Water deficit modulates gene expression in growing zones of soybean seedlings. Analysis of differentially expressed cDNAs, a new beta-tubulin gene, and expression of genes encoding cell wall proteins.

Science.gov (United States)

Creelman, R A; Mullet, J E

1991-10-01

Transfer of soybean seedlings to low-water-potential vermiculite (psi w = -0.3 MPa) results in a reversible decrease in hypocotyl growth and modulation of several polysomal mRNAs (Plant Physiol 92: 205-214). We report here the isolation of two cDNA clones (pGE16 and pGE95) which correspond to genes whose mRNA levels are increased, and one cDNA clone (pGE23) which corresponds to a gene whose mRNA level is decreased in the hypocotyl zone of cell elongation by water deficit. In well-watered seedlings mRNAs hybridizing to pGE16 and pGE95 are most abundant in mature regions of the seedling, but in water-deficient seedlings mRNA levels are reduced in mature regions and enhanced in elongating regions. RNA corresponding to soybean proline-rich protein 1 (sbPRP1) shows a similar tissue distribution and response to water deficit. In contrast, in well-watered seedlings, the gene corresponding to pGE23 was highly expressed in the hypocotyl and root growing zones. Transfer of seedlings to low-water-potential vermiculite caused a rapid decrease in mRNA hybridizing to pGE23. Sequence analysis revealed that pGE23 has high homology with beta-tubulin. Water deficit also reduced the level of mRNA hybridizing to JCW1, an auxin-modulated gene, although with different kinetics. Furthermore, mRNA encoding actin, glycine-rich proteins (GRPs), and hydroxyproline-rich glycoproteins (HRGPs) were down-regulated in the hypocotyl zone of elongation of seedlings exposed to water deficit. No effect of water deficit was observed on the expression of chalcone synthase. Decreased expression of beta-tubulin, actin, JCW1, HRGP and GRP and increased expression of sbPRP1, pGE95 and pGE16 in the hypocotyl zone of cell elongation could participate in the reversible growth inhibition observed in water-deficient soybean seedlings.

[Influence of physiologic 17 beta-estradiol concentrations on gene E6 expression in HVP type 18 in vitro].

Science.gov (United States)

Dziubińska-Parol, Izabella; Gasowska, Urszula; Rzymowska, Jolanta; Kwaśniewska, Anna

2003-09-01

Many recent studies indicate that long term use of contraceptives is a strong risk factor in the development of cervical cancer. Steroid hormones, in persistent papilloma virus infection act on various levels, one of them is enhancing transforming activity of the virus. The aim of the study was to estimate if physiological concentrations of 17 beta-estradiol could influence expression of viral transforming genes. HeLa cell lines were incubated with three different physiological concentrations and and on the third day of incubation the level of E6 gene expression was determined. Results show no differences in expression between the control culter, and cultures incubated with physiological concentrations. It indicates that normal levels of 17 beta-estradiol don't play role in transforming process but it also shows need to analyse higher levels of hormones by quantitative analyses in prospective studies.

Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

Science.gov (United States)

Takala, T M; Saris, P E J; Tynkkynen, S S H

2003-01-01

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

Transforming growth factor. beta. sub 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis

Energy Technology Data Exchange (ETDEWEB)

Broekelmann, T.J.; Limper, A.H.; McDonald, J.A. (Washington Univ., St. Louis, MO (United States)); Colby, T.V. (Mayo Clinic, Rochester, MN (United States))

1991-08-01

Idiopathic pulmonary fibrosis is an inexorably fatal disorder characterized by connective tissue deposition within the terminal air spaces resulting in loss of lung function and eventual respiratory failure. Previously, the authors demonstrated that foci of activated fibroblasts expressing high levels of fibronectin, procollagen, and smooth muscle actin and thus resembling those found in healing wounds are responsible for the connective tissue deposition and scarring in idiopathic pulmonary fibrosis. Using in situ hybridization and immunohistochemistry, they now demonstrate the presence of transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), a potent profibrotic cytokine, in the foci containing these activated fibroblasts. These results suggest that matrix-associated TGF-{beta}{sub 1} may serve as a stimulus for the persistent expression of connective tissue genes. One potential source of the TGF-{beta}{sub 1} is the alveolar macrophage, and they demonstrate the expression of abundant TGF-{beta}{sub 1} mRNA in alveolar macrophages in lung tissue from patients with idiopathic pulmonary fibrosis.

TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Chung, Eun Jee [Department of Ophthalmology, National Health Insurance Corporation Ilsan Hospital, Gyeonggi-do (Korea, Republic of); Chun, Ji Na; Jung, Sun-Ah [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of); Cho, Jin Won [Department of Biology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Lee, Joon H., E-mail: joonhlee@konyang.ac.kr [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of)

2011-11-18

Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information

Long-term high-level expression of human beta-globin occurs following transplantation of transgenic marrow into irradiated mice.

Science.gov (United States)

Himelstein, A; Ward, M; Podda, S; de la Flor Weiss, E; Costantini, F; Bank, A

1993-03-01

When the human beta-globin gene is transferred into the bone marrow cells of live mice, its expression is very low. To investigate the reason for this, we transferred the bone marrow of transgenic mice containing and expressing the human beta-globin into irradiated recipients. We demonstrate that long-term high level expression of the human beta-globin gene can be maintained in the marrow and blood of irradiated recipients following transplantation. Although expression decreased over time in most animals because of host marrow reconstitution, the ratio of human beta-globin transgene expression to endogenous mouse beta-globin gene expression in donor-derived erythroid cells remained constant over time. We conclude that there is no inherent limitation to efficient expression of an exogenous human beta-globin gene in mouse bone marrow cells following marrow transplantation.

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

Science.gov (United States)

Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-04-16

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

Science.gov (United States)

Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Homologous expression of a mutated beta-tubulin gene does not confer benomyl resistance on Trichoderma virens.

Science.gov (United States)

Mukherjee, M; Hadar, R; Mukherjee, P K; Horwitz, B A

2003-01-01

To clone the beta-tubulins and to induce resistance to benzimidazoles in the biocontrol fungus Trichoderma virens through site-directed mutagenesis. Two beta-tubulin genes have been cloned using PCR amplification followed by the screening of a T. virens cDNA library. The full-length cDNA clones, coding for 445 and 446 amino acids, have been designated as T. virens tub1 and T. virens tub2. A sequence alignment of these two tubulins with tubulins from other filamentous fungi has shown the presence of some unique amino acid sequences not found in those positions in other beta-tubulins. Constitutive expression of the tub2 gene with a histidine to tyrosine substitution at position 6 (known to impart benomyl/methyl benzimadazol-2-yl carbamate resistance in other fungi), under the Pgpd promoter of Aspergillus nidulans, did not impart resistance to benomyl. The homologous expression of tub2 gene with a histidine to tyrosine mutation at position +6, which is known to impart benomyl tolerance in other fungi, does not impart resistance in T. virens. Unlike other Trichoderma spp., T. virens, has been difficult to mutate for benomyl tolerance. The present study, through site-directed mutagenesis, shows that a mutation known to impart benomyl tolerance in T. viride and other fungi does not impart resistance in this fungus. Understanding the mechanisms of this phenomenon will have a profound impact in plant-disease management, as many plant pathogenic fungi develop resistance to this group of fungicides forcing its withdrawal after a short period of use.

TGF-beta induces connexin43 gene expression in normal murine mammary gland epithelial cells via activation of p38 and PI3K/AKT signaling pathways.

Science.gov (United States)

Tacheau, Charlotte; Fontaine, Juliette; Loy, Jennifer; Mauviel, Alain; Verrecchia, Franck

2008-12-01

One of the shared physiological roles between TGF-beta and connexin family members is to inhibit epithelial cell cycle progression and consequently, to provide protection against malignant transformation. Herein, we demonstrated that TGF-beta1 induces the expression of connexin43 (Cx43) in normal murine mammary gland (NMuMG) cell lines at the protein and mRNA levels, and transcriptionally. Using overexpression of a truncated dominant-negative form of Cx43, we determined that the modulation of gap junctional communication by TGF-beta1 plays a key role in the control of NMuMG cells proliferation by TGF-beta1. In addition, using overexpression of truncated dominant-negative forms of either Smad2 or Smad3, and MDA-MB-468 human breast carcinoma cells deficient for Smad4, we determined that the Smad cascade is not implicated in TGF-beta1 effect on Cx43 expression. Using specific pharmacologic inhibitors for JNK, ERK, p38, and PI3K/AKT signaling pathways, we demonstrated the cooperative role of p38 and PI3K/AKT signaling in TGF-beta1-induced Cx43 expression and gap junctional communication. Furthermore, transfection of a c-jun antisense expression vector significantly prevented TGF-beta1-induced Cx43 gene expression demonstrating the involvement of c-Jun/AP-1 pathway together with p38 and PI3K/AKT pathways in mediating TGF-beta1-induced Cx43 gene expression.

Phenotypic and gene expression changes between low (glucose-responsive) and High (glucose non-responsive) MIN-6 beta cells

DEFF Research Database (Denmark)

O´Driscoll, L.; Gammell, p.; McKierman, E.

2006-01-01

The long-term potential to routinely use replacement beta cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous beta cell studies, by ourselves and other researchers, have indicated...... that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H......, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies...

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

Energy Technology Data Exchange (ETDEWEB)

Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M. [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia); Cleasby, Mark E. [Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent' s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010 (Australia); Millard, Susan; Leong, Gary M. [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia); Cooney, Gregory J. [Diabetes and Obesity Research Program, Garvan Institute of Medical Research, St. Vincent' s Hospital, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010 (Australia); Muscat, George E.O., E-mail: g.muscat@imb.uq.edu.au [The University of Queensland, Institute for Molecular Bioscience, St. Lucia, Qld 4072 (Australia)

2009-10-30

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

Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

DEFF Research Database (Denmark)

Efrat, S; Linde, S; Kofod, Hans

1988-01-01

Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The ...... both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype....

Oxidative Metabolism Genes Are Not Responsive to Oxidative Stress in Rodent Beta Cell Lines

Directory of Open Access Journals (Sweden)

Faer Morrison

2012-01-01

Full Text Available Altered expression of oxidative metabolism genes has been described in the skeletal muscle of individuals with type 2 diabetes. Pancreatic beta cells contain low levels of antioxidant enzymes and are particularly susceptible to oxidative stress. In this study, we explored the effect of hyperglycemia-induced oxidative stress on a panel of oxidative metabolism genes in a rodent beta cell line. We exposed INS-1 rodent beta cells to low (5.6 mmol/L, ambient (11 mmol/L, and high (28 mmol/L glucose conditions for 48 hours. Increases in oxidative stress were measured using the fluorescent probe dihydrorhodamine 123. We then measured the expression levels of a panel of 90 oxidative metabolism genes by real-time PCR. Elevated reactive oxygen species (ROS production was evident in INS-1 cells after 48 hours (P<0.05. TLDA analysis revealed a significant (P<0.05 upregulation of 16 of the 90 genes under hyperglycemic conditions, although these expression differences did not reflect differences in ROS. We conclude that although altered glycemia may influence the expression of some oxidative metabolism genes, this effect is probably not mediated by increased ROS production. The alterations to the expression of oxidative metabolism genes previously observed in human diabetic skeletal muscle do not appear to be mirrored in rodent pancreatic beta cells.

Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

Science.gov (United States)

Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

2014-03-01

Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

Quantitative gene-expression of the tumor angiogenesis markers vascular endothelial growth factor, integrin alphaV and integrin beta3 in human neuroendocrine tumors

DEFF Research Database (Denmark)

Oxboel, Jytte; Binderup, Tina; Knigge, Ulrich

2009-01-01

, in neuroendocrine tumors. We used quantitative real-time PCR for measuring mRNA gene-expression of vascular endothelial growth factor (VEGF), integrin alphaV, and integrin beta3, and CD34 for a group of patients with neuroendocrine tumors (n=13). Tissue from patients with colorectal cancer liver metastases (n=14...... compared to both colorectal liver metastases (p=0.10) and normal liver tissue (p=0.06). In neuroendocrine tumors, gene-expression was highly variable of VEGF (530-fold), integrin alphaV (23-fold) and integrin beta3 (106-fold). Quantitative gene-expression levels of the key angiogenesis molecules VEGF......Anti-angiogenesis treatment is a promising new therapy for cancer that recently has also been suggested for patients with neuroendocrine tumors. The aim of the present study was therefore to investigate the level of tumor angiogenesis, and thereby the molecular basis for anti-angiogenesis treatment...

Short- and long-term changes in sugarbeet (Beta vulgaris L. gene expression due to postharvest jasmonic acid treatment - Data

Directory of Open Access Journals (Sweden)

Lucilene Silva de Oliveira

2017-04-01

Full Text Available Jasmonic acid is a natural plant hormone that induces native defense responses in plants. Sugarbeet (Beta vulgaris L. root unigenes that were differentially expressed 2 and 60 days after a postharvest jasmonic acid treatment are presented. Data include changes in unigene expression relative to water-treated controls, unigene annotations against nonredundant (Nr, Swiss-Prot, Clusters of Orthologous Groups (COG, and Kyoto Encyclopedia of Genes and Genomes (KEGG protein databases, and unigene annotations with Gene Ontology (GO terms. Putative defense unigenes are compiled and annotated against the sugarbeet genome. Differential gene expression data were generated by RNA sequencing. Interpretation of the data is available in the research article, “Jasmonic acid causes short- and long-term alterations to the transcriptome and the expression of defense genes in sugarbeet roots” (K.K. Fugate, L.S. Oliveira, J.P. Ferrareze, M.D. Bolton, E.L. Deckard, F.L. Finger, 2017 [1]. Public dissemination of this dataset will allow further analyses of the data.

Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.

Directory of Open Access Journals (Sweden)

Ashley T Martino

2009-08-01

Full Text Available Hepatic gene transfer, in particular using adeno-associated viral (AAV vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic beta-galactosidase (beta-gal was performed in immune competent mice, followed by a secondary beta-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in approximately 2% of hepatocytes almost completely protected from inflammatory T cell responses against beta-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, approximately 10% of hepatocytes continued to express beta-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8(+ T cell responses to beta-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer.These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines

DEFF Research Database (Denmark)

Damstrup, L; Rygaard, K; Spang-Thomsen, M

1993-01-01

A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis......(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell...... lines express TGF beta-receptors and also produce TGF beta mRNAs....

Expression and functional assessment of candidate type 2 diabetes susceptibility genes identify four new genes contributing to human insulin secretion

Directory of Open Access Journals (Sweden)

Fatou K. Ndiaye

2017-06-01

Full Text Available Objectives: Genome-wide association studies (GWAS have identified >100 loci independently contributing to type 2 diabetes (T2D risk. However, translational implications for precision medicine and for the development of novel treatments have been disappointing, due to poor knowledge of how these loci impact T2D pathophysiology. Here, we aimed to measure the expression of genes located nearby T2D associated signals and to assess their effect on insulin secretion from pancreatic beta cells. Methods: The expression of 104 candidate T2D susceptibility genes was measured in a human multi-tissue panel, through PCR-free expression assay. The effects of the knockdown of beta-cell enriched genes were next investigated on insulin secretion from the human EndoC-βH1 beta-cell line. Finally, we performed RNA-sequencing (RNA-seq so as to assess the pathways affected by the knockdown of the new genes impacting insulin secretion from EndoC-βH1, and we analyzed the expression of the new genes in mouse models with altered pancreatic beta-cell function. Results: We found that the candidate T2D susceptibility genes' expression is significantly enriched in pancreatic beta cells obtained by laser capture microdissection or sorted by flow cytometry and in EndoC-βH1 cells, but not in insulin sensitive tissues. Furthermore, the knockdown of seven T2D-susceptibility genes (CDKN2A, GCK, HNF4A, KCNK16, SLC30A8, TBC1D4, and TCF19 with already known expression and/or function in beta cells changed insulin secretion, supporting our functional approach. We showed first evidence for a role in insulin secretion of four candidate T2D-susceptibility genes (PRC1, SRR, ZFAND3, and ZFAND6 with no previous knowledge of presence and function in beta cells. RNA-seq in EndoC-βH1 cells with decreased expression of PRC1, SRR, ZFAND6, or ZFAND3 identified specific gene networks related to T2D pathophysiology. Finally, a positive correlation between the expression of Ins2 and the

Validation of commonly used reference genes for sleep-related gene expression studies

Directory of Open Access Journals (Sweden)

Castro Rosa MRPS

2009-05-01

Full Text Available Abstract Background Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin, beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, and hypoxanthine guanine phosphoribosyl transferase (HPRT. Results Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF and glycerol-3-phosphate dehydrogenase1 (GPD1 was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance. Conclusion This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results.

Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene.

Science.gov (United States)

Castresana, C; de Carvalho, F; Gheysen, G; Habets, M; Inzé, D; Van Montagu, M

1990-01-01

The Nicotiana plumbaginifolia gn1 gene encoding a beta-1,3-glucanase isoform has been characterized. The gn1 product represents an isoform distinct from the previously identified tobacco beta-1,3-glucanases. By expressing gn1 in Escherichia coli, we have determined directly that the encoded protein does, indeed, correspond to a beta-1,3-glucanase. In N. plumbaginifolia, gn1 was found to be expressed in roots and older leaves. Transgenic tobacco plants containing the 5'-noncoding region of gn1 fused to the beta-glucuronidase (GUS) reporter gene also showed maximum levels of GUS activity in roots and older leaves. No detectable activity was present in the upper part of the transgenic plants with the exception of stem cells at the bases of emerging shoots. The expression conferred by the gn1 promoter was differentially induced in response to specific plant stress treatments. Studies of three plant-bacteria interactions showed high levels of GUS activity when infection resulted in a hypersensitive reaction. Increased gene expression was confined to cells surrounding the necrotic lesions. The observed expression pattern suggests that the characterized beta-1,3-glucanase plays a role both in plant development and in the defense response against pathogen infection. PMID:2152158

Effect of beta2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A.

Science.gov (United States)

Kaur, Manminder; Holden, Neil S; Wilson, Sylvia M; Sukkar, Maria B; Chung, Kian Fan; Barnes, Peter J; Newton, Robert; Giembycz, Mark A

2008-09-01

In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE(2), forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) beta(2)-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1beta in ASM cells. IL-1beta-induced IL-8 release was also repressed by PGE(2) and forskolin, whereas the beta(2)-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKIalpha, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-kappaB-dependent genes, we used an adenoviral-delivered NF-kappaB-dependent luciferase reporter to examine the effects of forskolin and the beta(2)-adrenoceptor agonists on NF-kappaB activation. There was no effect on luciferase activity measured in the presence of forskolin or beta(2)-adrenoceptor agonists. This finding is consistent with the observation that IL-1beta-induced expression of IL-6, a known NF-kappaB-dependent gene in ASM, was also unaffected by beta(2)-adrenoceptor agonists, forskolin, PGE(2), 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1beta-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including beta(2)-adrenoceptor agonists, may not occur through a generic effect on NF-kappaB.

Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling.

Science.gov (United States)

Grimmig, Bernhard; Gonzalez-Perez, Maria N; Leubner-Metzger, Gerhard; Vögeli-Lange, Regina; Meins, Fred; Hain, Rüdiger; Penuelas, Josep; Heidenreich, Bernd; Langebartels, Christian; Ernst, Dieter; Sandermann, Heinrich

2003-03-01

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I beta-1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5'-deletion analysis of the class I beta-1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I beta-1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I beta-1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I beta-1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these datastrongly suggest that ethylene is signalling ozone-induced expression of class I beta-l,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an

Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls.

