cohort was associated with stage 4S (p = 0.02, HAND2 expression was not associated with tumor stage. In contrast, both HAND2 and DEIN transcript levels were highly associated with age at diagnosis DEIN orientation, an average 3.4 fold increase in activity was observed as compared to the promoterless vector, whereas an average 15.4 fold activation was detected in HAND2 orientation. The presence of two highly conserved putative regulatory elements, one of which was shown to enhance HAND2 expression in branchial arches previously, displayed weak repressor activity for both genes. Conclusion HAND2 and DEIN represent a gene pair that is tightly linked by a bidirectional promoter in an evolutionary highly conserved manner. Expression of both genes in NB is co-regulated by asymmetrical activity of this promoter and modulated by the activity of two cis-regulatory elements acting as weak repressors. The concurrent quantitative and tissue specific expression of HAND2 and DEIN suggests a functional link between both genes.

  17. Promoter methylation of CDKN2A and lack of p16 expression characterize patients with hepatocellular carcinoma

    International Nuclear Information System (INIS)

    The product of CDKN2A, p16 is an essential regulator of the cell cycle controlling the entry into the S-phase. Herein, we evaluated CDKN2A promoter methylation and p16 protein expression for the differentiation of hepatocellular carcinoma (HCC) from other liver tumors. Tumor and corresponding non-tumor liver tissue samples were obtained from 85 patients with liver tumors. CDKN2A promoter methylation was studied using MethyLight technique and methylation-specific PCR (MSP). In the MethyLight analysis, samples with ≥ 4% of PMR (percentage of methylated reference) were regarded as hypermethylated. p16 expression was evaluated by immunohistochemistry in tissue sections (n = 148) obtained from 81 patients using an immunoreactivity score (IRS) ranging from 0 (no expression) to 6 (strong expression). Hypermethylation of the CDKN2A promoter was found in 23 HCCs (69.7%; mean PMR = 42.34 ± 27.8%), six (20.7%; mean PMR = 31.85 ± 18%) liver metastases and in the extralesional tissue of only one patient. Using MSP, 32% of the non-tumor (n = 85), 70% of the HCCs, 40% of the CCCs and 24% of the liver metastases were hypermethylated. Correspondingly, nuclear p16 expression was found immunohistochemically in five (10.9%, mean IRS = 0.5) HCCs, 23 (92%; mean IRS = 4.9) metastases and only occasionally in hepatocytes of non-lesional liver tissues (mean IRS = 1.2). The difference of CDKN2A-methylation and p16 protein expression between HCCs and liver metastases was statistically significant (p < 0.01, respectively). Promoter methylation of CDKN2A gene and lack of p16 expression characterize patients with HCC

  18. IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer

    OpenAIRE

    Abiko, K; Matsumura, N; Hamanishi, J; Horikawa, N.(Nagoya University, Nagoya, 464, Japan); Murakami, R; K. Yamaguchi; Yoshioka, Y; Baba, T; Konishi, I; Mandai, M

    2015-01-01

    Background: PD-L1 (programmed cell death 1 ligand 1) on tumour cells suppresses host immunity through binding to its receptor PD-1 on lymphocytes, and promotes peritoneal dissemination in mouse models of ovarian cancer. However, how PD-L1 expression is regulated in ovarian cancer microenvironment remains unclear. Methods: The number of CD8-positive lymphocytes and PD-L1 expression in tumour cells was assessed in ovarian cancer clinical samples. PD-L1 expression and tumour progression in mouse...

  19. The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression

    OpenAIRE

    Sadasivam, Subhashini; Duan, Shenghua; DeCaprio, James A.

    2012-01-01

    Gene expression is tightly regulated during cell cycle progression. DeCaprio and colleagues now provide a comprehensive model of the MuvB complex's dynamic control of cell cycle gene expression from quiescence through mitosis. They find that MuvB sequentially recruits B-Myb and then FoxM1 to gene promoters during G2/M. The study provides a definitive description of MuvB's central role as a part of three distinct transcription complexes in the coordinated expression of a large number of genes ...

  20. A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

    Directory of Open Access Journals (Sweden)

    Lu Ying

    2004-04-01

    Full Text Available Abstract Background TGM1(transglutaminase 1 is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. Methods In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. Results In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the

  1. Gene expression and promoter analysis of a novel tomato aldo-keto reductase in response to environmental stresses.

    Science.gov (United States)

    Suekawa, Marina; Fujikawa, Yukichi; Inada, Shuhei; Murano, Asako; Esaka, Muneharu

    2016-08-01

    The functional role of an uncharacterized tomato (Solanum lycopersicum) aldo-keto reductase 4B, denoted as SlAKR4B, was investigated. The gene expression of tomato SlAKR4B was detected at a high level in the senescent leaves and the ripening fruits of tomato. Although d-galacturonic acid reductase activities tended to be higher in tomato SlAKR4B-overexpressing transgenic tobacco BY-2 cell lines than those in control cell lines, SlAKR4B gene expression was not well correlated with l-ascorbic acid content among the cell lines. The analysis of the transgenic cell lines showed that tomato SlAKR4B has enzyme activities toward d-galacturonic acid as well as glyceraldehyde and glyoxal, suggesting that the SlAKR4B gene encodes a functional enzyme in tomato. Gene expression of SlAKR4B was induced by NaCl, H2O2, and plant hormones such as salicylic acid and jasmonic acid, suggesting that SlAKR4B is involved in the stress response. The transient expression assay using protoplasts showed the promoter activity of the SlAKR4B gene was as high as that of the cauliflower mosaic virus 35S promoter. Also, the promoter region of the SlAKR4B gene was suggested to contain cis-element(s) for abiotic stress-inducible expression. PMID:27337067

  2. Human herpesvirus 6 major immediate early promoter has strong activity in T cells and is useful for heterologous gene expression

    Directory of Open Access Journals (Sweden)

    Yamanishi Koichi

    2011-01-01

    Full Text Available Abstract Background Human herpesvirus-6 (HHV-6 is a beta-herpesvirus. HHV-6 infects and replicates in T cells. The HHV-6-encoded major immediate early gene (MIE is expressed at the immediate-early infection phase. Human cytomegalovirus major immediate early promoter (CMV MIEp is commercially available for the expression of various heterologous genes. Here we identified the HHV-6 MIE promoter (MIEp and compared its activity with that of CMV MIEp in various cell lines. Methods The HHV-6 MIEp and some HHV-6 MIEp variants were amplified by PCR from HHV-6B strain HST. These fragments and CMV MIEp were subcloned into the pGL-3 luciferase reporter plasmid and subjected to luciferase reporter assay. In addition, to investigate whether the HHV-6 MIEp could be used as the promoter for expression of foreign genes in a recombinant varicella-zoster virus, we inserted HHV-6 MIEp-DsRed expression casette into the varicella-zoster virus genome. Results HHV-6 MIEp showed strong activity in T cells compared with CMV MIEp, and the presence of intron 1 of the MIE gene increased its activity. The NF-κB-binding site, which lies within the R3 repeat, was critical for this activity. Moreover, the HHV-6 MIEp drove heterologous gene expression in recombinant varicella-zoster virus-infected cells. Conclusions These data suggest that HHV-6 MIEp functions more strongly than CMV MIEp in various T-cell lines.

  3. Novel system uses probasin-based promoter, transcriptional silencers and amplification loop to induce high-level prostate expression

    Directory of Open Access Journals (Sweden)

    Yu Hong

    2007-02-01

    Full Text Available Abstract Background Despite several effective treatment options available for prostate cancer, it remains the second leading cause of cancer death in American men. Thus, there is a great need for new treatments to improve outcomes. One such strategy is to eliminate cancer through the expression of cytotoxic genes specifically in prostate cells by gene therapy vectored delivery. To prevent systemic toxicity, tissue- and/or cancer-specific gene expression is required. However, the use of tissue- or cancer-specific promoters to target transgene expression has been hampered by their weak activity. Results To address this issue, we have developed a regulation strategy that includes feedback amplification of gene expression along with a differentially suppressible tetracycline regulated expression system (DiSTRES. By differentially suppressing expression of the tetracycline-regulated transcriptional activator (tTA and silencer (tTS genes based on the cell origin, this leads to the activation and silencing of the TRE promoter, respectively. In vitro transduction of LNCaP cells with Ad/GFPDiSTRES lead to GFP expression levels that were over 30-fold higher than Ad/CMV-GFP. Furthermore, Ad/FasL-GFPDiSTRES demonstrated cytotoxic effects in prostate cancer cells known to be resistant to Fas-mediated apoptosis. Conclusion Prostate-specific regulation from the DiSTRES system, therefore, serves as a promising new regulation strategy for future applications in the field of cancer gene therapy and gene therapy as a whole.

  4. Assessment of clusters of transcription factor binding sites in relationship to human promoter, CpG islands and gene expression

    Directory of Open Access Journals (Sweden)

    Sakaki Yoshiyuki

    2004-02-01

    Full Text Available Abstract Background Gene expression is regulated mainly by transcription factors (TFs that interact with regulatory cis-elements on DNA sequences. To identify functional regulatory elements, computer searching can predict TF binding sites (TFBS using position weight matrices (PWMs that represent positional base frequencies of collected experimentally determined TFBS. A disadvantage of this approach is the large output of results for genomic DNA. One strategy to identify genuine TFBS is to utilize local concentrations of predicted TFBS. It is unclear whether there is a general tendency for TFBS to cluster at promoter regions, although this is the case for certain TFBS. Also unclear is the identification of TFs that have TFBS concentrated in promoters and to what level this occurs. This study hopes to answer some of these questions. Results We developed the cluster score measure to evaluate the correlation between predicted TFBS clusters and promoter sequences for each PWM. Non-promoter sequences were used as a control. Using the cluster score, we identified a PWM group called PWM-PCP, in which TFBS clusters positively correlate with promoters, and another PWM group called PWM-NCP, in which TFBS clusters negatively correlate with promoters. The PWM-PCP group comprises 47% of the 199 vertebrate PWMs, while the PWM-NCP group occupied 11 percent. After reducing the effect of CpG islands (CGI against the clusters using partial correlation coefficients among three properties (promoter, CGI and predicted TFBS cluster, we identified two PWM groups including those strongly correlated with CGI and those not correlated with CGI. Conclusion Not all PWMs predict TFBS correlated with human promoter sequences. Two main PWM groups were identified: (1 those that show TFBS clustered in promoters associated with CGI, and (2 those that show TFBS clustered in promoters independent of CGI. Assessment of PWM matches will allow more positive interpretation of TFBS in

  5. Technology Solutions Case Study: Preventing Thermal Bypass

    Energy Technology Data Exchange (ETDEWEB)

    none,

    2012-10-01

    This project highlights the importance of continuous air barriers in full alignment with insulation to prevent thermal bypasses and achieve high energy performance, and recommends use of ENERGY STAR's Thermal Bypass Inspection Checklist.

  6. Nonselective digital subtraction angiography of aortocoronary bypasses

    International Nuclear Information System (INIS)

    Intra-arterial DSA was performed on 225 patients with a total of 552 coronary bypasses (515 aorto-coronary venous bypasses and 37 internal mammary artery bypasses). Four hundred and ninety-five bypasses were examined in the four weeks following surgery; of these, 428 (85.9%) were patent. Demonstration of the distal anastomosis was obtained in 40.4% of bypasses of the right anterior interventricular artery and in 36.1% of the right coronary artery, at least in their proximal parts. Bypasses of smaller branches showed filling in 12.8 to 19.2%. Because of the unsatisfactory demonstration of distal vessels by non-selective intra-arterial DSA, this method is suitable only for showing the patency of a bypass in the postoperative phase, but should not be used for investigating cardiac signs and symptoms following a bypass examination. (orig.)

  7. Pineal expression-promoting element (PIPE), a cis-acting element, directs pineal-specific gene expression in zebrafish

    OpenAIRE

    Asaoka, Yoichi; Mano, Hiroaki; Kojima, Daisuke; Fukada, Yoshitaka

    2002-01-01

    The pineal gland, sharing morphological and biochemical similarities with the retina, plays a unique and central role in the photoneuroendocrine system. The unique development of the pineal gland is directed by a specific combination of the expressed genes, but little is known about the regulatory mechanism underlying the pineal-specific gene expression. We isolated a 1.1-kbp fragment upstream of the zebrafish exo-rhodopsin (exorh) gene, which is expressed specifically in the pineal gland. Tr...

  8. Tumor-promoting macrophages induce the expression of the macrophage-specific receptor CD163 in malignant cells

    DEFF Research Database (Denmark)

    Maniecki, Maciej Bogdan; Etzerodt, Anders; Ulhøi, Benedicte Parm; Steiniche, Torben; Borre, Michael; Andersen, Lars Dyrskjøt; Orntoft, Torben Falck; Moestrup, Søren Kragh; Møller, Holger Jon

    2012-01-01

    Tumor-associated macrophages (TAMs) represent a distinct malignancy-promoting phenotype suggested to play a key role in tumor formation and metastasis. We aimed to investigate the expression of the monocyte/macrophage-restricted receptor CD163 in bladder tumor biopsies and assess the potential...... and histologically advanced disease. In 39% of the biopsies, CD163 immunoreactivity was also observed in tumor cells, and CD163-expressing metastatic cells were identified in lymph node biopsies (n = 8). Bladder cancer cell lines did not express CD163; however, when cocultured with macrophages the...... expressed by a significant portion of the malignant cells in both tumors and lymph nodes. CD163 expressing tumor cells may constitute a subpopulation of tumor cells with a phenotypic shift associated with epithelial-to-mesenchymal transition (EMT) and increased metastatic activity induced by TAMs....

  9. Enhancement of extracellular expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis: Effects of promoter and host.

    Science.gov (United States)

    Song, Wan; Nie, Yao; Mu, Xiao Qing; Xu, Yan

    2016-08-01

    Pullulanase plays an important role in industrial applications of starch processing. However, extracellular production of pullulanase from recombinant Bacillus subtilis is yet limited due to the issues on regulatory elements of B. subtilis expression system. In this study, the gene encoding B. naganoensis pullulanase (PUL) was expressed in B. subtilis WB800 under the promoter PHpaII in the shuttle vector pMA0911. The extracellular activity of expressed pullulanase was 3.9 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. To further enhance the yield of PUL, the promoter PHpaII in pMA0911 was replaced by a stronger constitutive promoter P43. Then the activity was increased to 8.7 U ml(-1) from the recombinant B. subtilis WB800/pMA0911-P43-pul. Effect of host on pullulanase expression was further investigated by comparison between B. subtilis WB600 and B. subtilis WB800. In addition to the available B. subtilis WB800 recombinants, the constructed plasmids pMA0911-PHpaII-pul and pMA0911-P43-pul were transformed into B. subtilis WB600, respectively. Consequently, the extracellular production of PUL was significantly enhanced by B. subtilis WB600/pMA0911-P43-pul, resulting in the extracellular pullulanase activity of 24.5 U ml(-1). Therefore, promoter and host had an impact on pullulanase expression and their optimization would be useful to improve heterologous protein expression in B. subtilis. PMID:27109467

  10. Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer

    Directory of Open Access Journals (Sweden)

    E Ferreira

    2012-07-01

    Full Text Available Transplantation of mesenchymal stem cells (MSCs with electrotransferred bone morphogenetic protein-2 (BMP-2 transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

  11. Photon-induced cell migration and integrin expression promoted by DNA integration of HPV16 genome

    Energy Technology Data Exchange (ETDEWEB)

    Rieken, Stefan; Simon, Florian; Habermehl, Daniel; Dittmar, Jan Oliver; Combs, Stephanie E.; Weber, Klaus; Debus, Juergen; Lindel, Katja [University Hospital of Heidelberg, Department of Radiation Therapy and Radiation Oncology, Heidelberg (Germany)

    2014-10-15

    Persistent human papilloma virus 16 (HPV16) infections are a major cause of cervical cancer. The integration of the viral DNA into the host genome causes E2 gene disruption which prevents apoptosis and increases host cell motility. In cervical cancer patients, survival is limited by local infiltration and systemic dissemination. Surgical control rates are poor in cases of parametrial infiltration. In these patients, radiotherapy (RT) is administered to enhance local control. However, photon irradiation itself has been reported to increase cell motility. In cases of E2-disrupted cervical cancers, this phenomenon would impose an additional risk of enhanced tumor cell motility. Here, we analyze mechanisms underlying photon-increased migration in keratinocytes with differential E2 gene status. Isogenic W12 (intact E2 gene status) and S12 (disrupted E2 gene status) keratinocytes were analyzed in fibronectin-based and serum-stimulated migration experiments following single photon doses of 0, 2, and 10 Gy. Quantitative FACS analyses of integrin expression were performed. Migration and adhesion are increased in E2 gene-disrupted keratinocytes. E2 gene disruption promotes attractability by serum components, therefore, effectuating the risk of local infiltration and systemic dissemination. In S12 cells, migration is further increased by photon RT which leads to enhanced expression of fibronectin receptor integrins. HPV16-associated E2 gene disruption is a main predictor of treatment-refractory cancer virulence. E2 gene disruption promotes cell motility. Following photon RT, E2-disrupted tumors bear the risk of integrin-related infiltration and dissemination. (orig.) [German] Persistierende Infektionen mit humanen Papillomaviren 16 (HPV16) sind ein Hauptausloeser des Zervixkarzinoms. Die Integration der viralen DNS in das Wirtszellgenom fuehrt zum Integritaetsverlust des E2-Gens, wodurch in der Wirtszelle Apoptose verhindert und Motilitaet gesteigert werden. In

  12. Multimodality imaging of coronary artery bypass grafts

    OpenAIRE

    Salm, Liesbeth Pauline

    2006-01-01

    This thesis describes multiple imaging modalities to examine coronary artery bypass grafts, and the research which was performed to further develop noninvasive imaging techniques to detect stenoses in native coronary arteries and bypass grafts in patients who experienced recurrent chest pain after coronary artery bypass grafting (CABG).

  13. Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line.

    Science.gov (United States)

    Vázquez-Martínez, Edgar Ricardo; Camacho-Arroyo, Ignacio; Zarain-Herzberg, Angel; Rodríguez, María Carmen; Mendoza-Garcés, Luciano; Ostrosky-Wegman, Patricia; Cerbón, Marco

    2016-06-01

    Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12 h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6 h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12 h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells. PMID:26676302

  14. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Kai, E-mail: gk161@163.com [Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Department of Respiration, 161th Hospital, PLA, Wuhan 430015 (China); Jin, Faguang, E-mail: jinfag@fmmu.edu.cn [Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)

    2015-09-25

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells.

  15. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

    International Nuclear Information System (INIS)

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells

  16. Cilostazol promotes mitochondrial biogenesis in human umbilical vein endothelial cells through activating the expression of PGC-1α

    International Nuclear Information System (INIS)

    Highlights: ► First time to show that cilostazol promotes the expressions of PGC-1α. ► First time to show that cilostazol stimulates mitochondrial biogenesis in HUVECs. ► PKA/CREB pathway mediates the effect of cilostazol on PGC-1α expression. ► Suggesting the roles of cilostazol in mitochondrial dysfunction related disease. -- Abstract: Mitochondrial dysfunction is frequently observed in vascular diseases. Cilostazol is a drug approved by the US Food and Drug Administration for the treatment of intermittent claudication. Cilostazol increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibition of type III phosphodiesterase. The effects of cilostazol in mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. Cilostazol treated HUVECs displayed increased levels of ATP, mitochondrial DNA/nuclear DNA ratio, expressions of cytochrome B, and mitochondrial mass, suggesting an enhanced mitochondrial biogenesis induced by cilostazol. The promoted mitochondrial biogenesis could be abolished by Protein kinase A (PKA) specific inhibitor H-89, implying that PKA pathway played a critical role in increased mitochondrial biogenesis after cilostazol treatment. Indeed, expression levels of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), NRF 1 and mitochondrial transcription factor A (TFAM) were significantly increased in HUVECs after incubation with cilostazol at both mRNA levels and protein levels. Importantly, knockdown of PGC-1α could abolish cilostazol-induced mitochondrial biogenesis. Enhanced expression of p-CREB and PGC-1α induced by cilostazol could be inhibited by H-89. Moreover, the increased expression of PGC-1α induced by cilostazol could be inhibited by downregulation of CREB using CREB siRNA at both mRNA and protein levels. All the results indicated that cilostazol promoted mitochondrial biogenesis through activating the expression of PGC-1α in

  17. Cilostazol promotes mitochondrial biogenesis in human umbilical vein endothelial cells through activating the expression of PGC-1α

    Energy Technology Data Exchange (ETDEWEB)

    Zuo, Luning [Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China); Department of Cardiology, Yantaishan Hospital, Yantai, Shandong 264001 (China); Li, Qiang; Sun, Bei; Xu, Zhiying [Department of Cardiology, Yantaishan Hospital, Yantai, Shandong 264001 (China); Ge, Zhiming, E-mail: zhimingge2000@hotmail.com [Department of Cardiology, Qilu Hospital, Shandong University, 44 Wenhua Xi Road, Jinan, Shandong 250012 (China)

    2013-03-29

    Highlights: ► First time to show that cilostazol promotes the expressions of PGC-1α. ► First time to show that cilostazol stimulates mitochondrial biogenesis in HUVECs. ► PKA/CREB pathway mediates the effect of cilostazol on PGC-1α expression. ► Suggesting the roles of cilostazol in mitochondrial dysfunction related disease. -- Abstract: Mitochondrial dysfunction is frequently observed in vascular diseases. Cilostazol is a drug approved by the US Food and Drug Administration for the treatment of intermittent claudication. Cilostazol increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibition of type III phosphodiesterase. The effects of cilostazol in mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. Cilostazol treated HUVECs displayed increased levels of ATP, mitochondrial DNA/nuclear DNA ratio, expressions of cytochrome B, and mitochondrial mass, suggesting an enhanced mitochondrial biogenesis induced by cilostazol. The promoted mitochondrial biogenesis could be abolished by Protein kinase A (PKA) specific inhibitor H-89, implying that PKA pathway played a critical role in increased mitochondrial biogenesis after cilostazol treatment. Indeed, expression levels of peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), NRF 1 and mitochondrial transcription factor A (TFAM) were significantly increased in HUVECs after incubation with cilostazol at both mRNA levels and protein levels. Importantly, knockdown of PGC-1α could abolish cilostazol-induced mitochondrial biogenesis. Enhanced expression of p-CREB and PGC-1α induced by cilostazol could be inhibited by H-89. Moreover, the increased expression of PGC-1α induced by cilostazol could be inhibited by downregulation of CREB using CREB siRNA at both mRNA and protein levels. All the results indicated that cilostazol promoted mitochondrial biogenesis through activating the expression of PGC-1α in

  18. Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression.

    Science.gov (United States)

    Gao, Peng; Wang, Yanli; Villanueva, Iván; Ho, Pak Leung; Davies, Julian; Kao, Richard Yi Tsun

    2016-01-01

    As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm. PMID:27625639

  19. Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

    OpenAIRE

    Shah, Nameeta; Lin, Biaoyang; Sibenaller, Zita; Ryken, Timothy; Lee, Hwahyung; Yoon, Jae-Geun; Rostad, Steven; Foltz, Greg

    2011-01-01

    O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site met...

  20. Interventions in Infrainguinal Bypass Grafts

    International Nuclear Information System (INIS)

    The interventional radiologist plays an important role in the detection and prevention of infrainguinal bypass failure. Early detection and evaluation of flow-limiting lesions effectively preserve graft (venous bypass and polyester or expanded polytetrafluoroethylene bypass) patency by identifying stenoses before occlusion occurs. Delay in treatment of the at-risk graft may result in graft failure and a reduced chance of successful revascularization. For this reason, surveillance protocols form an important part of follow-up after infrainguinal bypass surgery. As well as having an understanding of the application of imaging techniques including ultrasound, MR angiography, CT angiography and digital subtraction angiography, the interventional radiologist should have detailed knowledge of the minimally invasive therapeutic options. Percutaneous transluminal angioplasty (PTA), or alternatively cutting balloon angioplasty, is the interventional treatment of choice in prevention of graft failure and occlusion. Further alternatives include metallic stent placement, fibrinolysis, and mechanical thrombectomy. Primary assisted patency rates following PTA can be up to 65% at 5 years. When the endovascular approach is unsuccessful, these therapeutic options are complemented by surgical procedures including vein patch revision, jump grafting, or placement of a new graft

  1. Induced Pib Expression and Resistance to Magnaporthe grisea are Compromised by Cytosine Demethylation at Critical Promoter Regions in Rice

    Institute of Scientific and Technical Information of China (English)

    Yuan Li; Qiong Xia; Hongping Kou; Dan Wang; Xiuyun Lin; Ying Wu; Chunming Xu; Shaochen Xing

    2011-01-01

    Pib is a well-characterized rice blast-resistance gene belonging to the nucleotide binding site (NBS) and leucine-rich repeat (LRR) superfamily.Expression of Pib was low under non-challenged conditions,but strongly induced by the blast-causing fungal pathogen Magnaporthe grisea,thereby conferring resistance to the pathogen.It is generally established that cytosine methylation of the promoter-region often plays a repressive role in modulating expression of the gene in question.We report here that two critical regions of the Pib promoter were heavily CG cytosine-methylated in both cultivars studied.Surprisingly,induced expression of Pib by M.grisea infection did not entail its promoter demethylation,and partial demethylation by 5-azacytidine-treatment actually reduced Pib expression relative to wildtype plants.Accordingly,the blast disease-resistance was compromised in the 5’-azaC-treated plants relative to wild-type.In contrast,the disease susceptibility was not affected by the 5’-azaC treatment in another two rice cultivars that did not contain the Pib gene,ruling out effects of other R genes and non-specific genotoxic effects by the drug-treatment as a cause for the compromised Pib-conditioned blast-resistance.Taken together,our results suggest that promoter DNA methylation plays a novel enhancing role in conditioning high-level of induced expression of the Pib gene in times of M.grisea infection,and its conferred resistance to the pathogen.

  2. Transient expression in tobacco Bright Yellow 2 cells and pollen grains: A fast, efficient and reliable system for functional promoter analysis of plant genes

    Directory of Open Access Journals (Sweden)

    Bratić Ana M.

    2010-01-01

    Full Text Available Gene expression is mediated by DNA sequences directly upstream from the coding sequences, recruited transcription factors and RNA polymerase in a spatially-defined manner. Understanding promoter strength and regulation would enhance our understanding of gene expression. The goal of this study was to develop a fast, efficient and reliable method for testing basal promoter activity and identifying core sequences within its pollen specific elements. In this paper we examined the functionality of buckwheat metallothionein promoter by a histochemical GUS assay in two transient expression systems: BY2 cells and pollen grains. Strong promoter activity was observed in both systems.

  3. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Qiang [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhao, Zhi-Ning [Clinical Laboratory, 451 Hospital of Chinese PLA, Xi' an 710054 (China); Cheng, Jing-Tao [Department of Special Dentistry, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Zhang, Bin [Department of Orthodontics, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Xu, Jie [Department of Periodontology, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Huang, Fei; Zhao, Rui-Ni [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China); Chen, Yong-Jin, E-mail: cyj1229@fmmu.edu.cn [Department of General Dentistry and Emergency, College of Stomatology, Fourth Military Medical University, Xi' an, Shaanxi 710032 (China)

    2011-01-07

    Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation

  4. Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

    International Nuclear Information System (INIS)

    Research highlights: → Ibandronate significantly promote the proliferation of PDLSC cells. → Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. → The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. → Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. → Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. They suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact

  5. Role of cytokines in promoting immune escape of FasL-expressing human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Tong Xu; Bao-Cun Sun; Qiang Li; Xi-Shan Hao

    2005-01-01

    AIM: To investigate the potential role of cytokines in promoting Fas ligand (FasL)-expressing colon cancer cells.METHODS: Immunohistochemical SABC method was used to observe the expression of Fas receptor and ligand in SW620 colon cancer cell line and Jurkat T cells in order to provide the morphological evidence for the functions of Fas receptor and ligand. To examine the cytotoxicity of effector cells, CytoTox96(R) non-radioactive cytotoxicity assay was adopted to measure the lactate dehydrogenase-releasing value after SW620 cells were co-cultured with Jurkat T lymphocytes.RESULTS: The FasL of colon cancer SW620 cells was positive. The positive substances were distributed in the cell membrane and cytoplasm. The Fas receptor of colon cancer SW620 cells was negative. The Fas receptor and ligand of Jurkat T lymphocytes tumed out to be positive. The positive substances were distributed in the cell membrane.After phytohemagglutinin (PHA)-stimulated Jurkat T lymphocytes were co-cultured with phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin-stimulated (for 48 h) SW620ceils or tumor necrosis factor-alpha (TNF-α)-stimulated (for 48 h) SW620 cells or unstimulated SW620 cells for 4 h,the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-to-target ratios of 10:1, 5:1, 2.5:1, and 1.25:1was 74.6%, 40.8%, 32.4%, and 10.9% (F= 8.19, P<0.05);or 54.9%, 35.3%, 22.0%, and 10.3% (F= 11.12, P<0.05);or 14.9%, 10.5%, 6.9%, and 5.8% (F= 3.45, P<0.05).After PHA-stimulated Jurkat T lymphocytes were co-cultured with unstimulated SW620 cells for 8 h, the cytotoxicity of SW620 cells to PHA-stimulated Jurkat cells at effector-totarget ratios of 5:1, 2.5:1, and 1.25:1 from the experiment was 83.9%, 74.1%, and 28.5% (F= 137.04, P<0.05)respectively. Non-radioactive cytotoxicity assay showed that the apoptotic rate of Jurkat cells remarkably increased with the increase of planting concentration of SW620 cells and co-culture time after the SW620 cells were co

  6. Testis hormone-sensitive lipase expression in spermatids is governed by a short promoter in transgenic mice.

    Science.gov (United States)

    Blaise, R; Guillaudeux, T; Tavernier, G; Daegelen, D; Evrard, B; Mairal, A; Holm, C; Jégou, B; Langin, D

    2001-02-16

    A testicular form of hormone-sensitive lipase (HSL(tes)), a triacylglycerol lipase, and cholesterol esterase, is expressed in male germ cells. Northern blot analysis showed HSL(tes) mRNA expression in early spermatids. Immunolocalization of the protein in human and rodent seminiferous tubules indicated that the highest level of expression occurred in elongated spermatids. We have previously shown that 0.5 kilobase pairs of the human HSL(tes) promoter directs testis-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice and determined regions binding nuclear proteins expressed in testis but not in liver (Blaise, R., Grober, J., Rouet, P., Tavernier, G., Daegelen, D., and Langin, D. (1999) J. Biol. Chem. 274, 9327-9334). Mutation of a SRY/Sox-binding site in one of the regions did not impair in vivo testis-specific expression of the reporter gene. Further transgenic analyses established that 95 base pairs upstream of the transcription start site were sufficient for correct testis expression. In gel retardation assays using early spermatid nuclear extracts, a germ cell-specific DNA-protein interaction was mapped between -46 and -29 base pairs. The DNA binding nuclear protein showed properties of zinc finger transcription factors. Mutation of the region abolished reporter gene activity in transgenic mice, showing that it is necessary for testis expression of HSL(tes). PMID:11076952

  7. Loss of the NKX3.1 tumorsuppressor promotes the TMPRSS2-ERG fusion gene expression in prostate cancer

    International Nuclear Information System (INIS)

    In normal prostate epithelium the TMPRSS2 gene encoding a type II serine protease is directly regulated by male hormones through the androgen receptor. In prostate cancer ERG protooncogene frequently gains hormonal control by seizing gene regulatory elements of TMPRSS2 through genomic fusion events. Although, the androgenic activation of TMPRSS2 gene has been established, little is known about other elements that may interact with TMPRSS2 promoter sequences to modulate ERG expression in TMPRSS2-ERG gene fusion context. Comparative genomic analyses of the TMPRSS2 promoter upstream sequences and pathway analyses were performed by the Genomatix Software. NKX3.1 and ERG genes expressions were evaluated by immunoblot or by quantitative Real-Time PCR (qRT-PCR) assays in response to siRNA knockdown or heterologous expression. QRT-PCR assay was used for monitoring the gene expression levels of NKX3.1-regulated genes. Transcriptional regulatory function of NKX3.1 was assessed by luciferase assay. Recruitment of NKX3.1 to its cognate elements was monitored by Chromatin Immunoprecipitation assay. Comparative analysis of the TMPRSS2 promoter upstream sequences among different species revealed the conservation of binding sites for the androgen inducible NKX3.1 tumor suppressor. Defects of NKX3.1, such as, allelic loss, haploinsufficiency, attenuated expression or decreased protein stability represent established pathways in prostate tumorigenesis. We found that NKX3.1 directly binds to TMPRSS2 upstream sequences and negatively regulates the expression of the ERG protooncogene through the TMPRSS2-ERG gene fusion. These observations imply that the frequently noted loss-of-function of NKX3.1 cooperates with the activation of TMPRSS2-ERG fusions in prostate tumorigenesis

  8. Epigenetic Modifications of the PGC-1α Promoter during Exercise Induced Expression in Mice.

    Directory of Open Access Journals (Sweden)

    Timothy L Lochmann

    Full Text Available The transcriptional coactivator, PGC-1α, is known for its role in mitochondrial biogenesis. Although originally thought to exist as a single protein isoform, recent studies have identified additional promoters which produce multiple mRNA transcripts. One of these promoters (promoter B, approximately 13.7 kb upstream of the canonical PGC-1α promoter (promoter A, yields alternative transcripts present at levels much lower than the canonical PGC-1α mRNA transcript. In skeletal muscle, exercise resulted in a substantial, rapid increase of mRNA of these alternative PGC-1α transcripts. Although the β2-adrenergic receptor was identified as a signaling pathway that activates transcription from PGC-1α promoter B, it is not yet known what molecular changes occur to facilitate PGC-1α promoter B activation following exercise. We sought to determine whether epigenetic modifications were involved in this exercise response in mouse skeletal muscle. We found that DNA hydroxymethylation correlated to increased basal mRNA levels from PGC-1α promoter A, but that DNA methylation appeared to play no role in the exercise-induced activation of PGC-1α promoter B. The level of the activating histone mark H3K4me3 increased with exercise 2-4 fold across PGC-1α promoter B, but remained unaltered past the canonical PGC-1α transcriptional start site. Together, these data show that epigenetic modifications partially explain exercise-induced changes in the skeletal muscle mRNA levels of PGC-1α isoforms.