Science.gov (United States)

Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T V; Alyethodi, Rafeeque R; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B

2015-12-01

Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly (P ATPase Β1, ATPase B2, and ATPase B3 is highly correlated (P ATPase beta family genes for cellular thermotolerance in cattle.

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells

International Nuclear Information System (INIS)

Wallden, Brett; Emond, Mary; Swift, Mari E; Disis, Mary L; Swisshelm, Karen

2005-01-01

The retinoic acid receptor beta 2 (RARβ2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARβ2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype. RNA from MDA-MB-435 human breast cancer cells transduced with RARβ2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors. RARβ2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 × 10 -8 ), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARβ2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007). Antimetastatic RARβ2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARβ2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN

Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene

DEFF Research Database (Denmark)

Larsen, Leif K; Amri, Ez-Zoubir; Mandrup, Susanne

2002-01-01

Peroxisome proliferator-activated receptor (PPAR) beta/delta is ubiquitously expressed, but the level of expression differs markedly between different cell types. In order to determine the molecular mechanisms governing PPARbeta/delta gene expression, we have isolated and characterized the mouse...

Activation of the human beta interferon gene by the adenovirus type 12 E1B gene

International Nuclear Information System (INIS)

Shiroki, K.; Toth, M.

1988-01-01

The transcription of endogenous beta interferon mRNA was activated in human embryo kidney (HEK) cells infected with adenovirus 12 (Ad12) but was activated only inefficiently or not at all in HEK cells infected with Ad5 and rc-1 (Ad5 dl312 containing the Ad12 E1A region). The analysis with Ad12 mutants showed that Ad12 E1B products, especially the 19K protein, were important for the expression of the endogenous beta interferon gene and Ad12 E1A products were not involved in the expression. The expression of exogeneously transfected pIFN-CAT (a hybrid plasmid having the human beta interferon promoter fused with the CAT gene) was activated in HEK and chicken embryo fibroblast (CEF) cells infected with either Ad12 or Ad5. The analysis of cotransfection of CEF cells with pIFN-CAT and plasmids containing fragments of Ad12 or Ad5 DNA showed that Ad12 or Ad5 E1B (possibly the 19K protein) was and E1A was not involved in the expression of the exogenous pIFN-CAT

11Beta-HSD type 1 expression in human adipose tissue: impact of gender, obesity, and fat localization

DEFF Research Database (Denmark)

Paulsen, Søren Kildeberg; Pedersen, Steen Bønløkke; Fisker, Sanne

2007-01-01

of the metabolic syndrome. Our objective was to compare 11beta-HSD1 gene expression in different fat depots (visceral, subcutaneous abdominal, and subcutaneous gluteal) in lean and obese men and women. RESEARCH METHODS AND PROCEDURES: A cross-sectional study design was used for healthy patients undergoing minor...... abdominal surgery (lean men, 10), minor gynecological surgery (lean woman, 10), or gastric banding operations (obese men, 10; and obese women, 10). Gene expressions of 11beta-HSD1 in adipose tissue samples were determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Lean...... women had lower 11beta-HSD1 gene expression in subcutaneous adipose tissue compared with men (62% lower, p women. 11Beta-HSD1 mRNA in human adipose tissue was higher in obese subjects compared with lean subjects in both women...

Molecular Characterization and Expression Analysis of Adrenergic Receptor Beta 2 ( Gene before and after Exercise in the Horse

Directory of Open Access Journals (Sweden)

Hyun-Woo Cho

2015-05-01

Full Text Available The adrenergic receptor beta 2 (ADRB2 plays a role in various physiological responses of the muscle to exercise, such as contraction and relaxation. Given its important role in muscle function, we investigated the structure of the horse ADRB2 gene and its expression pattern after exercise to determine if it can serve as a putative biomarker for recovery. Evolutionary analyses using synonymous and non-synonymous mutation ratios, were compared with other species (human, chimpanzee, mouse, rat, cow, pig, chicken, dog, and cat, and revealed the occurrence of positive selection in the horse ADRB2 gene. In addition, expression analyses by quantitative polymerase chain reaction exhibited ubiquitous distribution of horse ADRB2 in various tissues including lung, skeletal muscle, kidney, thyroid, appendix, colon, spinal cord and heart, with the highest expression observed in the lung. The expression of ADRB2 in skeletal muscle was significantly up-regulated about four folds 30 minutes post-exercise compared to pre-exercise. The expression level of ADRB2 in leukocytes, which could be collected with convenience compared with other tissues in horse, increased until 60 min after exercise but decreased afterward until 120 min, suggesting the ADRB2 expression levels in leukocytes could be a useful biomarker to check the early recovery status of horse after exercise. In conclusion, we identified horse ADRB2 gene and analyzed expression profiles in various tissues. Additionally, analysis of ADBR2 gene expression in leukocytes could be a useful biomarker useful for evaluation of early recovery status after exercise in racing horses.

Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

Directory of Open Access Journals (Sweden)

Cheng-Tao Yang

2009-07-01

Full Text Available The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels.To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm.These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

Wnt and TGF-beta expression in the sponge Amphimedon queenslandica and the origin of metazoan embryonic patterning.

Directory of Open Access Journals (Sweden)

Maja Adamska

2007-10-01

Full Text Available The origin of metazoan development and differentiation was contingent upon the evolution of cell adhesion, communication and cooperation mechanisms. While components of many of the major cell signalling pathways have been identified in a range of sponges (phylum Porifera, their roles in development have not been investigated and remain largely unknown. Here, we take the first steps toward reconstructing the developmental signalling systems used in the last common ancestor to living sponges and eumetazoans by studying the expression of genes encoding Wnt and TGF-beta signalling ligands during the embryonic development of a sponge.Using resources generated in the recent sponge Amphimedon queenslandica (Demospongiae genome project, we have recovered genes encoding Wnt and TGF-beta signalling ligands that are critical in patterning metazoan embryos. Both genes are expressed from the earliest stages of Amphimedon embryonic development in highly dynamic patterns. At the time when the Amphimedon embryos begin to display anterior-posterior polarity, Wnt expression becomes localised to the posterior pole and this expression continues until the swimming larva stage. In contrast, TGF-beta expression is highest at the anterior pole. As in complex animals, sponge Wnt and TGF-beta expression patterns intersect later in development during the patterning of a sub-community of cells that form a simple tissue-like structure, the pigment ring. Throughout development, Wnt and TGF-beta are expressed radially along the anterior-posterior axis.We infer from the expression of Wnt and TGF-beta in Amphimedon that the ancestor that gave rise to sponges, cnidarians and bilaterians had already evolved the capacity to direct the formation of relatively sophisticated body plans, with axes and tissues. The radially symmetrical expression patterns of Wnt and TGF-beta along the anterior-posterior axis of sponge embryos and larvae suggest that these signalling pathways

Patterns of expression of cell wall related genes in sugarcane

Directory of Open Access Journals (Sweden)

Lima D.U.

2001-01-01

Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

11Beta-HSD type 1 expression in human adipose tissue: impact of gender, obesity, and fat localization

DEFF Research Database (Denmark)

Paulsen, Søren Kildeberg; Pedersen, Steen Bønløkke; Fisker, Sanne

2007-01-01

of the metabolic syndrome. Our objective was to compare 11beta-HSD1 gene expression in different fat depots (visceral, subcutaneous abdominal, and subcutaneous gluteal) in lean and obese men and women. RESEARCH METHODS AND PROCEDURES: A cross-sectional study design was used for healthy patients undergoing minor...... women had lower 11beta-HSD1 gene expression in subcutaneous adipose tissue compared with men (62% lower, p difference was found between obese men and women. 11Beta-HSD1 mRNA in human adipose tissue was higher in obese subjects compared with lean subjects in both women...... and men and in both subcutaneous and visceral adipose tissue. No difference in mRNA expression of 11beta-HSD1 between visceral and subcutaneous adipose tissue or between subcutaneous adipose tissue from different depots was found. CONCLUSIONS: 11Beta-HSD1 in adipose tissue is increased in obesity in both...

Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes

Directory of Open Access Journals (Sweden)

Øvstebø Reidun

2010-05-01

Full Text Available Abstract Background Gene expression in lipopolysaccharide (LPS-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase or ACTB (beta-actin as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system. Results Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B, B2M (beta-2-microglobulin and PPIA (cyclophilin A as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-α (tumor necrosis factor-alpha and IL10 (interleukin 10 expression. Moreover, a significant difference in TNF-α expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH. Conclusions Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

Suppression of cancer growth in mice by adeno-associated virus vector-mediated IFN-beta expression driven by hTERT promoter.

Science.gov (United States)

He, Ling Feng; Wang, Yi Gang; Xiao, Tian; Zhang, Kang Jiang; Li, Gong Chu; Gu, Jin Fa; Chu, Liang; Tang, Wen Hao; Tan, Wen-Song; Liu, Xin Yuan

2009-12-28

Adeno-associated virus (AAV) has rapidly become a promising gene delivery vehicle for its excellent advantages of non-immunogenic, low pathogenicity and long-term gene expression in vivo. However, a major obstacle in development of effective AAV vector is the lack of tissue specificity, which caused low efficiency of AAV transfer to target cells. The application of human telomerase reverse transcriptase (hTERT) promoter is a prior targeting strategy for AAV in cancer gene therapy as hTERT activity is transcriptionally upregulated in most cancer cells. In the present work, we investigated whether AAV-mediated human interferon beta (IFN-beta) gene driven by hTERT promoter could specifically express in tumor cells and suppress tumor cell growth. Our data demonstrated that hTERT promoter-driven IFN-beta expression was the tumor-specific, decreased the cell viability of tumor cells but not normal cells, and induced tumor cell apoptosis via activation of caspase pathway and release of cytochrome c. AAV-mediated IFN-beta expression driven by hTERT promoter significantly suppressed the growth of colorectal cancer and lung cancer xenograft in mice and resulted in tumor cells death in vivo. These data suggested that AAVs in combination with hTERT-mediated IFN-beta expression could exert potential antitumor activity and provide a novel targeting approach to clinical gene therapy of varieties of cancers.

Common ADRB2 haplotypes derived from 26 polymorphic sites direct beta2-adrenergic receptor expression and regulation phenotypes.

Directory of Open Access Journals (Sweden)

Alfredo Panebra

2010-07-01

Full Text Available The beta2-adrenergic receptor (beta2AR is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions.We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5'UTR, coding, and 3'UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that beta2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. "Whole-gene" transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface beta2AR protein expression compared to the others. Agonist-promoted downregulation of beta2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression.Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of beta-agonist may be influenced by certain beta2AR haplotypes (expression phenotypes, while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes. An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where beta2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection

Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

Directory of Open Access Journals (Sweden)

Zhong Tao P

2007-07-01

Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

DEFF Research Database (Denmark)

Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

1996-01-01

In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...... and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

Science.gov (United States)

Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

1998-12-01

Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

Prolonged peritoneal gene expression using a helper-dependent adenovirus.

Science.gov (United States)

Liu, Limin; Shi, Chang-Xin; Ghayur, Ayesha; Zhang, Claire; Su, Je Yen; Hoff, Catherine M; Margetts, Peter J

2009-01-01

Encapsulating peritoneal sclerosis (EPS) is a rare complication of peritoneal dialysis. The causes of EPS are not well defined and are likely multifactorial. A suitable animal model would facilitate research into the pathophysiology and treatment of EPS. We developed a helper-dependent adenovirus that expresses both green fluorescent protein (GFP) and active transforming growth factor-beta (TGF-beta1; HDAdTGF-beta1). Mice were administered HDAdTGF-beta1 via intraperitoneal injection and the response was compared with mice administered either first-generation adenovirus expressing TGF-beta1 (AdTGF-beta1) or control adenovirus (AdGFP). HDAdTGF-beta1-treated mice continued to express the GFP reporter transgene to day 74, the end of the observation period. Transgene expression lasted less than 28 days in the animals treated with first-generation adenoviruses. Animals treated with first-generation AdTGF-beta1 demonstrated submesothelial thickening and angiogenesis at day 7, with almost complete resolution by day 28. The HDAdTGF-beta1-treated mice demonstrated progressive peritoneal fibrosis with adhesion formation and encapsulation of bowels. Weight gain was significantly reduced in animals treated with HDAdTGF-beta1 compared to both the control-treated animals and the AdTGF-beta1-treated animals. Inflammation was not a major component of the fibroproliferative response. Peritoneal administration of a first-generation AdTGF-beta1 leads to transient gene expression, resulting in a resolving fibrotic response and histology similar to that seen in simple peritoneal sclerosis. Prolonged TGF-beta1 expression induced by the helper-dependent HDAdTGF-beta1 led to changes in peritoneal morphology resembling EPS. This suggests that TGF-beta1 may be a contributing factor in both simple peritoneal sclerosis and EPS. This model will be useful for elucidation of the mechanism of EPS and evaluation of potential treatment.

Transcriptional Mechanisms Controlling miR-375 Gene Expression in the Pancreas

Directory of Open Access Journals (Sweden)

Tali Avnit-Sagi

2012-01-01

Full Text Available MicroRNAs (miRNAs are a class of small non-coding RNAs that play an important role in mediating a broad and expanding range of biological activities. miR-375 is expressed selectively in the pancreas. We have previously shown that selective expression of miR-375 in pancreatic beta cells is controlled by transcriptional mechanisms operating through a TATA box-containing promoter. Expression of miR-375 has been reported in non-beta cells within the endocrine pancreas, and indeed inactivation of miR-375 leads to perturbation in cell mass and number of both alpha and beta cells. Consistent with its expression throughout the endocrine pancreas, we now show that the promoter of the miR-375 gene shows selective activity in pancreatic endocrine alpha cells, comparable to that observed in beta cells. We previously identified a novel negative regulatory element located downstream of the miR-375 gene transcription start site. By generating luciferase reporter genes, we now show that the sequence is functional also when positioned upstream of a heterologous promoter, thus proving that the repressor effect is mediated at least in part at the level of transcription. Further characterization of the transcriptional control mechanism regulating expression of miR-375 and other pancreatic miRNAs will contribute to a better understanding of pancreas development and function.

Acquired TGF beta 1 sensitivity and TGF beta 1 expression in cell lines established from a single small cell lung cancer patient during clinical progression

DEFF Research Database (Denmark)

Nørgaard, P; Damstrup, L; Rygaard, K

1996-01-01

Three small cell lung cancer cell lines established from a single patient during longitudinal follow-up were examined for in vitro expression of TGF beta and TGF beta receptors, i.e. the components of an autocrine loop. GLC 14 was established prior to treatment, GLC 16 on relapse after chemotherapy...... was found in GLC 16 and GLC 19. These cell lines were also growth inhibited by exogenously administrated TGF beta 1. TGF beta 1 mRNA and protein in its latent form was only expressed in the radiotherapy-resistant cell line, GLC 19. The results indicate that disease progression in this patient was paralleled...... II receptor gene, as examined by Southern blotting. Also, the type I receptor could not be detected by ligand binding assay in this cell line, despite expression of mRNA for this receptor. This agrees with previous findings that type I receptor cannot bind TGF beta 1 without co-expression of the type...

Expression of streptavidin gene in bacteria and plants

International Nuclear Information System (INIS)

Guan, Xueni; Wurtele, E.S.; Nikolau, B.J.

1990-01-01

Six biotin-containing proteins are present in plants, representing at least four different biotin enzymes. The physiological function of these biotin enzymes is not understood. Streptavidin, a protein from Streptomyces avidinii, binds tightly and specifically to biotin causing inactivation of biotin enzymes. One approach to elucidating the physiological function of biotin enzymes in plant metabolism is to create transgenic plants expressing the streptavidin gene. A plasmid containing a fused streptavidin-beta-galactosidase gene has been expressed in E. coli. We also have constructed various fusion genes that include an altered CaMV 35S promoter, signal peptides to target the streptavidin protein to specific organelles, and the streptavidin coding gene. We are examining the expression of these genes in cells of carrot

Gastrointestinal hyperplasia with altered expression of DNA polymerase beta.

Directory of Open Access Journals (Sweden)

Katsuhiko Yoshizawa

2009-08-01

Full Text Available Altered expression of DNA polymerase beta (Pol beta has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol beta over-expression has not yet been evaluated in a mouse model.We have recently developed a novel transgenic mouse model that over-expresses Pol beta. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner's gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol beta over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol beta expression. We observed elevated expression of Pol beta in stomach adenomas and thyroid follicular carcinomas, but reduced Pol beta expression in esophageal adenocarcinomas and squamous carcinomas.These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation.

Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

Directory of Open Access Journals (Sweden)

Becker Sven

2008-01-01

Full Text Available Abstract Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG, depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. In the three-spined stickleback, Gasterosteus aculeatus, other HKG than the one for beta-actin have not been established so far. Results To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and NormFinder. Our results showed that in most of the tissues and treatments HKG that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin. Conclusion As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

Thymosin beta 4 and thymosin beta 10 expression in hepatocellular carcinoma

Directory of Open Access Journals (Sweden)

W. Theunissen

2014-03-01

Full Text Available Thymosin beta 4 (Tβ4 and thymosin beta 10 (Tβ10 are two members of the beta-thymosin family involved in many cellular processes such as cellular motility, angiogenesis, inflammation, cell survival and wound healing. Recently, a role for beta-thymosins has been proposed in the process of carcinogenesis as both peptides were detected in several types of cancer. The aim of the present study was to investigate the expression pattern of Tβ4 and Tβ10 in hepatocellular carcinoma (HCC. To this end, the expression pattern of both peptides was analyzed in liver samples obtained from 23 subjects diagnosed with HCC. Routinely formalin-fixed and paraffin-embedded liver samples were immunostained by indirect immunohistochemistry with polyclonal antibodies to Tβ4 and Tβ10. Immunoreactivity for Tβ4 and Tβ10 was detected in the liver parenchyma of the surrounding tumor area. Both peptides showed an increase in granular reactivity from the periportal to the periterminal hepatocytes. Regarding HCC, Tβ4 reactivity was detected in 7/23 cases (30% and Tβ10 reactivity in 22/23 (97% cases analyzed, adding HCC to human cancers that express these beta-thymosins. Intriguing finding was seen looking at the reactivity of both peptides in tumor cells infiltrating the surrounding liver. Where Tβ10 showed a strong homogeneous expression, was Tβ4 completely absent in cells undergoing stromal invasion. The current study shows expression of both beta-thymosins in HCC with marked differences in their degree of expression and frequency of immunoreactivity. The higher incidence of Tβ10 expression and its higher reactivity in tumor cells involved in stromal invasion indicate a possible major role for Tβ10 in HCC progression.

Intracellular Wnt/Beta-Catenin Signaling Underlying 17beta-Estradiol-Induced Matrix Metalloproteinase 9 Expression in Human Endometriosis.

Science.gov (United States)

Zhang, Ling; Xiong, Wenqian; Xiong, Yao; Liu, Hengwei; Li, Na; Du, Yu; Liu, Yi

2016-03-01

Extracellular matrix remodeling is necessary for ectopic endometrium implantation. Many studies have shown an increased expression of matrix metalloproteinase 9 (MMP9) in the ectopic endometrium of endometriosis. However, the signaling pathways and cellular effects related to this process remain incompletely elucidated. The objective of our study was to investigate the association between MMP9 and the Wnt signaling pathway under the regulation of 17beta-estradiol (E2) in endometrial stromal cells. We found that MMP9 was elevated in tissues from women with endometriosis compared with normal women. Furthermore, MMP9 and beta-catenin increased concurrently in a time- and dose-dependent manner after E2 treatment. To clarify the relationship between MMP9 and beta-catenin, we performed luciferase promoter reporter and chromatin immunoprecipitation assays. A beta-catenin/TCF3/LEF1 complex bound to a specific site on the MMP9 promoter that promoted MMP9 gene and protein expression. The promotion of MMP9 by the Wnt signaling pathway under the regulation of E2 may contribute to the pathophysiology of this disease. © 2016 by the Society for the Study of Reproduction, Inc.