  9. Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts

    Science.gov (United States)

    Huang, Yong-Zhen; Zhang, Liang-Zhi; Lai, Xin-Sheng; Li, Ming-Xun; Sun, Yu-Jia; Li, Cong-Jun; Lan, Xian-Yong; Lei, Chu-Zhao; Zhang, Chun-Lei; Zhao, Xin; Chen, Hong

    2014-04-01

    Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

  10. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  11. Isolation and expression analysis of a LEAFY/FLORICAULA homolog and its promoter from London plane (Platanus acerifolia Willd.).

    Science.gov (United States)

    Lu, Shunjiao; Li, Zhineng; Zhang, Jiaqi; Yi, Shuangshuang; Liu, Lei; Bao, Manzhu; Liu, Guofeng

    2012-10-01

    The LEAFY/FLORICAULA (LFY/FLO) homologous genes are necessary for normal flower development in diverse angiosperm species. To understand the genetic and molecular mechanisms underlying floral initiation and development in Platanaceae, an early divergent eudicot family consisting of large monoecious trees, we isolated a homolog of LFY/FLO, PlacLFY, and its promoter from London plane (Platanus acerifolia). PlacLFY is 1,419 bp in length, with an ORF of 1,122 bp encoding a predicted polypeptide of 374 amino acids and 5'/3'-UTR of 54 and 213 bp, respectively. The putative PlacLFY protein showed a high degree of identity (56-84 %) with LFY/FLO homologs from other species, including two highly conserved regions, the N and C domains, and a less conserved amino-terminal proline-rich region. Real-time PCR analysis showed that PlacLFY was expressed mainly in male inflorescences from May of the first year to March of next year, with the highest expression level in December, and in female inflorescences from June to April of next year. PlacLFY mRNA was also detected strongly in subpetiolar buds of December from 4-year-old and adult trees, and slightly in stem of young seedling and young leaf of adult plant. Additionally, we cloned 1,138 bp promoter sequence of PlacLFY and we drove GUS expression in transgenic tobacco by the chimerical pPlacLFY::GUS construction. Histological GUS staining analysis indicated that PlacLFY promoter can drive GUS gene expression in shoot apex, stem, young leaf and petiole, flower stalk, petal tip, and young/semi-mature fruits of transgenic tobacco, which is almost identical to the expression pattern of PlacLFY in London plane. The results revealed that the PlacLFY gene isolated from London plane is expressed not only in reproductive organ but also in vegetative organs. Moreover, this expression pattern is consistent with the expression pattern in tobacco of a GUS reporter gene under the control of the potential promoter region of PlacLFY. PMID

  12. Integration of Creative Expression into Community Based Participatory Research and Health Promotion with Native American/span>s

    Science.gov (United States)

    Gray, Norma; Oré de Boehm, Christina; Farnsworth, Angela; Wolf, Denise

    2010-01-01

    Involvement in creative expression has the potential of engaging individuals in personal and community level change through reflection, empowerment, and the facilitation of connectedness. It is a process that can be a powerful component of community based participatory research as it can facilitate and support the principles of co-learning, egalitarian relationships, and respect for non-academic knowledge. It is also a valuable means of appreciating culture and strengthening identity, which enhances health. This article reviews and discusses methods and benefits of incorporating creative expression into health promotion programs and community based participatory research with Native Americans. PMID:20531099

  13. Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

    Directory of Open Access Journals (Sweden)

    Manganelli Riccardo

    2005-01-01

    Full Text Available Abstract Background In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria. Results The aim of this work was to select a strong chromosomal promoter from Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat, was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the

  14. Synthetic gene involving azobenzene-tethered T7 promoter for the photocontrol of gene expression by visible light.

    Science.gov (United States)

    Kamiya, Yukiko; Takagi, Toshiki; Ooi, Hideaki; Ito, Hiroshi; Liang, Xingguo; Asanuma, Hiroyuki

    2015-04-17

    In the present study, we demonstrate photoregulation of gene expression in a cell-free translation system from a T7 promoter containing two azobenzene derivatives at specific positions. As photoswitches, we prepared azobenzene-4'-carboxlyic acid (Azo) and 2,6-dimethylazobenzene-4'-carboxylic acid (DM-Azo), which were isomerized from trans to cis upon irradiation with UV light (λ protein from a promoter modified with S-Azo or S-DM-Azo could be induced by harmless visible light whereas that from a promoter modified with Azo or DM-Azo was induced only by UV light (340-360 nm). Thus, efficient photoregulation of green fluorescent protein production was achieved in a cell-free translation system with visible light without photodamage. PMID:25144622

  15. Hepatitis B Virus X Protein Driven Alpha Fetoprotein Expression to Promote Malignant Behaviors of Normal Liver Cells and Hepatoma Cells

    Science.gov (United States)

    Zhu, Mingyue; Lu, Yan; Li, Wei; Guo, Junli; Dong, Xu; Lin, Bo; Chen, Yi; Xie, Xieju; Li, Mengsen

    2016-01-01

    Background: The infection of Hepatitis B virus (HBV) is closely associated with the development of hepatocellular carcinoma(HCC), HBV-X protein(HBx) is able to induce expression of alpha-fetoprotein(AFP) in normal liver cells, and AFP harbors a function to promote malignant transformation of normal liver cells, but the role AFP playing in malignant behaviors of HCC cells is still unclear. Methods: Fifty-six liver tissue samples were collected from the clinical patients through hepatectomy(include normal liver tissues, HBV-related hepatitis liver tissues and HBV-related HCC tissues), and diagnosis of these tissues by pathology section, expression of AFP, Ras and CXCR4 were evidenced by immunohisochemical staining and Western blotting; The proliferation of human normal liver cells line L-02 cells and human hepatoma cells line, HLE cells(non AFP-producing) were performed by MTT method; Repaired capacity of L-02 and HLE cells were compared by wound healing assay; Migration and invasion of these cells were analyzed by Transwell chamber assay; HBx expressed vectors(pcDNA3.1-HBx) were constructed and transfected into L-02 and HLE cells, effects of pcDNA3.1-HBx on the malignant behaviors were also detected by MTT, Transwell chamber assay and the expression of AFP, Ras and CXCR4 were evidenced by Western blotting. Results: we found that expression of AFP, Ras and CXCR4 in HBV-related HCC and lymph nodes metastasis tissues were significantly elevated compared with HBV-related HCC, non metastasis tissues and HBV-related hepatitis tissues; Expression of AFP, Ras and CXCR4 in HBV-related hepatitis tissues were significantly enhanced compared with normal liver tissues; The growth ratio, migratory and invasive ability, expression of AFP, Ras and CXCR4 of the cells were outstanding promoted while L-02 and HLE cells were transfected with pcDNA3.1-HBx vectors. The proliferation ratio, migration and invasion ability, and expression of Ras and CXCR4 were significantly inhibited while

  16. Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease models.

    Science.gov (United States)

    Boy, Jana; Leergaard, Trygve B; Schmidt, Thorsten; Odeh, Francis; Bichelmeier, Ulrike; Nuber, Silke; Holzmann, Carsten; Wree, Andreas; Prusiner, Stanley B; Bujard, Hermann; Riess, Olaf; Bjaalie, Jan G

    2006-11-01

    We present a digital atlas system that allows mapping of molecular expression patterns at cellular resolution through large series of histological sections. Using this system, we have mapped the distribution of a distinct marker, encoded by the LacZ reporter gene driven by the tetracycline-responsive prion protein promoter in double transgenic mice. The purpose is to evaluate the suitability of this promoter mouse line for targeting genes of interest to specific brain regions, essential for construction of inducible transgenic disease models. Following processing to visualize the promoter expression, sections were counterstained to simultaneously display cytoarchitectonics. High-resolution mosaic images covering entire coronal sections were collected through the mouse brain at intervals of 200 microm. A web-based application provides access to a customized virtual microscopy tool for viewing and navigation within and across the section images. For each section image, the nearest section in a standard atlas is defined, and annotations of key structures and regions inserted. Putative categorization of labeled cells was performed with use of distribution patterns, followed by cell size and shape, as parameters that were compared to legacy data. Among the ensuing results were expression of this promoter in putative glial cells in the cerebellum (and not in Purkinje cells), in putative glial cells in the substantia nigra, in pallidal glial cells or interneurons, and in distinct cell layers and regions of the hippocampus. The study serves as a precursor for a database resource allowing evaluation of the suitability of different promoter mouse lines for generating disease models. PMID:16931059

  17. Intraarterial digital subtraction angiography of aortofemoral bypasses

    International Nuclear Information System (INIS)

    In a retrospective study, 214 digital subtraction angiography (DSA) examinations of aortofemoral and femorocrural bypasses were reviewed. In 90% of cases intravenous DSA was diagnostic for aortofemoral bypasses, and in 95% of cases intrarterial DSA angiograms of excellent image quality were obtained. In 82% arterial stenoses proximal to the bypass, in 62% stenoses distal to the bypass, and in only 15% stenoses involving the bypass itself could be detected. In 54% the bypass was regularly perfused. In 26% a complete occlusion could be seen. All angiograms were obtained after infusion of the low-osmolar nonionic contrast medium Iopromide, 150mg/ml. All examinations were painless, and no heat-induced motion artifacts were registered. No disturbances of vital signs were observed. DSA with a nonionic low-osmolarity contrast medium (Iopromide) is a safe and reliable technique for the examination of all surgical aortofemoral bypasses

  18. Endogenous Expression of Matriptase in Neural Progenitor Cells Promotes Cell Migration and Neuron Differentiation*

    OpenAIRE

    Fang, Jung-Da; Chou, Hsiao-Chin; Tung, Hsiu-Hui; Huang, Pao-Yi; Lee, Sheau-Ling

    2010-01-01

    Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted...

  19. An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila

    OpenAIRE

    Larsen, Camilla; Franch-Marro, Xavier; Hartenstein, Volker; Alexandre, Cyrille; Vincent, Jean-Paul

    2006-01-01

    Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic male...

  20. Bypass materials in vascular surgery

    Directory of Open Access Journals (Sweden)

    Willich, Stephan N.

    2006-03-01

    Full Text Available Introduction: Arteriosclerotic changes can lead to circulatory disturbances in various areas of the human vascular system. In addition to pharmacological therapy and the management of risk factors (e. g. hypertension, diabetes, lipid metabolism disorders, and lifestyle, surgical interventions also play an important role in the treatment of arteriosclerosis. Long-segment arterial occlusions, in particular, can be treated successfully with bypass sur-gery. A number of different materials are available for this type of operation, such as autologous vein or pros-thetic grafts comprised of polytetrafluoroethylene (PTFE or Dacron®. Prosthetic materials are used especially in the treatment of peripheral artery disease, such as in aortoiliac or femoropopliteal bypass surgery. The present report will thus focus on this area in order to examine the effectiveness of different bypass materials. Among the efforts being made to refine the newly introduced DRG system in Germany, analysing the different bypass materials used in vascular surgery is particularly important. Indeed, in its current version the German DRG system does not distinguish between bypass materials in terms of reimbursement rates. Differences in cost structures are thus of especial interest to hospitals in their budget calculations, whereas both private and statutory health insurance funds are primarily interested in long-term results and their costs. Objectives: The goal of this HTA is to compare the different bypass materials used in vascular surgery in terms of their medical efficiency and cost-effectiveness, as well as with regard to their ethical, social and legal implications. In addition, this report aims to point out the areas in which further medical, epidemiological and health economic research is still needed. Methods: Relevant publications were identified by means of a structured search of databases accessed through the German Institute of Medical Documentation and Information

  1. Vesicular Stomatitis Virus Infection Promotes Immune Evasion by Preventing NKG2D-Ligand Surface Expression

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Nielsen, Jens; Christensen, Jan Pravsgaard; Skov, Søren

    2011-01-01

    leads to a robust induction of MICA mRNA expression, however the subsequent surface expression is potently hindered. Thus, VSV lines up with human cytomegalovirus (HCMV) and adenovirus, which actively subvert the immune system by negatively affecting NKG2D-ligand surface expression. VSV infection caused...

  2. Bmi1 promotes prostate tumorigenesis via inhibiting p16INK4A and p14ARF expression

    OpenAIRE

    Fan, Catherine; He, Lizhi; Kapoor, Anil; Gillis, Aubrey; Rybak, Adrian P.; Cutz, Jean-Claude; Tang, Damu

    2008-01-01

    Bmi1 promotes prostate tumorigenesis via inhibiting p16INK4A and p14ARF expression correspondence: Corresponding authors. Anil Kapoor is to be contacted at Tel.: +1 (905) 522 1155, ext. 33218; fax: +1 (905) 521 6195. Damu Tang, T3310, St. Joseph?s Hospital, 50 Charlton Ave East, Hamilton, ON, Canada L8N 4A6. Tel.: +1 (905) 522 1155, ext. 35168; fax: +1 (905) 521 6181. (Kapoor, Anil) correspondence: Corresponding authors. Anil Ka...

  3. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.

    Science.gov (United States)

    Olivera, A; Kohama, T; Edsall, L; Nava, V; Cuvillier, O; Poulton, S; Spiegel, S

    1999-11-01

    Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of

  4. Exercise promotes the expression of brain derived neurotrophic factor (BDNF) through the action of the ketone body β-hydroxybutyrate

    Science.gov (United States)

    Sleiman, Sama F; Henry, Jeffrey; Al-Haddad, Rami; El Hayek, Lauretta; Abou Haidar, Edwina; Stringer, Thomas; Ulja, Devyani; Karuppagounder, Saravanan S; Holson, Edward B; Ratan, Rajiv R; Ninan, Ipe; Chao, Moses V

    2016-01-01

    Exercise induces beneficial responses in the brain, which is accompanied by an increase in BDNF, a trophic factor associated with cognitive improvement and the alleviation of depression and anxiety. However, the exact mechanisms whereby physical exercise produces an induction in brain Bdnf gene expression are not well understood. While pharmacological doses of HDAC inhibitors exert positive effects on Bdnf gene transcription, the inhibitors represent small molecules that do not occur in vivo. Here, we report that an endogenous molecule released after exercise is capable of inducing key promoters of the Mus musculus Bdnf gene. The metabolite β-hydroxybutyrate, which increases after prolonged exercise, induces the activities of Bdnf promoters, particularly promoter I, which is activity-dependent. We have discovered that the action of β-hydroxybutyrate is specifically upon HDAC2 and HDAC3, which act upon selective Bdnf promoters. Moreover, the effects upon hippocampal Bdnf expression were observed after direct ventricular application of β-hydroxybutyrate. Electrophysiological measurements indicate that β-hydroxybutyrate causes an increase in neurotransmitter release, which is dependent upon the TrkB receptor. These results reveal an endogenous mechanism to explain how physical exercise leads to the induction of BDNF. DOI: http://dx.doi.org/10.7554/eLife.15092.001 PMID:27253067

  5. Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey

    Directory of Open Access Journals (Sweden)

    Tung Shu-Yun

    2011-04-01

    Full Text Available Abstract Background Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied. Results Several molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR. A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI genes (OnTI1, OnTI2 and OnTI3, which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses. Conclusions By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.

  6. TIPE2 Inhibits Lung Cancer Growth Attributing to Promotion of Apoptosis by Regulating Some Apoptotic Molecules Expression.

    Directory of Open Access Journals (Sweden)

    Qing-Qing Liu

    Full Text Available Recent studies found that TIPE2 was involved in cancer development. However, little is known about TIPE2 in lung cancer. Our study aims to clarify the role of TIPE2 in lung carcinogenesis. We examined the expression of TIPE2 in lung squamous cancer (LSC, small cell lung cancer and lung adenocarcinoma (AdC tissues and found that TIPE2 expression was lost in small cell lung cancer, compared with adjacent non-tumor tissues. Overexpression of TIPE2 significantly inhibited the growth of lung cancer cell H446 in vitro and even suppressed tumor formation in vivo. Flow cytometry analysis found TIPE2 overexpression promoted apoptosis of H446. In TIPE2 over-expression cells, caspase-3, caspase-9, and Bax were significantly up-regulated while Bcl-2 was down-regulated. Moreover, coincident results were shown by immunohistochemistry in tumors from nude mice. TIPE2 inhibited the phosphorylation of Akt, while promoting the phosphorylation of P38, but had no effect on IκBα and ERK pathway. Taken together, TIPE2 promoted lung cancer cell apoptosis through affecting apoptosis-related molecules caspase-3, caspase-9, Bcl-2 and Bax, possibly via regulating P38 and Akt pathways, indicating that TIPE2 might be a novel marker for lung cancer diagnosis and therapy.

  7. Association of diminished expression of RASSF1A with promoter methylation in primary gastric cancer from patients of central China

    International Nuclear Information System (INIS)

    Although methylation-mediated inactivation of expression of RASSF1A, a candidate tumor suppressor gene, has been observed in several human cancers, the data concerning alteration of RASSF1A expression and methylation in Chinese primary gastric cancer are scarce. Moreover, direct evidence showing the association between protein expression of RASSF1A and primary human cancers is lacking. The aim of this study was to investigate RASSF1A expression in tissue of primary gastric cancer (GC) at mRNA and protein levels, and to establish the possible relationship between DNA methylation status and protein expression of RASSF1A in Chinese. Fifty-four patients with primary gastric cancers were included in the study of RASSF1A mRNA expression and methylation status between the cancer tissue and the corresponding adjacent normal tissue. 20 out of 54 patients were included for study of RASSF1A protein expression. The expression of RASSF1A at mRNA and protein levels was determined by RT-PCR and Western-blotting, respectively. The RASSF1A promoter methylation was detected by methylation-specific PCR. RASSF1A mRNA and protein expressions in GC were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2589 ± 0.2407 vs 0.5448 ± 0.2971, P < 0.0001; 0.1874 ± 0.0737 vs 0.6654 ± 0.2201, P < 0.0001, respectively). Methylation frequency of RASSF1A in primary GC is higher than that in the corresponding normal tissues (66.7% vs. 14.8%, P < 0.0001). Furthermore, RASSF1A mRNA expression in methylation group of GC was further reduced when compared to the unmethylation group of GC (0.1384 ± 0.1142 vs. 0.5018 ± 0.2463, P < 0.0001). Expression of RASSF1A was reduced in tissue of GC at mRNA and protein levels. Diminished expression of RASSF1A was associated with the promoter methylation

  8. Rho GTPase signaling promotes constitutive expression and release of TGF-β2 by human trabecular meshwork cells.

    Science.gov (United States)

    Pervan, Cynthia L; Lautz, Jonathan D; Blitzer, Andrea L; Langert, Kelly A; Stubbs, Evan B

    2016-05-01

    Elevated intraocular pressure (IOP) is causally implicated in the pathophysiology of primary open-angle glaucoma (POAG). The molecular mechanisms responsible for elevated IOP remain elusive, but may involve aberrant expression and signaling of transforming growth factor (TGF)-β2 within the trabecular meshwork (TM). Consistent with previously published studies, we show here that exogenous addition of TGF-β2 to cultured porcine anterior segments significantly attenuates outflow facility in a time-dependent manner. By comparison, perfusing segments with a TGFβRI/ALK-5 antagonist (SB-431542) unexpectedly elicited a significant and sustained increase in outflow facility, implicating a role for TM-localized constitutive expression and release of TGF-β2. Consistent with this thesis, cultured primary or transformed (GTM3) quiescent human TM cells were found to constitutively express and secrete measurable amounts of biologically-active TGF-β2. Disrupting monomeric GTPase post-translational prenylation and activation with lovastatin or GGTI-298 markedly reduced constitutive TGF-β2 expression and release. Specifically, inhibiting the Rho subfamily of GTPases with C3 exoenzyme similarly reduced constitutive expression and secretion of TGF-β2. These findings suggest that Rho GTPase signaling, in part, regulates constitutive expression and release of biologically-active TGF-β2 from human TM cells. Localized constitutive expression and release of TGF-β2 by TM cells may promote or exacerbate elevation of IOP in POAG. PMID:26743044

  9. Cellular fibronectin 1 promotes VEGF-C expression, lymphangiogenesis and lymph node metastasis associated with human oral squamous cell carcinoma.

    Science.gov (United States)

    Morita, Yoshihiro; Hata, Kenji; Nakanishi, Masako; Omata, Tetsuji; Morita, Nobuo; Yura, Yoshiaki; Nishimura, Riko; Yoneda, Toshiyuki

    2015-10-01

    Lymph node metastasis (LNM) is associated with poor survival in patients with oral squamous cell carcinoma (OSCC). Vascular endothelial growth factor-C (VEGF-C) is thought to be responsible for increased lymphangiogenesis and LNM. Understanding of the mechanism by which VEGF-C expression is regulated in OSCC is thus important to design logic therapeutic interventions. We showed that inoculation of the SAS human OSCC cells expressing the venus GFP (V-SAS cells) into the tongue in nude mice developed LNM. V-SAS cells in LNM were isolated by FACS and re-inoculated into the tongue. This procedure was repeated eight times, establishing V-SAS-LM8 cells. Differential metastasis PCR array between the parental V-SAS and V-SAS-LM8 was performed to identify a molecule responsible for lymphangiogenesis and LNM. Fibronectin 1 (FN1) expression was elevated in V-SAS-LM8 cells compared to V-SAS-cells. V-SAS-LM8 tongue tumor showed increased expression of FN1 and VEGF-C, and promoted lymphangiogenesis and LNM compared with V-SAS tumor. Further, phosphorylation of focal adhesion kinase (FAK), a main downstream signaling molecule of FN1, was up-regulated, and epithelial-mesenchymal transition (EMT) was promoted in V-SAS-LM8 cells. Silencing of FN1 by shRNA in V-SAS-LM8 cells decreased FAK phosphorylation, VEGF-C expression and inhibited lymphangiogenesis and LNM. EMT was also reversed. The FAK phosphorylation inhibitor PF573228 also decreased VEGF-C expression and reversed EMT in V-SAS-LM8 cells. Finally, we detected intense FN1 expression in some clinical specimens obtained from OSCC patients with LNM. These results demonstrate that elevated expression of cellular FN1 and following activation of FAK lead to increased VEGF-C expression, lymphangiogenesis and LNM and promoted EMT in SAS human OSCC cells and suggest that FN1-phosphorylated FAK signaling cascade is a potential therapeutic target in the treatment of LNM in OSCC. PMID:26319373

  10. Cloning, expression and mo-lecular characterization of promoter elements from Ba-cillus pumilus

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Promoter elements from random chromosomal DNA of a rice epiphytic Bacillus pumilus were cloned into promoter probe shuttle vector ECE7 and sequenced. The results showed that these elements were all new DNA sequences. Six strong promoter elements were obtained by determination of CAT enzyme activity in both E. coli and B. pumilus. Transcription start sites of the cat mRNA were located by primer extension using total RNA. Comparison of the promoter sequences indicated that three of them contain -10 and -35 regions like B. pumilus s43 consensus sequence and another one is similar to B. pumilus s29. The other two have no typical consensus sequences of known sigma factors so far.

  11. Expressing activity of promoter elements of large intergenic region from cotton leaf curl virus in host plant*

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Cotton leaf curl virus (CLCuV) is a type of single-stranded DNAvirus, belonging to geminivirus of subgroup III. In order to determine the function of CLCuV large intergenic region (LIR), total DNA of CLCuV-infected cotton leaves was used as template, and fragment of LIR was obtained by PCR and inserted into clone vector. The fragment of LIR was fused with gus reporter gene and nos terminator in the orientation of transcription of virion sense and complementary sense respectively, and the plant expression vectors were constructed. GUS activity of Agrobacterium-mediated transgenic tobacco was measured. The result indicated that LIR showed strong promoter activity in complementary sense gene orientation. Average GUS activity of the complementary sense promoter was 5-6 times that of CaMV 35S promoter, and the highest GUS activity of individual plant was ten times of that of CaMV 35S promoter. Histochemical localization confirmed its activity in both mesophyll and vascular tissues. Activity of virion sense of LIR was rather low. Thus LIR isolated from CLCuV could be used as a novel strong promoter in plant genetic manipulation.

  12. Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer

    OpenAIRE

    V Shilpa; Rahul Bhagat; C S Premalata; V R Pallavi; Ramesh, G.; Lakshmi Krishnamoorthy

    2014-01-01

    Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O 6 -methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O 6 -position of guanine induced by alkylating agents. MGMT promoter hyperme...

  13. Isolation and molecular characterization of a stationary phase promoter useful for gene expression in Gordonia.

    Science.gov (United States)

    Singh, Pooja; Chachan, Sahil; Singhi, Divya; Srivastava, Preeti

    2016-10-10

    Gordonia are gram-positive bacteria belonging to Actinomycetes family with a wide variety of industrial and environmental applications. The genetic toolbox, however, is limited for manipulation of these organisms. In the present study, a new promoter has been isolated from Gordonia sp. IITR 100 and characterized in detail. The promoter was found to be functional in Escherichia coli. The minimal promoter was identified in a 166bp fragment by deletion mapping. The putative -35 and -10 hexamer showed four and five nucleotide matches respectively with the E. coli consensus sequence. Three direct repeats and an imperfect inverted repeat upstream to -35 were found. The isolated promoter was found to be six times stronger than the Pkan promoter observed by cloning lacZ downstream to each of them in a plasmid in E. coli. The β-galactosidase activity was maximum at stationary phase and found to be ~800MU for Gordonia sp. IITR 100 and E. coli. This is the first report of a stationary phase promoter isolated and characterized from Gordonia. PMID:27395430

  14. Berberine promotes the development of atherosclerosis and foam cell formation by inducing scavenger receptor A expression in macrophage

    Institute of Scientific and Technical Information of China (English)

    Ke Li; Wenqi Yao; Xiudan Zheng; Kan Liao

    2009-01-01

    Berberine is identified to lower the serum cholesterol level in human and hamster through the induction of low density lipoproteins (LDL) receptor in hepatic cells. To evaluate its potential in preventing atherosclerosis, the effect of berberine on atherosclerosis development in apolipoprotein E-deficient (apoE-/-) mice was investigated, in apoE-/-mice, berberine induced in vivo foam cell formation and promoted atherosclerosis development. The foam cell for-mation induced by berberine was also observed in mouse RAW264.7 cells, as well as in mouse and human primary macrophages. By inducing scavenger receptor A (SR-A) expression in macrophages, berberine increased the uptake of modified LDL (DiO-Ac-LDL). Berberine-induced SR-A expression was also observed in macrophage foam cells in vivo and in the cells at atherosclerotic lesion. Analysis in RAW264.7 cells indicated that berberine induced SR-A ex-pression by suppressing PTEN expression, which led to sustained Akt activation. Our results suggest that to evaluate the potential of a cholesterol-reducing compound in alleviating atherosclerosis, its effect on the cells involved in ath-erosclerosis development, such as macrophages, should also be considered. Promotion of foam cell formation could counter-balance the beneficial effect of lowering serum cholesterol.

  15. In vivo promoter analysis on refeeding response of hepatic sterol regulatory element-binding protein-1c expression

    International Nuclear Information System (INIS)

    Sterol regulatory element-binding protein (SREBP)-1c is the master regulator of lipogenic gene expression in liver. The mRNA abundance of SREBP-1c is markedly induced when animals are refed after starvation, although the regulatory mechanism is so far unknown. To investigate the mechanism of refeeding response of SREBP-1c gene expression in vivo, we generated a transgenic mouse model that carries 2.2 kb promoter region fused to the luciferase reporter gene. These transgenic mice exhibited refeeding responses of the reporter in liver and adipose tissues with extents essentially identical to those of endogenous SREBP-1c mRNA. The same results were obtained from experiments using adenovirus-mediated SREBP-1c-promoter-luciferase fusion gene transduction to liver. These data demonstrate that the regulation of SREBP-1c gene expression is at the transcription level, and that the 2.2 kb 5'-flanking region is sufficient for this regulation. Moreover, when these transgenic or adenovirus-infected mice were placed on insulin-depleted state by streptozotocin treatment, the reporter expression was upregulated as strongly as in control mice, demonstrating that this regulation is not dominated by serum insulin level. These mice are the first models to provide the mechanistic insight into the transcriptional regulation of SREBP-1c gene in vivo

  16. Up-regulation of TIMP-2 expression promotes SHI-1 leukemic cells proliferation and infiltration in immunodeficiency mice

    Institute of Scientific and Technical Information of China (English)

    Li Zhenjiang; Chen Zixing; Cen Jiannong; He Jun; Qiu Qiaocheng; Xue Yongquan

    2014-01-01

    Background MMPs and TIMPs play important roles in tumor angiogenesis and invasion.Studies have shown that TIMP-2 has two roles in tumor invasion.However,its role in leukemic infiltration has not been well investigated.This study explored the roles of TIMP-2 in extramedullary infiltration of acute monocytic leukemic SHI-1 cells both in vitro and in vitro.Methods A retroviral vector carrying the human TIMP-2 cDNA was constructed and transfected into the monocytic leukemic cell line SHI-1.The expression of TIMP-2 in the positive clones was determined.The proliferation of SHI-1 cells was examined by MTT assay.Trans-Matrigel invasion assays were used to investigate the infiltration ability in vitro.SHI-1 cells were intravenously injected into pre-treated nu/nu mice to investigate the infiltration ability feature in vitro.Results The expression of TIMP-2 on the cell membrane was significantly elevated in SHI-1/TIMP-2 cells.Over-expression of TIMP-2 promoted the cells proliferation and the invasions in vitro.The SHI-1/TIMP-2 cells demonstrated higher infiltration ability when intravenously injected into nu/nu mice.Conclusion Over-expression of TIMP-2,especially on the cell membrane,may play important roles in promoting the proliferation and infiltration of SHI-1 leukemic cells.

  17. Mini cardiopulmonary bypass: Anesthetic considerations

    OpenAIRE

    Alsatli, Raed A.

    2012-01-01

    This review article is going to elaborate on the description, components, and advantages of mini-cardiopulmonary bypass (mini-CPB), with special reference to the anesthetic management and fast track anesthesia with mini-CPB. There are several clinical advantages of mini-CPB like, reduced inflammatory reaction to the pump, reduced need for allogenic blood transfusion and lower incidence of postoperative neurological complications. There are certainly important points that have to be considered...

  18. Promoters of Cancer Genes for Recombinant Protein Expression in Human Cancer Cell Lines

    OpenAIRE

    Behrouz Farhadi; Tamouchin Moharrami; Mohammad Pourhassan-Moghaddam; Kazem Nejati-Koshki

    2012-01-01

    Introduction: Production of complex human recombinant proteins is an important issue in medical biotechnology. These proteins are mostly expressed in non-human mammalian host cells. This has some problems including non-human post-translational modifications, application of high-cost agents for inducing protein expression and low yields. Therefore, it is necessary to use new expression systems to overcome the indicated challenges. Methods: In this paper, we hypothesize the application of promo...

  19. Local expression of B7-H1 promotes organ-specific autoimmunity and transplant rejection

    OpenAIRE

    Subudhi, Sumit K.; Zhou, Ping; Yerian, Lisa M.; Chin, Robert K.; Lo, James C.; Anders, Robert A.; Sun, Yonglian; Chen, Lieping; Wang, Yang; Alegre, Maria-Luisa; Fu, Yang-Xin

    2004-01-01

    A number of studies have suggested B7-H1, a B7 family member, inhibits T cell responses. Therefore, its expression on nonlymphoid tissues has been proposed to prevent T cell–mediated tissue destruction. To test this hypothesis, we generated transgenic mice that expressed B7-H1 on pancreatic islet β cells. Surprisingly, we observed accelerated rejection of transplanted allogeneic B7-H1–expressing islet β cells. Furthermore, transgenic B7-H1 expression broke immune tolerance, as some of the mic...

  20. Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

    Directory of Open Access Journals (Sweden)

    Nannenga Brent L

    2011-12-01

    Full Text Available Abstract Background Membrane proteins (MPs populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function. Results Using the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells, toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host. Conclusions Our system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

  1. Myeloperoxidase Promoter Polymorphism −463G Is Associated With More Severe Clinical Expression of Cystic Fibrosis Pulmonary Disease

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    Wanda F. Reynolds

    2006-01-01

    Full Text Available The severity of cystic fibrosis (CF pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the −463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation.

  2. Hypomethylation of proximal CpG motif of interleukin-10 promoter regulates its expression in human rheumatoid arthritis

    Institute of Scientific and Technical Information of China (English)

    Li-hong FU; Chun-ling MA; Bin CONG; Shu-jin LI; Hai-ying CHEN; Jing-ge ZHANG

    2011-01-01

    Aim:The promoter of human interleukin-10 (IL10),a cytokine crucial for suppressing inflammation and regulating immune responses,contains an interspecies-conserved sequence with CpG motifs.The aim of this study was to investigate whether methylation of CpG motifs could regulate the expression of IL10 in rheumatoid arthritis (RA).Methods:Bioinformatic analysis was conducted to identify the interspecies-conserved sequence in human,macaque and mouse IL10 genes.Peripheral blood mononuclear cells (PBMCs) from 20 RA patients and 20 health controls were collected.The PBMCs from 6 patients were cultured in the presence or absence of 5-azacytidine (5 μmol/L).The mRNA and protein levels of IL10 were examined using RT-PCR and ELISA,respectively.The methylation of CpGs in the IL10 promoter was determined by pyrosequencing.Chromatin immunoprecipitation (CHIP) assays were performed to detect the cyclic AMP response element-binding protein (CREB)-DNA interactions.Results:One interspecies-conserved sequence was found within the IL10 promoter.The upstream CpGs at -408,-387,-385,and -355 bp were hypermethylated in PBMCs from both the RA patients and healthy controls.In contrast,the proximal CpG at -145 was hypomethylated to much more extent in the RA patients than in the healthy controls (P=0.016),which was correlated with higher IL10 mRNA and serum levels.In the 5-azacytidine-treated PBMCs,the CpG motifs were demethylated,and the expression levels of IL10 mRNA and protein was significantly increased.CHIP assays revealed increased phospho-CREB binding to the IL10 promoter.Conclusion:The methylation of the proximal CpGs in the IL10 promoter may regulate gene transcription in RA.

  3. High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter

    Directory of Open Access Journals (Sweden)

    D’Urzo Nunzia

    2013-02-01

    Full Text Available Abstract Background In past years research has focused on the development of alternative Gram positive bacterial expression systems to produce industrially relevant proteins. Brevibacillus choshinensis is an easy to handle non-sporulating bacterium, lacking extracellular proteases, that has been already shown to provide a high level of recombinant protein expression. One major drawback, limiting the applicability of the Brevibacillus expression system, is the absence of expression vectors based on inducible promoters. Here we used the PxylA inducible promoter, commonly employed in other Bacillae expression systems, in Brevibacillus. Results Using GFP, α-amylase and TcdA-GT as model proteins, high level of intracellular protein expression (up to 250 mg/L for the GFP was achieved in Brevibacillus, using the pHis1522 vector carrying the B. megaterium xylose-inducible promoter (PxylA. The GFP expression yields were more than 25 fold higher than those reported for B. megaterium carrying the same vector. All the tested proteins show significant increment in their expression levels (2-10 folds than those obtained using the available plasmids based on the P2 constitutive promoter. Conclusion Combining the components of two different commercially available Gram positive expression systems, such as Brevibacillus (from Takara Bio and B. megaterium (from Mobitec, we demonstrate that vectors based on the B. megaterium PxylA xylose inducible promoter can be successfully used to induce high level of intracellular expression of heterologous proteins in Brevibacillus.

  4. Over-expression of LSD1 promotes proliferation, migration and invasion in non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Tangfeng Lv

    Full Text Available BACKGROUND: Lysine specific demethylase 1 (LSD1 has been identified and biochemically characterized in epigenetics, but the pathological roles of its dysfunction in lung cancer remain to be elucidated. The aim of this study was to evaluate the prognostic significance of LSD1 expression in patients with non-small cell lung cancer (NSCLC and to define its exact role in lung cancer proliferation, migration and invasion. METHODS: The protein levels of LSD1 in surgically resected samples from NSCLC patients were detected by immunohistochemistry or Western blotting. The mRNA levels of LSD1 were detected by qRT-PCR. The correlation of LSD1 expression with clinical characteristics and prognosis was determined by statistical analysis. Cell proliferation rate was assessed by MTS assay and immunofluorescence. Cell migration and invasion were detected by scratch test, matrigel assay and transwell invasion assay. RESULTS: LSD1 expression was higher in lung cancer tissue more than in normal lung tissue. Our results showed that over-expression of LSD1 protein were associated with shorter overall survival of NSCLC patients. LSD1 was localized mainly to the cancer cell nucleus. Interruption of LSD1 using siRNA or a chemical inhibitor, pargyline, suppressed proliferation, migration and invasion of A549, H460 and 293T cells. Meanwhile, over-expression of LSD1 enhanced cell growth. Finally, LSD1 was shown to regulate epithelial-to-mesenchymal transition in lung cancer cells. CONCLUSIONS: Over-expression of LSD1 was associated with poor prognosis in NSCLC, and promoted tumor cell proliferation, migration and invasion. These results suggest that LSD1 is a tumor-promoting factor with promising therapeutic potential for NSCLC.

  5. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-κB (RANK)

    International Nuclear Information System (INIS)

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A-/- mice. The femoral osseous density of SR-A-/- mice was higher than that of SR-A+/+ mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A-/- mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  6. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    Energy Technology Data Exchange (ETDEWEB)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  7. Angiopoietin 2 stimulates TIE2-expressing monocytes to suppress T cell activation and to promote regulatory T cell expansion.

    Science.gov (United States)

    Coffelt, Seth B; Chen, Yung-Yi; Muthana, Munitta; Welford, Abigail F; Tal, Andrea O; Scholz, Alexander; Plate, Karl H; Reiss, Yvonne; Murdoch, Craig; De Palma, Michele; Lewis, Claire E

    2011-04-01

    Angiopoietin 2 (ANGPT2) is a proangiogenic cytokine whose expression is often upregulated by endothelial cells in tumors. Expression of its receptor, TIE2, defines a highly proangiogenic subpopulation of myeloid cells in circulation and tumors called TIE2-expressing monocytes/macrophages (TEMs). Genetic depletion of TEMs markedly reduces tumor angiogenesis in various tumor models, emphasizing their essential role in driving tumor progression. Previously, we demonstrated that ANGPT2 augments the expression of various proangiogenic genes, the potent immunosuppressive cytokine, IL-10, and a chemokine for regulatory T cells (Tregs), CCL17 by TEMs in vitro. We now show that TEMs also express higher levels of IL-10 than TIE2(-) macrophages in tumors and that ANGPT2-stimulated release of IL-10 by TEMs suppresses T cell proliferation, increases the ratio of CD4(+) T cells to CD8(+) T cells, and promotes the expansion of CD4(+)CD25(high)FOXP3(+) Tregs. Furthermore, syngeneic murine tumors expressing high levels of ANGPT2 contained not only high numbers of TEMs but also increased numbers of Tregs, whereas genetic depletion of tumor TEMs resulted in a marked reduction in the frequency of Tregs in tumors. Taken together, our data suggest that ANGPT2-stimulated TEMs represent a novel, potent immunosuppressive force in tumors. PMID:21368233

  8. Activation of corticotropin releasing factor receptors up regulates collagen production by hepatic stellate cells via promoting p300 expression.

    Science.gov (United States)

    Wang, Changzhen; Yang, Shan; Huang, Jingjing; Chen, Songlin; Li, Yuan; Li, Quanqiang

    2016-05-01

    Liver fibrosis is characterized with the over expression and excessive accumulation of extracellular matrix proteins, including collagens. The causative factors in the over production of collagens are not fully understood. This study aims to test a hypothesis that activation of corticotropin releasing factor receptors up regulates the expression of collagen in hepatic stellate cells. In this study, human hepatic stellate cell line, LX-2 cells were cultured. Expression of collagens by LX-2 cells was assessed by real time RT-PCR, Western blotting. The results showed that, upon exposure to urocortin in the culture, LX-2 cells (a human hepatic stellate cell line) increased the expression of collagen IV (Col4) markedly. The exposure to urocortin also enhanced the levels of pTip60, H3K9, RNA polymerase II and forkhead box protein 3 at the collagen promoter locus as well as increase in the expression of Col4 mRNA and protein in the cells. Blocking p300 efficiently suppressed the urocortin-induced Col4 expression in LX-2 cells and unveiled an apoptosis-inducing effect of urocortin. In conclusion, activation of CRF receptors is capable of enforcing the production of Col4 by LX-2 cells via up regulating the p300 pathway, which may contribute to the development of liver fibrosis. PMID:26756093

  9. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yu, E-mail: xuyu1001@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liu, Zhengchun, E-mail: l135027@126.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Kong, Haiyan, E-mail: suppleant@163.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Sun, Wenjie, E-mail: wendy11240325@163.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liao, Zhengkai, E-mail: fastbeta@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: happyzhoufx@sina.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  10. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    International Nuclear Information System (INIS)

    Highlights: → A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. → The promoter was characterized with radiation-inducibility and tumor-specificity. → Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. → Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  11. A comparative analysis of green fluorescent protein and -glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

    Indian Academy of Sciences (India)

    P Kavita; Pradeep Kumar Burma

    2008-09-01

    The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, -glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

  12. MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy

    Directory of Open Access Journals (Sweden)

    Melguizo Consolación

    2012-12-01

    Full Text Available Abstract Background The CD133 antigen is a marker of radio- and chemo-resistant stem cell populations in glioblastoma (GBM. The O6-methylguanine DNA methyltransferase (MGMT enzyme is related with temozolomide (TMZ resistance. Our propose is to analyze the prognostic significance of the CD133 antigen and promoter methylation and protein expression of MGMT in a homogenous group of GBM patients uniformly treated with radiotherapy and TMZ. The possible connection between these GBM markers was also investigated. Methods Seventy-eight patients with GBM treated with radiotherapy combined with concomitant and adjuvant TMZ were analyzed for MGMT and CD133. MGMT gene promoter methylation was determined by methylation-specific polymerase chain reaction after bisulfite treatment. MGMT and CD133 expression was assessed immunohistochemically using an automatic quantification system. Overall and progression-free survival was calculated according to the Kaplan–Meier method. Results The MGMT gene promoter was found to be methylated in 34 patients (44.7% and unmethylated in 42 patients (55.3%. A significant correlation was observed between MGMT promoter methylation and patients’ survival. Among the unmethylated tumors, 52.4% showed low expression of MGMT and 47.6% showed high-expression. Among methylated tumors, 58.8% showed low-expression of MGMT and 41.2% showed high-expression. No correlation was found between MGMT promoter methylation and MGMT expression, or MGMT expression and survival. In contrast with recent results, CD133 expression was not a predictive marker in GBM patients. Analyses of possible correlation between CD133 expression and MGMT protein expression or MGMT promoter methylation were negative. Conclusions Our results support the hypothesis that MGMT promoter methylation status but not MGMT expression may be a predictive biomarker in the treatment of patients with GBM. In addition, CD133 should not be used for prognostic evaluation of these

  13. Effects of Gene Orientation and Use of Multiple Promoters on the Expression of XYL1 and XYL2 in Saccharomyces cerevisiae

    Science.gov (United States)

    Bae, Ju Yun; Laplaza, José; Jeffries, Thomas W.

    Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We assembled d-xylose reductase (XYL1) and d-xylitol dehydrogenase (XYL2) in four ways. Each pair of genes was placed in two different tandem (l→2→ or √1√2), convergent (1→√2), and divergent (√1 2→) orientations in autonomous plasmids. The TEF1 promoter was used to drive XYL1 and the TDH3 promoter to drive XYL2 in each of the constructs. The effects of gene orientation on growth, transcription, and enzyme activity were analyzed. The transcription level as measured by quantitative PCR (q-PCR) correlated with enzyme activities, but our data did not show a significant effect of gene orientation. To test the possible dilution of promoter strength due to multiple use of the same promoter, we examined the level of expression of XYL1 driven by either the TEF1 or TDH3 promoter when carried on a single copy plasmid. We then coexpressed XYL2 from either a single or multicopy plasmid, which was also driven by the same promoter. XYL2 transcript and enzyme expression increased with plasmid copy number, while the expression of XYLl was constant regardless of the number of other TEF1 or TDH3 promoters present in the cell. According to our data, there is no significant effect of gene orientation or multiple promoter use on gene transcription and translation when genes are expressed from plasmids; however, other factors could affect expression of adjacent genes in chromosomes.

  14. Slug-upregulated miR-221 promotes breast cancer progression through suppressing E-cadherin expression.

    Science.gov (United States)

    Pan, Yi; Li, Jing; Zhang, Yaqin; Wang, Nan; Liang, Hongwei; Liu, Yuan; Zhang, Chen-Yu; Zen, Ke; Gu, Hongwei

    2016-01-01

    It is generally regarded that E-cadherin is downregulated during tumorigenesis via Snail/Slug-mediated E-cadherin transcriptional reduction. However, this transcriptional suppressive mechanism cannot explain the failure of producing E-cadherin protein in metastatic breast cancer cells after overexpressing E-cadherin mRNA. Here we reveal a novel mechanism that E-cadherin is post-transcriptionally regulated by Slug-promoted miR-221, which serves as an additional blocker for E-cadherin expression in metastatic tumor cells. Profiling the predicted E-cadherin-targeting miRNAs in breast cancer tissues and cells showed that miR-221 was abundantly expressed in breast tumor and metastatic MDA-MB-231 cells and its level was significantly higher in breast tumor or MDA-MB-231 cells than in distal non-tumor tissue and low-metastatic MCF-7 cells, respectively. MiR-221, which level inversely correlated with E-cadherin level in breast cancer cells, targeted E-cadherin mRNA open reading frame (ORF) and suppressed E-cadherin protein expression. Depleting or increasing miR-221 level in breast cancer cells induced or decreased E-cadherin protein level, leading to suppressing or promoting tumor cell progression, respectively. Moreover, miR-221 was specifically upregulated by Slug but not Snail. TGF-β treatment enhanced Slug activity and thus increased miR-221 level in MCF-7 cells. In summary, our results provide the first evidence that Slug-upregulated miR-221 promotes breast cancer progression via reducing E-cadherin expression. PMID:27174021

  15. Over-expression of ST3Gal-I promotes mammary tumorigenesis

    DEFF Research Database (Denmark)

    Picco, Gianfranco; Julien, Sylvain; Brockhausen, Inka; Beatson, Richard; Antonopoulos, Aristotelis; Haslam, Stuart; Mandel, Ulla; Dell, Anne; Pinder, Sarah; Taylor-Papadimitriou, Joyce; Burchell, Joy

    2010-01-01

    3Gal-I adds sialic acid to the galactose residue of core 1 (Galbeta1,3GalNAc) O-glycans and this enzyme is over-expressed in breast cancer resulting in the expression of sialylated core 1 glycans. In order to study the role of ST3Gal-I in mammary tumor development, we developed transgenic mice that...

  16. The positives of negative emotions: willingness to express negative emotions promotes relationships.

    Science.gov (United States)

    Graham, Steven M; Huang, Julie Y; Clark, Margaret S; Helgeson, Vicki S

    2008-03-01

    Four studies support the hypothesis that expressing negative emotion is associated with positive relationship outcomes, including elicitation of support, building of new close relationships, and heightening of intimacy in the closest of those relationships. In Study 1, participants read vignettes in which another person was experiencing a negative emotion. Participants reported they would provide more help when the person chose to express the negative emotion. In Study 2, participants watched a confederate preparing for a speech. Participants provided more help to her when she expressed nervousness. In Study 3, self-reports of willingness to express negative emotions predicted having more friends, controlling for demographic variables and extraversion. In Study 4, self-reports of willingness to express negative emotion measured prior to arrival at college predicted formation of more relationships, greater intimacy in the closest of those relationships, and greater received support from roommates across participants' first semester of college. PMID:18272807

  17. Increased expression of the dsRNA-activated protein kinase PKR in breast cancer promotes sensitivity to doxorubicin.

    Directory of Open Access Journals (Sweden)

    Richard L Bennett

    Full Text Available It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H(2O(2-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR

  18. Intron V, not intron I of human thrombopoietin, improves expression in the milk of transgenic mice regulated by goat beta-casein promoter

    OpenAIRE

    Yan Li; Hu Hao; Mingqian Zhou; Hongwei Zhou; Jianbin Ye; Lijun Ning; Yunshan Ning

    2015-01-01

    Introns near 5′ end of genes generally enhance gene expression because of an enhancer /a promoter within their sequence or as intron-mediated enhancement. Surprisingly, our previous experiments found that the vector containing the last intron (intron V) of human thromobopoietin (hTPO) expressed higher hTPO in cos-1 cell than the vector containing intron I regulated by cytomegalovirus promoter. Moreover, regulated by 1.0 kb rat whey acidic protein promoter, hTPO expression was higher in transg...

  19. Simian virus 40 promoters direct expression of the tetracycline gene in plasmid pACYC184.

    OpenAIRE

    Jenkins, F J; Howett, M K; Rapp, F

    1983-01-01

    Insertion of HindIII DNA fragments into the HindIII site of plasmid pACYC184 destroys the promoter of the plasmid tetracycline resistance gene and causes Escherichia coli cells harboring recombinant plasmids to be tetracycline sensitive and chloramphenicol resistant. The HindIII-C DNA fragment of simian virus 40 contains the two virus promoters and the virus origin of replication. We report the isolation of recombinant plasmids that contained the simian virus 40 HindIII-C DNA fragment at the ...

  20. Placenta-specific Expression of the Interleukin-2 (IL-2) Receptor β Subunit from an Endogenous Retroviral Promoter*

    Science.gov (United States)

    Cohen, Carla J.; Rebollo, Rita; Babovic, Sonja; Dai, Elizabeth L.; Robinson, Wendy P.; Mager, Dixie L.

    2011-01-01

    The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the β subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rβ protein in the placenta, suggesting that IL-2Rβ undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression. PMID:21865161

  1. Bypass of hexavalent chromium-induced growth arrest by a protein tyrosine phosphatase inhibitor: Enhanced survival and mutagenesis

    International Nuclear Information System (INIS)

    Although the consequences of genotoxic injury include cell cycle arrest and apoptosis, cell survival responses after genotoxic injury can produce intrinsic death-resistance and contribute to the development of a transformed phenotype. Protein tyrosine phosphatases (PTPs) are integral components of key survival pathways, and are responsible for their inactivation, while PTP inhibition is often associated with enhanced cell proliferation. Our aim was to elucidate signaling events that modulate cell survival after genotoxin exposure. Diploid human lung fibroblasts (HLF) were treated with Cr(VI) (as Na2CrO4), the soluble oxyanionic dissolution product of certain particulate chromates, which are well-documented human respiratory carcinogens. In vitro soluble Cr(VI) induces a wide spectrum of DNA damage, in both the presence and absence of a broad-range PTP inhibitor, sodium orthovanadate (SOV). Notably, SOV abrogated Cr(VI)-induced clonogenic lethality. The enhanced survival of Cr(VI)-exposed cells after SOV treatment was predominantly due to a bypass of cell cycle arrest, as there was no effect of the PTP inhibitor on Cr-induced apoptosis. Moreover, the SOV effect was not due to decreased Cr uptake as evidenced by unchanged Cr-DNA adduct burden. Additionally, the bypass of Cr-induced growth arrest by SOV was accompanied by a decrease in Cr(VI)-induced expression of cell cycle inhibiting genes, and an increase in Cr(VI)-induced expression of cell cycle promoting genes. Importantly, SOV resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of certain types of DNA damage may lead to increased genomic instability, via bypass of cell cycle checkpoints

  2. Bypass of hexavalent chromium-induced growth arrest by a protein tyrosine phosphatase inhibitor: Enhanced survival and mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Dongsoon; Camilli, Tura C. [Department of Pharmacology and Physiology, George Washington University Medical Center, Washington, DC (United States); Chun, Gina; Lal, Madhu; Wright, Kristen [Department of Pharmacology and Physiology, George Washington University Medical Center, Washington, DC (United States); Program in Molecular Medicine, George Washington University Medical Center, Washington, DC (United States); O' Brien, Travis J. [Department of Pharmacology and Physiology, George Washington University Medical Center, Washington, DC (United States); Program in Molecular Medicine, George Washington University Medical Center, Washington, DC (United States); GW Cancer Institute, George Washington University Medical Center, Washington, DC (United States); Patierno, Steven R. [Department of Pharmacology and Physiology, George Washington University Medical Center, Washington, DC (United States); Department of Medicine, George Washington University Medical Center, Washington, DC (United States); Program in Molecular Medicine, George Washington University Medical Center, Washington, DC (United States); GW Cancer Institute, George Washington University Medical Center, Washington, DC (United States); Ceryak, Susan [Department of Pharmacology and Physiology, George Washington University Medical Center, Washington, DC (United States); Department of Medicine, George Washington University Medical Center, Washington, DC (United States); Program in Molecular Medicine, George Washington University Medical Center, Washington, DC (United States); GW Cancer Institute, George Washington University Medical Center, Washington, DC (United States)], E-mail: phmsmc@gwumc.edu

    2009-01-15

    Although the consequences of genotoxic injury include cell cycle arrest and apoptosis, cell survival responses after genotoxic injury can produce intrinsic death-resistance and contribute to the development of a transformed phenotype. Protein tyrosine phosphatases (PTPs) are integral components of key survival pathways, and are responsible for their inactivation, while PTP inhibition is often associated with enhanced cell proliferation. Our aim was to elucidate signaling events that modulate cell survival after genotoxin exposure. Diploid human lung fibroblasts (HLF) were treated with Cr(VI) (as Na{sub 2}CrO{sub 4}), the soluble oxyanionic dissolution product of certain particulate chromates, which are well-documented human respiratory carcinogens. In vitro soluble Cr(VI) induces a wide spectrum of DNA damage, in both the presence and absence of a broad-range PTP inhibitor, sodium orthovanadate (SOV). Notably, SOV abrogated Cr(VI)-induced clonogenic lethality. The enhanced survival of Cr(VI)-exposed cells after SOV treatment was predominantly due to a bypass of cell cycle arrest, as there was no effect of the PTP inhibitor on Cr-induced apoptosis. Moreover, the SOV effect was not due to decreased Cr uptake as evidenced by unchanged Cr-DNA adduct burden. Additionally, the bypass of Cr-induced growth arrest by SOV was accompanied by a decrease in Cr(VI)-induced expression of cell cycle inhibiting genes, and an increase in Cr(VI)-induced expression of cell cycle promoting genes. Importantly, SOV resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of certain types of DNA damage may lead to increased genomic instability, via bypass of cell cycle checkpoints.

  3. GalNAc-T14 promotes metastasis through Wnt dependent HOXB9 expression in lung adenocarcinoma.

    Science.gov (United States)

    Kwon, Ok-Seon; Oh, Ensel; Park, Jeong-Rak; Lee, Ji-Seon; Bae, Gab-Yong; Koo, Jae-Hyung; Kim, Hyongbum; Choi, Yoon L; Choi, Young Soo; Kim, Jhingook; Cha, Hyuk-Jin

    2015-12-01

    While metastasis, the main cause of lung cancer-related death, has been extensively studied, the underlying molecular mechanism remains unclear. A previous clinicogenomic study revealed that expression of N-acetylgalactosaminyltransferase (GalNAc-T14), is highly inversely correlated with recurrence-free survival in those with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism(s) has not been determined. Here, we showed that GalNAc-T14 expression was positively associated with the invasive phenotype. Microarray and biochemical analyses revealed that HOXB9, the expression of which was increased in a GalNAc-T14-dependent manner, played an important role in metastasis. GalNAc-T14 increased the sensitivity of the WNT response and increased the stability of the β-catenin protein, leading to induced expression of HOXB9 and acquisition of an invasive phenotype. Pharmacological inhibition of β-catenin in GalNAc-T14-expressing cancer cells suppressed HOXB9 expression and invasion. A meta-analysis of clinical genomics data revealed that expression of GalNAc-T14 or HOXB9 was strongly correlated with reduced recurrence-free survival and increased hazard risk, suggesting that targeting β-catenin within the GalNAc-T14/WNT/HOXB9 axis may be a novel therapeutic approach to inhibit metastasis in NSCLC. PMID:26544896

  4. Stromelysin-1 (MMP-3) expression driven by a macrophage-specific promoter results in reduced viability in transgenic mice.

    Science.gov (United States)

    Fabunmi, R P; Moore, K J; Libby, P; Freeman, M W

    2000-02-01

    Macrophage expression of matrix degrading metalloproteinases (MMPs) in human atheroma has been found to occur in rupture-prone areas of plaques. To investigate the effect of metalloproteinase activity on plaque stability, we attempted to generate mice that expressed a stromelysin-1 (MMP-3) transgene specifically in macrophages. Promoter sequences taken from a macrophage-tropic lentivirus (visna) were used to drive transgene expression. The transgene construct was expressed in macrophages in vitro and its autoactivation was established by casein zymography. Transgenic mice generated with this construct died at or before birth. No gross anatomical changes were observed in these mice. Embryos arising from a second round of oocyte injections with the transgene were examined at day 16 of gestation. Of the products of conception, approximately 40% resulted in vacant conceptuses. Only one animal of 38 examined carried the transgene and its expression of MMP-3 mRNA at E16 was faintly detected by RT-PCR. When a non-toxic reporter gene, luciferase, was substituted for the MMP-3 cDNA, healthy transgenic mice were produced that expressed the reporter gene in a wide variety of tissue macrophages, including those located in the brain, testis, lung, and thymus. These studies suggest that constitutive expression of MMP-3 in diverse populations of tissue macrophages leads to prenatal or neonatal death in the mouse. It appears likely that more sophisticated transcriptional control of MMP-3 expression will be required in order to generate stromelysin-1 transgenic mice that could be useful models for studying overexpression of this metalloproteinase's activity in the lesional macrophages of atherosclerotic plaques. PMID:10657574

  5. Epicardial ultrasound in coronary artery bypass surgery

    OpenAIRE

    Budde, R.P.J.