Crosstalk between histone modifications maintains the developmental pattern of gene expression on a tissue-specific locus.

Science.gov (United States)

Hosey, Alison M; Chaturvedi, Chandra-Prakash; Brand, Marjorie

2010-05-16

Genome wide studies have provided a wealth of information related to histone modifications. Particular modifications, which can encompass both broad and discrete regions, are associated with certain genomic elements and gene expression status. Here we focus on how studies on the beta-globin gene cluster can complement the genome wide effort through the thorough dissection of histone modifying protein crosstalk. The beta-globin locus serves as a model system to study both regulation of gene expression driven at a distance by enhancers and mechanisms of developmental switching of clustered genes. We investigate recent studies, which uncover that histone methyltransferases, recruited at the beta-globin enhancer, control gene expression by long range propagation on chromatin. Specifically, we focus on how seemingly antagonistic complexes, such as those including MLL2, G9a and UTX, can cooperate to functionally regulate developmentally controlled gene expression. Finally, we speculate on the mechanisms of chromatin modifying complex propagation on genomic domains.

MicroRNA Expression Changes during Interferon-Beta Treatment in the Peripheral Blood of Multiple Sclerosis Patients

Directory of Open Access Journals (Sweden)

Georg Füllen

2013-08-01

Full Text Available MicroRNAs (miRNAs are small non-coding RNA molecules acting as post-transcriptional regulators of gene expression. They are involved in many biological processes, and their dysregulation is implicated in various diseases, including multiple sclerosis (MS. Interferon-beta (IFN-beta is widely used as a first-line immunomodulatory treatment of MS patients. Here, we present the first longitudinal study on the miRNA expression changes in response to IFN-beta therapy. Peripheral blood mononuclear cells (PBMC were obtained before treatment initiation as well as after two days, four days, and one month, from patients with clinically isolated syndrome (CIS and patients with relapsing-remitting MS (RRMS. We measured the expression of 651 mature miRNAs and about 19,000 mRNAs in parallel using real-time PCR arrays and Affymetrix microarrays. We observed that the up-regulation of IFN-beta-responsive genes is accompanied by a down-regulation of several miRNAs, including members of the mir-29 family. These differentially expressed miRNAs were found to be associated with apoptotic processes and IFN feedback loops. A network of miRNA-mRNA target interactions was constructed by integrating the information from different databases. Our results suggest that miRNA-mediated regulation plays an important role in the mechanisms of action of IFN-beta, not only in the treatment of MS but also in normal immune responses. miRNA expression levels in the blood may serve as a biomarker of the biological effects of IFN-beta therapy that may predict individual disease activity and progression.

Effects of transforming growth factor-beta1 and vascular endothelial growth factor 165 gene transfer on Achilles tendon healing.

Science.gov (United States)

Hou, Yu; Mao, ZeBin; Wei, XueLei; Lin, Lin; Chen, LianXu; Wang, HaiJun; Fu, Xin; Zhang, JiYing; Yu, Changlong

2009-07-01

Repaired Achilles tendons typically take weeks before they are strong enough to handle physiological loads. Gene therapy is a promising treatment for Achilles tendon defects. The aim of the present study was to evaluate the histological/biomechanical effects of Transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor 165 (VEGF(165)) gene transfer on Achilles tendon healing in rabbits. Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) were transduced with adenovirus carrying human TGF-beta1 cDNA (Ad-TGF-beta1), human VEGF(165) cDNA (Ad-VEGF(165)), or both (PIRES-TGF-beta1/VEGF(165)) Viruses, no cDNA (Ad-GFP), and the BMSCs without gene transfer and the intact tendon were used as control. BMSCs were surgically implanted into the experimentally injured Achilles tendons. TGF-beta1 distribution, cellularity, nuclear aspect ratio, nuclear orientation angle, vascular number, collagen synthesis, and biomechanical features were measured at 1, 2, 4, and 8 weeks after surgery. The TGF-beta1 and TGF beta 1/VEGF(165) co-expression groups exhibited improved parameters compared with other groups, while the VEGF(165) expression group had a negative impact. In the co-expression group, the angiogenesis effects of VEGF(165) were diminished by TGF-beta1, while the collagen synthesis effects of TGF-beta1 were unaltered by VEGF(165). Thus treatment with TGF-beta1 cDNA-transduced BMSCs grafts is a promising therapy for acceleration and improvement of tendon healing, leading to quicker recovery and improved biomechanical properties of Achilles tendons.

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

Science.gov (United States)

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

2009-08-07

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

Co-Cultures of Pseudomonas aeruginosa and Roseobacter denitrificans Reveal Shifts in Gene Expression Levels Compared to Solo Cultures

Directory of Open Access Journals (Sweden)

Crystal A. Conway

2012-01-01

Full Text Available Consistent biosynthesis of desired secondary metabolites (SMs from pure microbial cultures is often unreliable. In a proof-of-principle study to induce SM gene expression and production, we describe mixed “co-culturing” conditions and monitoring of messages via quantitative real-time PCR (qPCR. Gene expression of model bacterial strains (Pseudomonas aeruginosa PAO1 and Roseobacter denitrificans Och114 was analyzed in pure solo and mixed cocultures to infer the effects of interspecies interactions on gene expression in vitro, Two P. aeruginosa genes (PhzH coding for portions of the phenazine antibiotic pathway leading to pyocyanin (PCN and the RhdA gene for thiosulfate: cyanide sulfurtransferase (Rhodanese and two R. denitrificans genes (BetaLact for metallo-beta-lactamase and the DMSP gene for dimethylpropiothetin dethiomethylase were assessed for differential expression. Results showed that R. denitrificans DMSP and BetaLact gene expression became elevated in a mixed culture. In contrast, P. aeruginosa co-cultures with R. denitrificans or a third species did not increase target gene expression above control levels. This paper provides insight for better control of target SM gene expression in vitro and bypass complex genetic engineering manipulations.

[Analysis of resistant genes of beta-lactam antibiotics from Pseudomonas aeruginosa in pediatric patients].

Science.gov (United States)

Dong, Fang; Xu, Xi-wei; Song, Wen-qi; Lü, Ping; Yang, Yong-hong; Shen, Xu-zhuang

2008-11-18

To analyze the antibiotic resistance of the Pseudomonas aeruginosa (PA) isolated from pediatric patients and the resistant genes of beta-lactam antibiotics thereof. 146 PA strains were isolated from pediatric patients. Agar dilution method recommended by the Clinical and Laboratory Standards Institute was used to examine the minimum inhibitory concentrations (MICs) of 12 antimicrobial agents, including the penicillins, third and fourth genet ration cephalosporins, carbapenemase, Aztreonam, beta-lactamase inhibitors, quinolones, and aminoglycosides. PCR was used to detect the expression of the genes TEM, SHV, OXA, PER, GES, CTX-M, IMP, VIM, DHA, MIR, FOX, and oprD2. The multi-drug resistance rates against different antibiotic were high among the 146 PA strains. The rates of imipenem and meropenem resistance were 41.1% and 35.6% respectively. Among the 146 PA strains, 46 (31.5%) were positive for the MBL genotype; 38 (82.6%) carried the blaIMP gene, 8 (17.4%) carried the blaVIM gene, and 114 (78.1%) were oprD2 negative. The genes TEM, SHV, OXA, CTX-M, PER, VEB, GES, FOX, MIR, and DHA were not found in all strains. Many PA isolated from pediatric patients carry the genes IMP or VIM and losses oprD2 gene related to the expression of the outer membrane porin OprD2. The loss of the gene oprD2 is essential mechanism of beta-lactam antibiotics resistance in PA.

Rosiglitazone stimulates the release and synthesis of insulin by enhancing GLUT-2, glucokinase and BETA2/NeuroD expression

International Nuclear Information System (INIS)

Kim, Hyo-Sup; Noh, Jung-Hyun; Hong, Seung-Hyun; Hwang, You-Cheol; Yang, Tae-Young; Lee, Myung-Shik; Kim, Kwang-Won; Lee, Moon-Kyu

2008-01-01

Peroxisome proliferator-activated receptor (PPAR)-γ is a member of the nuclear receptor superfamily, and its ligands, the thiazolidinediones, might directly stimulate insulin release and insulin synthesis in pancreatic β-cells. In the present study, we examined the effects of rosiglitazone (RGZ) on insulin release and synthesis in pancreatic β-cell (INS-1). Insulin release and synthesis were stimulated by treatment with RGZ for 24 h. RGZ upregulated the expressions of GLUT-2 and glucokinase (GCK). Moreover, it was found that RGZ increased the expression of BETA2/NeuroD gene which could regulate insulin gene expression. These results suggest that RGZ could stimulate the release and synthesis of insulin through the upregulation of GLUT-2, GCK, and BETA2/NeuroD gene expression

Expression of inwardly rectifying potassium channels (GIRKs) and beta-adrenergic regulation of breast cancer cell lines

International Nuclear Information System (INIS)

Plummer, Howard K III; Yu, Qiang; Cakir, Yavuz; Schuller, Hildegard M

2004-01-01

Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling. Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting. Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 μM) increased the GIRK1 mRNA levels and decreased beta 2 -adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K + flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells. Our data suggests β-adrenergic receptors and GIRK channels may play a role in breast cancer

Cytoskeletal actin genes function downstream of HNF-3beta in ascidian notochord development.

Science.gov (United States)

Jeffery, W R; Ewing, N; Machula, J; Olsen, C L; Swalla, B J

1998-11-01

We have examined the expression and regulation of cytoskeletal actin genes in ascidians with tailed (Molgula oculata) and tailless larvae (Molgula occulta). Four cDNA clones were isolated representing two pairs of orthologous cytoskeletal actin genes (CA1 and CA2), which encode proteins differing by five amino acids in the tailed and tailless species. The CA1 and CA2 genes are present in one or two copies, although several related genes may also be present in both species. Maternal CA1 and CA2 mRNA is present in small oocytes but transcript levels later decline, suggesting a role in early oogenesis. In the tailed species, embryonic CA1 and CA2 mRNAs first appear in the presumptive mesenchyme and muscle cells during gastrulation, subsequently accumulate in the presumptive notochord cells, and can be detected in these tissues through the tadpole stage. CA1 mRNAs accumulate initially in the same tissues in the tailless species but subsequently disappear, in concert with the arrest of notochord and tail development. In contrast, CA2 mRNAs were not detected in embryos of the tailless species. Fertilization of eggs of the tailless species with sperm of the tailed species, which restores the notochord and the tail, also results in the upregulation of CA1 and CA2 gene expression in hybrid embryos. Antisense oligodeoxynucleotide experiments suggest that CA1 and CA2 expression in the notochord, but not in the muscle cells, is dependent on prior expression of Mocc FHI, an ascidian HNF-3beta-like gene. The expression of the CA1 and CA2 genes in the notochord in the tailed species, downregulation in the tailless species, upregulation in interspecific hybrids, and dependence on HNF-3beta activity is consistent with a role of these genes in development of the ascidian notochord.

Hepatic gene expression profiling using GeneChips in zebrafish exposed to 17{alpha}-methyldihydrotestosterone

Energy Technology Data Exchange (ETDEWEB)

Hoffmann, J.L.; Thomason, R.G.; Lee, D.M.; Brill, J.L.; Price, B.B.; Carr, G.J. [Miami Valley Innovation Center, Procter and Gamble Company, P.O. Box 538707, Cincinnati, OH 45253-8707 (United States); Versteeg, D.J. [Miami Valley Innovation Center, Procter and Gamble Company, P.O. Box 538707, Cincinnati, OH 45253-8707 (United States)], E-mail: versteeg.dj@pg.com

2008-04-28

Concentration and time-dependent changes in hepatic gene expression were examined in adult, female zebrafish (Danio rerio) exposed to 0, 0.1, 0.7, 4.9 {mu}g/L of a model androgen, 17{alpha}-methyldihydrotestosterone (MDHT). At 24 and 168 h, fish were sacrificed and liver was extracted for gene expression analysis using custom Affymetrix GeneChip Zebrafish Genome Microarrays. In an effort to link gene expression changes to higher levels of biological organization, blood was collected for measurement of plasma steroid hormones (17{beta}-estradiol (E2), testosterone (T)) and vitellogenin (VTG) using ELISA. Body and ovary weight were also measured. A significant reduction in E2 occurred at 24 h (0.7 and 4.9 {mu}g/L) and 168 h (4.9 {mu}g/L) following MDHT exposure. In contrast, T was significantly increased at 24 h (4.9 {mu}g/L) and 168 h (0.1, 0.7, 4.9 {mu}g/L). 171 and 575 genes were significantly affected in a concentration-dependent manner at either 24 or 168 h by MDHT exposure at p {<=} 0.001 and p {<=} 0.01, respectively. Genes involved in retinoic acid metabolism (e.g. aldehyde dehydrogenase 8, member A1; retinol dehydrogenase 12), steroid biosynthesis and metabolism (e.g. hydroxysteroid (11{beta}) dehydrogenase 2; hydroxy-delta-5-steroid dehydrogenase, 3 beta-), hormone transport (e.g. sex hormone binding globulin), and regulation of cell growth and proliferation (e.g. N-myc downstream regulated gene 1; spermidinespermine N(1)-acetyltransferase) were affected by MDHT exposure. In this study, we identified genes involved in a variety of biological processes that have the potential to be used as markers of exposure to androgenic substances. Genes identified in this study provide information on the potential mode of action of strong androgens in female fish. In addition, when used for screening of EDC's, these genes may also serve as sensitive markers of exposure to androgenic compounds.

Ethylene regulation of carotenoid accumulation and carotenogenic gene expression in colour-contrasted apricot varieties (Prunus armeniaca).

Science.gov (United States)

Marty, I; Bureau, S; Sarkissian, G; Gouble, B; Audergon, J M; Albagnac, G

2005-07-01

In order to elucidate the regulation mechanisms of carotenoid biosynthesis in apricot fruit (Prunus armeniaca), carotenoid content and carotenogenic gene expression were analysed as a function of ethylene production in two colour-contrasted apricot varieties. Fruits from Goldrich (GO) were orange, while Moniqui (MO) fruits were white. Biochemical analysis showed that GO accumulated precursors of the uncoloured carotenoids, phytoene and phytofluene, and the coloured carotenoid, beta-carotene, while Moniqui (MO) fruits only accumulated phytoene and phytofluene but no beta-carotene. Physiological analysis showed that ethylene production was clearly weaker in GO than in MO. Carotenogenic gene expression (Psy-1, Pds, and Zds) and carotenoid accumulation were measured with respect to ethylene production which is initiated in mature green fruits at the onset of the climacteric stage or following exo-ethylene or ethylene-receptor inhibitor (1-MCP) treatments. Results showed (i) systematically stronger expression of carotenogenic genes in white than in orange fruits, even for the Zds gene involved in beta-carotene synthesis that is undetectable in MO fruits, (ii) ethylene-induction of Psy-1 and Pds gene expression and the corresponding product accumulation, (iii) Zds gene expression and beta-carotene production independent of ethylene. The different results obtained at physiological, biochemical, and molecular levels revealed the complex regulation of carotenoid biosynthesis in apricots and led to suggestions regarding some possible ways to regulate it.

Development of library preparation method able to correct gene expression levels in rice anther and isolate a trace expression gene mediated in cold-resistance

Energy Technology Data Exchange (ETDEWEB)

Yamaguchi, Tomoya; Koike, Setsuo [Tohoku National Agricultural Experiment Station, Morioka (Japan)

2000-02-01

When cDNA library is prepared by a previously developed method, genes of which expression level is high are apt to be cloned at a high frequency, whereas genes of which expression level are low, are difficult to be cloned. A low-expression gene has been cloned at very low frequency. Therefore, the gene encoding the key enzyme that is involved in growth disturbance of rice pollen has not been identified. In this study, development of a library preparing method able to correct the expression level was attempted using highly sensitive detection method with radioisotope and some genes related to cold-resistance of rice were isolated. Double strand DNAs were synthesized using mRNA extract from rice anthers and annealed following heat-denaturation. It has been known that single strand DNA molecules abundantly existing in DNA solution can easily aggregate to form double strand DNA, but single stranded DNA molecules poor in the solution are apt to still remain as single strand after annealing. Thus, the amount of single strand DNA would be balanced in the solution between abundant DNA and poor DNA species. The authors succeeded to prepare a gene library including low and high expression genes at similar proportions. Moreover, spin trap method that allows RI labeling of DNA bound to latex particle, was developed to detect with high sensitivity, especially for genes that are expressed at low level. The present method could be used for recovery, detection and quantitative analysis of radiolabeled single strand DNA. Thus, it was demonstrated that the stage from tetrad sperm to small sperm might be easily affected by cold stress. The present results suggest that the expressions of {beta}-1 and {beta}-3 glucanase, which are involved in the release of small sperms following meiosis in the pollen formation, might be easily affected by cold stress. (M.N.)

Estrogen regulates estrogen receptors and antioxidant gene expression in mouse skeletal muscle.

Directory of Open Access Journals (Sweden)

Kristen A Baltgalvis

Full Text Available BACKGROUND: Estrogens are associated with the loss of skeletal muscle strength in women with age. Ovarian hormone removal by ovariectomy in mice leads to a loss of muscle strength, which is reversed with 17beta-estradiol replacement. Aging is also associated with an increase in antioxidant stress, and estrogens can improve antioxidant status via their interaction with estrogen receptors (ER to regulate antioxidant gene expression. The purpose of this study was to determine if ER and antioxidant gene expression in skeletal muscle are responsive to changes in circulating estradiol, and if ERs regulate antioxidant gene expression in this tissue. METHODOLOGY/PRINCIPAL FINDINGS: Adult C57BL/6 mice underwent ovariectomies or sham surgeries to remove circulating estrogens. These mice were implanted with placebo or 17beta-estradiol pellets acutely or chronically. A separate experiment examined mice that received weekly injections of Faslodex to chronically block ERs. Skeletal muscles were analyzed for expression of ER genes and proteins and antioxidant genes. ERalpha was the most abundant, followed by Gper and ERbeta in both soleus and EDL muscles. The loss of estrogens through ovariectomy induced ERalpha gene and protein expression in the soleus, EDL, and TA muscles at both the acute and chronic time points. Gpx3 mRNA was also induced both acutely and chronically in all 3 muscles in mice receiving 17beta-estradiol. When ERs were blocked using Faslodex, Gpx3 mRNA was downregulated in the soleus muscle, but not the EDL and TA muscles. CONCLUSIONS/SIGNIFICANCE: These data suggest that Gpx3 and ERalpha gene expression are sensitive to circulating estrogens in skeletal muscle. ERs may regulate Gpx3 gene expression in the soleus muscle, but skeletal muscle regulation of Gpx3 via ERs is dependent upon muscle type. Further work is needed to determine the indirect effects of estrogen and ERalpha on Gpx3 expression in skeletal muscle, and their importance in the

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells

DEFF Research Database (Denmark)

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe

2009-01-01

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell......-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However......, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell...