    2005-01-01

    Chapter 1 Coronary artery bypass surgery (CABG) is traditionally performed via a median sternotomy approach on cardiopulmonary bypass (arrested heart). Since the mid 1990ties, beating heart, minimally invasive and even totally endoscopic CABG are (re)explored. In all approaches to CABG, the surgeon may face several intraoperative difficulties: 1. Localization of the target coronary artery for bypass grafting. 2. Selection of the optimal anastomotic site on the target coronary artery. 3. Asses...

  6. RASSF1A promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis

    Directory of Open Access Journals (Sweden)

    Eilers Tyark

    2007-07-01

    Full Text Available Abstract Background Epigenetic silencing of the RAS association domain family 1A (RASSF1A tumor suppressor gene promoter has been demonstrated in renal cell carcinoma (RCC as a result of promoter hypermethylation. Contradictory results have been reported for RASSF1A methylation in normal kidney, thus it is not clear whether a significant difference between RASSF1A methylation in normal and tumor cells of the kidney exists. Moreover, RASSF1A expression has not been characterized in tumors or normal tissue as yet. Results Using combined bisulfite restriction analysis (COBRA we compared RASSF1A methylation in 90 paired tissue samples obtained from primary kidney tumors and corresponding normal tissue. Bisulfite sequence analysis was carried out using both pooled amplicons from the tumor and normal tissue groups and subclones obtained from a single tissue pair. Expression of RASSF1A was analyzed by the use of tissue arrays and immunohistochemistry. We found significantly increased methylation in tumor samples (mean methylation, 20% compared to corresponding normal tissues (mean methylation, 11%; P Conclusion Our methylation and expression data support the hypothesis that RASSF1A is involved in early tumorigenesis of renal cell carcinoma.

  7. Interleukin 17A Promotes Hepatocellular Carcinoma Metastasis via NF-kB Induced Matrix Metalloproteinases 2 and 9 Expression

    Science.gov (United States)

    Li, Jian; Lau, George Ka-Kit; Chen, Leilei; Dong, Sui-sui; Lan, Hui-Yao; Huang, Xiao-Ru; Li, Yan; Luk, John M.; Yuan, Yun-Fei; Guan, Xin-yuan

    2011-01-01

    Background  IL-17A is a pro-inflammatory cytokine that plays important role in inflammatory disease pathology and tumor microenvironment. The aim of this study is to investigate the effect of IL-17A on the progression of hepatocellular carcinoma (HCC). Methodology and Principal Finding Expression pattern of IL-17A in clinical HCC samples (n = 43) was determined by immunohistochemistry staining. Transcript levels of MMP2, MMP9 and IL-17A were measured in another 50 pairs (including tumor and related non-tumor tissues) HCC samples. Cell growth, focus formation, cell migration, invasion and western blot assays were used to characterize the functional and signaling mechanisms in IL-17A-treated HCC. Association study was used to identify clinical significance of IL-17A in HCC. Compared with paired non-tumor tissue, higher frequency of IL-17A-positive cells was detected in tumor tissues in HCCs with metastasis, and the frequency of IL-17A-positive cells was also significantly associated with poor prognosis of HCC (P = 0.01). Functional study found that IL-17A could promote HCC cell migration and invasion. Further molecular analysis also showed that IL-17A could upregulate MMP2 and MMP9 expression via NF-κB signaling activation. Conclusions  IL-17A could promote HCC metastasis by the upregulation of MMP2 and MMP9 expression via activating NF-κB signaling pathway. PMID:21760911

  8. Interleukin-1β induces fibroblast growth factor 2 expression and subsequently promotes endothelial progenitor cell angiogenesis in chondrocytes.

    Science.gov (United States)

    Chien, Szu-Yu; Huang, Chun-Yin; Tsai, Chun-Hao; Wang, Shih-Wei; Lin, Yu-Min; Tang, Chih-Hsin

    2016-05-01

    Arthritis is a process of chronic inflammation that results in joint damage. IL (interleukin)-1β is an inflammatory cytokine that acts as a key mediator of cartilage degradation, and is abundantly expressed in arthritis. Neovascularization is one of the pathological characteristics of arthritis. However, the role of IL-1β in the angiogenesis of chondrocytes remains unknown. In the present study, we demonstrate that stimulating chondrocytes (ATDC5) with IL-1β increased the expression of FGF (fibroblast growth factor)-2, a potent angiogenic inducer, and then promoted EPC (endothelial progenitor cell) tube formation and migration. In addition, FGF-2-neutralizing antibody abolished ATDC5-conditional medium-mediated angiogenesis in vitro, as well as its angiogenic effects in the CAM (chick chorioallantoic membrane) assay and Matrigel plug nude mice model in vivo. IHC (immunohistochemistry) staining from a CIA (collagen-induced arthritis) mouse model also demonstrates that arthritis increased the expression of IL-1β and FGF-2, as well as EPC homing in articular cartilage. Moreover, IL-1β-induced FGF-2 expression via IL-1RI (type-1 IL-1 receptor), ROS (reactive oxygen species) generation, AMPK (AMP-activated protein kinase), p38 and NF-κB (nuclear factor κB) pathway has been demonstrated. On the basis of these findings, we conclude that IL-1β promotes FGF-2 expression in chondrocytes through the ROS/AMPK/p38/NF-κB signalling pathway and subsequently increases EPC angiogenesis. Therefore IL-1β serves as a link between inflammation and angiogenesis during arthritis. PMID:26811540

  9. Expression of aquaporin-1 in SMMC-7221 liver carcinoma cells promotes cell migration

    Institute of Scientific and Technical Information of China (English)

    LI Yongming; FENG Xuechao; YANG Hong; MA Tonghui

    2006-01-01

    Migration of tumor cells is a crucial step in tumor invasion and metastasis. Here we provide evidence that aquaporin expression is involved in tumor cell migration. RT-PCR, immunofluorescence and Western blot analysis demonstrated the AQP1 protein expression on the plasma membrane of SMMC-7221 human hepatoma cells. SMMC-7221 cell clones with high (SMMC-7221hPf) and low (SMMC-7221/Pf) water permeability were identified by functional assays with corresponding high and low AQP1 expression. Cell migration rate was remarkably higher in SMMC-7221hPf cells than SMMC-7221/Pf cells, assessed by Boyden chamber and wound healing assays, whereas cell growth and adhesion were not different. Adenovirus-mediated AQP1 expression in SMMC-7221/Pf cells increased their water permeability and migration rate. These results provide the first evidence that aquaporin-mediated membrane water permeability enhances tumor cell migration and may be associated with tumor invasion and metastasis.

  10. Klf10 regulates odontoblast differentiation and mineralization via promoting expression of dentin matrix protein 1 and dentin sialophosphoprotein genes.

    Science.gov (United States)

    Chen, Zhuo; Li, Wentong; Wang, Han; Wan, Chunyan; Luo, Daoshu; Deng, Shuli; Chen, Hui; Chen, Shuo

    2016-02-01

    Klf10, a member of the Krüppel-like family of transcription factors, is critical for osteoblast differentiation, bone formation and mineralization. However, whether Klf10 is involved in odontoblastic differentiation and tooth development has not been determined. In this study, we investigate the expression patterns of Klf10 during murine tooth development in vivo and its role in odontoblastic differentiation in vitro. Klf10 protein was expressed in the enamel organ and the underlying mesenchyme, ameloblasts and odontoblasts at early and later stages of murine molar formation. Furthermore, the expression of Klf10, Dmp1, Dspp and Runx2 was significantly elevated during the process of mouse dental papilla mesenchymal differentiation and mineralization. The overexpression of Klf10 induced dental papilla mesenchymal cell differentiation and mineralization as detected by alkaline phosphatase staining and alizarin red S assay. Klf10 additionally up-regulated the expression of odontoblastic differentiation marker genes Dmp1, Dspp and Runx2 in mouse dental papilla mesenchymal cells. The molecular mechanism of Klf10 in controlling Dmp1 and Dspp expression is thus to activate their regulatory regions in a dosage-dependent manner. Our results suggest that Klf10 is involved in tooth development and promotes odontoblastic differentiation via the up-regulation of Dmp1 and Dspp transcription. PMID:26310138

  11. microRNA-218 inhibits prostate cancer cell growth and promotes apoptosis by repressing TPD52 expression

    International Nuclear Information System (INIS)

    Highlights: • miR-218 expression is downregulated in prostate cancer. • miR-218 inhibits prostate tumor cells proliferation partially through promoting apoptosis. • miR-218 targets TPD52 by binding to its 3′-UTR. • miR-218 suppresses prostate cancer cell growth through inhibiting TPD52 expression. - Abstract: The tumor protein D52 (TPD52) is an oncogene overexpressed in prostate cancer (PC) due to gene amplification. Although the oncogenic effect of TPD52 is well recognized, how its expression is regulated is still not clear. This study tried to explore the regulative role of miR-218, a tumor suppressing miRNA on TPD52 expression and prostate cancer cell proliferation. We found the expression of miR-218 was significantly lower in PC specimens. Based on gain and loss of function analysis, we found miR-218 significantly inhibit cancer cell proliferation by inducing apoptosis. These results strongly suggest that miR-218 plays a tumor suppressor role in PC cells. In addition, our data firstly demonstrated that miR-218 directly regulates oncogenic TPD52 in PC3 cells and the miR-218-TPD52 axis can regulate growth of this prostate cancer cell line. Knockdown of TPD52 resulted in significantly increased cancer cell apoptosis. Clearly understanding of oncogenic TPD52 pathways regulated by miR-218 might be helpful to reveal new therapeutic targets for PC

  12. Klf10 regulates odontoblast differentiation and mineralization via promoting expression of dentin matrix protein 1 and dentin sialophosphoprotein genes

    Science.gov (United States)

    Chen, Zhuo; Li, Wentong; Wang, Han; Wan, Chunyan; Luo, Daoshu; Deng, Shuli

    2016-01-01

    Klf10, a member of the Krüppel-like family of transcription factors, is critical for osteoblast differentiation, bone formation and mineralization. However, whether Klf10 is involved in odontoblastic differentiation and tooth development has not been determined. In this study, we investigate the expression patterns of Klf10 during murine tooth development in vivo and its role in odontoblastic differentiation in vitro. Klf10 protein was expressed in the enamel organ and the underlying mesenchyme, ameloblasts and odontoblasts at early and later stages of murine molar formation. Furthermore, the expression of Klf10, Dmp1, Dspp and Runx2 was significantly elevated during the process of mouse dental papilla mesenchymal differentiation and mineralization. The overexpression of Klf10 induced dental papilla mesenchymal cell differentiation and mineralization as detected by alkaline phosphatase staining and alizarin red S assay. Klf10 additionally up-regulated the expression of odontoblastic differentiation marker genes Dmp1, Dspp and Runx2 in mouse dental papilla mesenchymal cells. The molecular mechanism of Klf10 in controlling Dmp1 and Dspp expression is thus to activate their regulatory regions in a dosage-dependent manner. Our results suggest that Klf10 is involved in tooth development and promotes odontoblastic differentiation via the up-regulation of Dmp1 and Dspp transcription. PMID:26310138

  13. Correlation between ECT2 gene expression and methylation change of ECT2 promoter region in pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Mang-Li Zhang; Sen Lu; Lin Zhou; Shu-Sen Zheng

    2008-01-01

    BACKGROUND: Pancreatic cancer is closely related to epigenetic abnormality. The epithelial cell transforming sequence 2 gene (ECT2) plays a critical role in Rho activation during cytokinesis, and thus may play a role in the pathogenesis of pancreatic cancer. In this study, we investigated the relationships between aberrant expression and epigenetic changes of the ECT2 gene in pancreatic cancer. METHODS: Four cell lines (PANC-1, Colo357, T3M-4 and PancTuⅠ) and pancreatic ductal adenocarcinoma (PDAC) tissues were used for mRNA detection. After restriction isoschizomer endonucleases (MspⅠ/HpaⅡ) were used to digest the DNA sequence (5'-CCGG-3'), PCR was made to amplify the product. And RT-PCR was applied to determine the expression of the gene. RESULTS: The mRNA expression of the ECT2 gene was higher in pancreatic tumor tissue than in normal tissue. The gene was also expressed in the 4 PDAC cell lines. The methylation states of the upstream regions of the ECT2 gene were almost identical in normal, tumor pancreatic tissues, and the 4 PDAC cell lines. Some of the 5'-CCGG-3' areas in the upstream region of ECT2 were methylated, while others were unmethylated. CONCLUSIONS: The oncogene ECT2 is overexpressed in pancreatic tumor tissues as veriifed by RT-PCR detection. The methylation status of DNA in promoter areas is involved in the gene expression, along with other factors, in pancreatic cancer.

  14. Betatrophin expression is promoted in obese hyperinsulinemic type 2 but not type 1 diabetic mice.

    Science.gov (United States)

    Li, EnXu; Nakata, Masanori; Shinozaki, Atsumi; Yang, Yifei; Zhang, Boyang; Yada, Toshihiko

    2016-07-30

    Regeneration of pancreatic β-cell mass benefits both type 1 and type 2 diabetic patients. A recent study identified betatrophin as a β-cell proliferation factor. However, the expressional regulation of betatrophin remains less defined. In this study, we aimed to clarify the regulation of betatrophin expression in obese type 2 vs. type 1 diabetes model animals. We experimented type 2 diabetes models, diet-induced-obesity (DIO) mice and db/db mice, and type 1 diabetes models, C57B6 mice receiving streptozotocin (STZ) or 70% pancreatectomy to destroy or remove β-cells. Serum betatrophin levels and betatrophin mRNA expressions in the liver, white adipose tissue (WAT) and brown adipose tissue (BAT) were measured. In DIO mice and db/db mice, serum betatrophin and betatrophin mRNA expressions in the liver, WAT and BAT were elevated in parallel with increases in body weight and plasma insulin. These elevated betatrophin mRNA expressions were not altered by treatment with SGLT2 inhibitor that ameliorated hyperglycemia. In pancreatectomized mice, betatrophin expression in WAT decreased in parallel with reductions in weight and insulin. In STZ-treated mice, betatrophin expressions in the liver, WAT and BAT were reduced. However, when the mouse liver slices were cultured with STZ, betatrophin expression was significantly reduced, indicating a direct action of STZ on the liver. These results indicate that the expression of betatrophin is upregulated in the liver, WAT and BAT in obese hyperinsulinemic type 2 diabetes but decreased in WAT in hypoinsulinemic type 1 diabetes, suggesting its positive correlation with body weight and plasma insulin but not blood glucose. PMID:27097546

  15. Autocrine HBEGF expression promotes breast cancer intravasation, metastasis and macrophage-independent invasion in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Z. N.; Sharma, V. P.; Beaty, B. T.; Roh-Johnson, M.; Peterson, E. A.; Van Rooijen, N.; Kenny, P. A.; Wiley, H. S.; Condeelis, J. S.; Segall, J. E.

    2014-10-13

    Increased expression of HBEGF in estrogen receptor-negative breast tumors is correlated with enhanced metastasis to distant organ sites and more rapid disease recurrence upon removal of the primary tumor. Our previous work has demonstrated a paracrine loop between breast cancer cells and macrophages in which the tumor cells are capable of stimulating macrophages through the secretion of colony-stimulating factor-1 while the tumor-associated macrophages (TAMs), in turn, aid in tumor cell invasion by secreting epidermal growth factor. To determine how the autocrine expression of epidermal growth factor receptor (EGFR) ligands by carcinoma cells would affect this paracrine loop mechanism, and in particular whether tumor cell invasion depends on spatial ligand gradients generated by TAMs, we generated cell lines with increased HBEGF expression. We found that autocrine HBEGF expression enhanced in vivo intravasation and metastasis and resulted in a novel phenomenon in which macrophages were no longer required for in vivo invasion of breast cancer cells. In vitro studies revealed that expression of HBEGF enhanced invadopodium formation, thus providing a mechanism for cell autonomous invasion. The increased invadopodium formation was directly dependent on EGFR signaling, as demonstrated by a rapid decrease in invadopodia upon inhibition of autocrine HBEGF/EGFR signaling as well as inhibition of signaling downstream of EGFR activation. HBEGF expression also resulted in enhanced invadopodium function via upregulation of matrix metalloprotease 2 (MMP2) and MMP9 expression levels. We conclude that high levels of HBEGF expression can short-circuit the tumor cell/macrophage paracrine invasion loop, resulting in enhanced tumor invasion that is independent of macrophage signaling.

  16. Expression of endoglucanases in Pichia pastoris under control of the GAP promoter

    OpenAIRE

    Varnai, Aniko; Tang, Campbell; Bengtsson, Oskar; Atterton, Andrew; Mathiesen, Geir; Eijsink, Vincent

    2014-01-01

    Background Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selec...

  17. Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas

    OpenAIRE

    Everhard, Sibille; Tost, Jörg; Abdalaoui, Hafida El; Crinière, Emmanuelle; Busato, Florence; Marie, Yannick; Gut, Ivo G.; Sanson, Marc; Mokhtari, Karima; Laigle-Donadey, Florence; Hoang-Xuan, Khê; Delattre, Jean-Yves; Thillet, Joëlle

    2009-01-01

    The O6-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sit...

  18. Prognostic impact of MGMT promoter methylation and MGMT and CD133 expression in colorectal adenocarcinoma

    OpenAIRE

    Oliver, Jaime Antonio; Ortiz Quesada, Ra??l; Melguizo Alonso, Consolaci??n; ??lvarez, Pablo Juan; G??mez-Mill??n, Jaime; Prados, Jos??

    2014-01-01

    Background: New biomarkers are needed for the prognosis of advanced colorectal cancer, which remains incurable by conventional treatments. O6-methylguanine DNA methyltransferase (MGMT) methylation and protein expression have been related to colorectal cancer treatment failure and tumor progression. Moreover, the presence in these tumors of cancer stem cells, which are characterized by CD133 expression, has been associated with chemoresistance, radioresistance, metastasis, and local recurr...

  19. Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone

    Institute of Scientific and Technical Information of China (English)

    Li YANG; Ta Yuan CHANG; Bo Liang LI; Jin Bo YANG; Jia CHEN; Guang Yao YU; Pei ZHOU; Lei LEI; Zhen Zhen WANG; Catherine CY CHANG; XinYing YANG

    2004-01-01

    In macrophages, the accumulation of cholesteryl esters synthesized by the activated acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) results in the foam cell formation, a hallmark of early atherosclerotic lesions. In this study,with the treatment of a glucocorticoid hormone dexamethasone (Dex), lipid staining results clearly showed the large accumulation of lipid droplets containing cholesteryl esters in THP- 1-derived macrophages exposed to lower concentration of the oxidized low-density lipoprotein (ox-LDL). More notably, when treated together with specific anti-ACAT inhibitors, the abundant cholesteryl ester accumulation was markedly diminished in THP-l-derived macrophages, confirming that ACAT is the key enzyme responsible for intracellular cholesteryl ester synthesis. RT-PCR and Western blot results indicated that Dex caused up-regulation of human ACAT1 expression at both the mRNA and protein levels in THP-1 and THP- 1-derived macrophages. The luciferase activity assay demonstrated that Dex could enhance the activity of human ACAT1 gene P1 promoter, a major factor leading to the ACAT1 activation, in a cell-specific manner.Further experimental evidences showed that a glucocorticoid response element (GRE) located within human ACAT1gene P1 promoter to response to the elevation of human ACAT1 gene expression by Dex could be functionally bound with glucocorticoid receptor (GR) proteins. These data supported the hypothesis that the clinical treatment with Dex,which increased the incidence of atherosclerosis, may in part due to enhancing the ACAT1 expression to promote the accumulation of cholesteryl esters during the macrophage-derived foam cell formation, an early stage of atherosclerosis.

  20. BRCA1 gene expression and promoter methylation patterns in early-onset breast cancers among A-bomb survivors

    International Nuclear Information System (INIS)

    Full text: Early-onset breast cancer is one of the most obviously radiogenic cancers among A-bomb survivors. Our initial hypothesis was that A-bomb radiation exposure might have enhanced the risk of early-onset breast cancer in initially heterozygous BRCA1 mutation carriers by mutationally inactivating the normal copy of the BRCA1 gene. To test this hypothesis, we began by examining the changes in BRCA1 expression immunohistochemically using the formalin-fixed, paraffin-embedded breast cancer tissues that had been stored at various hospitals in Hiroshima and Nagasaki for several decades. We then developed a highly-sensitive methylation-specific PCR method to test for hypermethylation of the BRCA1 gene promoter as a possible explanation for BRCA1 expression having been reduced in breast cancer cases. The breast cancer tissues studied so far were from a group of A-bomb survivors who were <45 years of age at diagnosis (31cases), and from a non-exposed group who were also <45 (21 cases). Our observations indicate that BRCA1 protein expression levels were reduced by ∼90% in many tumor tissues from both groups. Review of these reduced cases indicated that methylation silencing of the BRCA1 gene promoter was present in 40-50% of both exposed and unexposed cases. By contrast, fully unmethylated BRCA1 promoter sequences were present in as few as 15-20% of cases in the non-exposed group and 30-35% in the exposed group. Clearly we need to examine many more of the 700 samples that we aim to collect before we will be able to draw any conclusions about the possible role of methylation silencing in the development of early-onset breast cancers among A-bomb survivors

  1. Overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating chemokine expression

    Science.gov (United States)

    Duckworth, C; Zhang, L; Carroll, S L; Ethier, S P; Cheung, H W

    2016-01-01

    We previously found that the scaffold adapter GRB2-associated binding protein 2 (GAB2) is amplified and overexpressed in a subset of primary high-grade serous ovarian cancers and cell lines. Ovarian cancer cells overexpressing GAB2 are dependent on GAB2 for activation of the phosphatidylinositol 3-kinase (PI3K) pathway and are sensitive to PI3K inhibition. In this study, we show an important role of GAB2 overexpression in promoting tumor angiogenesis by upregulating expression of multiple chemokines. Specifically, we found that suppression of GAB2 by inducible small hairpin RNA in ovarian cancer cells inhibited tumor cell proliferation, angiogenesis and peritoneal tumor growth in immunodeficient mice. Overexpression of GAB2 upregulated the secretion of several chemokines from ovarian cancer cells, including CXCL1, CXCL2 and CXCL8. The secreted chemokines not only signal through endothelial CXCR2 receptor in a paracrine manner to promote endothelial tube formation, but also act as autocrine growth factors for GAB2-induced transformation of fallopian tube secretory epithelial cells and clonogenic growth of ovarian cancer cells overexpressing GAB2. Pharmacological inhibition of inhibitor of nuclear factor kappa-B kinase subunit β (IKKβ), but not PI3K, mechanistic target of rapamycin (mTOR) or mitogen-activated protein kinase (MEK), could effectively suppress GAB2-induced chemokine expression. Inhibition of IKKβ augmented the efficacy of PI3K/mTOR inhibition in suppressing clonogenic growth of ovarian cancer cells with GAB2 overexpression. Taken together, these findings suggest that overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating expression of CXCL1, CXCL2 and CXCL8 that is IKKβ-dependent. Co-targeting IKKβ and PI3K pathways downstream of GAB2 might be a promising therapeutic strategy for ovarian cancer that overexpresses GAB2. PMID:26657155

  2. OCT4 Remodels the Phenotype and Promotes Angiogenesis of HUVECs by Changing the Gene Expression Profile

    Science.gov (United States)

    Mou, Yan; Yue, Zhen; Wang, Xiaotong; Li, Wenxue; Zhang, Haiying; Wang, Yang; Li, Ronggui; Sun, Xin

    2016-01-01

    It has been shown that forced expression of four mouse stem cell factors (OCT4, Sox2, Klf4, and c-Myc) changed the phenotype of rat endothelial cells to vascular progenitor cells. The present study aimed to explore whether the expression of OCT4 alone might change the phenotype of human umbilical vein endothelial cells (HUVECs) to endothelial progenitor cells and, if so, to examine the possible mechanism involved. A Matrigel-based in vitro angiogenesis assay was used to evaluate the angiogenesis of the cells; the gene expression profile was analyzed by an oligonucleotide probe-based gene array chip and validated by RT-QPCR. The cellular functions of the mRNAs altered by OCT4 were analyzed with Gene Ontology. We found that induced ectopic expression of mouse OCT4 in HUVECs significantly enhanced angiogenesis of the cells, broadly changed the gene expression profile and particularly increased the expression of CD133, CD34, and VEGFR2 (KDR) which are characteristic marker molecules for endothelial progenitor cells (EPCs). Furthermore by analyzing the cellular functions that were targeted by the mRNAs altered by OCT4 we found that stem cell maintenance and cell differentiation were among the top functional response targeted by up-regulated and down-regulated mRNAs upon forced expression of OCT4. These results support the argument that OCT4 remodels the phenotype of HUVECs from endothelial cells to EPCs by up-regulating the genes responsible for stem cell maintenance and down-regulating the genes for cell differentiation.

  3. Promoter methylation and expression levels of selected hematopoietic genes in pediatric B-cell acute lymphoblastic leukemia

    OpenAIRE

    Musialik, Ewa; Bujko, Mateusz; Kober, Paulina; Agnieszka WYPYCH; Gawle-Krawczyk, Karolina; Matysiak, Michal; Siedlecki, Janusz Aleksander

    2015-01-01

    Background Precursor B-cell acute lymphoblastic leukemia (B-cell ALL) is the most common neoplasm in children and is characterized by genetic and epigenetic aberrations in hematopoietic transcription factor (TF) genes. This study evaluated promoter DNA methylation and aberrant expression levels of early- and late-acting hematopoietic TF genes homeobox A4 and A5 (HOXA4 and HOXA5), Meis homeobox 1 (MEIS1), T-cell acute lymphocytic leukemia 1 (TAL1), and interferon regulatory factors 4 and 8 (IR...

  4. Crimean-Congo hemorrhagic fever virus nucleoprotein suppresses IFN-beta-promoter-mediated gene expression.

    Science.gov (United States)

    Fajs, Luka; Resman, Katarina; Avšič-Županc, Tatjana

    2014-02-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the family Bunyaviridae and is a causative agent of severe hemorrhagic disease. Knowledge regarding the pathogenesis of CCHFV is limited due to the requirement for high-containment laboratories and the lack of an immunocompetent animal host. Previous studies have shown that CCHFV delays the activation of the human innate immune response, specifically, the type I interferon response. Our study results show that antagonism of the interferon-beta promoter is mediated by the nucleoprotein of CCHFV strain Hoti, while strains IbAr10200 and AP92 do not suppress the activity of the IFN-beta promoter. Our results also suggest that several viral factors may provide antagonistic action against the type I interferon response. PMID:23990053

  5. Bypasses of the antimycin a block of mitochondrial electron transport in relation to ubisemiquinone function.

    Science.gov (United States)

    Alexandre, A; Lehninger, A L

    1984-10-26

    Two different bypasses around the antimycin block of electron transport from succinate to cytochrome c via the ubiquinol-cytochrome c oxidoreductase of intact rat liver mitochondria were analyzed, one promoted by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and the other by 2,6-dichlorophenolindophenol (DCIP). Both bypasses are inhibited by myxothiazol, which blocks electron flow from ubiquinol to the Rieske iron-sulfur center, and by 2-hydroxy-3-undecyl-1,4-naphthoquinone, which inhibits electron flow from the iron-sulfur center to cytochrome c1. In the bypass promoted by TMPD its oxidized form (Wurster's blue) acts as an electron acceptor from some reduced component prior to the antimycin block, which by exclusion of other possibilities is ubisemiquinone. In the DCIP bypass its reduced form acts as an electron donor, by reducing ubisemiquinone to ubiquinol; reduced DCIP is regenerated again at the expense of either succinate or ascorbate. The observations described are consistent with and support current models of the Q cycle. Bypasses promoted by artificial electron carriers provide an independent approach to analysis of electron flow through ubiquinol-cytochrome c oxidoreductase. PMID:6091750

  6. Human papillomavirus type 16 E7 perturbs DREAM to promote cellular proliferation and mitotic gene expression

    OpenAIRE

    DeCaprio, James A.

    2013-01-01

    Study of the small DNA tumor viruses continues to provide valuable new insights into oncogenesis and fundamental biological processes. While much has already been revealed about how the human papillomaviruses (HPVs) can transform cells and contribute to cervical and oropharyngeal cancer, there clearly is much more to learn. In this issue of Oncogene, Pang et al. demonstrate that the high-risk HPV16 E7 oncogene can promote cellular proliferation by interacting with the DREAM (DP, RB-like, E2F ...

  7. Demethylation of the human eotaxin-3 gene promoter leads to the elevated expression of eotaxin-3

    OpenAIRE

    Lim, Eunjin; Rothenberg, Marc E.

    2013-01-01

    DNA demethylation has been primarily studied in the context of development biology, cell fate, and cancer, with less attention on inflammation. Herein, we investigate the association between DNA methylation and production of the chemoattractant cytokine eotaxin-3 in the tissue of patients with allergic disease. Regions of the human eotaxin-3 promoter were found to be hypomethylated in primary epithelial cells obtained from allergic tissue compared with normal control tissue (CTL). The demethy...

  8. Expression of the myosin heavy chain IIB gene in porcine skeletal muscle: the role of the CArG-Box promoter response element.

    Directory of Open Access Journals (Sweden)

    David M Brown

    Full Text Available Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC IIB gene (MYH4 exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter.

  9. Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens

    Directory of Open Access Journals (Sweden)

    Nätt Daniel

    2012-02-01

    Full Text Available Abstract Background Variations in gene expression, mediated by epigenetic mechanisms, may cause broad phenotypic effects in animals. However, it has been debated to what extent expression variation and epigenetic modifications, such as patterns of DNA methylation, are transferred across generations, and therefore it is uncertain what role epigenetic variation may play in adaptation. Results In Red Junglefowl, ancestor of domestic chickens, gene expression and methylation profiles in thalamus/hypothalamus differed substantially from that of a domesticated egg laying breed. Expression as well as methylation differences were largely maintained in the offspring, demonstrating reliable inheritance of epigenetic variation. Some of the inherited methylation differences were tissue-specific, and the differential methylation at specific loci were little changed after eight generations of intercrossing between Red Junglefowl and domesticated laying hens. There was an over-representation of differentially expressed and methylated genes in selective sweep regions associated with chicken domestication. Conclusions Our results show that epigenetic variation is inherited in chickens, and we suggest that selection of favourable epigenomes, either by selection of genotypes affecting epigenetic states, or by selection of methylation states which are inherited independently of sequence differences, may have been an important aspect of chicken domestication.

  10. Long-term inducible expression in striatal neurons from helper virus-free HSV-1 vectors that contain the tetracycline-inducible promoter system

    OpenAIRE

    Gao, Qingshen; Sun, Mei; Wang, Xiaodan; Zhang, Guo-rong; Geller, Alfred I.