Gene expression profiling of placentas affected by pre-eclampsia

DEFF Research Database (Denmark)

Hoegh, Anne Mette; Borup, Rehannah; Nielsen, Finn Cilius

2010-01-01

Several studies point to the placenta as the primary cause of pre-eclampsia. Our objective was to identify placental genes that may contribute to the development of pre-eclampsia. RNA was purified from tissue biopsies from eleven pre-eclamptic placentas and eighteen normal controls. Messenger RNA...... expression from pooled samples was analysed by microarrays. Verification of the expression of selected genes was performed using real-time PCR. A surprisingly low number of genes (21 out of 15,000) were identified as differentially expressed. Among these were genes not previously associated with pre-eclampsia...... as bradykinin B1 receptor and a 14-3-3 protein, but also genes that have already been connected with pre-eclampsia, for example, inhibin beta A subunit and leptin. A low number of genes were repeatedly identified as differentially expressed, because they may represent the endpoint of a cascade of events...

Human CRF{sub 2} {alpha} and {beta} splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay

Energy Technology Data Exchange (ETDEWEB)

Ardati, A. [Rhone-Poulenc Rorer, Cardiovascular Biology, NW4, 500 Arcola Road, Collegeville, PA (United States); Goetschy, V.; Gottowick, J.; Henriot, S.; Deuschle, U.; Kilpatrick, G.J. [Central Nervous System, Pharma Division, F. Hoffmann-La Roche AG, CH-4070 Basel (Switzerland); Valdenaire, O. [Cardiovascular Research, Pharma Division, F. Hoffmann-La Roche AG, CH-4070 Basel (Switzerland)

1999-03-14

Corticotropin releasing factor (CRF) receptors belong to the super-family of G protein-coupled receptors. These receptors are classified into two subtypes (CRF{sub 1} and CRF{sub 2}). Both receptors are positively coupled to adenylyl cyclase but they have a distinct pharmacology and distribution in brain. Two isoforms belonging to the CRF{sub 2} subtype receptors, CRF{sub 2{alpha}} and CRF{sub 2{beta}}, have been identified in rat and man. The neuropeptides CRF and urocortin mediate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recently identified hCRF{sub 2{beta}} receptor. We have used radioligand binding with [{sup 125}I]-tyr{sup 0}-sauvagine and a gene expression assay in which the firefly luciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [{sup 125}I]-tyr{sup 0}-sauvagine binding to the hCRF{sub 2{beta}} receptor were monophasic while dissociation kinetics were biphasic, in agreement with the kinetics results obtained with the hCRF{sub 2{alpha}} receptor. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and with parameters obtained with the hCRF{sub 2{alpha}} receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [{sup 125}I]-tyr{sup 0}-sauvagine to both hCRF{sub 2} receptor isoforms in a similar manner. The rank order of potency of CRF agonist peptides in competition experiments was identical for both hCRF{sub 2}{alpha}-helical CRF{sub (9-41)}oCRF). Similarly, agonist potency was similar for the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist {alpha}-helical CRF{sub (9-41)} exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF{sub 2} receptor isoforms. Taken together, these results indicate that the

Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

Energy Technology Data Exchange (ETDEWEB)

Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

2011-09-09

Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

The effect of the colostral cells on gene expression of cytokines in cord blood cells.

Science.gov (United States)

Hrdý, Jiří; Novotná, Olga; Kocourková, Ingrid; Prokešová, Ludmila

2017-11-01

Beneficial effect of maternal milk is acknowledged, but there is still question whether maternal milk from allergic mother is as good as from healthy one. In our study, we have assayed the effect of cells from colostrum of healthy and allergic mothers on gene expression of cytokines in cord blood cells of newborns of healthy and allergic mothers. Cytokines typical for Th1 (IL-2, IFN-gamma), Th2 (IL-4, IL-13), Tregs (IL-10, TGF-beta), and IL-8 were followed. We were not able to detect significant influence of colostral cells on gene expression of cytokines in cord blood after 2-day coculture using Transwell system. There was no difference in gene expression of cytokines in nonstimulated cord blood cells of newborns of healthy and allergic mothers, but generally increased gene expression of cytokines except IL-10 and TGF-beta after polyclonal stimulation was detected in cord blood cells of children of allergic mothers. There was no difference in IL-10 expression in stimulated cord blood cells of children of healthy and allergic mothers. Gene expression of TGF-beta was even decreased in stimulated cord blood cells of children of allergic mothers in comparison to healthy ones. We have not observed difference in the capacity of colostral cells of healthy and allergic mothers to influence gene expression of cytokines in cord blood cells, but we have described difference in the reactivity of cord blood cells between children of allergic and healthy mothers.

In silico identification of NF-kappaB-regulated genes in pancreatic beta-cells

Directory of Open Access Journals (Sweden)

Eizirik Decio L

2007-02-01

Full Text Available Abstract Background Pancreatic beta-cells are the target of an autoimmune attack in type 1 diabetes mellitus (T1DM. This is mediated in part by cytokines, such as interleukin (IL-1β and interferon (IFN-γ. These cytokines modify the expression of hundreds of genes, leading to beta-cell dysfunction and death by apoptosis. Several of these cytokine-induced genes are potentially regulated by the IL-1β-activated transcription factor (TF nuclear factor (NF-κB, and previous studies by our group have shown that cytokine-induced NF-κB activation is pro-apoptotic in beta-cells. To identify NF-κB-regulated gene networks in beta-cells we presently used a discriminant analysis-based approach to predict NF-κB responding genes on the basis of putative regulatory elements. Results The performance of linear and quadratic discriminant analysis (LDA, QDA in identifying NF-κB-responding genes was examined on a dataset of 240 positive and negative examples of NF-κB regulation, using stratified cross-validation with an internal leave-one-out cross-validation (LOOCV loop for automated feature selection and noise reduction. LDA performed slightly better than QDA, achieving 61% sensitivity, 91% specificity and 87% positive predictive value, and allowing the identification of 231, 251 and 580 NF-κB putative target genes in insulin-producing INS-1E cells, primary rat beta-cells and human pancreatic islets, respectively. Predicted NF-κB targets had a significant enrichment in genes regulated by cytokines (IL-1β or IL-1β + IFN-γ and double stranded RNA (dsRNA, as compared to genes not regulated by these NF-κB-dependent stimuli. We increased the confidence of the predictions by selecting only evolutionary stable genes, i.e. genes with homologs predicted as NF-κB targets in rat, mouse, human and chimpanzee. Conclusion The present in silico analysis allowed us to identify novel regulatory targets of NF-κB using a supervised classification method based on

Cytokines interleukin-1beta and tumor necrosis factor-alpha regulate different transcriptional and alternative splicing networks in primary beta-cells

DEFF Research Database (Denmark)

Ortis, Fernanda; Naamane, Najib; Flamez, Daisy

2010-01-01

by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells. RESEARCH DESIGN AND METHODS: Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h......-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression...... of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine...

Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

Directory of Open Access Journals (Sweden)

Lee Yeon-Su

2010-05-01

Full Text Available Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. Methods We assessed the suitability of six possible reference genes, beta-actin (ACTB, glyceraldehydes-3-phosphate dehydrogenase (GAPDH, hypoxanthine phosphoribosyl transferase 1 (HPRT1, beta-2-microglobulin (B2M, ribosomal subunit L29 (RPL29 and 18S ribosomal RNA (18S rRNA in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. Results This RT-qPCR study showed that there are statistically significant (p Conclusion This study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.

beta. -Amyloid gene dosage in Alzheimer's disease

Energy Technology Data Exchange (ETDEWEB)

Murdoch, G H; Manuelidis, L; Kim, J H; Manuelidis, E E

1988-01-11

The 4-5 kd amyloid ..beta..-peptide is a major constituent of the characteristic amyloid plaque of Alzheimer's disease. It has been reported that some cases of sporatic Alzheimer's disease are associated with at least a partial duplication of chromosome 21 containing the gene corresponding to the 695 residue precursor of this peptide. To contribute to an understanding of the frequency to such a duplication event in the overall Alzheimer's population, the authors have determined the gene dosage of the ..beta..-amyloid gene in this collection of cases. All cases had a clinical diagnosis of Alzheimer's confirmed neuropathologically. Each Alzheimer's case had an apparent normal diploid ..beta..-amyloid gene dosage, while control Down's cases had the expected triploid dosage. Thus partial duplication of chromosome 21 may be a rare finding in Alzheimer's disease. Similar conclusions were just reported in several studies of the Harvard Alzheimer collection.

Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1-producing fibroblasts.

Science.gov (United States)

Lee, K H; Song, S U; Hwang, T S; Yi, Y; Oh, I S; Lee, J Y; Choi, K B; Choi, M S; Kim, S J

2001-09-20

Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

Transforming growth factor beta stimulation of biglycan gene expression is potentially mediated by sp1 binding factors

DEFF Research Database (Denmark)

Heegaard, Anne-Marie; Xie, Zhongjian; Young, Marian Frances

2004-01-01

. In this study, we have investigated the mechanism by which TGF-beta(1), TGF-beta(2) and TGF-beta(3) stimulate biglycan mRNA expression in the osteoblastic cell line MG-63. The cells were transfected with a series of deletional human biglycan promoter constructs and a region in the biglycan 5' DNA was found...... to respond to TGF-beta(1) with increased transcriptional activity in a dose-dependent manner. Also TGF-beta(2) and TGF-beta(3), two structurally highly related TGF-beta isoforms stimulated biglycan transcription. A TGF-beta responsive region was identified within the first 218 bp of the human biglycan...... was abrogated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. A mutation in the Sp1 site at -216 to -208 within the -218 biglycan promoter construct substantially diminished the transcriptional up-regulation by TGF-beta(1). Taken together this data shows for the first time that TGF-beta(1...

The Effect of 5 FU on the Expression of Transforming Growth Factor Beta-1 (Tgf-1 in Cultured Tendon Cells

Directory of Open Access Journals (Sweden)

Zeynep Karacor Altuntas

2014-10-01

Full Text Available This study investigated the effect of the treatment with 1 min exposures to 5-fluorouracil (5-FU  on the expression of TGF-beta 1 in cultured tendon cells. Fibroblasts cultured from the flexor tendons of dog paws were treated with 3 different doses of 5-FU ( control, 5-15-25 mgr /ml for 1 minute.  After 5-FU exposure  the expression of TGF –beta 1 were tested by real time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR at 3rd and 7th days. There were no statistically significant differences in the expression levels of the TGF-b1 gene between the control group and all other groups on day 3 and 7 (p>0.05.However, when the percentage changes in the TGF-BETA1 gene expression on days 3–7 were compared, there were statistically significant differences and this was maximally observed with 89% +12 (p<0.05 in the group treated with 25-mg/ml 5-FU.  We conclude that 1min. 5-FU application may be  sufficient to prevent adhesions in tendon healing by limiting the expression of TGF-BETA1. 

Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

Science.gov (United States)

Takahashi, Yuki; Nishikawa, Makiya; Suehara, Tetsuya; Takiguchi, Naomi; Takakura, Yoshinobu

2008-11-15

Altered expression of beta-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of beta-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of beta-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshbeta-catenin) markedly suppressed their growth in vivo, gene silencing of beta-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of beta-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshbeta-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to beta-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshbeta-catenin, suggesting that cadherin is shed from the cell surface when the expression of beta-catenin is reduced. The mobility of B16-BL6 cells transfected with pshbeta-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshbeta-catenin (B16/pshbeta-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshbeta-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of beta-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells. (c) 2008 Wiley-Liss, Inc.

Trichostatin A inhibits beta-casein expression in mammary epithelial cells

International Nuclear Information System (INIS)

Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

2002-01-01

Many aspects of cellular behavior are affected by information derived from association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. Previously, we defined the minimal ECM- and Prl-responsive enhancer element BCE-1 from the upstream region of the beta-casein gene. We also found that BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous b-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of b-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM could mediate rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses.

Directory of Open Access Journals (Sweden)

Dongli Wan

Full Text Available Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2 were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.

Differential expression of largemouth bass (Micropterus salmoides) estrogen receptor isotypes alpha, beta, and gamma by estradiol.

Science.gov (United States)

Sabo-Attwood, Tara; Kroll, Kevin J; Denslow, Nancy D

2004-04-15

The expression levels of three estrogen receptor (ER) isotypes alpha, beta, and gamma were quantified in female largemouth bass (Micropterus salmoides) (LMB) liver, ovary, brain, and pituitary tissues. ER alpha and beta expression predominated in the liver, while ERs beta and gamma predominated in the other tissues. Temporally in females, ER alpha was highly up-regulated, ER gamma was slightly up-regulated, and ER beta levels remained unchanged in the liver when plasma 17-beta estradiol (E2) and vitellogenin (Vtg) levels were elevated in the spring. In ovarian tissue from these same fish, all three ERs were maximally expressed in the fall, during early oocyte development and prior to peak plasma E2 levels. When males were injected with E2, ER alpha was highly inducible, ER gamma was moderately up-regulated, and ER beta levels were not affected. None of the ER isotypes were induced by E2 in gonadal tissues. These results combined suggest that the ERs themselves are not regulated in the same manner by E2, and furthermore, do not contribute equally to the transcriptional regulation of genes involved in fish reproduction such as Vtg.

Clonal expansion of T-cell receptor beta gene segment in the retrocochlear lesions of EAE mice.

Science.gov (United States)

Cheng, K C; Lee, K M; Yoo, T J

1998-01-01

It has been reported that the T cell receptor V beta 8.2 (TcrbV8.2) gene segment is predominantly expressed in encephalomyelitic T cells responding to myelin basic protein (MBP) in experimental allergic encephalomyelitis (EAE) mice. We have demonstrated retrocochlear hearing loss in EAE mice in previous studies. Administration of a monoclonal antibody specific to the T cell receptor V beta 8 (TcrbV8) subfamily prevented both this type of hearing loss and the central nerve disease. In this study, we examined the role of the TcrbV8.2 gene segment in the retrocochlear lesions of EAE mice. A clonal expression of T cell receptor beta chain gene segment (TcrbV8.2-TcrbD2-TcrbJ2.7) was identified in the retrocochlear lesions. The TcrbV8.2 gene segment appears to recombine only with TcrbJ2.1 (32.1%) and TcrbJ2.7 (67.9%) gene segments. The TcrbJ2.7 gene segment has also been previously identified as the dominant TcrbJ gene in the lymph nodes of EAE mice. Only TcrbD2, with a length of 4 amino acids, was observed recombining with these TcrbV8.2 sequences. G and C nucleotides are predominantly expressed at the N regions between the V-D and D-J junctions. This dominant TcrbV gene segment (TcrbV8.2-TcrbD2-TcrbJ2.7) observed in the retrocochlear lesions has been identified in the MBP-specific T cells from the lymph nodes of EAE mice. These results suggest that a small subset of antigen-specific T cells migrate to, and expand at, the retrocochlear lesions, which leads to hearing loss.

MSX-1 gene expression and regulation in embryonic palatal tissue.

Science.gov (United States)

Nugent, P; Greene, R M

1998-01-01

The palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.

Gene expression profiles in adenosine-treated human mast cells ...

African Journals Online (AJOL)

Gene expression profiles in adenosine-treated human mast cells. ... SW Kang, JE Jeong, CH Kim, SH Choi, SH Chae, SA Jun, HJ Cha, JH Kim, YM Lee, YS ... beta 4, ring finger protein, high-mobility group, calmodulin 2, RAN binding protein, ...

Expression of P-aPKC-iota, E-cadherin, and beta-catenin related to invasion and metastasis in hepatocellular carcinoma.

Science.gov (United States)

Du, Guang-Sheng; Wang, Jian-Ming; Lu, Jin-Xi; Li, Qiang; Ma, Chao-Qun; Du, Ji-Tao; Zou, Sheng-Quan

2009-06-01

Atypical protein kinase C iota (aPKC-iota) and its associated intracellular molecules, E-cadherin and beta-catenin, are important for cell polarization in tumorigenesis and progression. Expression of aPKC-iota, P-aPKC-iota (activated aPKC-iota), E-cadherin, and beta-catenin in hepatocellular carcinoma (HCC) was measured, and correlation with clinicopathological characteristics of HCC was analyzed. Paraffin-embedded tumor tissue was obtained from patients with HCC after resection without preoperative radiotherapy or chemotherapy. Gene expression was detected by polymerase chain reaction (PCR), and protein expression was detected by immunohistochemistry and Western blot analysis. Expressions of aPKC-iota, P-aPKC-iota, E-cadherin, and beta-catenin were analyzed with relation to the clinicopathological data. The gene and protein expression of aPKC-iota are obviously higher in HCC tissues than that in peritumoral tissues and normal tissues by semiquantitative PCR and immunohistochemistry methods. Accumulation of aPKC-iota in HCC cytoplasm and nucleolus inhibited the later formation of belt-like adherens junctions (AJs) and/or tight junctions (TJs) in cell-cell contact. E-cadherin was reduced and accumulation of cytoplasm beta-catenin was increased in HCC. The expression of aPKC-iota was closely related to pathological differentiation, tumor size, invasion, and metastasis of HCC. Accumulation of cytoplasm aPKC-iota may reflect pathological differentiation, invasion, and metastasis potential of HCC. In this regard, our study on HCC revealed the potential usefulness of aPKC-iota, E-cadherin, and beta-catenin as a prognostic marker, closely related to pathological differentiation, invasion, metastasis, and prognosis of HCC.

Frequency of alpha- and beta-haemolysin in Staphylococcus aureus of bovine and human origin - A comparison between pheno- and genotype and variation in phenotypic expression

DEFF Research Database (Denmark)

Aarestrup, Frank Møller; Larsen, H.D.; Eriksen, N.H.R.

1999-01-01

The phenotypic expression of haemolysins and the presence of genes encoding alpha and beta-haemolysin were determined in 105 Sraphylococcus aureus isolates from bovine mastitis, 100 isolates from the nostrils of healthy humans, and 60 isolates from septicaemia in humans. Furthermore, the possible...... change in expression of haemolysins after subcultivation in human and bovine blood and milk was studied in selected isolates. alpha-haemolysin was expressed phenotypically in 39 (37%) of the bovine isolates, in 59 (59%) of the human carrier isolates, and in 40 (67%) of the isolates from septicaemia. beta......-haemolysin was expressed in 76 (72%) bovine, 11 (11%) carrier, and 8 (13%) septicaemia isolates. Significantly more bovine than human isolates expressed beta-haemolysin and significantly fewer expressed alpha-haemolysin. Genotypically, the gene encoding alpha-haemolysin was detected in all isolates. A significant...

Gene expression profile of the fibrotic response in the peritoneal cavity.