    2006-01-01

    Direct gene transfer into neurons in the brain via a virus vector system has potential for both examining neuronal physiology and for developing gene therapy treatments for neurological diseases. Many of these applications require precise control of the levels of recombinant gene expression. The preferred method for controlling the levels of expression is by use of an inducible promoter system, and the tetracycline (tet)-inducible promoter system is the preferred system. Helper virus-free Her...

  11. Human TMEM174 that is highly expressed in kidney tissue activates AP-1 and promotes cell proliferation

    International Nuclear Information System (INIS)

    Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174ΔTM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.

  12. ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression

    DEFF Research Database (Denmark)

    Varmanen, P.; Vogensen, F.K.; Hammer, Karin;

    2003-01-01

    The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp...... ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in......R homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR....

  13. Cloning and Analyzing of Xenopus Mespo Promoter in Retinoic Acid Regulated Mespo Expression

    Institute of Scientific and Technical Information of China (English)

    Jin-Hu WANG; Xiao-Yan DING

    2006-01-01

    Juring vertebrate embryogenesis, presomitic mesoderm cells enter a segmental program to generate somite, a process termed somitogenesis. Mespo, a member of the bHLH transcription factor family,plays important roles in this process. However, how Mespo expression is regulated remains unclear. To address this question, we isolated a genomic DNA sequence containing 4317 bp of Mespo 5' flanking region in Xenopus. Luciferase assays show that this upstream sequence has transcription activity. Transgenic assay shows that this genomic contig is sufficient to recapitulate the dynamic stage- and tissue-specific expression pattern of endogenous Mespo from the gastrula to the tailbud stage. We further mapped a 326 bp DNA sequence responding to retinoic acid signaling. These results shed light on how Mespo expression is regulated,and suggest that retinoic acid signaling pathways play roles in somitogenesis through regulating Mespo.

  14. Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encoding human erythropoietin (hEPO) and governed by regulatory sequences of mouse whey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%. hEPO was expressed in the milk of 16 mice of 39 mice measured by hEPO ELISA kit .The expression level gets over 15 m g/mL.

  15. Promoter Hypermethylation and Decreased Expression of Syncytin-1 in Pancreatic Adenocarcinomas.

    Directory of Open Access Journals (Sweden)

    Qinsheng Lu

    Full Text Available Syncytin-1 is a member of human endogenous retroviral W gene family (HERVW1. Known to be expressed in human placental trophoblast, syncytin-1 protein mediates the fusion of cytotrophoblasts for the formation of syncytiotrophoblasts, the terminally differentiated form of trophoblast lineage. In addition, in vitro studies indicate that syncytin-1 possessed nonfusogenic functions such as those for immune suppression, cell cycle regulation and anti-apoptotic activities. Overexpression of syncytin-1 has been observed in various malignant tissues including breast, endometrial and ovarian cancers. It was reported that syncytin-1 gene expression is associated with dynamic changes of DNA hypomethylation in the 5' LTR. In this study, applying the real-time PCR, Western blot analysis and immunohistochemistry methods, we demonstrate a constitutive expression of syncytin-1 in normal pancreas tissues as well as normal tissues adjacent to cancer lesions. Moreover, a reduced expression is found in the pancreatic adenocarcinoma tissues. The expression levels of syncytin-1 are not correlated with the stage, historical grade and gender, but inversely correlated with patients' age. Furthermore, COBRA and bisulfite sequencing results indicated that the lower expression of syncytin-1 is correlated with the hypermethylation of two CpG dinucleotides in the 5' LTR of syncytin-1 gene. The nonfusogenic function of syncytin-1 in normal pancreas as well as its role(s in the pathogenesis and progression of pancreatic cancers remains to be investigated. Identification of the two CpG dinucleotides around transcription start site as key epigenetic elements has provided valuable information for further studies on the epigenetic regulation of syncytin-1 in pancreatic cancer cells.

  16. IL-15 expression on RA synovial fibroblasts promotes B cell survival.

    Directory of Open Access Journals (Sweden)

    Marta Benito-Miguel

    Full Text Available INTRODUCTION: The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib IL-15 expression on B cell survival. METHODS: Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib. RESULTS: RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/-8% (p<0.001. IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/-6% (p<0.05. Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures. CONCLUSION: IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.

  17. Prenatal Stress Down-Regulates Reelin Expression by Methylation of Its Promoter and Induces Adult Behavioral Impairments in Rats

    Science.gov (United States)

    Palacios-García, Ismael; Lara-Vásquez, Ariel; Montiel, Juan F.; Díaz-Véliz, Gabriela F.; Sepúlveda, Hugo; Utreras, Elías; Montecino, Martín; González-Billault, Christian; Aboitiz, Francisco

    2015-01-01

    Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS) upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that can yield new

  18. Prenatal stress down-regulates Reelin expression by methylation of its promoter and induces adult behavioral impairments in rats.

    Directory of Open Access Journals (Sweden)

    Ismael Palacios-García

    Full Text Available Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that

  19. Inhibition of Nogo expression to promote repair after spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    SUN Hong-hui; GAO Feng; LIU Bin; YU Hai-tao; KONG Ning; LIU Guo-min

    2012-01-01

    Background One of the reasons for poor neuroregeneration after central nervous system injury is the presence of inhibitory factors such as Nogo.Here,we tested the inhibition of Nogo by RNA interference both in vitro and in vivo,using recombinant adenovirus-mediated transfection of short hairpin RNAs,to explore a new method of treatment for spinal cord injury.Methods We designed and cloned two Nogo-specific short hairpin RNAs and an unrelated short hairpin RNA,packaged the clones into adenovirus,and amplified the recombinant virus in 293 cells.We then tested the inhibition of Nogo expression both in vitro in adenovirus-transfected oligodendrocytes and in vivo in spinal cord tissue from adenovirus-transfected spinal cord injury model rats.We tested Nogo expression at the mRNA level by reverse-transcription PCR and at the protein level by Western blotting and immunohistochemistry.Results In vitro,the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 51% and 49%,respectively,compared with Nogo expression in ceils transfected with the unrelated control small hairpin RNA(P<0.005).Similarly,Nogo protein expression decreased by 50% and 48%,respectively(P<0.005).In vivo,in spinal cord injury model rats,the two specific Nogo short hairpin RNAs decreased Nogo mRNA expression by 45% and 40%,respectively,compared with Nogo expression in spinal cord injury model rats transfected with the unrelated control short hairpin RNA(P<0.005).The Nogo protein level was similarly decreased.Conclusions We were successful in specifically downregulating Nogo at the mRNA and protein levels by adenovirus-mediated delivery of short hairpin RNAs,both in vitro and in vivo.This confirms the effectiveness of RNA interference for the inhibition of Nogo gene expression and the efficiency of using adenovirus for delivery.Thus gene therapy may be an effective treatment for spinal cord injury.

  20. THE BASIC LAWS AND FEATURES OF CYTOKINE DYNAMICS IN PROCESS AND EARLY TERMS AFTER CARDIOPULMONARY BYPASS

    Directory of Open Access Journals (Sweden)

    S. I. Suskov

    2011-01-01

    Full Text Available The basic variants of cytokines reactions defining type of organ dysfunctions are revealed in the course of car- diopulmonary bypass and in the early postoperative period. Their character and expression, depends on gravity preoperative an immunodeficiency and initial degree of heart insufficiency. Diphasic dynamics of development of system inflammatory reaction is confirmed after cardiopulmonary bypass: increase of levels proinflammatory cytokines is in the first phase and anti-inflammatory cytokines with development immunodepression and cellular anergy in is the second phase. Also, key role IL-1Ra is revealed in restraint of hyperactivation of system inflam- matory reaction. Blood whey levels IL-6, IL-8, G-CSF, TNF-α and IL-1Ra should be defined to cardiopulmonary bypass, in 10–12 hours, 24 hours and 3 days after cardiopulmonary bypass and may be used as prognostic criteria of development of postoperative complications. 

  1. Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter.

    Science.gov (United States)

    Sablowski, R W; Baulcombe, D C; Bevan, M

    1995-07-18

    The promoter of the bean PAL2 gene (encoding phenylalanine ammonia-lyase; EC 4.3.1.5) is a model for studies of tissue-restricted gene expression in plants. Petal epidermis is one of the tissues in which this promoter is activated in tobacco. Previous work suggested that a major factor establishing the pattern of PAL2 expression in tobacco petals is the tissue distribution of a protein closely related to Myb305, which is a Myb-like transcriptional activator from snapdragon. In the present work, we show that Myb305 expression in tobacco leaves causes ectopic activation of the PAL2 promoter. To achieve Myb305 expression in planta, a viral expression vector was used. This approach combines the utility of transient assays with the possibility of direct biochemical detection of the introduced factor and may have wider application for studying the function of plant transcription factors. PMID:7624340

  2. Homocysteine and copper interact to promote type 5 phosphodiesterase expression in rabbit cavernosal smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Matthew Hotston; Jamie Y.Jeremy; Jonathon Bloor; Nick S.Greaves; Raj Persad; Giarmi Angelini; Nilima Shukla

    2008-01-01

    Aim: To study the effects of homocysteine and copper on type 5 phosphodiesterase (PDE5) expression in cavernosal vascular smooth muscle cells (CVSMCs) and to investigate superoxide (O2) derived from nicotinamide adenine dinucleotide phosphate oxidase as homocysteine and copper generate O2, and O2- upregulates PDE5 expression.Methods: CVSMCs derived from rabbit penis were incubated with homocysteine or copper chloride with or without superoxide dismutase (SOD), catalase, sildenafil citrate, or apocynin (nicotinamide adenine dinucleotide phosphate inhibitor) for 16 h. The expression of PDE5 and of glyceraldehyde-3-phosphate dehydrogenase (internal standard) was assessed using Western blot analysis. In parallel, O2 was measured spectrophotometrically. Results: CuCl2alone (up to 10 μmol/L) and homocysteine alone (up to 100 μmol/L) had no effect on O2 formation in CVSMCs compared to controls. In combination, however, homocysteine and CuCl2 arkedly increased O2 formation, an effect blocked by SOD, catalase, apocynin, and sildenafil (1 μmol/L) when co-incubated over the same time course.PDE5 expression was also significantly increased in CVSMCs incubated with homocysteine and CuCl2, compared to controls. This effect was also negated by 16-h co-incubation with SOD, catalase, apocynin and sildenafil. Conclusion:This represents a novel pathogenic mechanism underlying ED, and indicates that the therapeutic actions of prolonged sildenafil use are mediated in part through inhibition of this pathway.

  3. Progesterone Promotes HOXa-10 Expression in Mouse During Embryo Implantation Period

    Directory of Open Access Journals (Sweden)

    Shuhong Zhao

    2012-01-01

    Full Text Available HOXa-10 (HomeoboxA10 gene plays an important role in embryo implantation and development regulated by Progesterone (P4 which is secreted mainly through corpus luteum cells to regulate the embryo development. The study mainly investigated the effect of progesterone on the expression of HOXa-10 in the endometrium during the embryo implantation. Kunming mice were injected progesterone (2 or 4 mg/each per day, respectively from early pregnant D1 (the 1st day of pregnancy to D4 (the 4th day of pregnancy or D6 (the 6th day of pregnancy by intraperitoneal injection. The endometrium samples of early pregnant D4, D5 (the 5th day of pregnance and D6, from 3 groups were collected with each group containing 3 mice. Real-time fluorescence Quantitative PCR (QPCR was used to analyze the expression of the mouse HOXa-10 gene. The result demonstrated that the expression level of HOXa-10 was increased at pregnant D4 and there was a peak at pregnant D5 then it began to decrease at pregnant D6. Besides, the expression level of HOXa-10 was dependent on the dose of progesterone.

  4. Novel Implant Coating Agent Promotes Gene Expression of Osteogenic Markers in Rats during Early Osseointegration

    Directory of Open Access Journals (Sweden)

    Kostas Bougas

    2012-01-01

    Full Text Available The aim of this study was to evaluate the early bone response around laminin-1-coated titanium implants. Forty-five rats distributed in three equally sized groups were provided with one control (turned and one test (laminin-1-coated implant and were sacrificed after 3, 7, and 21 days. Real-time reverse-transcriptase polymerase chain reaction was performed for osteoblast markers (alkaline phosphatase, runt-related transcription factor 2, osteocalcin, type I collagen, and bone morphogenic protein 2, osteoclast markers (cathepsin K and tartrate-resistant acid phosphatase, inflammation markers (tumor necrosis factor α, interleukin 1β and interleukin 10, and integrin β1. Bone implant contact (BIC and bone area (BA were assessed and compared to the gene expression. After 3 days, the expression of bone markers was higher for the control group. After 7 days, the expression of integrin β1 and osteogenic markers was enhanced for the test group, while cathepsin K and inflammation markers were down-regulated. No significant differences in BIC or BA were detected between test and control at any time point. As a conclusion, implant coating with laminin-1 altered gene expression in the bone-implant interface. However, traditional evaluation methods, as histomorphometry, were not adequately sensitive to detect such changes due to the short follow-up time.

  5. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Science.gov (United States)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  6. Combinatorial control of temporal gene expression in the Drosophila wing by enhancers and core promoters

    OpenAIRE

    O’Keefe, David D.; Thomas, Sean R; Bolin, Kelsey; Griggs, Ellen; Edgar, Bruce A.; Buttitta, Laura A

    2012-01-01

    Abstract Background The transformation of a developing epithelium into an adult structure is a complex process, which often involves coordinated changes in cell proliferation, metabolism, adhesion, and shape. To identify genetic mechanisms that control epithelial differentiation, we analyzed the temporal patterns of gene expression during metamorphosis of the Drosophila wing. Results We found...

  7. BMP-6 promotes E-cadherin expression through repressing δEF1 in breast cancer cells

    International Nuclear Information System (INIS)

    Bone morphogenetic protein-6 (BMP-6) is critically involved in many developmental processes. Recent studies indicate that BMP-6 is closely related to tumor differentiation and metastasis. Quantitative RT-PCR was used to determine the expression of BMP-6, E-cadherin, and δEF1 at the mRNA level in MCF-7 and MDA-MB-231 breast cancer cells, as well as in 16 breast cancer specimens. Immunoblot analysis was used to measure the expression of δEF1 at the protein level in δEF1-overexpressing and δEF1-interfered MDA-MB-231 cells. Luciferase assay was used to determine the rhBMP-6 or δEF1 driven transcriptional activity of the E-cadherin promoter in MDA-MB-231 cells. Quantitative CHIP assay was used to detect the direct association of δEF1 with the E-cadherin proximal promoter in MDA-MB-231 cells. MCF-7 breast cancer cells, an ER+ cell line that expressed high levels of BMP-6 and E-cadherin exhibited very low levels of δEF1 transcript. In contrast, MDA-MB-231 cells, an ER- cell line had significantly reduced BMP-6 and E-cadherin mRNA levels, suggesting an inverse correlation between BMP-6/E-cadherin and δEF1. To determine if the same relationship exists in human tumors, we examined tissue samples of breast cancer from human subjects. In 16 breast cancer specimens, the inverse correlation between BMP-6/E-cadherin and δEF1 was observed in both ER+ cases (4 of 8 cases) and ER- cases (7 of 8 cases). Further, we found that BMP-6 inhibited δEF1 transcription, resulting in an up-regulation of E-cadherin mRNA expression. This is consistent with our analysis of the E-cadherin promoter demonstrating that BMP-6 was a potent transcriptional activator. Interestingly, ectopic expression of δEF1 was able to block BMP-6-induced transactivation of E-cadherin, whereas RNA interference-mediated down-regulation of endogenous δEF1 in breast cancer cells abolished E-cadherin transactivation by BMP-6. In addition to down-regulating the expression of δEF1, BMP-6 also physically

  8. Non-canonical NFκB activation promotes chemokine expression in podocytes

    Science.gov (United States)

    Valiño-Rivas, Lara; Gonzalez-Lafuente, Laura; Sanz, Ana B.; Ruiz-Ortega, Marta; Ortiz, Alberto; Sanchez-Niño, Maria D.

    2016-01-01

    TNF-like weak inducer of apoptosis (TWEAK) receptor Fn14 is expressed by podocytes and Fn14 deficiency protects from experimental proteinuric kidney disease. However, the downstream effectors of TWEAK/Fn14 in podocytes are poorly characterized. We have explored TWEAK activation of non-canonical NFκB signaling in cultured podocytes. In cultured podocytes, TWEAK increased the expression of the chemokines CCL21, CCL19 and RANTES in a time-dependent manner. The inhibitor of canonical NFκB activation parthenolide inhibited the CCL19 and the early RANTES responses, but not the CCL21 or late RANTES responses. In this regard, TWEAK induced non-canonical NFκB activation in podocytes, characterized by NFκB2/p100 processing to NFκB2/p52 and nuclear migration of RelB/p52. Silencing by a specific siRNA of NIK, the upstream kinase of the non-canonical NFκB pathway, prevented CCL21 upregulation but did not modulate CCL19 or RANTES expression in response to TWEAK, thus establishing CCL21 as a non-canonical NFκB target in podocytes. Increased kidney Fn14 and CCL21 expression was also observed in rat proteinuric kidney disease induced by puromycin, and was localized to podocytes. In conclusion, TWEAK activates the non-canonical NFκB pathway in podocytes, leading to upregulation of CCL21 expression. The non-canonical NFκB pathway should be explored as a potential therapeutic target in proteinuric kidney disease. PMID:27353019

  9. Tissue-specific expression from a compound TATA-dependent and TATA-independent promoter

    OpenAIRE

    Garrity, P A; Wold, B J

    1990-01-01

    We have found that the mouse metallothionein-I (MT-I) gene promoter functions in an unusual, compound manner. It directs both TATA-dependent and TATA-independent modes of transcription in vivo. The TATA-dependent message is initiated at the previously characterized +1 transcription start site and is the predominant species in most tissues. In many cell types it is metal inducible. The TATA-independent initiation sites are distributed over the 160 bp upstream of the previously characterized +1...

  10. The stearoyl-acyl-carrier-protein desaturase promoter (Des) from oil palm confers fruit-specific GUS expression in transgenic tomato.

    Science.gov (United States)

    Saed Taha, Rima; Ismail, Ismanizan; Zainal, Zamri; Abdullah, Siti Nor Akmar

    2012-09-01

    The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5' deletions of the Des promoter (Des1-4) and the β-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between -590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits. PMID:22658816

  11. Expression of auxin synthesis gene tms1 under control of tuber-specific promoter enhances potato tuberization in vitro

    Institute of Scientific and Technical Information of China (English)

    Oksana O Kolachevskaya; Valeriya V Alekseeva; Lidiya I Sergeeva; Elena B Rukavtsova; Irina A Getman; Dick Vreugdenhil; Yaroslav I Buryanov; Georgy A Romanov

    2015-01-01

    Phytohormones, auxins in particular, play an important role in plant development and productivity. Earlier data showed positive impact of exogenous auxin on potato (Solanum tuberosum L.) tuberization. The aim of this study was to generate potato plants with increased auxin level predominantly in tubers. To this end, a pBinB33-tms1 vector was constructed harboring the Agrobacterium auxin biosynthesis gene tms1 fused to tuber-specific promoter of the class I patatin gene (B33-promoter) of potato. Among numerous independently generated B33:tms1 lines, those without visible differences from control were selected for detailed studies. In the majority of transgenic lines, tms1 gene transcription was detected, mostly in tubers rather than in shoots. Indoleacetic acid (IAA) content in tubers and the auxin tuber-to-shoot ratio were increased in tms1-expressing transformants. The organ-specific increase in auxin synthesis in B33:tms1-transformants accelerated and intensified the process of tuber formation, reduced the dose of carbohydrate supply required for in vitro tuber-ization, and decreased the photoperiodic dependence of tuber initiation. Overall, a positive correlation was observed between tms1 expression, IAA content in tubers, and stimulation of tuber formation. The revealed proper-ties of B33:tms1 transformants imply an important role for auxin in potato tuberization and offer prospects to magnify potato productivity by a moderate organ-specific enhance-ment of auxin content.

  12. Enforced Expression of Hoxa3 Inhibits Classical and Promotes Alternative Activation of Macrophages In Vitro and In Vivo

    Science.gov (United States)

    Al Sadoun, Hadeel; Burgess, Matthew; Hentges, Kathryn E.

    2016-01-01

    The regulated differentiation of macrophages (mφs) and their subsequent activation into proinflammatory or prohealing subtypes is critical for efficient wound healing. Chronic wounds such as diabetic (db) ulcers are associated with dysregulation of macrophage function. Whereas non-db mφs polarize to an M2-like, prohealing phenotype during the late stages of healing, db-derived mφs continue to display an M1-like, proinflammatory, or a mixed M1-like/M2-like phenotype. We have previously shown that sustained expression of Hoxa3 reduces the excessive number of leukocytes within the db wound; however, the effect of Hoxa3 on mφ polarization was unknown. In this study, we show that Hoxa3 protein transduction of mφs in vitro enhances macrophage maturation, inhibits M1 polarization, and promotes M2 polarization, in part via regulation of Pu.1/Spi1 and Stat6. Sustained expression of Hoxa3 in vivo in db wounds reduces the number of Nos2+ (M1-like) mφs, increases the number of Arg1+ and VEGF+ (M2-like) mφs, and accelerates healing in a DNA-binding independent manner. Our findings suggest a role for Hox protein activity in promoting M1-to-M2-like phenotypic switching via interactions with myeloid transcription factors and provide insight into mechanisms regulating this process in db wound healing. PMID:27342843

  13. Promoter hypomethylation and expression is conserved in mouse chronic lymphocytic leukemia induced by decreased or inactivated Dnmt3a

    Science.gov (United States)

    Haney, Staci L.; Upchurch, G. Michael; Opavska, Jana; Klinkebiel, David; Hlady, Ryan A.; Suresh, Abhinav; Pirruccello, Samuel J.; Shukla, Vipul; Lu, Runqing; Costinean, Stefan; Rizzino, Angie; Karpf, Adam R.; Joshi, Shantaram; Swanson, Patrick; Opavsky, Rene

    2016-01-01

    SUMMARY DNA methyltransferase 3a (DNMT3A) catalyzes the formation of 5-methyl-cytosine in mammalian genomic DNA and it is frequently mutated in human hematologic malignancies. Bi-allelic loss of Dnmt3a in mice results in leukemia and lymphoma, including chronic lymphocytic leukemia (CLL). Here we investigate whether mono-allelic loss of Dnmt3a is sufficient to induce disease. We show that by 16 months of age, 65% of Dnmt3a+/− mice develop a CLL-like disease and 15% of mice develop non-malignant myeloproliferation. Genome-wide methylation analysis reveals that reduced Dnmt3a levels induce promoter hypomethylation at similar loci in Dnmt3a+/− and Dnmt3aΔ/Δ CLL, suggesting that promoters are particularly sensitive to Dnmt3a levels. Gene-expression analysis identified 26 hypomethylated and over-expressed genes common to both Dnmt3a+/− and Dnmt3aΔ/Δ CLL as putative oncogenic drivers. Our data provide evidence that Dnmt3a is a haplo-insufficient tumor suppressor in CLL and highlights the importance of deregulated molecular events in disease pathogenesis. PMID:27134162

  14. A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter.

    Science.gov (United States)

    Omidvar, V; Siti Nor Akmar, A; Marziah, M; Maheran, A A

    2008-09-01

    The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms. PMID:18563415

  15. Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Knudsen, Jan Dines; Johanson, Ted; Eliasson Lantz, Anna; Carlquist, Magnus

    2015-01-01

    A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by p...... mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.......A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by...... growth rate of 0.3 h-1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry...

  16. TECAB - Totally Endoscopic Coronary Artery Bypass

    Medline Plus

    Full Text Available ... And the bypass vessel is the so-called “internal mammary artery.” That’s an artery, as opposed to ... you very much. -- while I’m preparing the internal mammary artery for bypass. Good. All right. We ...

  17. Epicardial ultrasound in coronary artery bypass surgery

    NARCIS (Netherlands)

    Budde, R.P.J.

    2005-01-01

    Chapter 1 Coronary artery bypass surgery (CABG) is traditionally performed via a median sternotomy approach on cardiopulmonary bypass (arrested heart). Since the mid 1990ties, beating heart, minimally invasive and even totally endoscopic CABG are (re)explored. In all approaches to CABG, the surgeo

  18. TECAB - Totally Endoscopic Coronary Artery Bypass

    Medline Plus

    Full Text Available ... as doctors use for this operation is “TECAB,” meaning “Totally Endoscopic Coronary Artery Bypass.” This procedure will ... make sure his vessels are able for bypass, meaning doing the CT angiograph for the peripheral arteries. ...

  19. Atrial fibrillation post cardiac bypass surgery

    OpenAIRE

    Mostafa, Ashraf; EL-Haddad, Mohamed A.; Shenoy, Maithili; Tuliani, Tushar

    2012-01-01

    Atrial fibrillation occurs in 5-40% patients after coronary artery bypass graft surgery. Atrial fibrillation increases mortality and morbidity in the post-operative period. We sought to conduct a comprehensive review of literature focusing on pathophysiology, risk factors, prevention and treatment of post coronary artery bypass graft atrial fibrillation.

  20. TECAB - Totally Endoscopic Coronary Artery Bypass

    Medline Plus

    Full Text Available ... going to thread that up to the patient’s bypass graft using X-ray, and there you are. So what we’re going to do is manipulate this catheter into actually the artery that leads to the arm, the subclavian, which is where the bypass comes off. Let me just saw it for ...

  1. IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells.

    Science.gov (United States)

    Curran, Terry-Ann; Jalili, Reza Baradar; Farrokhi, Ali; Ghahary, Aziz

    2014-01-01

    Regulatory CD4(+)CD25(+)Foxp3(+) T cells (Tregs) can be induced and expanded by dendritic cells (DCs) in the presence of the enzyme indoleamine 2,3-dioxygenase (IDO). Here we report that a possible alternative to DCs are IDO expressing dermal fibroblasts (DFs), which are easier to isolate and sustain in culture compared to DCs. When mouse splenocytes were co-cultured with IDO expressing DFs, a significant increase in frequency and the number of Tregs was found compared to those of control group (13.16%±1.8 vs. 5.53%±1.2, pa subset of Tregs which can be used to generate antigen-specific immune tolerance. PMID:23891282

  2. A hyper-dynamic nature of bivalent promoter states underlies coordinated developmental gene expression modules

    OpenAIRE

    Shah, Akshay; Oldenburg, Anja; Collas, Philippe

    2014-01-01

    Background Chromatin remodeling is crucial for proper programing of developmental gene expression. Recent work provides a dynamic view of post-translational histone modifications during differentiation; however there is little insight on the evolution of combinatorial genome-wide patterns of chromatin marks, excluding an essential aspect of developmental gene regulation. Results We report here a 15-chromatin state Hidden Markov Model which describes changes in chromatin signatures in relation...

  3. Schwann cells genetically modified to express neurotrophins promote spiral ganglion neuron survival in vitro

    OpenAIRE

    Pettingill, Lisa N.; Minter, Ricki L.; Shepherd, Robert K.

    2008-01-01

    The intracochlear infusion of neurotrophic factors via a mini-osmotic pump has been shown to prevent deafness-induced spiral ganglion neuron (SGN) degeneration; however, the use of pumps may increase the incidence of infection within the cochlea, making this technique unsuitable for neurotrophin administration in a clinical setting. Cell- and gene-based therapies are potential therapeutic options. This study investigated whether Schwann cells which were genetically modified to over-express th...

  4. Nonlinear Dynamics in Gene Regulation Promote Robustness and Evolvability of Gene Expression Levels

    Science.gov (United States)

    Steinacher, Arno; Bates, Declan G.; Akman, Ozgur E.; Soyer, Orkun S.

    2016-01-01

    Cellular phenotypes underpinned by regulatory networks need to respond to evolutionary pressures to allow adaptation, but at the same time be robust to perturbations. This creates a conflict in which mutations affecting regulatory networks must both generate variance but also be tolerated at the phenotype level. Here, we perform mathematical analyses and simulations of regulatory networks to better understand the potential trade-off between robustness and evolvability. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics, through the creation of regions presenting sudden changes in phenotype with small changes in genotype. For genotypes embedding low levels of nonlinearity, robustness and evolvability correlate negatively and almost perfectly. By contrast, genotypes embedding nonlinear dynamics allow expression levels to be robust to small perturbations, while generating high diversity (evolvability) under larger perturbations. Thus, nonlinearity breaks the robustness-evolvability trade-off in gene expression levels by allowing disparate responses to different mutations. Using analytical derivations of robustness and system sensitivity, we show that these findings extend to a large class of gene regulatory network architectures and also hold for experimentally observed parameter regimes. Further, the effect of nonlinearity on the robustness-evolvability trade-off is ensured as long as key parameters of the system display specific relations irrespective of their absolute values. We find that within this parameter regime genotypes display low and noisy expression levels. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics. Our results provide a possible solution to the robustness-evolvability trade-off, suggest an explanation for

  5. Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish

    DEFF Research Database (Denmark)

    Koltowska, Katarzyna; Lagendijk, Anne Karine; Pichol-Thievend, Cathy; Fischer, Johanna C; Francois, Mathias; Ober, Elke A; Yap, Alpha S; Hogan, Benjamin M

    2015-01-01

    Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics...... cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division....

  6. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

    Science.gov (United States)

    Xu, Juan; Zhang, Chunhua

    2016-01-01

    Abstract Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies.

  7. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein.

    Science.gov (United States)

    Xu, Juan; Zhang, Chunhua

    2016-05-01

    Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies. PMID:27533931

  8. MiR-183 Regulates ITGB1P Expression and Promotes Invasion of Endometrial Stromal Cells

    Directory of Open Access Journals (Sweden)

    Jie Chen

    2015-01-01

    miR-183 expression induced the invasiveness and inhibition of apoptosis in endometrial stromal cells. The current study aims to identify the miR-183 targets with relevance to cell functions in endometrial stromal cells, to verify the interaction of miR-183 with its target genes, and to confirm the role of miR-183 in the process of endometriosis. Using microarray analysis, we identified 27 differentially expressed genes (19 were upregulated and 8 downregulated, from which we selected 4 downregulated genes (ITGB1, AMIGO2, VAV3, and PSEN2 based on GO databases for functional analysis and significant pathway analysis. Western blotting analyses showed that integrin β1 (ITGB1, but not AMIGO2, was affected by miR-183 overexpression, whereas no protein expression of VAV3 and PSEN2 was detected. Luciferase reporter assay verified that ITGB1 is a target gene of miR-183. Moreover, we found that ITGB1 is overexpressed in the endometrium of endometriosis patients. Furthermore, overexpression of ITGB1 rescued the repressive effects of miR-183 on the invasiveness of endometrial stromal cells. These findings, together with the fact that ITGB1 is a critical factor for cell adhesion and invasiveness, suggest that miR-183 may be involved in the development of endometriosis by regulating ITGB1 in endometrial stromal cells.

  9. Numerical simulation of an alternative to prevent hydrates formation in a bypass section

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, Lucilla Coelho; Oliveira Junior, Joao Americo Aguirre; Fonte, Clarissa Bergman [Engineering Simulation and Scientific Software Ltda. (ESSS), Florianopolis, SC (Brazil); Silva, Fabricio Soares da; Moraes, Carlos Alberto Capela [Petroleo Brasileiro S.A. (PETROBRAS), Rio de Janeiro, RJ (Brazil)

    2012-07-01

    This work presents the use of Computational Fluid Dynamics to evaluate the feasibility of MEG (monoethylene glycol) injection as an alternative to prevent hydrate formation in a bypass section, present in an inlet module of a separation device of a subsea separation system. As the bypass section is open to the main pipeline, MEG will probably be dragged due to secondary flows generated by the main flow stream. The MEG removal rate is estimated, as well as the internal heat transfer between the currents and the heat loss to the external environment in order to estimate the temperature in the equipment. In a first step, the MEG removal was evaluated considering the heat transfer between the liquid phase (composed of water, oil and MEG) and the gas phase as well as the heat transfer by forced convection to the external environment. In a second step, the influence of a thermal insulation layer around the bypass line, reducing the heat loss to the external environment, was studied. Both simulations (with or without thermal insulation) showed the establishment of secondary flows in the open connection between the main line and bypass line, promoting the removal of MEG from the bypass section and enabling other components of the liquid phase and/or gas to enter in the bypass line. This MEG removal is faster when thermal isolation was considered, due to the fact that higher temperatures are established in the bypass, maintaining the liquid phase with lower densities and viscosities. With regard to temperature, the insulation was able to keep higher temperatures at the bypass line than those obtained without insulation, indicating that the combination of MEG injection and thermal insulation may be able to avoid the critical condition for hydrate formation. (author)

  10. Surgical treatment of 82 patients with diabetic lower limb ischemia by distal arterial bypass

    Institute of Scientific and Technical Information of China (English)

    GU Yong-quan; WANG Zhong-gao; ZHANG Jian; QI Li-xing; YU Heng-xi; LI Jian-xin; LI Xue-feng; GUO Lian-rui; LUO Tao; CUI Shi-jun

    2007-01-01

    Background Diabetic lower limb ischemia is a serious complication of diabetes mellitus.This study was conducted to investigate the effectiveness of distal arterial bypass treatment in diabetic patients with lower limb ischemia. Methods From July 2000 to July 2004, 96 lower limbs of 82 diabetic patients (type 2) with severe lower limb ischemia were treated in Xuan Wu Hospital. Arterial bypass with femoro-popliteal polytetrafluoroethylene (PTFE) and graft-tibial autologous grafts was performed on 311 limbs (32.3%). Popliteal-tibial artery bypass alone was performed on 22 limbs (22.9%). Combined iliac artery stenting, femoro-popliteal artery PTFE graft bypass, and graft-tibial artery autologous graft bypass was performed on 12 limbs (12.5%), and femoro-tibial artery graft bypass was performed on 10 limbs (10.4%). Popliteal-tibial-pedal artery graft bypass was performed on 7 limbs (7.3%). Results Arterial grafts in 92 limbs of 79 patients were patent on discharge. Three patients with 4 ischemic limbs (3.7%)died of respiratory failure 12 hours, 3 days and 7 days after operation respectively. Early operation success rate was 96.3% (79/82). Graft patency rate of patients on discharge was 95.8% (92/96). The short-term total effectiveness rate was 83.3% (80/96). Foot ulcer healing rate was 35.7% (10/28). 97.4% (75/77) patients were followed up for a mean of 13.5 months. The long-term total effective rate was 80.7% (71/88). The total amputation rate was 4.5% (4/88). Mortality was 4.5%. The total graft patency rate was 90.9% (80/88).Conclusion In the treatment of diabetic foot, distal lower limb arterial bypass can help to avoid amputation or lower the amputation level, and may promote foot ulcer healing and improve patient's quality of life.

  11. Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize

    Science.gov (United States)

    Hi II maize (Zea mays) plants were engineered to express maize p1 cDNA, a Myb transcription factor, controlled by a putative silk specific promoter, for secondary metabolite production and corn earworm resistance. Transgene expression did not enhance silk color, but about half of the transformed p...

  12. Piceatannol promotes apoptosis via up-regulation of microRNA-129 expression in colorectal cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Haogang [Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081 (China); Jia, Ruichun [Department of Blood Transfusion, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081 (China); Wang, Chunjing; Hu, Tianming [Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081 (China); Wang, Fujing, E-mail: wangfujing-hyd@163.com [Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081 (China)

    2014-09-26

    Highlights: • Piceatannol induces apoptosis in cultured CRC cells. • Piceatannol promotes expression of miR-129. • miR-129 mediates proapoptotic effects of piceatannol. - Abstract: Piceatannol, a naturally occurring analog of resveratrol, has been confirmed as an antitumor agent by inhibiting proliferation, migration, and metastasis in diverse cancer. However, the effect and mechanisms of piceatannol on colorectal cancer (CRC) have not been well understood. This study aimed to test whether piceatannol could inhibit growth of CRC cells and reveal its underlying molecular mechanism. MTT assay was used to detect the cell viability in HCT116 and HT29 cells. Flow cytometry analysis was employed to measure apoptosis of CRC cells. Bcl-2, Bax and caspase-3 levels were analyzed by Western blot and miR-129 levels were determined by real-time RT-PCR. Our study showed that piceatannol inhibited HCT116 and HT29 cells growth in a concentration- and time-dependent manner. Piceatannol induced apoptosis by promoting expression of miR-129, and then inhibiting expression of Bcl-2, an known target for miR-129. Moreover, knock down of miR-129 could reverse the reduction of cell viability induced by piceatannol in HCT116 and HT29 cells. Taken together, our study unraveled the ability of piceatannol to suppress colorectal cancer growth and elucidated the participation of miR-129 in the anti-cancer action of piceatannol. Our findings suggest that piceatannol can be considered to be a promising anticancer agent for CRC.

  13. Piceatannol promotes apoptosis via up-regulation of microRNA-129 expression in colorectal cancer cell lines

    International Nuclear Information System (INIS)

    Highlights: • Piceatannol induces apoptosis in cultured CRC cells. • Piceatannol promotes expression of miR-129. • miR-129 mediates proapoptotic effects of piceatannol. - Abstract: Piceatannol, a naturally occurring analog of resveratrol, has been confirmed as an antitumor agent by inhibiting proliferation, migration, and metastasis in diverse cancer. However, the effect and mechanisms of piceatannol on colorectal cancer (CRC) have not been well understood. This study aimed to test whether piceatannol could inhibit growth of CRC cells and reveal its underlying molecular mechanism. MTT assay was used to detect the cell viability in HCT116 and HT29 cells. Flow cytometry analysis was employed to measure apoptosis of CRC cells. Bcl-2, Bax and caspase-3 levels were analyzed by Western blot and miR-129 levels were determined by real-time RT-PCR. Our study showed that piceatannol inhibited HCT116 and HT29 cells growth in a concentration- and time-dependent manner. Piceatannol induced apoptosis by promoting expression of miR-129, and then inhibiting expression of Bcl-2, an known target for miR-129. Moreover, knock down of miR-129 could reverse the reduction of cell viability induced by piceatannol in HCT116 and HT29 cells. Taken together, our study unraveled the ability of piceatannol to suppress colorectal cancer growth and elucidated the participation of miR-129 in the anti-cancer action of piceatannol. Our findings suggest that piceatannol can be considered to be a promising anticancer agent for CRC

  14. Effects of gamma rays, ultraviolet radiation, sunlight, microwaves and electromagnetic fields on gene expression mediated by human immunodeficiency virus promoter

    International Nuclear Information System (INIS)

    Previous work by our group and others has shown the modulation of human immunodeficiency virus (HIV) promoter or long terminal repeat (LTR) after exposure to neutrons and ultraviolet radiations. Using HeLa cells stably transfected with a construct containing the chloramphenicol acetyl transferase (CAT) gene, the transcription of which is mediated by the HIV-LTR, we designed experiments to examine the effects of exposure to different types of radiation (such as γ rays, ultraviolet and sunlight irradiations, electromagnetic fields and microwaves) in HIV-LTR-driven expression of CAT. These results demonstrated ultraviolet-light-induced transcription from the HIV promoter, as has been shown by others. Exposure to other DNA-damaging agents such as γ rays and sunlight (with limited exposures) had no significant effect on transcription mediated by HIV-LTR, suggesting that induction of HIV is not mediated by just any type of DNA damage but rather may require specific types of DNA damage. Microwaves did not cause cell killing when cells in culture were exposed in high volumes of medium, and the same cells showed no changes in expression. When microwave exposure was carried out in low volumes of medium (so that excessive heat was generated) induction of HIV-LTR transcription (as assayed by CAT activity) was evident. Electromagnetic field exposures had no effect on expression of HIV-LTR. These results demonstrate that not all types of radiation and not all DNA-damaging agents are capable of inducing HIV. We hypothesize that induction of HIV transcription may be mediated by several different signals exposure to radiation. 22 refs., 8 figs

  15. Hydrocellular foam dressing promotes wound healing along with increases in hyaluronan synthase 3 and PPARα gene expression in epidermis.

    Directory of Open Access Journals (Sweden)

    Takumi Yamane

    Full Text Available BACKGROUND: Hydrocellular foam dressing, modern wound dressing, induces moist wound environment and promotes wound healing: however, the regulatory mechanisms responsible for these effects are poorly understood. This study was aimed to reveal the effect of hydrocellular foam dressing on hyaluronan, which has been shown to have positive effects on wound healing, and examined its regulatory mechanisms in rat skin. METHODOLOGY/PRINCIPAL FINDINGS: We created two full-thickness wounds on the dorsolateral skin of rats. Each wound was covered with either a hydrocellular foam dressing or a film dressing and hyaluronan levels in the periwound skin was measured. We also investigated the mechanism by which the hydrocellular foam dressing regulates hyaluronan production by measuring the gene expression of hyaluronan synthase 3 (Has3, peroxisome proliferator-activated receptor α (PPARα, and CD44. Hydrocellular foam dressing promoted wound healing and upregulated hyaluronan synthesis, along with an increase in the mRNA levels of Has3, which plays a primary role in hyaluronan synthesis in epidermis. In addition, hydrocellular foam dressing enhanced the mRNA levels of PPARα, which upregulates Has3 gene expression, and the major hyaluronan receptor CD44. CONCLUSIONS/SIGNIFICANCE: These findings suggests that hydrocellular foam dressing may be beneficial for wound healing along with increases in hyaluronan synthase 3 and PPARα gene expression in epidermis. We believe that the present study would contribute to the elucidation of the mechanisms underlying the effects of hydrocellular foam dressing-induced moist environment on wound healing and practice evidence-based wound care.

  16. Boron promotes streptozotocin-induced diabetic wound healing: roles in cell proliferation and migration, growth factor expression, and inflammation.

    Science.gov (United States)

    Demirci, Selami; Doğan, Ayşegül; Aydın, Safa; Dülger, Esra Çikler; Şahin, Fikrettin

    2016-06-01

    Acute wounds do not generally require professional treatment modalities and heal in a predictable fashion, but chronic wounds are mainly accompanied with infection and prolonged inflammation, leading to healing impairments and continuous tissue degradation. Although a vast amount of products have been introduced in the market, claiming to provide a better optimization of local and systemic conditions of patients, they do not meet the expectations due to being expensive and not easily accessible, requiring wound care facilities, having patient-specific response, low efficiency, and severe side-effects. In this sense, developing new, safe, self-applicable, effective, and cheap wound care products with broad-range antimicrobial activity is still an attractive area of international research. In the present work, boron derivatives [boric acid and sodium pentaborate pentahydrate (NaB)] were evaluated for their antimicrobial activity, proliferation, migratory, angiogenesis, gene, and growth factor expression promoting effects on dermal cells in vitro. In addition, boron-containing hydrogel formulation was examined for its wound healing promoting potential using full-thickness wound model in streptozotocin-induced diabetic rats. The results revealed that while both boron compounds significantly increased proliferation, migration, vital growth factor, and gene expression levels of dermal cells along with displaying remarkable antimicrobial effects against bacteria, yeast, and fungi, NaB displayed greater antimicrobial properties as well as gene and growth factor expression inductive effects. Animal studies proved that NaB-containing gel formulation enhanced wound healing rate of diabetic animals and histopathological scores. Overall data suggest a potential promising therapeutic option for the management of chronic wounds but further studies are highly warranted to determine signaling pathways and target metabolisms in which boron is involved to elucidate the limitations

  17. Sequence Analysis and Potentials of the Native RbcS Promoter in the Development of an Alternative Eukaryotic Expression System Using Green Microalga Ankistrodesmus convolutus

    Directory of Open Access Journals (Sweden)

    Suhaimi Napis

    2012-02-01

    Full Text Available The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS has been used to drive the expression of β-glucuronidase (gusA gene in this microalga. Besides the determination of the transcription start site by 5¢-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2, which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif, which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.

  18. Ectopic Expression of Col2.3 and Col3.6 Promoters in the Brain and Association with Leptin Signaling

    OpenAIRE

    Scheller, Erica L.; Leinninger, Gina M.; Hankenson, Kurt D.; Myers, Martin G.; Paul H Krebsbach

    2011-01-01

    The collagen 2.3 and 3.6 promoters have been used to drive Cre expression for generation of conditional transgenic mutant mice. Within the bone, Col3.6 is expressed by mesenchymal precursor cells and their downstream progeny, while Col2.3 is more osteoblast specific. Our generation of transgenic mice with Col2.3-Cre- and Col3.6-Cre-driven deletion of the long-form leptin receptor (ObRb) necessitated a thorough analysis of the nonspecific expression of these promoters in the central nervous sy...

  19. EGFR signaling promotes β-cell proliferation and survivin expression during pregnancy.

    Directory of Open Access Journals (Sweden)

    Elina Hakonen

    Full Text Available Placental lactogen (PL induced serotonergic signaling is essential for gestational β-cell mass expansion. We have previously shown that intact Epidermal growth factor -receptor (EGFR function is a crucial component of this pathway. We now explored more specifically the link between EGFR and pregnancy-induced β-cell mass compensation. Islets were isolated from wild-type and β-cell-specific EGFR-dominant negative mice (E1-DN, stimulated with PL and analyzed for β-cell proliferation and expression of genes involved in gestational β-cell growth. β-cell mass dynamics were analyzed both with traditional morphometrical methods and three-dimensional optical projection tomography (OPT of whole-mount insulin-stained pancreata. Insulin-positive volume analyzed with OPT increased 1.4-fold at gestational day 18.5 (GD18.5 when compared to non-pregnant mice. Number of islets peaked by GD13.5 (680 vs 1134 islets per pancreas, non-pregnant vs. GD13.5. PL stimulated beta cell proliferation in the wild-type islets, whereas the proliferative response was absent in the E1-DN mouse islets. Serotonin synthesizing enzymes were upregulated similarly in both the wild-type and E1-DN mice. However, while survivin (Birc5 mRNA was upregulated 5.5-fold during pregnancy in the wild-type islets, no change was seen in the E1-DN pregnant islets. PL induced survivin expression also in isolated islets and this was blocked by EGFR inhibitor gefitinib, mTOR inhibitor rapamycin and MEK inhibitor PD0325901. Our 3D-volumetric analysis of β-cell mass expansion during murine pregnancy revealed that islet number increases during pregnancy. In addition, our results suggest that EGFR signaling is required for lactogen-induced survivin expression via MAPK and mTOR pathways.

  20. The C. elegans CSR-1 Argonaute pathway counteracts epigenetic silencing to promote germline gene expression

    OpenAIRE

    Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C

    2013-01-01

    Organisms can develop adaptive sequence-specific immunity by re-expressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piRNA pathway recruits RNA-dependent RNA polymerase RdRP to foreign sequences to amplify a trans-generational small RNA-induced epigenetic silencing signal (termed RNAe). Here we provide evidence that in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expres...

  1. Decreased fructose-1,6-bisphosphatase-2 expression promotes glycolysis and growth in gastric cancer cells

    OpenAIRE

    Li, He; Wang, Juan; Xu, Huiyu; Xing, Rui; Pan, Yuanming; Wenmei LI; Cui, Jiantao; Zhang, Hongbing; Lu, Youyong

    2013-01-01

    Background Increasing evidence suggests that cancer is a metabolic disease. Here, we investigated the potential role of fructose-1,6-bisphosphatase-2 (FBP2), the enzyme that catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate in glucose metabolism, in gastric cancer (GC) development. Results Our data indicated that FBP2 was downregulated in GC tissues (86.2%, 100/116), and absent or low FBP2 expression in GC tissues was correlated with poor su...

  2. Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression

    Directory of Open Access Journals (Sweden)

    Strain Alastair J

    2010-03-01

    Full Text Available Abstract The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%, and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC derived from human umbilical cord blood (UCB and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB.

  3. Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2

    Directory of Open Access Journals (Sweden)

    Gibson Joanne

    2007-10-01

    Full Text Available Abstract Background In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot. Results The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt was inserted between fused lacZ and luciferase (luc genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA. Conclusion This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.

  4. Expression of ipt gene driven by tomato fruit specific promoter and its effects on fruit development of tomato

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Fruit specific promoter (2A12) from Lycopersicom esculentum and cDNA of isopentenyl-transferase (ipt) from Ti plasmid of Agrobacterium tumerfaciens C58 were cloned by PCR procedure respectively. Two plant expression vectors with 2A12/gus or 2A12/ipt were respectively constructed. These two chimeric genes were transferred into tomato by Agrobacterium mediated procedure. The results of Southern hybridization showed that the fusion genes had been integrated into tomatoes. The result of gus histochemical staining showed that 2A12 had high fruit specific expressive capability in transgenic tomato. The ipt expression resulted in accumulation of high level of cytokinins (CTKs) in fruit lead to developmental changes in fruits and seeds. The fruit of ipt transformed tomato had the hyperplastic placenta with very few seeds or even seedless. The shelf life of transgenic fruits elongated for 1-2 weeks. The ratio of fruit set, the dry weight of fruit and the crude protein content in fruit were increased, while the soluble sugar of fruits decreased.

  5. Distinct repressing modules on the distal region of the SBP2 promoter contribute to its vascular tissue-specific expression in different vegetative organs.

    Science.gov (United States)

    Freitas, Rejane L; Carvalho, Claudine M; Fietto, Luciano G; Loureiro, Marcelo E; Almeida, Andrea M; Fontes, Elizabeth P B

    2007-11-01

    The Glycine max sucrose binding protein (GmSBP2) promoter directs vascular tissue-specific expression of reporter genes in transgenic tobacco. Here we showed that an SBP2-GFP fusion protein under the control of the GmSBP2 promoter accumulates in the vascular tissues of vegetative organs, which is consistent with the proposed involvement of SBP in sucrose transport-dependent physiological processes. Through gain-of-function experiments we confirmed that the tissue-specific determinants of the SBP2 promoter reside in the distal cis-regulatory domain A, CRD-A (position -2000 to -700) that is organized into a modular configuration to suppress promoter activity in tissues other than vascular tissues. The four analyzed CRD-A sub-modules, designates Frag II (-1785/-1508), Frag III (-1507/-1237), Frag IV (-1236/-971) and Frag V (-970/-700), act independently to alter the constitutive pattern of -92pSBP2-mediated GUS expression in different organs. Frag V fused to -92pSBP2-GUS restored the tissue-specific pattern of the full-length promoter in the shoot apex, but not in other organs. Likewise, Frag IV confined GUS expression to the vascular bundle of leaves, whereas Frag II mediated vascular specific expression in roots. Strong stem expression-repressing elements were located at positions -1485 to -1212, as Frag III limited GUS expression to the inner phloem. We have also mapped a procambium silencer to the consensus sequence CAGTTnCaAccACATTcCT which is located in both distal and proximal upstream modules. Fusion of either repressing element-containing module to the constitutive -92pSBP2 promoter suppresses GUS expression in the elongation zone of roots. Together our results demonstrate the unusual aspect of distal sequences negatively controlling tissue-specificity of a plant promoter. PMID:17710554

  6. Low-level laser therapy promotes dendrite growth via upregulating brain-derived neurotrophic factor expression

    Science.gov (United States)

    Meng, Chengbo; He, Zhiyong; Xing, Da

    2014-09-01

    Downregulation of brain-derived neurotrophic factor (BDNF) in the hippocampus occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival and dendrite growth, BDNF upregulation may contribute to rescue dendrite atrophy and cell loss in AD. Low-level laser therapy (LLLT) has been demonstrated to regulate neuronal function both in vitro and in vivo. In the present study, we found that LLLT rescued neurons loss and dendritic atrophy via the increase of both BDNF mRNA and protein expression. In addition, dendrite growth was improved after LLLT, characterized by upregulation of PSD95 expression, and the increase in length, branching, and spine density of dendrites in hippocampal neurons. Together, these studies suggest that upregulation of BDNF with LLLT can ameliorate Aβ-induced neurons loss and dendritic atrophy, thus identifying a novel pathway by which LLLT protects against Aβ-induced neurotoxicity. Our research may provide a feasible therapeutic approach to control the progression of Alzheimer's disease.

  7. Stem cell factor expression after renal ischemia promotes tubular epithelial survival.

    Directory of Open Access Journals (Sweden)

    Geurt Stokman

    Full Text Available BACKGROUND: Renal ischemia leads to apoptosis of tubular epithelial cells and results in decreased renal function. Tissue repair involves re-epithelialization of the tubular basement membrane. Survival of the tubular epithelium following ischemia is therefore important in the successful regeneration of renal tissue. The cytokine stem cell factor (SCF has been shown to protect the tubular epithelium against apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse model for renal ischemia/reperfusion injury, we studied how expression of c-KIT on tubular epithelium and its ligand SCF protect cells against apoptosis. Administration of SCF specific antisense oligonucleotides significantly decreased specific staining of SCF following ischemia. Reduced SCF expression resulted in impaired renal function, increased tubular damage and increased tubular epithelial apoptosis, independent of inflammation. In an in vitro hypoxia model, stimulation of tubular epithelial cells with SCF activated survival signaling and decreased apoptosis. CONCLUSIONS/SIGNIFICANCE: Our data indicate an important role for c-KIT and SCF in mediating tubular epithelial cell survival via an autocrine pathway.

  8. TNF-α-Induced VEGF and MMP-9 Expression Promotes Hemorrhagic Transformation in Pituitary Adenomas

    Directory of Open Access Journals (Sweden)

    Qin Liu

    2011-06-01

    Full Text Available Pituitary apoplexy is a clinical syndrome with unknown pathogenesis. Therefore, identifying the underlying mechanisms is of high clinical relevance. Tumor necrosis factor alpha (TNF-α is a critical cytokine mediating various hemorrhagic events, but little is known about its involvement in pituitary apoplexy. Here we show that TNF-α may be an important regulator of hemorrhagic transformation in pituitary adenomas. In this study, sixty surgical specimens of hemorrhagic and non-hemorrhagic human pituitary adenomas were examined. Hemorrhagic pituitary adenomas displayed higher protein and mRNA levels of TNF-α, vascular endothelial growth factor (VEGF and matrix metalloproteinase-9 (MMP-9 compared with those of non-hemorrhagic tumors. Exposure of MMQ pituitary adenoma cells to TNF-α induced VEGF and MMP-9 expression in vitro. Additionally, TNF-α administration caused hemorrhagic transformation and enhanced VEGF and MMP-9 expression in MMQ pituitary adenoma cell xenografts in mice. Blockers of VEGF or MMP-9, either alone or in combination, attenuated but not abrogated TNF-α mediated hemorrhagic transformation in xenografts. This study suggests that TNF-α may play a role in the development of intratumoral hemorrhage in pituitary adenomas via up-regulation of VEGF and MMP-9.

  9. MicroRNA-155 confers encephalogenic potential to Th17 cells by promoting effector gene expression.

    Science.gov (United States)

    Hu, Ruozhen; Huffaker, Thomas B; Kagele, Dominique A; Runtsch, Marah C; Bake, Erin; Chaudhuri, Aadel A; Round, June L; O'Connell, Ryan M

    2013-06-15

    Th17 cells are central to the pathogenesis of autoimmune disease, and recently specific noncoding microRNAs have been shown to regulate their development. However, it remains unclear whether microRNAs are also involved in modulating Th17 cell effector functions. Consequently, we examined the role of miR-155 in differentiated Th17 cells during their induction of experimental autoimmune encephalomyelitis. Using adoptive transfer experiments, we found that highly purified, myelin oligodendrocyte glycoprotein Ag-specific Th17 cells lacking miR-155 were defective in their capacity to cause experimental autoimmune encephalomyelitis. Gene expression profiling of purified miR-155(-/-)IL-17F(+) Th17 cells identified a subset of effector genes that are dependent on miR-155 for their proper expression through a mechanism involving repression of the transcription factor Ets1. Among the genes reduced in the absence of miR-155 was IL-23R, resulting in miR-155(-/-) Th17 cells being hyporesponsive to IL-23. Taken together, our study demonstrates a critical role for miR-155 in Th17 cells as they unleash autoimmune inflammation and finds that this occurs through a signaling network involving miR-155, Ets1, and the clinically relevant IL-23-IL-23R pathway. PMID:23686497

  10. Ectopic Expression of the Chinese Cabbage Malate Dehydrogenase Gene Promotes Growth and Aluminum Resistance in Arabidopsis.

    Science.gov (United States)

    Li, Qing-Fei; Zhao, Jing; Zhang, Jing; Dai, Zi-Hui; Zhang, Lu-Gang

    2016-01-01

    Malate dehydrogenases (MDHs) are key metabolic enzymes that play important roles in plant growth and development. In the present study, we isolated the full-length and coding sequences of BraMDH from Chinese cabbage [Brassica campestris L. ssp. pekinensis (Lour) Olsson]. We conducted bioinformatics analysis and a subcellular localization assay, which revealed that the BraMDH gene sequence contained no introns and that BraMDH is localized to the chloroplast. In addition, the expression pattern of BraMDH in Chinese cabbage was investigated, which revealed that BraMDH was heavily expressed in inflorescence apical meristems, as well as the effect of BraMDH overexpression in two homozygous transgenic Arabidopsis lines, which resulted in early bolting and taller inflorescence stems. Furthermore, the fresh and dry weights of aerial tissue from the transgenic Arabidopsis plants were significantly higher than those from the corresponding wild-type plants, as were plant height, the number of rosette leaves, and the number of siliques produced, and the transgenic plants also exhibited stronger aluminum resistance when treated with AlCl3. Therefore, our results suggest that BraMDH has a dramatic effect on plant growth and that the gene is involved in both plant growth and aluminum resistance. PMID:27536317

  11. Construction and expression of secreting type human TRAIL gene vector mediated by hypoxia/radiation double sensitive promoter

    International Nuclear Information System (INIS)

    Objective: To construct secreting type human TRAIL (shTRAIL) gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter, and observe the effect of hypoxia and radiation on shTRAIL. Methods: HRE upper and lower strands were gotten by chemical synthesis, double strands HRE was gotten by PCR; pMD19T-Egr1 was digested by Sac I and Hind III, then Egr1 was obtained, pshuttle-shTRAIL was digested by Kpn I and BamH I, then shTRAIL was obtained; HRE/Egr1 double sensitive promoter mediated shTRAIL expression vector pcDNA3.1-HRE/Egr1-shTRAIL was constructed by gene recombination technique, it was identified correctly by enzyme digestion, PCR and sequencing. A549 cells were divided into normal, hypoxia (0.1%), irradiation (6 Gy) and hypoxia + irradiation groups. Results: After enzyme digestion by BamH I and Sma I, the fragments which lengths were 1284 bp and 4 998 bp, 2 292 bp and 3 990 bp were obtained; the vector was amplified by PCR with Egr1 and shTRAIL primer, the products which lengthens were 469 bp and 820 bp were obtained; pcDNA3.1-HRE/Egr1-shTRAIL was sequenced, the result was same to designed, this demonstrated that the construction was right. The vectors were transfected into A549 cells of adenocarcinoma of lung, the expression levels of shTRAIL mRNA and protein were increased after treated with hypoxia and radiation, it had statistically significant differences compared with normal group (P<0.05), and when they were combinated, the effect was more obvious. Conclusion: Secreting type human TRAIL gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter is constructed successfully, and hypoxia and radiation could increase the expression of TRAIL, and they have synergetic effect. (authors)

  12. In situ bypass og diabetes

    DEFF Research Database (Denmark)

    Jensen, Leif Panduro; Schroeder, T V; Lorentzen, J E

    1993-01-01

    From 1986 through to 1990 a total of 483 in situ bypass procedures were performed in 444 patients. Preoperative risk-factors were equally distributed among diabetic (DM) and non-diabetic (NDM) patients, except for smoking habits (DM:48%, NDM:64%, p = 0.002) and cardiac disease (DM:45%, NDM:29%, p...... = 0.005). Critical limb-ischaemia was more often present in diabetic than non-diabetic patients (DM:57%, NDM:36%, p = 0.0002). Diabetic patients had a significantly lower distal anastomosis than non-diabetic patients (p = 0.00001). There were no differences among diabetic and non-diabetic patients...... regarding three years primary and secondary patency (58% and 64% respectively), and regarding major amputations. However, the rate of minor amputations was higher in insulin-dependent compared with non-insulin-dependent diabetics, who in turn had a higher rate than non-diabetics (p

  13. Mono-(2-Ethylhexyl) Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta

    Science.gov (United States)

    Parobchak, Nataliya; Rosen, Alex; Vetrano, Anna M.; Srinivasan, Aarthi; Wang, Bingbing; Rosen, Todd

    2016-01-01

    Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2) are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52) signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl)-phthalate (MEHP), increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth. PMID:26751383

  14. Mono-(2-Ethylhexyl Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta.

    Directory of Open Access Journals (Sweden)

    Ximi K Wang

    Full Text Available Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH and cyclooxygenase-2 (COX-2 are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52 signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl-phthalate (MEHP, increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth.

  15. A gain-of-function senescence bypass screen identifies the homeobox transcription factor DLX2 as a regulator of ATM-p53 signaling.