Science.gov (United States)

Le, S J; Gongora, M; Zhang, B; Grimmond, S; Campbell, G R; Campbell, J H; Rolfe, B E

2010-01-01

The cellular response to materials implanted in the peritoneal cavity has been utilised to produce tissue for grafting to hollow smooth muscle organs (blood vessels, bladder, uterus and vas deferens). To gain insight into the regulatory mechanisms involved in encapsulation of a foreign object, and subsequent differentiation of encapsulating cells, the present study used microarray technology and real-time RT-PCR to identify the temporal changes in gene expression associated with tissue development. Immunohistochemical analysis showed that 3-7 days post-implantation of foreign objects (cubes of boiled egg white) into rats, they were encapsulated by tissue comprised primarily of haemopoietic (CD45(+)) cells, mainly macrophages (CD68(+), CCR1(+)). By day 14, tissue capsule cells no longer expressed CD68, but were positive for myofibroblast markers alpha-smooth muscle (SM) actin and SM22. In accordance with these results, gene expression data showed that early capsule (days 3-7) development was dominated by the expression of monocyte/macrophage-specific genes (CD14, CSF-1, CSF-1R, MCP-1) and pro-inflammatory mediators such as transforming growth factor (TGF-beta). As tissue capsule development progressed (days 14-21), myofibroblast-associated and pro-fibrotic genes (associated with TGF-beta and Wnt/beta-catenin signalling pathways, including Wnt 4, TGFbetaRII, connective tissue growth factor (CTGF), SMADs-1, -2, -4 and collagen-1 subunits) were significantly up-regulated. The up-regulation of genes associated with Cardiovascular and Skeletal and Muscular System Development at later time-points suggests the capacity of cells within the tissue capsule for further differentiation to smooth muscle, and possibly other cell types. The identification of key regulatory pathways and molecules associated with the fibrotic response to implanted materials has important applications not only for optimising tissue engineering strategies, but also to control deleterious fibrotic

DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Kenyon, C.J.; Walker, G.C.

1988-05-01

Operon fusions in Escherichia coli were obtained that showed increased beta-galactosidase expression in response to treatment with the DNA-damaging agent mitomycin C. These fusions were generated by using the Mud(ApR, lac) vector to insert the lactose structural genes randomly into the bacterial chromosome. Induction of beta-galactosidase in these strains, which carried fusions of lac to these din (damage-inducible) loci, was (i) triggered by UV light as well as by mitomycin C and (ii) abolished by either a recA- or a lexA- mutation. Similar characteristics of induction were observed when the lactose genes were fused to a prophage lambda promoter by using Mud(ApR, lac). These results indicate that E. coli contains a set of genes that, like prophage lambda genes, are expressed in response to DNA-damaging agents and regulated by the recA and lexA gene products. These din genes map at five bacterial loci. One din::Mud(ApR, lac) insertion results in a UV-sensitive phenotype and may be within the uvrA transcriptional unit.

Method for Screening Compounds That Influence Virulence Gene Expression in Staphylococcus aureus

DEFF Research Database (Denmark)

Nielsen, A.; Nielsen, Kristian Fog; Frees, D.

2010-01-01

We present a simple assay to examine effects of compounds on virulence gene expression in the human pathogen Staphylococcus aureus. The assay employs transcriptional reporter strains carrying lacZ fused to central virulence genes. Compounds affecting virulence gene expression and activity...... of the agr locus are scored based on color change in the presence of a chromogenic beta-galactosidase substrate. The assay can be used to screen for novel antivirulence compounds from many different sources, such as fungi, as demonstrated here....

Temporal profile of estrogen-dependent gene expression in LHRH-producing GT1-7 cells.

Science.gov (United States)

Varju, Patricia; Chang, Ken C; Hrabovszky, Erik; Merchenthaler, István; Liposits, Zsolt

2009-02-01

The long-term cellular effects of estrogens are mediated by nuclear estrogen receptors which act as transcription factors to regulate gene expression. Hypothalamic targets of estrogen action include luteinizing hormone-releasing hormone-secreting neurons controlling reproduction in vertebrates. Microarray analysis and qRT-PCR studies were performed on GT1-7, immortalized LHRH neurons after 17beta-estradiol treatment to reveal the nature of estrogen-regulated genes and the time course of changes in their expression profile. More than 1000 transcripts showed robust responses to estrogen treatment and the majority of responding genes were up-regulated. Early-responding genes showed altered expression 0.5-2h after estrogen exposure, whereas late-responding genes changed after 24-48h treatment. Up-regulated genes encoded transcription factors, molecules involved in cellular movement, cell death, immune response, neurotransmitter and neuropeptide receptors, ion channels and transporters. The 17beta-estradiol modulation of 12 genes - representing characteristic gene clusters - has been confirmed by qRT-PCR. Our studies highlighted diverse gene networks, cell regulatory mechanisms and metabolic pathways through which estrogen may alter gene expression in immortalized LHRH neurons. The findings also support the notion that genomic effects of estrogen targeting in vivo directly the LHRH neuronal network of mammals play an important role in the central feedback regulation of the reproductive axis by estrogen.

Divergent expression of 11beta-hydroxysteroid dehydrogenase and 11beta-hydroxylase genes between male morphs in the central nervous system, sonic muscle and testis of a vocal fish.

Science.gov (United States)

Arterbery, Adam S; Deitcher, David L; Bass, Andrew H

2010-05-15

The vocalizing midshipman fish, Porichthys notatus, has two male morphs that exhibit alternative mating tactics. Only territorial males acoustically court females with long duration (minutes to >1h) calls, whereas sneaker males attempt to steal fertilizations. During the breeding season, morph-specific tactics are paralleled by a divergence in relative testis and vocal muscle size, plasma levels of the androgen 11-ketotestosterone (11KT) and the glucocorticoid cortisol, and mRNA expression levels in the central nervous system (CNS) of the steroid-synthesizing enzyme aromatase (estrogen synthase). Here, we tested the hypothesis that the midshipman's two male morphs would further differ in the CNS, as well as in the testis and vocal muscle, in mRNA abundance for the enzymes 11beta-hydroxylase (11betaH) and 11beta-hydroxysteroid dehydrogenase (11betaHSD) that directly regulate both 11KT and cortisol synthesis. Quantitative real-time PCR demonstrated male morph-specific profiles for both enzymes. Territorial males had higher 11betaH and 11betaHSD mRNA levels in testis and vocal muscle. By contrast, sneaker males had the higher CNS expression, especially for 11betaHSD, in the region containing an expansive vocal pacemaker circuit that directly determines the temporal attributes of natural calls. We propose for territorial males that higher enzyme expression in testis underlies its greater plasma 11KT levels, which in vocal muscle provides both gluconeogenic and androgenic support for its long duration calling. We further propose for sneaker males that higher enzyme expression in the vocal CNS contributes to known cortisol-specific effects on its vocal physiology. Copyright 2010 Elsevier Inc. All rights reserved.

A Hybrid One-Way ANOVA Approach for the Robust and Efficient Estimation of Differential Gene Expression with Multiple Patterns.

Directory of Open Access Journals (Sweden)

Mohammad Manir Hossain Mollah

Full Text Available Identifying genes that are differentially expressed (DE between two or more conditions with multiple patterns of expression is one of the primary objectives of gene expression data analysis. Several statistical approaches, including one-way analysis of variance (ANOVA, are used to identify DE genes. However, most of these methods provide misleading results for two or more conditions with multiple patterns of expression in the presence of outlying genes. In this paper, an attempt is made to develop a hybrid one-way ANOVA approach that unifies the robustness and efficiency of estimation using the minimum β-divergence method to overcome some problems that arise in the existing robust methods for both small- and large-sample cases with multiple patterns of expression.The proposed method relies on a β-weight function, which produces values between 0 and 1. The β-weight function with β = 0.2 is used as a measure of outlier detection. It assigns smaller weights (≥ 0 to outlying expressions and larger weights (≤ 1 to typical expressions. The distribution of the β-weights is used to calculate the cut-off point, which is compared to the observed β-weight of an expression to determine whether that gene expression is an outlier. This weight function plays a key role in unifying the robustness and efficiency of estimation in one-way ANOVA.Analyses of simulated gene expression profiles revealed that all eight methods (ANOVA, SAM, LIMMA, EBarrays, eLNN, KW, robust BetaEB and proposed perform almost identically for m = 2 conditions in the absence of outliers. However, the robust BetaEB method and the proposed method exhibited considerably better performance than the other six methods in the presence of outliers. In this case, the BetaEB method exhibited slightly better performance than the proposed method for the small-sample cases, but the the proposed method exhibited much better performance than the BetaEB method for both the small- and large

Positive selection within the Schizophrenia-associated GABA(A receptor beta(2 gene.

Directory of Open Access Journals (Sweden)

Wing-Sze Lo

Full Text Available The gamma-aminobutyric acid type-A (GABA(A receptor plays a major role in inhibitory neurotransmissions. Intronic SNPs and haplotypes in GABRB2, the gene for GABA(A receptor beta(2 subunit, are associated with schizophrenia and correlated with the expression of two alternatively spliced beta(2 isoforms. In the present study, using chimpanzee as an ancestral reference, high frequencies were observed for the derived (D alleles of the four SNPs rs6556547, rs187269, rs1816071 and rs1816072 in GABRB2, suggesting the occurrence of positive selection for these derived alleles. Coalescence-based simulation showed that the population frequency spectra and the frequencies of H56, the haplotype having all four D alleles, significantly deviated from neutral-evolution expectation in various demographic models. Haplotypes containing the derived allele of rs1816072 displayed significantly less diversity compared to haplotypes containing its ancestral allele, further supporting positive selection. The variations in DD-genotype frequencies in five human populations provided a snapshot of the evolutionary history, which suggested that the positive selections of the D alleles are recent and likely ongoing. The divergence between the DD-genotype profiles of schizophrenic and control samples pointed to the schizophrenia-relevance of positive selections, with the schizophrenic samples showing weakened selections compared to the controls. These DD-genotypes were previously found to increase the expression of beta(2, especially its long isoform. Electrophysiological analysis showed that this long beta(2 isoform favored by the positive selections is more sensitive than the short isoform to the inhibition of GABA(A receptor function by energy depletion. These findings represent the first demonstration of positive selection in a schizophrenia-associated gene.

Mutants of Agrobacterium tumefaciens with elevated vir gene expression

International Nuclear Information System (INIS)

Pazour, G.J.; Ta, C.N.; Das, A.

1991-01-01

Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Directory of Open Access Journals (Sweden)

Fei Mo

2014-09-01

Full Text Available Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus under various conditions (sex, age, water temperature, and drug treatments, seven reference genes, including beta actin (ACTB, beta-2 microglobulin (B2M, embryonic elongation factor-1 alpha (EEF1A, glyceraldehyde phosphate dehydrogenase (GAPDH, alpha tubulin (TUBA, ribosomal protein l8 (RPL8 and glucose-6-phosphate dehydrogenase (G6PDH, were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.

IL-1beta-induced chemokine and Fas expression are inhibited by suppressor of cytokine signalling-3 in insulin-producing cells

DEFF Research Database (Denmark)

Jacobsen, M L B; Rønn, S G; Bruun, C

2008-01-01

AIMS/HYPOTHESIS: Chemokines recruit activated immune cells to sites of inflammation and are important mediators of insulitis. Activation of the pro-apoptotic receptor Fas leads to apoptosis-mediated death of the Fas-expressing cell. The pro-inflammatory cytokines IL-1beta and IFN-gamma regulate...... the transcription of genes encoding the Fas receptor and several chemokines. We have previously shown that suppressor of cytokine signalling (SOCS)-3 inhibits IL-1beta- and IFN-gamma-induced nitric oxide production in a beta cell line. The aim of this study was to investigate whether SOCS-3 can influence cytokine......-induced Fas and chemokine expression in beta cells. METHODS: Using a beta cell line with inducible Socs3 expression or primary neonatal rat islet cells transduced with a Socs3-encoding adenovirus, we employed real-time RT-PCR analysis to investigate whether SOCS-3 affects cytokine-induced chemokine and Fas m...

Validation of suitable reference genes for quantitative gene expression analysis in Panax ginseng

Directory of Open Access Journals (Sweden)

Meizhen eWang

2016-01-01

Full Text Available Reverse transcription-qPCR (RT-qPCR has become a popular method for gene expression studies. Its results require data normalization by housekeeping genes. No single gene is proved to be stably expressed under all experimental conditions. Therefore, systematic evaluation of reference genes is necessary. With the aim to identify optimum reference genes for RT-qPCR analysis of gene expression in different tissues of Panax ginseng and the seedlings grown under heat stress, we investigated the expression stability of eight candidate reference genes, including elongation factor 1-beta (EF1-β, elongation factor 1-gamma (EF1-γ, eukaryotic translation initiation factor 3G (IF3G, eukaryotic translation initiation factor 3B (IF3B, actin (ACT, actin11 (ACT11, glyceraldehyde-3-phosphate dehydrogenase (GAPDH and cyclophilin ABH-like protein (CYC, using four widely used computational programs: geNorm, Normfinder, BestKeeper, and the comparative ΔCt method. The results were then integrated using the web-based tool RefFinder. As a result, EF1-γ, IF3G and EF1-β were the three most stable genes in different tissues of P. ginseng, while IF3G, ACT11 and GAPDH were the top three-ranked genes in seedlings treated with heat. Using three better reference genes alone or in combination as internal control, we examined the expression profiles of MAR, a multiple function-associated mRNA-like non-coding RNA (mlncRNA in P. ginseng. Taken together, we recommended EF1-γ/IF3G and IF3G/ACT11 as the suitable pair of reference genes for RT-qPCR analysis of gene expression in different tissues of P. ginseng and the seedlings grown under heat stress, respectively. The results serve as a foundation for future studies on P. ginseng functional genomics.

Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR

International Nuclear Information System (INIS)

Rho, Hyun-Wook; Lee, Byoung-Chan; Choi, Eun-Seok; Choi, Il-Ju; Lee, Yeon-Su; Goh, Sung-Ho

2010-01-01

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results. We assessed the suitability of six possible reference genes, beta-actin (ACTB), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), beta-2-microglobulin (B2M), ribosomal subunit L29 (RPL29) and 18S ribosomal RNA (18S rRNA) in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values. This RT-qPCR study showed that there are statistically significant (p < 0.05) differences in the expression levels of HPRT1 and 18S rRNA in 'normal-' versus 'tumor stomach tissues'. The stability analyses by geNorm suggest B2M-GAPDH, as best reference gene combination for 'stomach cancer cell lines'; RPL29-HPRT1, for 'all stomach tissues'; and ACTB-18S rRNA, for 'all stomach cell lines and tissues'. NormFinder also identified B2M as the best reference gene for 'stomach cancer cell lines', RPL29-B2M for 'all stomach tissues', and 18S rRNA-ACTB for 'all stomach cell lines and tissues'. The comparisons of normalized expression of the target gene, GPNMB, showed different interpretation of target gene expression depend on best single reference gene or combination. This study validated RPL29 and RPL29-B2M as the best single reference

Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon.

Science.gov (United States)

Kang, H G; Jun, S H; Kim, J; Kawaide, H; Kamiya, Y; An, G

1999-10-01

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.

How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

Directory of Open Access Journals (Sweden)

Barth Sandra

2005-04-01

Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

The Importance of REST for Development and Function of Beta Cells

DEFF Research Database (Denmark)

Martin, David; Grapin-Botton, Anne

2017-01-01

that are crucial for both neuronal and pancreatic endocrine function, through the recruitment of multiple transcriptional and epigenetic co-regulators. REST targets include genes encoding transcription factors, proteins involved in exocytosis, synaptic transmission or ion channeling, and non-coding RNAs. REST......Beta cells are defined by the genes they express, many of which are specific to this cell type, and ensure a specific set of functions. Beta cells are also defined by a set of genes they should not express (in order to function properly), and these genes have been called forbidden genes. Among...... these, the transcriptional repressor RE-1 Silencing Transcription factor (REST) is expressed in most cells of the body, excluding most populations of neurons, as well as pancreatic beta and alpha cells. In the cell types where it is expressed, REST represses the expression of hundreds of genes...

Testes and brain gene expression in precocious male and adult maturing Atlantic salmon (Salmo salar

Directory of Open Access Journals (Sweden)

Houeix Benoit

2010-03-01

Full Text Available Abstract Background The male Atlantic salmon generally matures in fresh water upon returning after one or several years at sea. Some fast-growing male parr develop an alternative life strategy where they sexually mature before migrating to the oceans. These so called 'precocious' parr or 'sneakers' can successfully fertilise adult female eggs and so perpetuate their line. We have used a custom-built cDNA microarray to investigate gene expression changes occurring in the salmon gonad and brain associated with precocious maturation. The microarray has been populated with genes selected specifically for involvement in sexual maturation (precocious and adult and in the parr-smolt transformation. Results Immature and mature parr collected from a hatchery-reared stock in January were significantly different in weight, length and condition factor. Changes in brain expression were small - never more than 2-fold on the microarray, and down-regulation of genes was much more pronounced than up-regulation. Significantly changing genes included isotocin, vasotocin, cathepsin D, anamorsin and apolipoprotein E. Much greater changes in expression were seen in the testes. Among those genes in the testis with the most significant changes in expression were anti-Mullerian hormone, collagen 1A, and zinc finger protein (Zic1, which were down-regulated in precocity and apolipoproteins E and C-1, lipoprotein lipase and anti-leukoproteinase precursor which were up-regulated in precocity. Expression changes of several genes were confirmed in individual fish by quantitative PCR and several genes (anti-Mullerian hormone, collagen 1A, beta-globin and guanine nucleotide binding protein (G protein beta polypeptide 2-like 1 (GNB2L1 were also examined in adult maturing testes. Down-regulation of anti-Mullerian hormone was judged to be greater than 160-fold for precocious males and greater than 230-fold for November adult testes in comparison to July testes by this method. For

Assessment of affinities of beta-CIT, beta-CIT-FE, and beta-CIT-FP for monoamine transporters permanently expressed in cell lines

International Nuclear Information System (INIS)

Okada, Tomoya; Fujita, Masahiro; Shimada, Shoichi; Sato, Kohji; Schloss, Patrick; Watanabe, Yoshiyuki; Itoh, Yasushi; Tohyama, Masaya; Nishimura, Tsunehiko

1998-01-01

We investigated the effects of three cocaine analogs, beta-CIT (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-tropane), beta-CIT-FE (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-N-(2-fluoroethyl)-nortropane), and beta-CIT-FP (2-beta-carbomethoxy-3-beta-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane), on the uptake of [ 3 H]dopamine(DA), serotonin(5-HT), and 1-norepinephrine (NE) using cell lines permanently expressing DA, 5-HT, and NE transporters, respectively, to determine their affinities for these three transporters. We generated cell lines stably expressing DA, 5-HT, and NE transporters, respectively, by the Chen-Okayama method, and then tested the abilities of (-)cocaine, beta-CIT, beta-CIT-FE, beta-CIT-FP, and clomipramine to inhibit the uptake of [ 3 H]DA, 5-HT, and 1-NE. Ki values of beta-CIT, beta-CIT-FE, and beta-CIT-FP for [ 3 H]DA, 5-HT, 1-NE uptake were 6, 29, and 33 nM, 91, 133, and 130 nM, and 28, 113 and 70 nM, respectively, whereas those of cocaine and clomipramine were 316, 581, and 176 nM and > 10,000, 437, and 851 nM, respectively. Beta-CIT, beta-CIT-FE, and beta-CIT-FP were shown to be potent DA, 5-HT, and NE uptake inhibitors. Beta-CIT and beta-CIT-FP were highly potent and selective dopamine uptake inhibitors, and therefore might be useful for imaging of DA transporter with single photon emission computed tomography (SPECT) or positron emission tomography (PET)

A comprehensive review of the prevalence of beta globin gene variations and the co-inheritance of related gene variants in Saudi Arabians with beta-thalassemia

Science.gov (United States)

Alaithan, Mousa A.; AbdulAzeez, Sayed; Borgio, J. Francis

2018-01-01

Beta-thalassemia is a genetic disorder that is caused by variations in the beta-hemoglobin (HBB) gene. Saudi Arabia is among the countries most affected by beta-thalassemia, and this is particularly problematic in the Eastern regions. This review article is an attempt to compile all the reported mutations to facilitate further national-level studies to prepare a Saudi repository of HBB gene variations. In Saudi Arabians, IVSI-5 (G>C) and Cd 39 (C>T) are the most prevalent HBB gene variations out of 42 variations. The coinheritance of HBB gene variations with ATRX, HBA1, HBA2, HBA12, AHSP, and KLF1 gene variations were observed to be common in the Saudi population. National surveys on the molecular nature of hemoglobinopathies should be set up through collaborations between research centers from various regions to create a well-documented molecular data bank. This data bank can be used to develop a premarital screening program and lead to the best treatment and prevention strategies for beta-thalassemia. PMID:29619482

Induction of gene expression in sheepshead minnows (Cyprinodon variegatus) treated with 17beta-estradiol, diethylstilbestrol, or ethinylestradiol: the use of mRNA fingerprints as an indicator of gene regulation.