    Science.gov (United States)

    Wang, Yifan; Xu, Qikai; Sack, Laura; Kang, Chanhee; Elledge, Stephen J

    2016-02-01

    Senescence stimuli activate multiple tumor suppressor pathways to initiate cycle arrest and a differentiation program characteristic of senescent cells. We performed a two-stage, gain-of-function screen to select for the genes whose enhanced expression can bypass replicative senescence. We uncovered multiple genes known to be involved in p53 and Rb regulation and ATM regulation, two components of the CST (CTC1-STN1-TEN1) complex involved in preventing telomere erosion, and genes such as REST and FOXO4 that have been implicated in aging. Among the new genes now implicated in senescence, we identified DLX2, a homeobox transcription factor that has been shown to be required for tumor growth and metastasis and is associated with poor cancer prognosis. Growth analysis showed that DLX2 expression led to increased cellular replicative life span. Our data suggest that DLX2 expression reduces the protein components of the TTI1/TTI2/TEL2 complex, a key complex required for the proper folding and stabilization of ATM and other members of the PIKK (phosphatidylinositol 3-kinase-related kinase) family kinase, leading to reduced ATM-p53 signaling and senescence bypass. We also found that the overexpression of DLX2 exhibited a mutually exclusive relationship with p53 alterations in cancer patients. Our functional screen identified novel players that may promote tumorigenesis by regulating the ATM-p53 pathway and senescence. PMID:26833729

  16. Sequential Infection with Common Pathogens Promotes Human-like Immune Gene Expression and Altered Vaccine Response.

    Science.gov (United States)

    Reese, Tiffany A; Bi, Kevin; Kambal, Amal; Filali-Mouhim, Ali; Beura, Lalit K; Bürger, Matheus C; Pulendran, Bali; Sekaly, Rafick-Pierre; Jameson, Stephen C; Masopust, David; Haining, W Nicholas; Virgin, Herbert W

    2016-05-11

    Immune responses differ between laboratory mice and humans. Chronic infection with viruses and parasites are common in humans, but are absent in laboratory mice, and thus represent potential contributors to inter-species differences in immunity. To test this, we sequentially infected laboratory mice with herpesviruses, influenza, and an intestinal helminth and compared their blood immune signatures to mock-infected mice before and after vaccination against yellow fever virus (YFV-17D). Sequential infection altered pre- and post-vaccination gene expression, cytokines, and antibodies in blood. Sequential pathogen exposure induced gene signatures that recapitulated those seen in blood from pet store-raised versus laboratory mice, and adult versus cord blood in humans. Therefore, basal and vaccine-induced murine immune responses are altered by infection with agents common outside of barrier facilities. This raises the possibility that we can improve mouse models of vaccination and immunity by selective microbial exposure of laboratory animals to mimic that of humans. PMID:27107939

  17. Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish

    Directory of Open Access Journals (Sweden)

    Katarzyna Koltowska

    2015-12-01

    Full Text Available Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.

  18. Small molecule screen for inhibitors of expression from canonical CREB response element-containing promoters

    Science.gov (United States)

    Mitton, Bryan; Hsu, Katie; Dutta, Ritika; Tiu, Bruce C.; Cox, Nick; McLure, Kevin G.; Chae, Hee-Don; Smith, Mark; Eklund, Elizabeth A.; Solow-Cordero, David E.; Sakamoto, Kathleen M.

    2016-01-01

    The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ∼114,000 candidate compounds from Stanford University's small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML. PMID:26840025

  19. RickA expression is not sufficient to promote actin-based motility of Rickettsia raoultii.

    Directory of Open Access Journals (Sweden)

    Premanand Balraj

    Full Text Available BACKGROUND: Rickettsia raoultii is a novel Rickettsia species recently isolated from Dermacentor ticks and classified within the spotted fever group (SFG. The inability of R. raoultii to spread within L929 cells suggests that this bacterium is unable to polymerize host cell actin, a property exhibited by all SFG rickettsiae except R. peacocki. This result led us to investigate if RickA, the protein thought to generate actin nucleation, was expressed within this rickettsia species. METHODOLOGY/PRINCIPAL FINDINGS: Amplification and sequencing of R. raoultii rickA showed that this gene encoded a putative 565 amino acid protein highly homologous to those found in other rickettsiae. Using immunofluorescence assays, we determined that the motility pattern (i.e. microcolonies or cell-to-cell spreading of R. raoultii was different depending on the host cell line in which the bacteria replicated. In contrast, under the same experimental conditions, R. conorii shares the same phenotype both in L929 and in Vero cells. Transmission electron microscopy analysis of infected cells showed that non-motile bacteria were free in the cytosol instead of enclosed in a vacuole. Moreover, western-blot analysis demonstrated that the defect of R. raoultii actin-based motility within L929 cells was not related to lower expression of RickA. CONCLUSION/SIGNIFICANCE: These results, together with previously published data about R. typhi, strongly suggest that another factor, apart from RickA, may be involved with be responsible for actin-based motility in bacteria from the Rickettsia genus.

  20. DHEA promotes osteoblast differentiation by regulating the expression of osteoblast-related genes and Foxp3(+) regulatory T cells.

    Science.gov (United States)

    Qiu, Xuemin; Gui, Yuyan; Xu, Yingping; Li, Dajin; Wang, Ling

    2015-10-01

    Several studies have reported that dehydroepiandrosterone (DHEA) promotes osteoblast proliferation and inhibits osteoblast apoptosis and that DHEA inhibits osteoclast maturation. However, whether DHEA regulates osteoblast differentiation remains unclear. The present study first examined the effect of DHEA on bone morphology in vivo. DHEA was found to increase bone volume (BV), bone mineral density (BMD), and the number of trabeculae in bone (Th.N) and it was found to decrease trabecular spacing in bone (Th.sp) in ovariectomized (OVX) mice. Next, the effect of DHEA on osteoblast differentiation was examined in vitro and osteoblastogenesis-related marker genes, such as Runx2, Osterix, Collagen1, and Osteocalcin, were also detected. DHEA increased osteoblast production in mesenchymal stem cells (MSCs) cultured in osteoblastogenic medium, and DHEA increased the expression of Runx2 and osterix, thereby increasing the expression of osteocalcin and collagen1. Immune cells and bone interact, so changes in immune cells were detected in vivo. DHEA increased the number of Foxp3(+) regulatory T cells (Tregs) in the spleen but it did not affect CTLA-4 or IL-10. When MSCs were treated with DHEA in the presence of Tregs, alkaline phosphatase (ALP) activity increased. Osteoblasts and adipocytes are both generated by MSCs. If osteoblast differentiation increases, adipocyte differentiation will decrease, and the reverse also holds true. DHEA was found to increase the number of adipocytes in osteoblastogenic medium but it had no effect on the number of adipocytes and expression of PPARγ mRNA in adipogenic medium. This finding suggests that osteoblasts may be involved in adipocyte production. In conclusion, the current results suggest that DHEA can improve postmenopausal osteoporosis (PMO) by up-regulating osteoblast differentiation via the up-regulation of the expression of osteoblastogenesis-related genes and via an increase in Foxp3(+) Tregs. PMID:26559023

  1. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy.

    Directory of Open Access Journals (Sweden)

    Brandon K Sack

    Full Text Available The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors against factor VIII (FVIII. The current method for eradicating inhibitors, termed immune tolerance induction (ITI, is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8 gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.

  2. Expression of the rat liver carnitine palmitoyltransferase I (CPT-Ialpha) gene is regulated by Sp1 and nuclear factor Y: chromosomal localization and promoter characterization.

    Science.gov (United States)

    Steffen, M L; Harrison, W R; Elder, F F; Cook, G A; Park, E A

    1999-01-01

    Carnitine palmitoyltransferase (CPT)-I catalyses the transfer of long-chain fatty acids from CoA to carnitine for translocation across the mitochondrial inner membrane. Expression of the 'liver' isoform of the CPT-I gene (CPT-Ialpha) is subject to developmental, hormonal and tissue-specific regulation. To understand the basis for control of CPT-Ialpha gene expression, we have characterized the proximal promoter of the CPT-Ialpha gene. Here, we report the sequence of 6839 base pairs of the promoter and the localization of the rat CPT-Ialpha gene to region q43 on chromosome 1. Our studies show that the first 200 base pairs of the promoter are sufficient to drive transcription of the CPT-Ialpha gene. Within this region are two sites that bind both Sp1 and Sp3 transcription factors. In addition, nuclear factor Y (NF-Y) binds the proximal promoter. Mutation at the Sp1 or NF-Y sites severely decreases transcription from the CPT-Ialpha promoter. Other protein binding sites were identified within the first 200 base pairs of the promoter by DNase I footprinting, and these elements contribute to CPT-Ialpha gene expression. Our studies demonstrate that CPT-Ialpha is a TATA-less gene which utilizes NF-Y and Sp proteins to drive basal expression. PMID:10333485

  3. An SNP polymorphism (-844C/T) in the promoter of catalase gene leads to differential expression

    Institute of Scientific and Technical Information of China (English)

    LI Yanping; ZHANG Xin; WANG Zhimin; LU Daru; HANG Wei; JIN Li

    2004-01-01

    @@ Imbalance of redox state has been associated with human diseases, such as cardiovascular diseases, Huntington's disease and Alzheimer's disease[1]. Catalase, an important antioxidant enzyme, decomposes H2O2 into O2and H2O, therefore limiting the deleterious effect of the reactive oxygen species (ROS)[1 -4]. Previous study showed that a C-T polymorphism, located at -844 bp from the translational start site of catalase, is strongly associated with essential hypertension in an isolated Chinese population of Xiangchang country, Anhui Province[5]. To explore the impact of SNP-844C/T on the expression of catalase, human catalase promoters containing either SNP-844C or T variant were subcloned into the pGL3-Basic luciferase vector.

  4. Collagen/Wollastonite nanowire hybrid scaffolds promoting osteogenic differentiation and angiogenic factor expression of mesenchymal stem cells.

    Science.gov (United States)

    Zhang, Qin; Nakamoto, Tomoko; Chen, Shangwu; Kawazoe, Naoki; Lin, Kaili; Chang, Jiang; Chen, Guoping

    2014-04-01

    Porous materials and scaffolds have wide applications in biomedical and biological fields. They can provide biological and physical cues to promote cell adhesion, proliferation, differentiation and extracellular matrix secretion to guide new tissue formation. Hybrid scaffolds of collagen and wollastonite nanowires with well controlled pore structures were prepared by using ice particulates as a porogen material. The hybrid scaffolds had interconnected large spherical pores with wollastonite nanowires embedded in the walls of the pores. The wollastonite nanowires reinforced the hybrid scaffolds and showed some stimulatory effects on cell functions. Human bone marrow-derived mesenchymal stem cells showed higher proliferation and osteogenic differentiation and expressed higher level of genes encoding angiogenesis-related genes in the hybrid scaffolds than did in the collagen scaf-. fold. The results suggest the hybrid scaffolds could facilitate osteogenic differentiation and induce angiogenesis and will be useful for bone tissue engineering. PMID:24734758

  5. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Cheng-hu; Cao, Guo-Fan [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Jiang, Qin, E-mail: Jqin710@vip.sina.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Yao, Jin, E-mail: dryaojin@yahoo.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  6. TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    International Nuclear Information System (INIS)

    Highlights: ► TNF-α induces MMP-9 expression and secretion to promote RPE cell migration. ► MAPK activation is not critical for TNF-α-induced MMP-9 expression. ► Akt and mTORC1 signaling mediate TNF-α-induced MMP-9 expression. ► SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-α. -- Abstract: Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  7. Secondary coolant purification system with demineralizer bypass

    International Nuclear Information System (INIS)

    Apparatus and method are provided for a nuclear stream supply system for adequately controlling the chemistry of the secondary coolant. The invention includes means for the addition of volatile chemicals, a full flow condensate demineralizer, continuous blowdown capability, radiation detection means, a condensate demineralizer bypass line, and an auxiliary demineralizer bypass line, and an auxiliary demineralizer sized to handle full blowdown flow. The auxiliary demineralizer is cut into the system and the steam generator feedwater flow is bypassed around the full flow condensate demineralizer whenever radioactivity is detected in the secondary coolant

  8. Ectopic expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis promotes seed dormancy and stress tolerance.

    Science.gov (United States)

    Lin, Pei-Chi; Hwang, San-Gwang; Endo, Akira; Okamoto, Masanori; Koshiba, Tomokazu; Cheng, Wan-Hsing

    2007-02-01

    Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mm NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2::beta-glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress. PMID:17189333

  9. Effect of glucocorticoids on the activity, expression and proximal promoter of type II deiodinase in rat brown adipocytes.

    Science.gov (United States)

    Martinez-deMena, Raquel; Calvo, Rosa-Maria; Garcia, Laura; Obregon, Maria Jesus

    2016-06-15

    Triiodothyronine (T3) is important for thermogenesis in brown adipose tissue (BAT). Type II deiodinase (DIO2) produces T3 required for intracellular needs in BAT. Brown adipocytes in culture require T3 for the adrenergic stimulation of DIO2. Glucocorticoids induce adipocyte differentiation (lipogenesis). We investigated the regulation of DIO2 activity, Dio2 mRNA and Dio2 promoter activity by glucocorticoids in primary cultures of rat brown adipocytes using dexamethasone (DEX) and hydrocortisone (HC). DEX and HC regulated the adrenergic stimulation of DIO2 activity in a dose- and time-dependent manner, inhibiting DIO2 activity at short treatment times and large doses (1-10 μM) and stimulating DIO2 at low HC doses (1-100 nM) and longer times (DEX). Insulin depletion reduced DIO2 activity but the response to glucocorticoids remained unchanged. DEX and HC inhibited basal DIO2 activity. DEX had no effect on DIO2 half-life, whereas HC stabilized DIO2 activity. DEX and HC inhibited the adrenergic stimulation of Dio2 mRNA expression (100-10000 nM, 14-96 h), but stabilized Dio2 mRNA, particularly DEX. DEX increased basal Dio2 mRNA levels, possibly through stabilization of Dio2 mRNA. An 807 bp construct of the murine Dio2 proximal promoter showed maximal reporter activity, with the cAMP response element (CRE) essential for transcriptional activity. DEX caused inhibition in most constructs containing the CRE element whereas HC stimulated reporter activity in the 807 bp construct. Glucocorticoids inhibited the adrenergic stimulation of Dio2 at the transcriptional level in brown adipocytes, although DIO2 activity increased with HC, possibly due to stabilization of Dio2 activity and mRNA. The CRE and cEBP elements of the Dio2 promoter seem involved in the regulation by glucocorticoids. PMID:26994513

  10. A plant 35S CaMV promoter induces long-term expression of luciferase in Atlantic salmon.

    Science.gov (United States)

    Seternes, Tore; Tonheim, Tom C; Myhr, Anne I; Dalmo, Roy A

    2016-01-01

    The long-term persistence and activity of a naked plasmid DNA (pGL3-35S) containing a luc gene (reporter gene) controlled by a plant 35S CaMV promoter was studied in Atlantic salmon (Salmo salar L.) after injection. Atlantic salmon (mean weight 70 grams) were injected intramuscularly with 100 μg of plasmid DNA. Blood, different tissues and organs were sampled at different time points up to day 535 after injection. Southern blot analysis suggested the presence of extra-chromosomally open circular, linear and supercoiled topoforms of pGL3-35S at day 150 after injection. At day 536 open circular and supercoiled topoforms were detected. Luciferase activity was detected at the injection site up to 536 days post-injection of pGL3-35S, where it peaked at day 150 and decreased to approximately 17% of its maximum activity by day 536. Our study demonstrated that a plasmid containing the 35S promoter was able to induce expression of a reporter gene/protein in fish in vivo and that the plasmid DNA persisted for a prolonged time after intramuscular injection. PMID:27114167

  11. Human fibrocytic myeloid-derived suppressor cells express IDO and promote tolerance via Treg-cell expansion.

    Science.gov (United States)

    Zoso, Alessia; Mazza, Emilia M C; Bicciato, Silvio; Mandruzzato, Susanna; Bronte, Vincenzo; Serafini, Paolo; Inverardi, Luca

    2014-11-01

    By restraining T-cell activation and promoting Treg-cell expansion, myeloid-derived suppressor cells (MDSCs) and tolerogenic DCs can control self-reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4-day culture with FDA-approved cytokines (recombinant human-GM-CSF and recombinant human-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fibrocyte-associated markers, promotes Treg-cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes. In order to exert their protolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection. PMID:25113564

  12. Expression of the human amylase genes: Recent origin of a salivary amylase promoter from an actin pseudogene

    Energy Technology Data Exchange (ETDEWEB)

    Samuelson, L.C.; Gumucio, D.L.; Meisler, M.H. (Univ. of Michigan, Ann Arbor (USA)); Wiebauer, K. (Friedrich Miescher Institut, Basel (Switzerland))

    1988-09-12

    The human genes encoding salivary amylase (AMY1) and pancreatic amylase (AMY2) are nearly identical in structure and sequence. The authors have used ribonuclease protection studies to identify the functional gene copies in this multigene family. Riboprobes derived from each gene were hybridized to RNA from human pancreas, parotid and liver. The sizes of the protected fragments demonstrated that both pancreatic genes are expressed in pancreas. One of the pancreatic genes, AMY2B, is also transcribed at a low level in liver, but not from the promoter used in pancreas. AMY1 transcripts were detected in parotid, but not in pancreas or liver. Unexpected fragments protected by liver RNA led to the discovery that the 5{prime} regions of the five human amylase genes contain a processed {gamma}-actin pseudogene. The promoter and start site for transcription of AMY1 are recently derived from the 3{prime} untranslated region of {gamma}-actin. In addition, insertion of an endogenous retrovirus has interrupted the {gamma}-actin pseudogene in four of the five amylase genes.

  13. Screening of microbial radiation-inducible promoter and study of its expression; Development of basic technique of radiogenic therapy

    International Nuclear Information System (INIS)

    In the search for new therapeutic modalities for cancer, gene therapy has attracted enormous interest over the last few years. Recently, the use of bacteria as a tumor specific protein transfer system has attracted interest. Attenuated Salmonella has been shown to provide selective colonization in tumors. This strategy to apply gene therapy for cancer has been defined as 'Radiogenic Therapy'. In this research, firstly, we screened a radiation inducible promoter of Salmonella responding to clinically relevant low dose of 10 Gy using microarray analysis. Of all genes showing a expression ratio of at least 2-fold changes relative to wild type, 168 genes were induced. To confirm the findings of the microarray by an alternative method, we investigated the transcriptional changes of radio-inducible genes using real time PCR analysis. To verify the ability of screened genes (fadB, narK, cyoA, STM1011, STM2617, and STM2632) to produce a downstream protein by irradiation, the reporter plasmids were constructed. Finally, we found that the promoter of fadB, cyoA, and STM2617 can be activated by irradiation within cancer cells. These results suggest that these genes may be the most probable candidate used in radiogenic therapy

  14. Screening of microbial radiation-inducible promoter and study of its expression; Development of basic technique of radiogenic therapy

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Sangyong; Kim Dongho; Yang, Jaeseung

    2007-02-15

    In the search for new therapeutic modalities for cancer, gene therapy has attracted enormous interest over the last few years. Recently, the use of bacteria as a tumor specific protein transfer system has attracted interest. Attenuated Salmonella has been shown to provide selective colonization in tumors. This strategy to apply gene therapy for cancer has been defined as 'Radiogenic Therapy'. In this research, firstly, we screened a radiation inducible promoter of Salmonella responding to clinically relevant low dose of 10 Gy using microarray analysis. Of all genes showing a expression ratio of at least 2-fold changes relative to wild type, 168 genes were induced. To confirm the findings of the microarray by an alternative method, we investigated the transcriptional changes of radio-inducible genes using real time PCR analysis. To verify the ability of screened genes (fadB, narK, cyoA, STM1011, STM2617, and STM2632) to produce a downstream protein by irradiation, the reporter plasmids were constructed. Finally, we found that the promoter of fadB, cyoA, and STM2617 can be activated by irradiation within cancer cells. These results suggest that these genes may be the most probable candidate used in radiogenic therapy.

  15. Adrenomedullin promotes differentiation of oligodendrocyte precursor cells into myelin-basic-protein expressing oligodendrocytes under pathological conditions in vitro

    Directory of Open Access Journals (Sweden)

    Takakuni Maki

    2015-07-01

    Full Text Available Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs. However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM promotes the in vitro differentiation of OPCs under pathological conditions. Primary OPCs were prepared from neonatal rat brains, and differentiated into myelin-basic-protein expressing oligodendrocytes over time. This in vitro OPC differentiation was inhibited by prolonged chemical hypoxic stress induced by non-lethal CoCl2 treatment. However, AM promoted the OPC differentiation under the hypoxic stress conditions, and the AM receptor antagonist AM22–52 canceled the AM-induced OPC differentiation. In addition, AM treatment increased the phosphorylation level of Akt in OPC cultures, and correspondingly, the PI3K/Akt inhibitor LY294002 blocked the AM-induced OPC differentiation. Taken together, AM treatment rescued OPC maturation under pathological conditions via an AM-receptor-PI3K/Akt pathway. Oligodendrocytes play critical roles in white matter by forming myelin sheath. Therefore, AM signaling may be a promising therapeutic target to boost oligodendrocyte regeneration in CNS disorders.

  16. Adrenomedullin promotes differentiation of oligodendrocyte precursor cells into myelin-basic-protein expressing oligodendrocytes under pathological conditions in vitro.

    Science.gov (United States)

    Maki, Takakuni; Takahashi, Yoko; Miyamoto, Nobukazu; Liang, Anna C; Ihara, Masafumi; Lo, Eng H; Arai, Ken

    2015-07-01

    Oligodendrocytes, which are the main cell type in cerebral white matter, are generated from their precursor cells (oligodendrocyte precursor cells: OPCs). However, the differentiation from OPCs to oligodendrocytes is disturbed under stressed conditions. Therefore, drugs that can improve oligodendrocyte regeneration may be effective for white matter-related diseases. Here we show that a vasoactive peptide adrenomedullin (AM) promotes the in vitro differentiation of OPCs under pathological conditions. Primary OPCs were prepared from neonatal rat brains, and differentiated into myelin-basic-protein expressing oligodendrocytes over time. This in vitro OPC differentiation was inhibited by prolonged chemical hypoxic stress induced by non-lethal CoCl(2) treatment. However, AM promoted the OPC differentiation under the hypoxic stress conditions, and the AM receptor antagonist AM(22-52) canceled the AM-induced OPC differentiation. In addition, AM treatment increased the phosphorylation level of Akt in OPC cultures, and correspondingly, the PI3K/Akt inhibitor LY294002 blocked the AM-induced OPC differentiation. Taken together, AM treatment rescued OPC maturation under pathological conditions via an AM-receptor-PI3K/Akt pathway. Oligodendrocytes play critical roles in white matter by forming myelin sheath. Therefore, AM signaling may be a promising therapeutic target to boost oligodendrocyte regeneration in CNS disorders. PMID:26002630

  17. Interleukins 4 and 13 modulate gene expression and promote proliferation of primary human tenocytes

    Directory of Open Access Journals (Sweden)

    Courneya Jean-Paul

    2010-06-01

    Full Text Available Abstract Background Tendon disorders (tendinopathies pose serious biomedical and socioeconomic problems. Despite diverse treatment approaches, the best treatment strategy remains unclear. Surgery remains the last resort because of the associated morbidity and inconsistent outcomes. We hypothesized that, similar to fibroblasts in various organs, tendon fibroblasts (tenocytes might be responsive to stimulation with interleukins (ILs, particularly IL-4 and IL-13. These two cytokines share sequence homology, receptor chains and functional effects, including stimulation of fibrogenesis. It is unknown whether tenocytes are responsive to stimulation with IL-4 or IL-13. If true, local use of these cytokines might be used to facilitate tendon repair in patients with tendinopathies or used for tendon tissue-engineering approaches to facilitate tenocyte growth on scaffolds in culture. Results Tendon tissues that would normally be discarded were obtained during reconstructive surgery procedures performed for clinical indications. Primary tenocytes were derived from Achilles, posterior tibial, flexor digitorum longus and flexor hallucis longus tendon tissue samples. Reverse transcriptase quantitative PCR (RT-qPCR experiments revealed that mRNAs for the receptor (R chains IL-4Rα, IL-13Rα1 and IL-13Rα2, but not the common γ-chain were present in all tested tendon tissues and in cultured tenocytes. Levels of IL-13R chain mRNAs were significantly higher than those of IL-4R mRNA. The cultures responded, in a dose-dependent fashion, to stimulation with recombinant human IL-4 or IL-13, by increasing proliferation rates 1.5 to 2.0-fold. The mRNA levels of 84 genes related to cell cycle regulation were measured by RT-qPCR after 6 h and 24 h of activation. The expression levels of several genes, notably CDK6 and CDKN2B changed more than twofold. In contrast to their effects on proliferation, stimulation with IL-4 or IL-13 had little if any effect on the levels of

  18. High-fat diets promote insulin resistance through cytokine gene expression in growing female rats.

    Science.gov (United States)

    Flanagan, Anne M; Brown, Jackie L; Santiago, Consuelo A; Aad, Pauline Y; Spicer, Leon J; Spicer, Maria T

    2008-08-01

    To determine if tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). IL-6 gene expression is influenced by amount and source of dietary fat, 30 weanling female rats were randomly assigned to a moderate-fat soybean oil (MFS; 22% of total energy fed as fat), high-fat (HF) soybean oil (HFS; 39% of total energy fed as fat), or HF tallow (HFT; 39% of total energy fed as fat) diet treatments. Oral glucose tolerance tests (OGTT) were conducted serially over 10 weeks of treatment. HFT and HFS rats gained more weight and had greater body fat than the MFS rats fed similar amounts of energy. Both groups of HF-fed rats had greater (PHFT compared to HFS and MFS rats. There were positive correlations (PHFT group, after which, on Week 9, insulin secretion was diminished in response to the OGTT, suggesting impaired pancreatic insulin secretion. HFS rats exhibited insulin resistance on Week 9 OGTT. In summary, an HFT diet fed to growing female rats caused insulin resistance associated with increased hepatic TNF-alpha mRNA leading to pancreatic insufficiency. Early-onset insulin resistance related to the inflammatory process in obesity is influenced by the amount and type of fat in the diet. PMID:17904344

  19. Enhanced gene expression promoted by hybrid magnetic/cationic block copolymer micelles.

    Science.gov (United States)

    Haladjova, E; Rangelov, S; Tsvetanov, Ch B; Posheva, V; Peycheva, E; Maximova, V; Momekova, D; Mountrichas, G; Pispas, S; Bakandritsos, A

    2014-07-15

    We report on novel gene delivery vector systems based on hybrid polymer-magnetic micelles. The hybrid micelles were prepared by codissolution of hydrophobically surface modified iron oxide and amphiphilic polystyrene-b-poly(quaternized 2-vinylpyridine) block copolymer (PS-b-P2QVP) in organic solvent. After extensive dialysis against water, micelles with positively charged hydrophilic corona of PQVP and hydrophobic PS core were prepared, in which magnetic nanoparticles were randomly distributed. The hybrid micelles were used to form complexes with linear (salmon sperm, 2000 bp, corresponding to M(w) of 1.32 × 10(6) Da) and plasmid (pEGFP-N1, 4730 bp, corresponding to M(w) of 3.12 × 10(6) Da) DNA. The resulting magnetopolyplexes of phosphate:amine (P/N) ratios in the 0.05-20 range were characterized by light scattering, ζ-potential measurements, and transmission electron microscopy as well as cytotoxicity and gel retardation assays. The investigated systems displayed a narrow size distribution, particle dimensions below 360 nm, whereas their ζ-potential values varied from positive to negative depending of the P/N ratio. The resulting vector nanosystems exhibited low toxicity. They were able to introduce pEGFP-N1 molecules into the cells. The application of a magnetic field markedly boosted the transgene expression efficiency of the magnetopolyplexes, which was even superior to those of commercial transfectants such as Lipofectamine and dendritic polyethylenimine. PMID:24945823

  20. Tumor-Promoting Desmoplasia Is Disrupted by Depleting FAP-Expressing Stromal Cells.

    Science.gov (United States)

    Lo, Albert; Wang, Liang-Chuan S; Scholler, John; Monslow, James; Avery, Diana; Newick, Kheng; O'Brien, Shaun; Evans, Rebecca A; Bajor, David J; Clendenin, Cynthia; Durham, Amy C; Buza, Elizabeth L; Vonderheide, Robert H; June, Carl H; Albelda, Steven M; Puré, Ellen

    2015-07-15

    Malignant cells drive the generation of a desmoplastic and immunosuppressive tumor microenvironment. Cancer-associated stromal cells (CASC) are a heterogeneous population that provides both negative and positive signals for tumor cell growth and metastasis. Fibroblast activation protein (FAP) is a marker of a major subset of CASCs in virtually all carcinomas. Clinically, FAP expression serves as an independent negative prognostic factor for multiple types of human malignancies. Prior studies established that depletion of FAP(+) cells inhibits tumor growth by augmenting antitumor immunity. However, the potential for immune-independent effects on tumor growth have not been defined. Herein, we demonstrate that FAP(+) CASCs are required for maintenance of the provisional tumor stroma because depletion of these cells, by adoptive transfer of FAP-targeted chimeric antigen receptor (CAR) T cells, reduced extracellular matrix proteins and glycosaminoglycans. Adoptive transfer of FAP-CAR T cells also decreased tumor vascular density and restrained growth of desmoplastic human lung cancer xenografts and syngeneic murine pancreatic cancers in an immune-independent fashion. Adoptive transfer of FAP-CAR T cells also restrained autochthonous pancreatic cancer growth. These data distinguish the function of FAP(+) CASCs from other CASC subsets and provide support for further development of FAP(+) stromal cell-targeted therapies for the treatment of solid tumors. PMID:25979873

  1. Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons

    KAUST Repository

    Yang, Jiangyan

    2014-07-28

    L-lactate is a product of aerobic glycolysis that can be used by neurons as an energy substrate. Here we report that in neurons L-lactate stimulates the expression of synaptic plasticity-related genes such as Arc, c-Fos, and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. L-lactate potentiates NMDA receptor-mediated currents and the ensuing increase in intracellular calcium. In parallel to this, L-lactate increases intracellular levels of NADH, thereby modulating the redox state of neurons. NADH mimics all of the effects of L-lactate on NMDA signaling, pointing to NADH increase as a primary mediator of L-lactate effects. The induction of plasticity genes is observed both in mouse primary neurons in culture and in vivo in the mouse sensory-motor cortex. These results provide insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived L-lactate in long-term memory and long-term potentiation in vivo. This set of data reveals a previously unidentified action of L-lactate as a signaling molecule for neuronal plasticity.

  2. Long non-coding RNA MINCR promotes gallbladder cancer progression through stimulating EZH2 expression.

    Science.gov (United States)

    Wang, Shou-Hua; Yang, Yong; Wu, Xiao-Cai; Zhang, Ming-Di; Weng, Ming-Zhe; Zhou, Di; Wang, Jian-Dong; Quan, Zhi-Wei

    2016-09-28

    The regulation of MYC-regulated long non-coding RNAs has been reported to contribute to certain types of cancers. However, the role of MYC-induced long non-coding RNA (MINCR) in the tumorigenesis of gallbladder cancer (GBC) is still largely unknown. In this study, we discovered that MINCR was markedly upregulated in GBC tissues compared with adjacent normal tissues. High MINCR expression levels in GBC were positively associated with tumor volume and lymph node metastasis and were negatively correlated with overall survival (OS). Upregulation of MINCR and enhancer of zeste homolog 2 (EZH2) in GBC coincided with the downregulation of miR-26a-5p in GBC. Mechanistically, MINCR/miR-26a-5p/EZH2 axis was found to be involved in cell proliferation, cell invasive and apoptosis in GBC cells. Moreover, knockdown of MINCR suppressed cell proliferation, decreased S-phase cell numbers, increased cell apoptosis, and inhibited cell invasion by inhibiting the epithelial-mesenchymal transition (EMT) phenomenon in GBC cells. In vivo, tumor volumes were significantly decreased in the MINCR silencing group compared with those in the control group. These results demonstrated that MINCR could potentially be a therapeutic target as well as a prognostic marker in GBC. PMID:27345740

  3. Methylation of a CpG Island within the Uroplakin Ib Promoter: A Possible Mechanism for Loss of Uroplakin lb Expression in Bladder Carcinoma

    Directory of Open Access Journals (Sweden)

    Andrea E. Varga

    2004-03-01

    Full Text Available Uroplakin Ib is a structural protein on the surface of urothelial cells. Expression of uroplakin Ib mRNA is reduced or absent in many transitional cell carcinomas (TCCs but molecular mechanisms underlying loss of expression remain to be determined. Analysis of the uroplakin Ib promoter identified a weak CpG island spanning the proximal promoter, exon 1, and the beginning of intron 1. This study examined the hypothesis that methylation of this CpG island regulates uroplakin Ib expression. Uroplakin Ib mRNA levels were determined by reverse transcription polymerase chain reaction and CpG methylation was assessed by bisulfite modification of DNA, PCR, and sequencing. A correlation was demonstrated in 15 TCC lines between uroplakin Ib mRNA expression and lack of CpG methylation. In support of a regulatory role for methylation, incubating uroplakin Ib-negative lines with 5-aza-2′ -deoxycytidine reactivated uroplakin Ib mRNA expression. A trend between uroplakin Ib mRNA expression and CpG methylation was also observed in normal urothelium and bladder carcinomas. In particular, loss of uroplakin Ib expression correlated with methylation of a putative Spi/NFκB binding motif. The data are consistent with the hypothesis that methylation of specific sites within the uroplakin Ib promoter may be an important factor in the loss of uroplakin Ib expression in TCCs.

  4. Use of the Lactococcal nisA Promoter To Regulate Gene Expression in Gram-Positive Bacteria : Comparison of Induction Level and Promoter Strength

    NARCIS (Netherlands)

    Eichenbaum, Zehava; Federle, Michael J.; Marra, Diana; Vos, Willem M. de; Kuipers, Oscar P.; Kleerebezem, Michiel; Scott, June R.

    1998-01-01

    We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and Nis

  5. A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells

    Directory of Open Access Journals (Sweden)

    Lejeune Laurence

    2006-11-01

    Full Text Available Abstract Background T-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2 promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes. Results First, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg. Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 μM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA. This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A. Conclusion These results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be

  6. Expression and purification of a new recombinant camel hepcidin able to promote the degradation of the iron exporter ferroportin1.

    Science.gov (United States)

    Boumaiza, Mohamed; Jaouen, Maryse; Deschemin, Jean-Christophe; Ezzine, Aymen; Ben Khalaf, Noureddine; Vaulont, Sophie; Marzouki, Mohamed Nèjib; Sari, Marie Agnès

    2015-11-01

    Hepcidin, a 25-amino-acid and highly disulfide bonded antimicrobial peptide, is the central regulator of iron homeostasis. This hormone is expressed in response to iron and inflammation and interacts with ferroportin1 (FPN1), the only known iron exporter in vertebrates, inducing its internalization and degradation. Thus, the export of iron from cells to plasma will be significantly diminished. Thereby, hepcidin has become the target of intense research studies due to its profound biomedical significance. This study describes the functional expression of recombinant camel hepcidin in Escherichia coli. Biologically active recombinant camel hepcidin was obtained thanks to the production of a hepcidin-thioredoxin fusion protein (TRX-HepcD) and a purified camel hepcidin, with an extra methionine at the N-terminus, was obtained after enterokinase cleavage of the fusion protein. Presence of the four disulfide bridges was verified using MALDI-ToF spectrometry. The recombinant camel hepcidin was compared to related synthetic bioactive peptides, including human hepcidin, and was found equally able to promote ferroportin degradation of mouse macrophages. Furthermore, camel hepcidins exhibits a high capacity to inhibit the growth of Leishmania major promastigotes. These results proved that production of functional camel hepcidin can be achieved in E. coli, this is a major interest for the production of cysteine rich peptides or proteins that can be purified under their functional form without the need of a refolding process. PMID:26169129

  7. Expression in Arabidopsis of a Strawberry Linalool Synthase Gene Under the Control of the Inducible Potato P12 Promoter

    Institute of Scientific and Technical Information of China (English)

    YANG Li-mei; Per Mercke; Joop J A van Loon; FANG Zhi-yuan; Marcel Dicke; Maarten A Jongsma

    2008-01-01

    To investigate the role of inducible linalool in Arabidopsis-insect interactions, the FANESl linalool synthase (LIS) cDNA from strawberry with plastid targeting and a synthetic intron (LIS') was placed under the control of the wound inducible proteinase inhibitor 2 (PI2) promoter from potato. The construct pBin-PP12-LIS' was transformed to Arabidopsis thaliana ecotype Columbia O. Kanamycin resistant T0 seedlings were confirmed for the presence and transcription of the LIS' gene by PCR analysis on genomic DNA and by RT-PCR analysis on RNA. Genomic and RT-PCR products were sequenced to confirm correct splicing of the synthetic intron. The expression of active linalool synthase by the PP12-LIS' gene construct in the transgenic lines was assessed by measuring linalool emission using solid phase micro-extraction (SPME) GC-MS measurements after induction with methyl jasmonate. Among 30 tested independent T2 transgenic lines, 10 exhibited linalool production.Linalool expression could be induced by methyl jasmonate treatment, but not by diamondback moth larvae.

  8. ModuleFinder and CoReg: alternative tools for linking gene expression modules with promoter sequences motifs to uncover gene regulation mechanisms in plants

    Directory of Open Access Journals (Sweden)

    Whelan James

    2006-04-01

    Full Text Available Abstract Background Uncovering the key sequence elements in gene promoters that regulate the expression of plant genomes is a huge task that will require a series of complementary methods for prediction, substantial innovations in experimental validation and a much greater understanding of the role of combinatorial control in the regulation of plant gene expression. Results To add to this larger process and to provide alternatives to existing prediction methods, we have developed several tools in the statistical package R. ModuleFinder identifies sets of genes and treatments that we have found to form valuable sets for analysis of the mechanisms underlying gene co-expression. CoReg then links the hierarchical clustering of these co-expressed sets with frequency tables of promoter elements. These promoter elements can be drawn from known elements or all possible combinations of nucleotides in an element of various lengths. These sets of promoter elements represent putative cis-acting regulatory elements common to sets of co-expressed genes and can be prioritised for experimental testing. We have used these new tools to analyze the response of transcripts for nuclear genes encoding mitochondrial proteins in Arabidopsis to a range of chemical stresses. ModuleFinder provided a subset of co-expressed gene modules that are more logically related to biological functions than did subsets derived from traditional hierarchical clustering techniques. Importantly ModuleFinder linked responses in transcripts for electron transport chain components, carbon metabolism enzymes and solute transporter proteins. CoReg identified several promoter motifs that helped to explain the patterns of expression observed. Conclusion ModuleFinder identifies sets of genes and treatments that form useful sets for analysis of the mechanisms behind co-expression. CoReg links the clustering tree of expression-based relationships in these sets with frequency tables of promoter

  9. Your diet after gastric bypass surgery

    Science.gov (United States)

    ... low-fat or nonfat hard cheeses, cottage cheese, milk, and yogurt. After gastric bypass surgery, your body will not absorb some important vitamins and minerals. You will need to take these vitamins and ...

  10. TECAB - Totally Endoscopic Coronary Artery Bypass

    Medline Plus

    Full Text Available ... perfect bypass. We are often asked how you learn this. Important question. Hours and hours of suturing ... OR-Live” makes it easy for you to learn more. Just click on the “Request information” button ...

  11. TECAB - Totally Endoscopic Coronary Artery Bypass

    Medline Plus

    Full Text Available ... Artery Bypass June 10, 2009 Welcome to the University of Maryland Medical Center in Baltimore, where you ... to Maryland. Welcome to Baltimore. Welcome to the University of Maryland Medical Center here in OR-26. ...

  12. TECAB - Totally Endoscopic Coronary Artery Bypass

    Medline Plus

    Full Text Available ... on for coronary surgery. A very durable bypass running here and supplying the chest wall with blood. ... case the branch, the typical branch that is running between the two heart chambers is located more ...

  13. TECAB - Totally Endoscopic Coronary Artery Bypass

    Medline Plus

    Full Text Available ... is pulsating behind the fascia. Here is some muscle. Here we have the apex or tip of ... tissue. It then turns into the transverse thoracic muscle here. This bypass vessel has an extremely long ...

  14. TECAB - Totally Endoscopic Coronary Artery Bypass

    Medline Plus

    Full Text Available ... sure that we preserve this bypass vessel very well in order to achieve this. So I go ... French quick-draw Venus cardiac visions cannula as well, which will draw back, go to the heart- ...

  15. Types of Coronary Artery Bypass Grafting

    Science.gov (United States)

    ... best option for you based on your needs. Traditional Coronary Artery Bypass Grafting Traditional CABG is used when at least one major ... Grafting This type of CABG is similar to traditional CABG because the chest bone is opened to ...

  16. Barley beta-glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

    Science.gov (United States)

    Agostini, Silvia; Chiavacci, Elena; Matteucci, Marco; Torelli, Michele; Pitto, Letizia; Lionetti, Vincenzo

    2015-01-01

    Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H2O2-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O2−) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O2−, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure. PMID:25388628

  17. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal

    OpenAIRE

    Niakan, K. K.; H. Ji; Maehr, R.; Vokes, S. A.; Rodolfa, K. T.; Sherwood, Richard Irving; Yamaki, M; Dimos, J. T.; Chen, A. E.; Melton, Douglas A.; McMahon, Andrew P.; Eggan, Kevin Carl

    2010-01-01

    In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and mainta...

  18. O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

    OpenAIRE

    Simone Kreth; Niklas Thon; Sabina Eigenbrod; Juergen Lutz; Carola Ledderose; Rupert Egensperger; Tonn, Joerg C.; Kretzschmar, Hans A.; Ludwig C Hinske; Kreth, Friedrich W.

    2011-01-01

    Background: We analyzed prospectively whether MGMT (O(6)-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylatio...

  19. Exhaust gas bypass valve control for thermoelectric generator

    Science.gov (United States)

    Reynolds, Michael G; Yang, Jihui; Meisner, Greogry P.; Stabler, Francis R.; De Bock, Hendrik Pieter Jacobus; Anderson, Todd Alan

    2012-09-04

    A method of controlling engine exhaust flow through at least one of an exhaust bypass and a thermoelectric device via a bypass valve is provided. The method includes: determining a mass flow of exhaust exiting an engine; determining a desired exhaust pressure based on the mass flow of exhaust; comparing the desired exhaust pressure to a determined exhaust pressure; and determining a bypass valve control value based on the comparing, wherein the bypass valve control value is used to control the bypass valve.

  20. Isolation, Expression, and Promoter Analysis of GbWRKY2: A Novel Transcription Factor Gene from Ginkgo biloba

    Directory of Open Access Journals (Sweden)

    Yong-Ling Liao

    2015-01-01

    Full Text Available WRKY transcription factor is involved in multiple life activities including plant growth and development as well as biotic and abiotic responses. We identified 28 WRKY genes from transcriptome data of Ginkgo biloba according to conserved WRKY domains and zinc finger structure and selected three WRKY genes, which are GbWRKY2, GbWRKY16, and GbWRKY21, for expression pattern analysis. GbWRKY2 was preferentially expressed in flowers and strongly induced by methyl jasmonate. Here, we cloned the full-length cDNA and genomic DNA of GbWRKY2. The full-length cDNA of GbWRKY2 was 1,713 bp containing a 1,014 bp open reading frame encoding a polypeptide of 337 amino acids. The GbWRKY2 genomic DNA had one intron and two exons. The deduced GbWRKY2 contained one WRKY domain and one zinc finger motif. GbWRKY2 was classified into Group II WRKYs. Southern blot analysis revealed that GbWRKY2 was a single copy gene in G. biloba. Many cis-acting elements related to hormone and stress responses were identified in the 1,363 bp-length 5′-flanking sequence of GbWRKY2, including W-box, ABRE-motif, MYBCOREs, and PYRIMIDINE-boxes, revealing the molecular mechanism of upregulated expression of GbWRKY2 by hormone and stress treatments. Further functional characterizations in transiently transformed tobacco leaves allowed us to identify the region that can be considered as the minimal promoter.

  1. Isolation, Expression, and Promoter Analysis of GbWRKY2: A Novel Transcription Factor Gene from Ginkgo biloba

    Science.gov (United States)

    Liao, Yong-Ling; Shen, Yong-Bao; Chang, Jie; Zhang, Wei-Wei; Cheng, Shui-Yuan; Xu, Feng

    2015-01-01

    WRKY transcription factor is involved in multiple life activities including plant growth and development as well as biotic and abiotic responses. We identified 28 WRKY genes from transcriptome data of Ginkgo biloba according to conserved WRKY domains and zinc finger structure and selected three WRKY genes, which are GbWRKY2, GbWRKY16, and GbWRKY21, for expression pattern analysis. GbWRKY2 was preferentially expressed in flowers and strongly induced by methyl jasmonate. Here, we cloned the full-length cDNA and genomic DNA of GbWRKY2. The full-length cDNA of GbWRKY2 was 1,713 bp containing a 1,014 bp open reading frame encoding a polypeptide of 337 amino acids. The GbWRKY2 genomic DNA had one intron and two exons. The deduced GbWRKY2 contained one WRKY domain and one zinc finger motif. GbWRKY2 was classified into Group II WRKYs. Southern blot analysis revealed that GbWRKY2 was a single copy gene in G. biloba. Many cis-acting elements related to hormone and stress responses were identified in the 1,363 bp-length 5′-flanking sequence of GbWRKY2, including W-box, ABRE-motif, MYBCOREs, and PYRIMIDINE-boxes, revealing the molecular mechanism of upregulated expression of GbWRKY2 by hormone and stress treatments. Further functional characterizations in transiently transformed tobacco leaves allowed us to identify the region that can be considered as the minimal promoter. PMID:26351628

  2. Phrenic Nerve Injury during Coronary Artery Bypass

    OpenAIRE

    Guinn, Gene A.; Beall, Arthur C.; Lamki, Neela; Heibig, Jacques; Thornby, John

    1990-01-01

    After coronary artery bypass, some patients have diaphragmatic elevation, usually on the left side. To test our hypothesis that this phenomenon is due to phrenic nerve injury resulting from either 1) dissection of the proximal portion of the left internal mammary artery or 2) topical cooling of the heart with icy slush, we performed the following 2-part study. First, we reviewed our hospital records of 99 coronary artery bypass patients, 55 of whom had received left internal mammary artery gr...

  3. Induced gene expression in industrial Saccharomyces pastorianus var. carlsbergensis TUM 34/70: evaluation of temperature and ethanol inducible native promoters.

    Science.gov (United States)

    Fischer, S; Engstler, C; Procopio, S; Becker, T

    2016-05-01

    Induced gene expression is an important trait in yeast metabolic engineering, but current regulations prevent the use of conventional expression systems, such as galactose and copper, in food and beverage fermentations. This article examines the suitability of temperature-inducible native promoters for use in the industrial yeast strain Saccharomyces pastorianus var. carlsbergensis TUM 34/70 under brewing conditions. Ten different promoters were cloned and characterized under varying temperature shifts and ethanol concentrations using a green fluorescent protein reporter. The activities of these promoters varied depending upon the stress conditions applied. A temperature shift to 4°C led to the highest fold changes of PSSA3, PUBI4 and PHSP104 by 5.4, 4.5 and 5.0, respectively. Ethanol shock at 24°C showed marked, concentration-dependent induction of the promoters. Here, PHSP104 showed its highest induction at ethanol concentrations between 4% (v/v) and 6% (v/v). The highest fold changes of PSSA3 and PUBI4 were found at 10% (v/v) ethanol. In comparison, the ethanol shock at a typical fermentation temperature (12°C) leads to lower induction patterns of these promoters. Taken together, the data show that three promoters (PHSP104, PUBI4 and PSSA3) have high potential for targeted gene expression in self-cloning brewing yeast using temperature shifts. PMID:26882929

  4. Bypass Pigging of Subsea Pipelines Suffering Wax Deposition

    OpenAIRE

    Galta, Tore

    2014-01-01

    Which criteria to pay attention to is important when finding the optimal pigging frequency. This thesis illustrates the forces acting on a bypass pig in operation. Expressions for both the frictional force and wax removal force have been presented. Results presented in this thesis show that the frictional forces are much higher than the forces for wax removal. The most important factor for the contact forces for a cleaning pig seems to be due to the oversize of the discs. However, it is diffi...

  5. A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid

    Directory of Open Access Journals (Sweden)

    Sinah Namita

    2012-08-01

    Full Text Available Abstract Background The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae. Results A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter and in S. cerevisiae (via the CUP1 or MET25 promoter. These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins. Conclusions The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.

  6. An experimental study of nerve bypass graft

    Institute of Scientific and Technical Information of China (English)

    XU Jie; LI Xue-shi

    2008-01-01

    Objective: To study the use of a nerve "bypass" graft as a possible alternative to neurolysis or segmental resection with interposition grafting in the treatment of neuroma-in-continuity. Methods: A sciatic nerve crush injury model was established in the Sprague-Dawley rat by compression with a straight hemostatic forceps. Epineurial windows were created proximal and distal to the injury site. An 8-mm segment of radial nerve was harvested and coaptated to the sciatic nerve at the epineurial window sites proximal and distal to the compressed segment (bypass group). A sciatic nerve crush injury without bypass served as a control. Nerve conduction studies were performed over an 8-week period. Sciatic nerves were then harvested and studied under transmission electron microscopy. Myelinated axon counts were obtained. Results: Nerve conduction velocity was significantly faster in the bypass group than in the control group at 8 weeks (63.57 m/s±5.83 m/s vs. 54.88 m/s±4.79m/s, P<0.01). Myelinated axon counts in distal segments were found more in the experimental sciatic nerve than in the control sciatic nerve. Significant axonal growth was noted in the bypass nerve segment itself. Conclusion: Nerve bypass may serve to augment peripheral axonal growth while avoiding further loss of the native nerve.

  7. Expression of lacZ from the Promoter of the Escherichia coli spc Operon Cloned into Vectors Carrying the W205 trp-lac Fusion

    OpenAIRE

    Liang, Sung-Tzu; Dennis, Patrick P.; Bremer, Hans

    1998-01-01

    The expression of lacZ has been analyzed and compared in a series of promoter cloning vectors by measuring the amount of lacZ mRNA by hybridization and the amount of β-galactosidase by standard enzymatic assay. Expression was driven by the promoter, Pspc, of the spc ribosomal protein operon. The vectors contained either the standard W205 trp-lac fusion with the trp operon transcription terminator, trpt, located in the lacZ leader sequence, or a deletion derivative that functionally inactivate...

  8. Symmetry in the Language of Gene Expression: A Survey of Gene Promoter Networks in Multiple Bacterial Species and Non-σ Regulons

    Directory of Open Access Journals (Sweden)

    Stefan M. Turcic

    2011-11-01

    Full Text Available The language of gene expression displays topological symmetry. An important step during gene expression is the binding of transcriptional proteins to DNA promoters adjacent to a gene. Some proteins bind to many promoters in a genome, defining a regulon of genes wherein each promoter might vary in DNA sequence relative to the average consensus. Here we examine the linguistic organization of gene promoter networks, wherein each node in the network represents a promoter and links between nodes represent the extent of base pair-sharing. Prior work revealed a fractal nucleus in several σ-factor regulons from Escherichia coli. We extend these findings to show fractal nuclei in gene promoter networks from three bacterial species, E. coli, Bacillus subtilis, and Pseudomonas aeruginosa. We surveyed several non-σ transcription factors from these species and found that many contain a nucleus that is both visually and numerically fractal. Promoter footprint size scaled as a negative power-law with both information entropy and fractal dimension, while the latter two parameters scaled positively and linearly. The fractal dimension of the diffuse networks (dB = ~1.7 was close to that expected of a diffusion limited aggregation process, confirming prior predictions as to a possible mechanism for development of this structure.

  9. Characterization of genome-wide enhancer-promoter interactions reveals co-expression of interacting genes and modes of higher order chromatin organization

    Institute of Scientific and Technical Information of China (English)

    Iouri Chepelev; Gang Wei; Dara Wangsa; Qingsong Tang; Keji Zhao

    2012-01-01

    Recent epigenomic studies have predicted thousands of potential enhancers in the human genome.However,there has not been systematic characterization of target promoters for these potential enhancers.Using H3K4me2 as a mark for active enhancers,we identified genome-wide EP interactions in human CD4+ T cells.Among the 6 520 longdistance chromatin interactions,we identify 2 067 enhancers that interact with 1 619 promoters and enhance their expression.These enhancers exist in accessible chromatin regions and are associated with various histone modifications and polymerase Ⅱ binding.The promoters with interacting enhancers are expressed at higher levels than those without interacting enhancers,and their expression levels are positively correlated with the number of interacting enhancers.Interestingly,interacting promoters are co-expressed in a tissue-specific manner.We also find that chromosomes are organized into multiple levels of interacting domains.Our results define a global view of EP interactions and provide a data set to further understand mechanisms of enhancer targeting and long-range chromatin organization.The Gene Expression Omnibus accession number for the raw and analyzed chromatin interaction data is GSE32677.

  10. Demethylation profile of the TNF-α promoter gene is associated with high expression of this cytokine in Dengue virus patients.

    Science.gov (United States)

    Gomes, Alessandra Vilas Boas Terra; de Souza Morais, Stella Maria; Menezes-Filho, Sergio Luiz; de Almeida, Luiz Gustavo Nogueira; Rocha, Raissa Prado; Ferreira, Jaqueline Maria Siqueira; Dos Santos, Luciana Lara; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2016-08-01

    Dengue is the most prevalent arthropod-borne viral illness in humans. The overexpression of cytokines by Dengue virus (DENV) infected cells is associated with the most severe forms of the disease. Unmethylated CpG islands are related to a transcriptionally active structure, whereas methylated DNA recruits methyl-binding proteins that inhibit gene expression. Several studies have described the importance of epigenetic events in the regulation and expression of many cytokines. The purpose of the present study was to evaluate the methylation status of the IFN-γ and TNF-α promoters in DNA extracted from dengue infected patients using methylation-specific polymerase chain reaction. A high frequency of demethylation was observed in the TNF-α promoter of DENV infected patients when compared to non-infected controls. The patients with an unmethylated profile showed higher expression of TNF-α mRNA than patients with the methylated status. No difference was found in the methylation frequency between the two analyzed groups regarding the IFN-γ promoter or in the expression of IFN-γ transcripts. The present study provides the first association of TNF-α promoter demethylation in DENV infected individuals and demonstrates a correlation between the methylation status of the region analyzed and the expression of TNF-α transcripts in DENV infected patients. J. Med. Virol. 88:1297-1302, 2016. © 2016 Wiley Periodicals, Inc. PMID:26792115

  11. Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

    Science.gov (United States)

    Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

    2016-06-01

    Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits. PMID:26976138

  12. Eomesodermin promotes interferon-γ expression and binds to multiple conserved noncoding sequences across the Ifng locus in mouse thymoma cell lines.

    Science.gov (United States)

    Fukuoka, Natsuki; Harada, Misuzu; Nishida, Ai; Ito, Yuko; Shiota, Hideki; Kataoka, Takao

    2016-02-01

    The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-γ (IFN-γ). However, in contrast to T-bet, the role of Eomes in the expression of IFN-γ remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-γ in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulation-dependent and stimulation-independent manners. PMID:26749212

  13. Feasibility of a novel positive feedback effect of 131I-promoted Bac-Egr1-hNIS expression in malignant glioma via baculovirus

    International Nuclear Information System (INIS)

    Purpose: As intracellular iodine is released rapidly, increased expression of sodium/iodide symporter (NIS) is required for effective radioiodine treatment of tumor. As Egr1 promoter is activated by 131I and may promote human NIS (hNIS) expression, hNIS also induces 131I uptake and activates Egr1, so the existence of a positive feedback effect of 131I-promoted Egr1-hNIS expression is possible. Our purpose was to investigate the possible existence of this positive feedback effect through a series of in vitro pioneer studies. Method: Recombinant baculovirus (Bac-Egr1-hNIS) encoding the hNIS gene under the control of a radiation-inducible Egrl promoter was constructed. To test 131I-promoted hNIS expression, human malignant glioma U87 cells were transfected with Bac-Egr1-hNIS, stimulated with or without 131I; the expression of hNIS protein was detected by immunofluorescence and flow cytometry test. In addition, the uptake and efflux of 131I were determined after the incubation of Bac-Egr1-hNIS-transfected U87 cells with or without 131I. Results: Immunocytochemical staining and flow cytometry test showed a higher hNIS protein expression in Bac-Egr1-hNIS-transfected U87 cells with 131I stimulation than in cells without stimulation. Bac-Egr1-hNIS-transfected U87 cells accumulated up to about 4.05 times of 131I after 131I stimulation. The amount of 131I uptake in both groups showed a baculovirus dose-dependent manner. However, rapid efflux of radioactivity was observed in both groups, with 50% lost during the first 2 min after the 131I-containing medium had been replaced by a nonradioactive medium. Conclusion: Our results indicated that an improved transgene expression of 131I-stimulated hNIS in U87 cells using a baculovirus vector containing the Egr1 promoter is possible, and the increased expression of hNIS is responsible for a higher 131I uptake. It might provide a reference for the existence of a positive feedback effect in 131I-promoted Bac-Egr1-hNIS expression in

  14. BdCESA7, BdCESA8, and BdPMT Utility Promoter Constructs for Targeted Expression to Secondary Cell-Wall-Forming Cells of Grasses

    OpenAIRE

    Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana; Foster, Cliff E.; Vogel, John P; Steven D. Karlen; Ralph, John; Sedbrook, John C

    2016-01-01

    Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned...

  15. Endothelin‑1 induces oncostatin M expression in osteoarthritis osteoblasts by trans‑activating the oncostatin M gene promoter via Ets‑1.

    Science.gov (United States)

    Wu, Ren; Wang, Wanchun; Huang, Guoliang; Mao, Xinzhan; Chen, You; Tang, Qi; Liao, Lele

    2016-04-01

    Oncostatin M (OSM) contributes to cartilage degeneration in osteoarthritis (OA) and was demonstrated to be expressed in OA osteoblasts. Endothelin‑1 (ET‑1) is implicated in the degradation of OA articular cartilage, and osteoblast proliferation and bone development. In the present study, the effects of ET‑1 on OSM expression in human OA osteoblasts were investigated, to the best of our knowledge, for the first time. Primary human OA osteoblasts were treated with ET‑1 (1, 5, 10, 20 and 30 nM) for 0.5, 1, 2, 3 and 4 h with or without the selective ETA receptor (ETAR) antagonist, BQ123, ETB receptor antagonist, BQ788 or the phosphatidylinositol 3‑kinase (PI3K) inhibitor, BKM120. ET‑1 treatment induced OSM mRNA expression, and the intracellular and secreted protein levels of OA osteoblasts in a dose‑dependent manner. This effect was suppressed by BQ123 and BKM120, but not BQ788 administration. In combination with electrophoretic mobility shift assays, deletional and mutational analyses on the activity of a human OSM promoter/luciferase reporter demonstrated that ET‑1 induced OSM expression in OA osteoblasts by trans‑activating the OSM gene promoter through specific binding of Ets‑1 to an Ets‑1 binding site in the OSM promoter in an ETAR‑ and PI3K‑dependent manner. Furthermore, ET‑1 treatment increased the expression of Ets‑1 in a dose‑dependent manner, however the knockdown of Ets‑1 suppressed the ET1‑induced expression of OSM in OA osteoblasts. In conclusion, the present study demonstrated that ET‑1 induces the expression of OSM in OA osteoblasts by trans‑activating the OSM gene promoter primarily through increasing the expression level of Ets‑1 in an ETAR‑ and PI3K‑dependent manner. The current study suggested novel insights into the mechanistic role of ET‑1 in the pathophysiology of OA. PMID:26934912

  16. A RARE of hepatic Gck promoter interacts with RARα, HNF4α and COUP-TFII that affect retinoic acid- and insulin-induced Gck expression.

    Science.gov (United States)

    Li, Rui; Zhang, Rui; Li, Yang; Zhu, Bing; Chen, Wei; Zhang, Yan; Chen, Guoxun

    2014-09-01

    The expression of hepatic glucokinase gene (Gck) is regulated by hormonal and nutritional signals. How these signals integrate to regulate the hepatic Gck expression is unclear. We have shown that the hepatic Gck expression is affected by Vitamin A status and synergistically induced by insulin and retinoids in primary rat hepatocytes. We hypothesized that this is mediated by a retinoic acid responsive element (RARE) in the hepatic Gck promoter. Here, we identified the RARE in the hepatic Gck promoter using standard molecular biology techniques. The single nucleotide mutations affecting the promoter activation by retinoic acid (RA) were also determined for detail analysis of protein and DNA interactions. We have optimized experimental conditions for performing electrophoresis mobility shift assay and demonstrated the interactions of the retinoic acid receptor α (RARα), retinoid X receptor α (RXRα), hepatocyte nuclear factor 4α (HNF4α) and chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) in the rat nuclear extract with this RARE, suggesting their roles in the regulation of Gck expression. Chromatin immunoprecipitation assays demonstrated that recombinant adenovirus-mediated overexpression of RARα, HNF4α and COUP-TFII, but not RXRα, significantly increased their occupancy in the hepatic Gck promoter in primary rat hepatocytes. Overexpression of RARα, HNF4α and COUP-TFII, but not RXRα, also affected the RA- and insulin-mediated Gck expression in primary rat hepatocytes. In summary, this