Science.gov (United States)

Denslow, N D; Bowman, C J; Ferguson, R J; Lee, H S; Hemmer, M J; Folmar, L C

2001-03-01

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variegatus) exposed in vivo to the natural and pharmaceutical estrogens 17beta-estradiol, ethinylestradiol, and diethylstilbestrol. This method used differential display reverse transcriptase polymerase chain reaction assays to compare the expression of individual mRNAs from control and estrogen-exposed fish. Forty-eight differentially expressed cDNAs were isolated by this method, including cDNAs for vitelline envelope proteins and vitellogenin. The mRNA expression patterns for fish injected with a pharmacological dose of estradiol (5 mg/kg) were identical to those obtained in fish receiving constant aqueous exposure to 212 ng estradiol/liter. Further, the cDNA "fingerprint" pattern observed in the estradiol-treated fish also matched that obtained in fish receiving continuous-flow aqueous exposures to 192 ng ethinyl estradiol/liter and a nominal concentration of 200 ng diethylstilbestrol/liter. The results demonstrate a characteristic expression pattern for genes upregulated by exposure to a variety of natural and anthropogenic estrogens and suggest this approach may be valuable to examine the potential effects of environmental contaminants on other endocrine-mediated pathways of reproduction, growth, and development. Copyright 2001 Academic Press.

The change of transforming growth factor {beta} 1 (TGF- {beta} 1) expression by melatonin in irradiated lung

Energy Technology Data Exchange (ETDEWEB)

Jang, Seong Soon; Choi, Ihl Bohng [College of Medicine, The Catholic University of Korea, Seoul (Korea, Republic of)

2005-09-15

The changed expressions of TGF- {beta} 1, as a key cytokine in the fibrotic process, due to melatonin with potent antioxidative effects, were investigated in the irradiated lung using fibrosis-sensitive C57BL/6 mice. Female C57BL/6 mice were divided into control irradiation-only, and melatonin (300 mg/kg i.p. 1 hr before irradiation) pretreatment groups. The thoraces of the mice were irradiated with a single dose of 12 Gy. The mRNA expressions of TGF-{beta} 1 in the lung tissue 2 and 4 weeks after irradiation were quantified using semiquantitive RT-PCR, and the cellular origin and expression levels of TGF- {beta} 1 protein were identified using immunohistochemical staining. The relative mRNA expression levels in the irradiation-only and melatonin pretreatment group 2 and 4 weeks after irradiation were 1.92- and 1.80-fold ({rho} = 0.064) and 2.38- and 1.94-fold ({rho} = 0.004) increased, respectively compared to those in the control group. Increased expressions of TGF- {beta} 1 protein were prominently detected in regions of histopathological radiation injury, with alveolar macrophages and septal epithelial cells serving as important sources of TGF- {beta} 1 expression. At 2 and 4 weeks after irradiation, the expression levels of protein were 15.8% vs. 16.9% ({rho} = 0.565) and 36.1% vs. 25.7% ({rho} = 0.009), respectively. The mRNA and protein expressions of TGF- {beta} 1 in the lung tissue following thoracic irradiation with 12 Gy were significantly decreased by melatonin pretreatment at 4 weeks. These results indicate that melatonin may have a possible application as an antifibrotic agent in radiation-induced lung injury.

Identification of specific gene expression profiles in fibroblasts derived from middle ear cholesteatoma.

Science.gov (United States)

Yoshikawa, Mamoru; Kojima, Hiromi; Wada, Kota; Tsukidate, Toshiharu; Okada, Naoko; Saito, Hirohisa; Moriyama, Hiroshi

2006-07-01

To investigate the role of fibroblasts in the pathogenesis of cholesteatoma. Tissue specimens were obtained from our patients. Middle ear cholesteatoma-derived fibroblasts (MECFs) and postauricular skin-derived fibroblasts (SFs) as controls were then cultured for a few weeks. These fibroblasts were stimulated with interleukin (IL) 1alpha and/or IL-1beta before gene expression assays. We used the human genome U133A probe array (GeneChip) and real-time polymerase chain reaction to examine and compare the gene expression profiles of the MECFs and SFs. Six patients who had undergone tympanoplasty. The IL-1alpha-regulated genes were classified into 4 distinct clusters on the basis of profiles differentially regulated by SF and MECF using a hierarchical clustering analysis. The messenger RNA expressions of LARC (liver and activation-regulated chemokine), GMCSF (granulocyte-macrophage colony-stimulating factor), epiregulin, ICAM1 (intercellular adhesion molecule 1), and TGFA (transforming growth factor alpha) were more strongly up-regulated by IL-1alpha and/or IL-1beta in MECF than in SF, suggesting that these fibroblasts derived from different tissues retained their typical gene expression profiles. Fibroblasts may play a role in hyperkeratosis of middle ear cholesteatoma by releasing molecules involved in inflammation and epidermal growth. These fibroblasts may retain tissue-specific characteristics presumably controlled by epigenetic mechanisms.

Selection of reference genes in different myocardial regions of an in vivo ischemia/reperfusion rat model for normalization of antioxidant gene expression

Directory of Open Access Journals (Sweden)

Vesentini Nicoletta

2012-02-01

Full Text Available Abstract Background Changes in cardiac gene expression due to myocardial injury are usually assessed in whole heart tissue. However, as the heart is a heterogeneous system, spatial and temporal heterogeneity is expected in gene expression. Results In an ischemia/reperfusion (I/R rat model we evaluated gene expression of mitochondrial and cytoplasmatic superoxide dismutase (MnSod, Cu-ZnSod and thioredoxin reductase (trxr1 upon short (4 h and long (72 h reperfusion times in the right ventricle (RV, and in the ischemic/reperfused (IRR and the remote region (RR of the left ventricle. Gene expression was assessed by Real-time reverse-transcription quantitative PCR (RT-qPCR. In order to select most stable reference genes suitable for normalization purposes, in each myocardial region we tested nine putative reference genes by geNorm analysis. The genes investigated were: Actin beta (actb, Glyceraldehyde-3-P-dehydrogenase (gapdh, Ribosomal protein L13A (rpl13a, Tyrosine 3-monooxygenase (ywhaz, Beta-glucuronidase (gusb, Hypoxanthine guanine Phosphoribosyltransferase 1 (hprt, TATA binding box protein (tbp, Hydroxymethylbilane synthase (hmbs, Polyadenylate-binding protein 1 (papbn1. According to our findings, most stable reference genes in the RV and RR were hmbs/hprt and hmbs/tbp/hprt respectively. In the IRR, six reference genes were recommended for normalization purposes; however, in view of experimental feasibility limitations, target gene expression could be normalized against the three most stable reference genes (ywhaz/pabp/hmbs without loss of sensitivity. In all cases MnSod and Cu-ZnSod expression decreased upon long reperfusion, the former in all myocardial regions and the latter in IRR alone. trxr1 expression did not vary. Conclusions This study provides a validation of reference genes in the RV and in the anterior and posterior wall of the LV of cardiac ischemia/reperfusion model and shows that gene expression should be assessed separately in

Expression of T cell antigen receptor genes in the thymus of irradiated mice after bone marrow transplantation

International Nuclear Information System (INIS)

Matsuzaki, G.; Yoshikai, Y.; Kishihara, K.; Nomoto, K.

1988-01-01

Sequential appearance of the expression of T cell antigen receptor genes was investigated in the thymus of irradiated mice at the early stage after transplantation of Thy-1 congeneic H-2 compatible allogeneic bone marrow cells. The first cells to repopulate the thymus on day 7 after bone marrow transplantation were intrathymic radioresistant T cell precursors, which expanded mainly to CD4+CD8+ host-type thymocytes by day 14. A high level of gamma gene expression but a much reduced level of alpha and beta gene expression were detected in the host-type thymocytes on day 7. During regeneration of these cells, gamma-chain messages fell to low level and alpha and beta mRNA levels increased. The thymus of the recipients began to be repopulated by donor-derived T cells about 2 wk after bone marrow transplantation and was almost completely replaced by the third week. An ordered expression of gamma then beta and alpha-chain gene transcript was also observed in the donor-type thymocytes at the early stage after bone marrow transplantation. The use of thymocytes at early stage in whole-body irradiated bone marrow chimera provides a pertinent source for investigating the molecular mechanism of T cell differentiation in adult thymus

CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

DEFF Research Database (Denmark)

Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O

2002-01-01

An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

Stabilization of mismatch repair gene PMS2 by glycogen synthase kinase 3beta is implicated in the treatment of cervical carcinoma.

Science.gov (United States)

Zhang, Yuan; Shu, Yi Min; Wang, Shu Fang; Da, Bang Hong; Wang, Ze Hua; Li, Hua Bin

2010-02-23

PMS2 expression loss was reported in a variety of human. However, its importance has not been fully understood in cervical carcinoma. The aim of this study was to determine the expression of PMS2 in cervical carcinoma and evaluate the significance of mismatch repair gene PMS2 regulated by glycogen synthase kinase 3beta (GSK-3beta) in chemosensitivity. We examined PMS2 and phosphorylated GSK-3beta(s9) expression in cervical carcinoma tissues using immunohistochemical staining. Furthermore, we detected PMS2 expression in HeLa cells and evaluate the interaction with GSK-3beta after transfection with GSK-3beta by small interference RNA (siRNA), co-immunoprecipitation and immunoblotting. We also evaluated the effect of PMS2 transfection on HeLa cells' chemosensitivity to cisplatin treatment. We found significant downregulation of PMS2 in cervical carcinoma, which was negatively associated with phosphorylated GSK-3beta (s9). Furthermore, we demonstrated GSK-3beta transfection was able to interact with PMS2 and enhance PMS2 production in HeLa cells, and increased PMS2 production was responsible for enhanced chemosensitivity. Our results provide the evidence that stabilization of PMS2 production by GSK-3beta was important to improve chemosensitization, indicating the significance of GSK-3beta-related PMS2 downregulation in the development of cervical carcinoma and in developing a potential strategy for chemotherapy.

Lack of the central nervous system- and neural crest-expressed forkhead gene Foxs1 affects motor function and body weight.

Science.gov (United States)

Heglind, Mikael; Cederberg, Anna; Aquino, Jorge; Lucas, Guilherme; Ernfors, Patrik; Enerbäck, Sven

2005-07-01

To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-beta-galactosidase reporter gene "knock-in" (Foxs1beta-gal/beta-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a beta-galactosidase reporter gene. Staining for beta-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1beta-gal/beta-gal mice perform significantly better (P fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1beta-gal/beta-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.

Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

Directory of Open Access Journals (Sweden)

Parra Edwin R

2010-01-01

Full Text Available Abstract Background The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods Female New Zealand rabbits (N = 12 were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM. After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day (IM-TOL daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p Results IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p p p = 0.026. The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002 and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009 collagen, in addition to decreased TGF-beta expression (p Conclusions Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

Reduced Expression of the Liver/Beta-Cell Glucose Transporter Isoform in Glucose-Insensitive Pancreatic Beta Cells of Diabetic Rats

Science.gov (United States)

Thorens, Bernard; Weir, Gordon C.; Leahy, John L.; Lodish, Harvey F.; Bonner-Weir, Susan

1990-09-01

Rats injected with a single dose of streptozocin at 2 days of age develop non-insulin-dependent diabetes 6 weeks later. The pancreatic beta islet cells of these diabetic rats display a loss of glucose-induced insulin secretion while maintaining sensitivity to other secretagogues such as arginine. We analyzed the level of expression of the liver/beta-cell glucose transporter isoform in diabetic islets by immunofluorescence staining of pancreas sections and by Western blotting of islet lysates. Islets from diabetic animals have a reduced expression of this beta-cell-specific glucose transporter isoform and the extent of reduction is correlated with the severity of hyperglycemia. In contrast, expression of this transporter isoform in liver is minimally modified by the diabetes. Thus a decreased expression of the liver/beta-cell glucose transporter isoform in beta cells is associated with the impaired glucose sensing characteristic of diabetic islets; our data suggest that this glucose transporter may be part of the beta-cell glucose sensor.

Changes in skeletal muscle gene expression following clenbuterol administration

Directory of Open Access Journals (Sweden)

McIntyre Lauren M

2006-12-01

Full Text Available Abstract Background Beta-adrenergic receptor agonists (BA induce skeletal muscle hypertrophy, yet specific mechanisms that lead to this effect are not well understood. The objective of this research was to identify novel genes and physiological pathways that potentially facilitate BA induced skeletal muscle growth. The Affymetrix platform was utilized to identify gene expression changes in mouse skeletal muscle 24 hours and 10 days after administration of the BA clenbuterol. Results Administration of clenbuterol stimulated anabolic activity, as indicated by decreased blood urea nitrogen (BUN; P P Conclusion Global evaluation of gene expression after administration of clenbuterol identified changes in gene expression and overrepresented functional categories of genes that may regulate BA-induced muscle hypertrophy. Changes in mRNA abundance of multiple genes associated with myogenic differentiation may indicate an important effect of BA on proliferation, differentiation, and/or recruitment of satellite cells into muscle fibers to promote muscle hypertrophy. Increased mRNA abundance of genes involved in the initiation of translation suggests that increased levels of protein synthesis often associated with BA administration may result from a general up-regulation of translational initiators. Additionally, numerous other genes and physiological pathways were identified that will be important targets for further investigations of the hypertrophic effect of BA on skeletal muscle.

Functional analysis of multiple carotenogenic genes from Lycium barbarum and Gentiana lutea L. for their effects on beta-carotene production in transgenic tobacco.

Science.gov (United States)

Ji, Jing; Wang, Gang; Wang, Jiehua; Wang, Ping

2009-02-01

Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are especially abundant in yellow-orange fruits and vegetables and dark green leafy vegetables. Carotenoids are essential for photosynthesis and photoprotection in plant life and also have different beneficial effects in humans and animals (van den Berg et al. 2000). For example, beta-carotene plays an essential role as the main dietary source of vitamin A. To obtain further insight into beta-carotene biosynthesis in two important economic plant species, Lycium barbarum and Gentiana lutea L., and to investigate and prioritize potential genetic engineering targets in the pathway, the effects of five carotenogenic genes from these two species, encoding proteins including geranylgeranyl diphosphate synthase, phytoene synthase and delta-carotene desaturase gene, lycopene beta-cyclase, lycopene epsilon-cyclase were functionally analyzed in transgenic tobacco (Nicotiana tabacum) plants. All transgenic tobacco plants constitutively expressing these genes showed enhanced beta-carotene contents in their leaves and flowers to different extents. The addictive effects of co-ordinate expression of double transgenes have also been investigated.

CLONING, SEQUENCE ANALYSIS, AND CHARACTERIZATION OF PUTATIVE BETA-LACTAMASE OF STENOTROPHOMONAS MALTOPHILIA

Directory of Open Access Journals (Sweden)

Chong Seng Shueh

2012-10-01

Full Text Available The main objective of current study was to explore the function of chromosomal putative beta-lactamase gene (smlt 0115 in clinical Stenotrophomonas maltophilia. Antibiotic susceptibility test (AST screening for current antimicrobial drugs was done and Minimum Inhibitory Concentration (MIC level towards beta-lactams was determined by E-test. Putative beta-lactamase gene of S. maltophilia was amplified via PCR, with specific primers, then cloned into pET-15 expression plasmid and transformed into Escherichia coli BL21. The gene was sequenced and analyzed. The expressed protein was purified by affinity chromatography and the kinetic assay was performed. S. maltophilia ATCC 13637 was included in this experiment. Besides, a hospital strain which exhibited resistant to a series of beta-lactams including cefepime was identified via AST and MIC, hence it was named as S2 strain and was considered in this study. Sequencing result showed that putative beta-lactamase gene obtained from ATCC 13637 and S2 strains were predicted to have cephalosporinase activity by National Center for Biotechnology Information (NCBI blast program. Differences in the sequences of both ATCC 13637 and S2 strains were found via ClustalW alignment software. Kinetic assay proved a cephalosporinase characteristic produced by E. coli BL21 clone that overexpressed the putative beta-lactamase gene cloned under the control of an external promoter. Yet, expressed protein purified from S2 strain had high catalytic activity against beta-lactam antibiotics which was 14-fold higher than expressed protein purified from ATCC 13637 strain. This study represents the characterization analysis of putative beta-lactamase gene (smlt 0115 of S. maltophilia. The presence of the respective gene in the chromosome of S. maltophilia suggested that putative beta-lactamase gene (smlt 0115 of S. maltophilia plays a role in beta-lactamase resistance.

Effects of 2G on Gene Expression of Stress-Related Hormones in Rat Placenta

Science.gov (United States)

Benson, S.; Talyansky, Y.; Moyer, E. L.; Lowe, M.; Baer, L. A.; Ronca, A. E.

2017-01-01

Understanding the effects of spaceflight on mammalian reproductive and developmental physiology is important to future human space exploration and permanent settlement beyond Earth orbit. Fetal developmental programming, including modulation of the HPA axis, is thought to originate at the placental-uterine interface, where both transfer of maternal hormones to the fetus and synthesis of endogenous hormones occurs. In healthy rats, fetal corticosterone levels are kept significantly lower by 11BetaHSD-2, which inactivates corticosterone by conversion into cortisone. Placental tissues express endogenous HPA axis-associated hormones including corticotropin-releasing hormone (CRH), pre-opiomelanocortin (POMC), and vasopressin, which may contribute to fetal programming alongside maternal hormones. DNA methylase 3A, 11BetaHSD-2, and 11BetaHSD-1, which are involved in the regulation of maternal cortisol transfer and modulation of the HPA axis, are also expressed in placental tissues along with glucocorticoid receptor and may be affected by differential gravity exposure during pregnancy. Fetuses may respond differently to maternal glucocorticoid exposure during gestation through sexually dimorphic expression of corticosterone-modulating hormones. To elucidate effects of altered gravity on placental gene expression, here we present a ground-based analogue study involving continuous centrifugation to produce 2g hypergravity. We hypothesized that exposure to 2g would induce a decrease in 11BetaHSD-2 expression through the downregulation of DNA methylase 3a and GC receptor, along with concurrent upregulation in endogenous CRH, POMC, and vasopressin expression. Timed pregnant female rats were exposed to 2G from Gestational day 6 to Gestational day 20, and comparisons made with Stationary Control (SC) and Vivarium Control (VC) dams at 1G. Dams were euthanized and placentas harvested on G20. We homogenized placental tissues, extracted and purified RNA, synthesized cDNA, and

Signaling pathway-focused gene expression profiling in pressure overloaded hearts

Directory of Open Access Journals (Sweden)

Marco Musumeci

2011-01-01

Full Text Available The β-blocker propranolol displays antihypertrophic and antifibrotic properties in the heart subjected to pressure overload. Yet the underlying mechanisms responsible for these important effects remain to be completely understood. The purpose of this study was to determine signaling pathway-focused gene expression profile associated with the antihypertrophic action of propranolol in pressure overloaded hearts. To address this question, a focused real-time PCR array was used to screen left ventricular RNA expression of 84 gene transcripts representative of 18 different signaling pathways in C57BL/6 mice subjected to transverse aortic constriction (TAC or sham surgery. On the surgery day, mice received either propranolol (80 mg/kg/day or vehicle for 14 days. TAC caused a 49% increase in the left ventricular weight-to-body weight (LVW/BW ratio without changing gene expression. Propranolol blunted LVW/BW ratio increase by approximately 50% while causing about a 3-fold increase in the expression of two genes, namely Brca1 and Cdkn2a, belonging to the TGF-beta and estrogen pathways, respectively. In conclusion, after 2 weeks of pressure overload, TAC hearts show a gene expression profile superimposable to that of sham hearts. Conversely, propranolol treatment is associated with an increased expression of genes which negatively regulate cell cycle progression. It remains to be established whether a mechanistic link between gene expression changes and the antihypertrophic action of propranolol occurs.

Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming {beta}-cyano-L-alanine

Energy Technology Data Exchange (ETDEWEB)

Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru [Gifu Univ. (Japan). Dept. of Biomolecular Science; Kuroda, Masako [Ikeda Food Research Co., Ltd., Fukuyama, Hiroshima (Japan); Kobayashi, Michihiko; Shimizu, Sakayu [Kyoto Univ. (Japan). Agricultural Sciences

2003-10-01

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable {beta}-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of {beta}-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various {beta}-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the {beta}-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the {beta}-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed {beta}-cyano-L-alanine synthase. Heat stable {beta}-cyano-L-alanine synthase can be applied to the synthesis of [4-{sup 11}C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

TGF-beta1 expression in EL4 lymphoma cells overexpressing growth hormone.

Science.gov (United States)

Farmer, John T; Weigent, Douglas A

2006-03-01

Our previous studies show that growth hormone overexpression (GHo) upregulates the expression of the IGF-1R and IGF-2R resulting in the protection of the EL4 lymphoma cell line from apoptosis. In this study, we report that GHo also increases TGF-beta1 protein expression measured by luciferase promoter assay, Western analysis, and ELISA. Further, the data show that antibody to TGF-betaR2 decreases TGF-beta1 promoter activity to the level of vector alone control cells. GHo cells treated with (125)I-rh-latent TGF-beta1 showed increased activation of latent TGF-beta1 as measured by an increase in the active 24kDa, TGF-beta1 compared to vector alone control cells. The ability of endogenous GH to increase TGF-beta1 expression is blocked in EL4 cells by antisense but not sense oligodeoxynucleotides or in cells cultured with antibody to growth hormone (GH). The data suggest that endogenous GH may protect from apoptosis through the IGF-1R receptor while limiting cellular growth through increased expression and activation of TGF-beta1.

Transcriptome Analysis of Beta macrocarpa and Identification of Differentially Expressed Transcripts in Response to Beet Necrotic Yellow Vein Virus Infection.

Directory of Open Access Journals (Sweden)

Huiyan Fan

Full Text Available Rhizomania is one of the most devastating diseases of sugar beet. It is caused by Beet necrotic yellow vein virus (BNYVV transmitted by the obligate root-infecting parasite Polymyxa betae. Beta macrocarpa, a wild beet species widely used as a systemic host in the laboratory, can be rub-inoculated with BNYVV to avoid variation associated with the presence of the vector P. betae. To better understand disease and resistance between beets and BNYVV, we characterized the transcriptome of B. macrocarpa and analyzed global gene expression of B. macrocarpa in response to BNYVV infection using the Illumina sequencing platform.The overall de novo assembly of cDNA sequence data generated 75,917 unigenes, with an average length of 1054 bp. Based on a BLASTX search (E-value ≤ 10-5 against the non-redundant (NR, NCBI protein, Swiss-Prot, the Gene Ontology (GO, Clusters of Orthologous Groups of proteins (COG and Kyoto Encyclopedia of Genes and Genomes (KEGG databases, there were 39,372 unigenes annotated. In addition, 4,834 simple sequence repeats (SSRs were also predicted, which could serve as a foundation for various applications in beet breeding. Furthermore, comparative analysis of the two transcriptomes revealed that 261 genes were differentially expressed in infected compared to control plants, including 128 up- and 133 down-regulated genes. GO analysis showed that the changes in the differently expressed genes were mainly enrichment in response to biotic stimulus and primary metabolic process.Our results not only provide a rich genomic resource for beets, but also benefit research into the molecular mechanisms of beet- BNYV Vinteraction.

Enhanced cortisol production rates, free cortisol, and 11beta-HSD-1 expression correlate with visceral fat and insulin resistance in men: effect of weight loss.

Science.gov (United States)

Purnell, Jonathan Q; Kahn, Steven E; Samuels, Mary H; Brandon, David; Loriaux, D Lynn; Brunzell, John D

2009-02-01

Controversy exists as to whether endogenous cortisol production is associated with visceral obesity and insulin resistance in humans. We therefore quantified cortisol production and clearance rates, abdominal fat depots, insulin sensitivity, and adipocyte gene expression in a cohort of 24 men. To test whether the relationships found are a consequence rather than a cause of obesity, eight men from this larger group were studied before and after weight loss. Daily cortisol production rates (CPR), free cortisol levels (FC), and metabolic clearance rates (MCR) were measured by stable isotope methodology and 24-h sampling; intra-abdominal fat (IAF) and subcutaneous fat (SQF) by computed tomography; insulin sensitivity (S(I)) by frequently sampled intravenous glucose tolerance test; and adipocyte 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1) gene expression by quantitative RT-PCR from subcutaneous biopsies. Increased CPR and FC correlated with increased IAF, but not SQF, and with decreased S(I). Increased 11beta-HSD-1 gene expression correlated with both IAF and SQF and with decreased S(I). With weight loss, CPR, FC, and MCR did not change compared with baseline; however, with greater loss in body fat than lean mass during weight loss, both CPR and FC increased proportionally to final fat mass and IAF and 11beta-HSD-1 decreased compared with baseline. These data support a model in which increased hypothalamic-pituitary-adrenal activity in men promotes selective visceral fat accumulation and insulin resistance and may promote weight regain after diet-induced weight loss, whereas 11beta-HSD-1 gene expression in SQF is a consequence rather than cause of adiposity.

Ins1 Cre knock-in mice for beta cell-specific gene recombination

OpenAIRE

Thorens Bernard; Tarussio David; Maestro Miguel Angel; Maestro Miguel Angel; Rovira Meritxell; Rovira Meritxell; Heikkilä Eija; Ferrer Jorge; Ferrer Jorge; Ferrer Jorge

2013-01-01

Aims/hypothesis Pancreatic beta cells play a central role in the control of glucose homeostasis by secreting insulin to stimulate glucose uptake by peripheral tissues. Understanding the molecular mechanisms that control beta cell function and plasticity has critical implications for the pathophysiology and therapy of major forms of diabetes. Selective gene inactivation in pancreatic beta cells, using the Cre-lox system, is a powerful approach to assess the role of particular genes in beta cel...

Effect of homeopathic treatment on gene expression in Copenhagen rat tumor tissues.

Science.gov (United States)

Thangapazham, Rajesh L; Rajeshkumar, N V; Sharma, Anuj; Warren, Jim; Singh, Anoop K; Ives, John A; Gaddipati, Jaya P; Maheshwari, Radha K; Jonas, Wayne B

2006-12-01

Increasing evidence suggests that the inability to undergo apoptosis is an important factor in the development and progression of prostate cancer. Agents that induce apoptosis may inhibit tumor growth and provide therapeutic benefit. In a recent study, the authors found that certain homeopathic treatments produced anticancer effects in an animal model. In this study, the authors examined the immunomodulating and apoptotic effects of these remedies. The authors investigated the effect of a homeopathic treatment regimen containing Conium maculatum, Sabal serrulata, Thuja occidentalis, and a MAT-LyLu Carcinosin nosode on the expression of cytokines and genes that regulate apoptosis. This was assessed in prostate cancer tissues, extracted from animals responsive to these drugs, using ribonuclease protection assay or reverse transcription polymerase chain reaction. There were no significant changes in mRNA levels of the apoptotic genes bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, FasL, or the cytokines interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-beta, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-alpha, IL-2, and interferon-gamma in prostate tumor and lung metastasis after treatment with homeopathic medicines. This study indicates that treatment with the highly diluted homeopathic remedies does not alter the gene expression in primary prostate tumors or in lung metastasis. The therapeutic effect of homeopathic treatments observed in the in vivo experiments cannot be explained by mechanisms based on distinct alterations in gene expression related to apoptosis or cytokines. Future research should explore subtle modulations in the expression of multiple genes in different biological pathways.

Gene expression profiling in respond to TBT exposure in small abalone Haliotis diversicolor.

Science.gov (United States)

Jia, Xiwei; Zou, Zhihua; Wang, Guodong; Wang, Shuhong; Wang, Yilei; Zhang, Ziping

2011-10-01

In this study, we investigated the gene expression profiling of small abalone, Haliotis diversicolor by tributyltin (TBT) exposure using a cDNA microarray containing 2473 unique transcripts. Totally, 107 up-regulated genes and 41 down-regulated genes were found. For further investigation of candidate genes from microarray data and EST analysis, quantitative real-time PCR was performed at 6 h, 24 h, 48 h, 96 h and 192 h TBT exposure. 26 genes were found to be significantly differentially expressed in different time course, 3 of them were unknown. Some gene homologues like cellulose, endo-beta-1,4-glucanase, ferritin subunit 1 and thiolester containing protein II CG7052-PB might be the good biomarker candidate for TBT monitor. The identification of stress response genes and their expression profiles will permit detailed investigation of the defense responses of small abalone genes. Published by Elsevier Ltd.

Phospholipase C-{delta}{sub 1} regulates interleukin-1{beta} and tumor necrosis factor-{alpha} mRNA expression

Energy Technology Data Exchange (ETDEWEB)

Chung, Eric; Jakinovich, Paul; Bae, Aekyung [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States); Rebecchi, Mario, E-mail: Mario.rebecchi@SBUmed.org [Department of Anesthesiology, Health Sciences Center L4 Rm 081, Stony Brook University, Stony Brook, NY 11794 (United States)

2012-10-01

Phospholipase C-{delta}{sub 1} (PLC{delta}{sub 1}) is a widely expressed highly active PLC isoform, modulated by Ca{sup 2+} that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLC{delta}{sub 1} modulated expression of the pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}), tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLC{delta}{sub 1} was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLC{delta}{sub 1} knockdown enhanced expression IL-1{beta} and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLC{delta}{sub 1} knock down caused persistently high Nf{kappa}b levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLC{delta}{sub 1} knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1{beta} and TNF-{alpha} mRNA's induced by PLC{delta}{sub 1} knockdown. Our results show that loss of PLC{delta}{sub 1} enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nf{kappa}b pathway. Our findings are consistent with the idea that PLC{delta}{sub 1} is a

Gene expression in the lignin biosynthesis pathway during soybean seed development.

Science.gov (United States)

Baldoni, A; Von Pinho, E V R; Fernandes, J S; Abreu, V M; Carvalho, M L M

2013-02-28

The study of gene expression in plants is fundamental, and understanding the molecular mechanisms involved in important biological processes, such as biochemical pathways or signaling that are used or manipulated in improvement programs, are key for the production of high-quality soybean seeds. Reports related to gene expression of lignin in seeds are scarce in the literature. We studied the expression of the phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase, 4-hydroxycinnamate 3-hydroxylase, and cinnamyl alcohol dehydrogenase genes involved in lignin biosynthesis during the development of soybean (Glycine max L. Merrill) seeds. As the endogenous control, the eukaryotic elongation factor 1-beta gene was used in two biological replicates performed in triplicate. Relative quantitative expression of these genes during the R4, R5, R6, and R7 development stages was analyzed. Real-time polymerase chain reaction was used for the gene expression study. The analyses were carried out in an ABI PRISM 7500 thermocycler using the comparative Ct method and SYBR Green to detect amplification. The seed samples at the R4 stage were chosen as calibrators. Increased expression of the cinnamate-4-hydroxylase and PAL genes occurred in soybean seeds at the R5 and R6 development stages. The cinnamyl alcohol dehydrogenase gene was expressed during the final development phases of soybean seeds. In low-lignin soybean cultivars, the higher expression of the PAL gene occurs at development stages R6 and R7. Activation of the genes involved in the lignin biosynthesis pathway occurs at the beginning of soybean seed development.

Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

Science.gov (United States)

Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C

2009-06-01

Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P cancer group (P prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.

Expression and functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

DEFF Research Database (Denmark)

Andreasen, Susanne Ø; Thomsen, Allan R; Koteliansky, Victor E

2003-01-01

decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect......Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using...... this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1...

Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

DEFF Research Database (Denmark)

Fromont-Racine, M; Bucchini, D; Madsen, O

1990-01-01

Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific...... of the transgene was observed in cell types other than beta-islet cells....

Characterization of differential gene expression in adrenocortical tumors harboring beta-catenin (CTNNB1) mutations.

Science.gov (United States)

Durand, Julien; Lampron, Antoine; Mazzuco, Tania L; Chapman, Audrey; Bourdeau, Isabelle

2011-07-01

Mutations of β-catenin gene (CTNNB1) are frequent in adrenocortical adenomas (AA) and adrenocortical carcinomas (ACC). However, the target genes of β-catenin have not yet been identified in adrenocortical tumors. Our objective was to identify genes deregulated in adrenocortical tumors harboring CTNNB1 genetic alterations and nuclear accumulation of β-catenin. Microarray analysis identified a dataset of genes that were differently expressed between AA with CTNNB1 mutations and wild-type (WT) tumors. Within this dataset, the expression profiles of five genes were validated by real time-PCR (RT-PCR) in a cohort of 34 adrenocortical tissues (six AA and one ACC with CTNNB1 mutations, 13 AA and four ACC with WT CTNNB1, and 10 normal adrenal glands) and two human ACC cell lines. We then studied the effects of suppressing β-catenin transcriptional activity with the T-cell factor/β-catenin inhibitors PKF115-584 and PNU74654 on gene expression in H295R and SW13 cells. RT-PCR analysis confirmed the overexpression of ISM1, RALBP1, and PDE2A and the down-regulation of PHYHIP in five of six AA harboring CTNNB1 mutations compared with WT AA (n = 13) and normal adrenal glands (n = 10). RALBP1 and PDE2A overexpression was also confirmed at the protein level by Western blotting analysis in mutated tumors. ENC1 was specifically overexpressed in three of three AA harboring CTNNB1 point mutations. mRNA expression and protein levels of RALBP1, PDE2A, and ENC1 were decreased in a dose-dependent manner in H295R cells after treatment with PKF115-584 or PNU74654. This study identified candidate genes deregulated in CTNNB1-mutated adrenocortical tumors that may lead to a better understanding of the role of the Wnt-β-catenin pathway in adrenocortical tumorigenesis.

No Effect of the Transforming Growth Factor {beta}1 Promoter Polymorphism C-509T on TGFB1 Gene Expression, Protein Secretion, or Cellular Radiosensitivity

Energy Technology Data Exchange (ETDEWEB)

Reuther, Sebastian; Metzke, Elisabeth [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Bonin, Michael [Department of Medical Genetics, University of Tuebingen (Germany); Petersen, Cordula [Clinic of Radiotherapy and Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Dikomey, Ekkehard, E-mail: dikomey@uke.de [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany); Raabe, Annette [Laboratory of Radiobiology and Experimental Radiooncology, University Hospital Hamburg-Eppendorf, Hamburg (Germany)

2013-02-01

Purpose: To study whether the promoter polymorphism (C-509T) affects transforming growth factor {beta}1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. Methods and Materials: Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. Results: The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). Conclusion: Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

Science.gov (United States)

Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

2009-08-15

To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

Directory of Open Access Journals (Sweden)

Cunyou Zhao

Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

Science.gov (United States)

Kholmanskikh, Olga; van Baren, Nicolas; Brasseur, Francis; Ottaviani, Sabrina; Vanacker, Julie; Arts, Nathalie; van der Bruggen, Pierre; Coulie, Pierre; De Plaen, Etienne

2010-10-01

We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1beta. This effect is NF-kappaB and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

Extracellular Matrix-Regulated Gene Expression RequiresCooperation of SWI/SNF and Transcription Factors

Energy Technology Data Exchange (ETDEWEB)

Xu, Ren; Spencer, Virginia A.; Bissell, Mina J.

2006-05-25

Extracellular cues play crucial roles in the transcriptional regulation of tissue-specific genes, but whether and how these signals lead to chromatin remodeling is not understood and subject to debate. Using chromatin immunoprecipitation (ChIP) assays and mammary-specific genes as models, we show here that extracellular matrix (ECM) molecules and prolactin cooperate to induce histone acetylation and binding of transcription factors and the SWI/SNF complex to the {beta}- and ?-casein promoters. Introduction of a dominant negative Brg1, an ATPase subunit of SWI/SNF complex, significantly reduced both {beta}- and ?-casein expression, suggesting that SWI/SNF-dependent chromatin remodeling is required for transcription of mammary-specific genes. ChIP analyses demonstrated that the ATPase activity of SWI/SNF is necessary for recruitment of RNA transcriptional machinery, but not for binding of transcription factors or for histone acetylation. Coimmunoprecipitation analyses showed that the SWI/SNF complex is associated with STAT5, C/EBP{beta}, and glucocorticoid receptor (GR). Thus, ECM- and prolactin-regulated transcription of the mammary-specific casein genes requires the concerted action of chromatin remodeling enzymes and transcription factors.

Gene Expression by PBMC in Primary Sclerosing Cholangitis: Evidence for Dysregulation of Immune Mediated Genes

Directory of Open Access Journals (Sweden)

Christopher A. Aoki

2006-01-01

Full Text Available Primary sclerosing cholangitis (PSC is a chronic disease of the bile ducts characterized by an inflammatory infiltrate and obliterative fibrosis. The precise role of the immune system in the pathogenesis of PSC remains unknown. We used RNA microarray analysis to identify immune-related genes and pathways that are differentially expressed in PSC. Messenger RNA (mRNA from peripheral blood mononuclear cells (PBMC was isolated from both patients with PSC and age and sex matched healthy controls. Samples from 5 PSC patients and 5 controls were analyzed by microarray and based upon rigorous statistical analysis of the data, relevant genes were chosen for confirmation by RT-PCR in 10 PSC patients and 10 controls. Using unsupervised hierarchical clustering, gene expression in PSC was statistically different from our control population. Interestingly, genes within the IL-2 receptor beta, IL-6 and MAP Kinase pathways were found to be differently expressed in patients with PSC compared to controls. Further, individual genes, TNF-α induced protein 6 (TNFaip6 and membrane-spanning 4-domains, subfamily A (ms4a were found to be upregulated in PSC while similar to Mothers against decapentaplegic homolog 5 (SMAD 5 was downregulated. In conclusion, several immune-related pathways and genes were differentially expressed in PSC compared to control patients, giving further evidence that this disease is systemic and immune-mediated.

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

Science.gov (United States)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

Energy Technology Data Exchange (ETDEWEB)

Kover, Karen, E-mail: kkover@cmh.edu [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States); Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States); Tasch, James; Hager, Melissa [Kansas City University Medical Biosciences, Kansas City, MO (United States); Clements, Mark; Moore, Wayne V. [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States)

2015-06-19

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

International Nuclear Information System (INIS)

Kover, Karen; Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu; Tasch, James; Hager, Melissa; Clements, Mark; Moore, Wayne V.

2015-01-01

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H 2 O 2 assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H 2 O 2 levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose-induced TXNIP

Mesenchymal stem cells maintain TGF-beta-mediated chondrogenic phenotype in alginate bead culture

DEFF Research Database (Denmark)

Mehlhorn, A T; Schmal, H; Kaiser, S

2006-01-01

cultured in osteogenic medium after TGF-beta-mediated chondroinduction. Gene expression of col2a1, aggrecan, COMP, alkaline phosphatase (AP), and correlating protein synthesis was analyzed. After short-term stimulation with TGF-beta, MSCs maintained a chondrogenic phenotype. Chondrogenic gene expression...

Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells

Science.gov (United States)

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W.; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S.; Troncoso, Patricia; Maity, Sankar N.; Navone, Nora M.

2012-01-01

Purpose To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction. Experimental Design We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs. Results Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling. Conclusion We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients. PMID:22298898

Molecular characterization of four beta-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica.

Science.gov (United States)

Yamamoto, E; Baird, W V

1999-01-01

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that alpha-tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297-308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four beta-tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four beta-tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four beta-tubulins was highest in inflorescences. This is in contrast to alpha-tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that beta-tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four beta-tubulins, together with three alpha-tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.

Estrogen induced {beta}-1,4-galactosyltransferase 1 expression regulates proliferation of human breast cancer MCF-7 cells

Energy Technology Data Exchange (ETDEWEB)

Choi, Hee-Jung [Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan (Korea, Republic of); Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan-city, Gyeongsangnam-do (Korea, Republic of); Chung, Tae-Wook; Kim, Cheorl-Ho [Department of Molecular and Cellular Glycobiology, College of Natural Science, Sungkyunkwan University, Suwon, Kyungki-do (Korea, Republic of); Jeong, Han-Sol; Joo, Myungsoo [Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan-city, Gyeongsangnam-do (Korea, Republic of); Youn, BuHyun, E-mail: bhyoun72@pusan.ac.kr [Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan (Korea, Republic of); Ha, Ki-Tae, E-mail: hagis@pusan.ac.kr [Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan-city, Gyeongsangnam-do (Korea, Republic of)

2012-10-05

Highlights: Black-Right-Pointing-Pointer We examined the regulation and biological functions of B4GALT1 expression induced by estrogen. Black-Right-Pointing-Pointer Estrogen-induced B4GALT1 expression through the direct binding of ER-{alpha} to ERE in MCF-7 cells. Black-Right-Pointing-Pointer B4GALT1 expression activates the proliferation of MCF-7 cells via its receptor function. Black-Right-Pointing-Pointer Thus, we suggest B4GALT1 as a molecular target for inhibiting breast cancer proliferation. -- Abstract: Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose {beta}-1,4-N-acetylglucosamine (Gal{beta}1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressed by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Gal{beta}1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-{alpha}-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells. Considering

Acetaminophen modulates the transcriptional response to recombinant interferon-beta.

Directory of Open Access Journals (Sweden)

Aaron Farnsworth

Full Text Available BACKGROUND: Recombinant interferon treatment can result in several common side effects including fever and injection-site pain. Patients are often advised to use acetaminophen or other over-the-counter pain medications as needed. Little is known regarding the transcriptional changes induced by such co-administration. METHODOLOGY/PRINCIPAL FINDINGS: We tested whether the administration of acetaminophen causes a change in the response normally induced by interferon-beta treatment. CD-1 mice were administered acetaminophen (APAP, interferon-beta (IFN-beta or a combination of IFN-beta+APAP and liver and serum samples were collected for analysis. Differential gene expression was determined using an Agilent 22 k whole mouse genome microarray. Data were analyzed by several methods including Gene Ontology term clustering and Gene Set Enrichment Analysis. We observed a significant change in the transcription profile of hepatic cells when APAP was co-administered with IFN-beta. These transcriptional changes included a marked up-regulation of genes involved in signal transduction and cell differentiation and down-regulation of genes involved in cellular metabolism, trafficking and the IkappaBK/NF-kappaB cascade. Additionally, we observed a large decrease in the expression of several IFN-induced genes including Ifit-3, Isg-15, Oasl1, Zbp1 and predicted gene EG634650 at both early and late time points. CONCLUSIONS/SIGNIFICANCE: A significant change in the transcriptional response was observed following co-administration of IFN-beta+APAP relative to IFN-beta treatment alone. These results suggest that administration of acetaminophen has the potential to modify the efficacy of IFN-beta treatment.

Interleukin-1beta gene polymorphisms in Taiwanese patients with gout.

Science.gov (United States)

Chen, Man-Ling; Huang, Chung-Ming; Tsai, Chang-Hai; Tsai, Fuu-Jen

2005-04-01

The purpose of this study was to examine whether interleukin-1 beta (IL-1beta) promoter and exon 5 gene polymorphisms are markers of susceptibility or clinical manifestations in Taiwanese patients with gout. The study included 196 patients in addition to 103 unrelated healthy control subjects living in central Taiwan. From genomic DNA, polymorphisms of the gene for IL-1beta promoter and IL-1beta exon 5 were typed. Allelic frequencies were compared between the two groups, and the relationship between allelic frequencies and clinical manifestations of gout was evaluated. No significant differences were observed in the allelic frequencies of the IL-1beta promoter between patients with gout and healthy control subjects. Additionally, we did not detect any association of the IL-1beta promoter genotype with the clinical and laboratory profiles of gout patients. However, there was a significant difference between the two groups in terms of hypertriglyceridemia (P=0.0004, chi(2)=12.52, OR 7.14, 95%CI 0.012-0.22). There was also a significant difference in the genotype of IL-1beta exon 5 polymorphism between patients with and without hypertriglyceridemia. Results of the present study suggest that polymorphisms of the IL-1beta promoter and IL-1beta exon 5 are not related to gout patients in central Taiwan.

Effects of Nebivolol on Endothelial Gene Expression during Oxidative Stress in Human Umbilical Vein Endothelial Cells

Directory of Open Access Journals (Sweden)

Ulisse Garbin

2008-01-01

Full Text Available The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L for 24 hours and oxidative stress was induced by the addition of oxidized (ox-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin; proteins linked to inflammation (IL-6 and TNFalpha, thrombotic state (tissue factor, PAI-1 and uPA, hypertension such as endothelin-1 (ET-1, and vascular remodeling such as metalloproteinases (MMP-2, MMP-9 and protease inhibitor (TIMP-1. The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.

Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.

Directory of Open Access Journals (Sweden)

Carlos F Solis

Full Text Available BACKGROUND: Modern RNA interference (RNAi methodologies using small interfering RNA (siRNA oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. PRINCIPAL FINDINGS: Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica beta-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. CONCLUSIONS: Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

Gene expression and gene therapy imaging

International Nuclear Information System (INIS)

Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

2007-01-01

The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

PARP-1 and YY1 are important novel regulators of CXCL12 gene transcription in rat pancreatic beta cells.

Directory of Open Access Journals (Sweden)

Jelena Marković

Full Text Available Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12 transcription. The roles of poly(ADP-ribose polymerase-1 (PARP-1 and transcription factor Yin Yang 1 (YY1 in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the

Upregulation of adipose 11-beta-hydroxysteroid dehydrogenase type 1 expression in ovariectomized rats is due to obesity rather than lack of estrogen.

Science.gov (United States)

Paulsen, Søren K; Nielsen, Maria P; Richelsen, Bjørn; Bruun, Jens M; Flyvbjerg, Allan; Pedersen, Steen B

2008-04-01

Increased tissue activity of cortisol induced by the activation of inert cortisone to active cortisol through 11-beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) may play a role in the metabolic syndrome. We recently found that 11beta-HSD1 in subcutaneous adipose tissue (AT) was lower in lean women compared with lean men. Estrogen suppresses hepatic and renal 11beta-HSD1 in rats; hence we investigated the in vitro effect of estrogen on human and rat AT, and the in vivo effects on rat AT 11beta-HSD1 expression. Wistar rats were divided into four groups of eight animals. One group was sham-operated (controls) and others were ovariectomized (OVX). One OVX group was left untreated (OVX-E), another (OVX+E) received estrogen treatment, and one received a hypo-caloric diet (OVX-E+D), matching the weight gain of the control group. AT from women undergoing liposuction or surgery and from killed male and female rats were incubated with estrogen alone or in the presence of IL-1beta. Gene expressions were determined by real-time reverse transcriptase PCR. Ovariectomy resulted in a 280% increase in adipose 11beta-HSD1 expression P < 0.05). 11beta-HSD1 expression in the (OVX+E)-group was significantly reduced compared with the nonsubstituted group (P < 0.05). 11beta-HSD1 expression in the (OVX-E+D)-group was reduced significantly (P < 0.05) when compared with the level of the estrogen-substituted group. No significant differences between the control group, the (OVX+E)-group, and the (OVX-E+D)-group were found. In the in vitro studies, no direct effect of estrogen on adipose 11beta-HSD1 was found. The upregulation of 11beta-HSD1 in ovariectomized rats was most likely due to changes in body composition rather than lack of estrogen.

Structural characterization and expression analysis of a beta-thymosin homologue (Tβ) in disk abalone, Haliotis discus discus.

Science.gov (United States)

Kasthuri, Saranya Revathy; Premachandra, H K A; Umasuthan, Navaneethaiyer; Whang, Ilson; Lee, Jehee

2013-09-15

Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus. The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon-2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates. Copyright © 2013 Elsevier B.V. All rights reserved.

Analysis of meniscal degeneration and meniscal gene expression

Directory of Open Access Journals (Sweden)

Norton James H

2010-01-01

Full Text Available Abstract Background Menisci play a vital role in load transmission, shock absorption and joint stability. There is increasing evidence suggesting that OA menisci may not merely be bystanders in the disease process of OA. This study sought: 1 to determine the prevalence of meniscal degeneration in OA patients, and 2 to examine gene expression in OA meniscal cells compared to normal meniscal cells. Methods Studies were approved by our human subjects Institutional Review Board. Menisci and articular cartilage were collected during joint replacement surgery for OA patients and lower limb amputation surgery for osteosarcoma patients (normal control specimens, and graded. Meniscal cells were prepared from these meniscal tissues and expanded in monolayer culture. Differential gene expression in OA meniscal cells and normal meniscal cells was examined using Affymetrix microarray and real time RT-PCR. Results The grades of meniscal degeneration correlated with the grades of articular cartilage degeneration (r = 0.672; P HLA-DPA1, integrin, beta 2 (ITGB2, ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1, ankylosis, progressive homolog (ANKH and fibroblast growth factor 7 (FGF7, were expressed at significantly higher levels in OA meniscal cells compared to normal meniscal cells. Importantly, many of the genes that have been shown to be differentially expressed in other OA cell types/tissues, including ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5 and prostaglandin E synthase (PTGES, were found to be expressed at significantly higher levels in OA meniscal cells. This consistency suggests that many of the genes detected in our study are disease-specific. Conclusion Our findings suggest that OA is a whole joint disease. Meniscal cells may play an active role in the development of OA. Investigation of the gene expression profiles of OA meniscal cells may reveal new therapeutic targets for OA therapy and also may uncover novel

Ginger extract mitigates ethanol-induced changes of alpha and beta - myosin heavy chain isoforms gene expression and oxidative stress in the heart of male wistar rats.

Science.gov (United States)

Shirpoor, Alireza; Zerehpoosh, Mitra; Ansari, Mohammad Hasan Khadem; Kheradmand, Fatemeh; Rasmi, Yousef

2017-09-01

The association between ethanol consumption and heart abnormalities, such as chamber dilation, myocyte damage, ventricular hypertrophy, and hypertension is well known. However, underlying molecular mediators involved in ethanol-induced heart abnormalities remain elusive. The aim of this study was to investigate the effect of chronic ethanol exposure on alpha and beta - myosin heavy chain (MHC) isoforms gene expression transition and oxidative stress in rats' heart. It was also planned to find out whether ginger extract mitigated the abnormalities induced by ethanol in rats' heart. Male wistar rats were divided into three groups of eight animals as follows: Control, ethanol, and ginger extract treated ethanolic (GETE) groups. After six weeks of treatment, the results revealed a significant increase in the β-MHC gene expression, 8- OHdG amount, and NADPH oxidase level. Furthermore, a significant decrease in the ratio of α-MHC/β-MHC gene expression to the amount of paraoxonase enzyme in the ethanol group compared to the control group was found. The consumption of Ginger extract along with ethanol ameliorated the changes in MHC isoforms gene expression and reduced the elevated amount of 8-OHdG and NADPH oxidase. Moreover, compared to the consumption of ethanol alone, it increased the paraoxonase level significantly. These findings indicate that ethanol-induced heart abnormalities may in part be associated with MHC isoforms changes mediated by oxidative stress, and that these effects can be alleviated by using ginger extract as an antioxidant molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

1995-12-01

Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

Estrogenic effect of soy isoflavones on mammary gland morphogenesis and gene expression profile

DEFF Research Database (Denmark)

Thomsen, Anni R.; Almstrup, Kristian; Nielsen, John E.

2006-01-01

We examined the effect of 17 beta-estradiol (E2) and soy isoflavones' exposure on morphogenesis and global gene expression in the murine mammary gland. Three exposure regimens were applied: isoflavones added to the diet throughout either the lactational period (via the dams) or the postweaning...... period and E2 administered orally during the lactational period. Whole mounts of mammary glands were evaluated both in juvenile and adult animals with respect to branching morphogenesis and terminal end bud (TEB) formation. At postnatal day (PND) 28, we observed a significant increase in branching...... isoflavone and E2 exposure was further substantiated by changes in gene expression, since the same groups of genes were up- and downregulated, particularly in the E2 and postweaning isoflavone regimen. All changes in gene expression correlated with changes in the cellular composition of the gland, i.e., more...

Identification of gene expression modifications in myostatin-stimulated myoblasts

International Nuclear Information System (INIS)

Yang Wei; Zhang Yong; Ma Guoda; Zhao Xinyi; Chen Yan; Zhu Dahai

2005-01-01

Myostatin belongs to the transforming growth factor beta superfamily and has been shown to function as an inhibitor of skeletal muscle proliferation and differentiation. To gain insight into the molecular mechanisms of myostatin function during myogenesis, differential display reverse transcription PCR was employed to identify altered gene expressions associated with myostatin inhibitory function in chicken fetal myoblasts (CFMs). In this work, we have identified seven up-regulated and 12 down-regulated genes in myostatin stimulated CFMs. Those genes are involved in myogenic differentiation, cell architecture, energy metabolism, signal transduction, and apoptosis. The down-regulation of muscle creatine kinase B, troponin C, and myosin regulatory light chain is in agreement with the myostatin negative role in myocyte differentiation. In addition, the expression alteration of skeletal muscle-specific cardiac ankyrin repeat protein and the bcl-2 related anti-apoptotic protein Nr-13 suggests possible unique roles for myostatin in regulating myogenesis by controlling cofactors participated transcriptional regulation and apoptosis

Molecular identification of TEM-116 beta-lactamase gene in isolates ...

African Journals Online (AJOL)

Purpose: Purpose: To determine TEM-116 beta-lactamase gene prevalence in drug-resistant Pseudomonas aeruginosa isolates from Pakistan. Methods: Sequence analysis of TEM beta-lactamase isolates and their antibiotic susceptibility patterns were carried out. Quantitative bacteriostatic concentrations for commonly ...

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation.

Science.gov (United States)

Fustiñana, Maria Sol; Ariel, Pablo; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

2010-09-01

Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

SATB1 tethers multiple gene loci to reprogram expression profiledriving breast cancer metastasis

Energy Technology Data Exchange (ETDEWEB)

Han, Hye-Jung; Kohwi, Yoshinori; Kohwi-Shigematsu, Terumi

2006-07-13

Global changes in gene expression occur during tumor progression, as indicated by expression profiling of metastatic tumors. How this occurs is poorly understood. SATB1 functions as a genome organizer by folding chromatin via tethering multiple genomic loci and recruiting chromatin remodeling enzymes to regulate chromatin structure and expression of a large number of genes. Here we show that SATB1 is expressed at high levels in aggressive breast cancer cells, and is undetectable in non-malignant breast epithelial cells. Importantly, RNAi-mediated removal of SATB1 from highly-aggressive MDA-MB-231 cells altered the expression levels of over 1200 genes, restored breast-like acinar polarity in three-dimensional cultures, and prevented the metastastic phenotype in vivo. Conversely, overexpression of SATB1 in the less-aggressive breast cancer cell line Hs578T altered the gene expression profile and increased metastasis dramatically in vivo. Thus, SATB1 is a global regulator of gene expression in breast cancer cells, directly regulating crucial metastasis-associated genes, including ERRB2 (HER2/NEU), TGF-{beta}1, matrix metalloproteinase 3, and metastasin. The identification of SATB1 as a protein that re-programs chromatin organization and transcription profiles to promote breast cancer metastasis suggests a new model for metastasis and may provide means of therapeutic intervention.

Age-Specific Gene Expression Profiles of Rhesus Monkey Ovaries Detected by Microarray Analysis

Directory of Open Access Journals (Sweden)

Hengxi Wei

2015-01-01

Full Text Available The biological function of human ovaries declines with age. To identify the potential molecular changes in ovarian aging, we performed genome-wide gene expression analysis by microarray of ovaries from young, middle-aged, and old rhesus monkeys. Microarray data was validated by quantitative real-time PCR. Results showed that a total of 503 (60 upregulated, 443 downregulated and 84 (downregulated genes were differentially expressed in old ovaries compared to young and middle-aged groups, respectively. No difference in gene expression was found between middle-aged and young groups. Differentially expressed genes were mainly enriched in cell and organelle, cellular and physiological process, binding, and catalytic activity. These genes were primarily associated with KEGG pathways of cell cycle, DNA replication and repair, oocyte meiosis and maturation, MAPK, TGF-beta, and p53 signaling pathway. Genes upregulated were involved in aging, defense response, oxidation reduction, and negative regulation of cellular process; genes downregulated have functions in reproduction, cell cycle, DNA and RNA process, macromolecular complex assembly, and positive regulation of macromolecule metabolic process. These findings show that monkey ovary undergoes substantial change in global transcription with age. Gene expression profiles are useful in understanding the mechanisms underlying ovarian aging and age-associated infertility in primates.

Gene expression

International Nuclear Information System (INIS)

Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

1983-01-01

We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

Neurotensin receptor 1 gene activation by the Tcf/beta-catenin pathway is an early event in human colonic adenomas.

Science.gov (United States)

Souazé, Frédérique; Viardot-Foucault, Véronique; Roullet, Nicolas; Toy-Miou-Leong, Mireille; Gompel, Anne; Bruyneel, Erik; Comperat, Eva; Faux, Maree C; Mareel, Marc; Rostène, William; Fléjou, Jean-François; Gespach, Christian; Forgez, Patricia

2006-04-01

Alterations in the Wnt/APC (adenomatous polyposis coli) signalling pathway, resulting in beta-catenin/T cell factor (Tcf)-dependent transcriptional gene activation, are frequently detected in familial and sporadic colon cancers. The neuropeptide neurotensin (NT) is widely distributed in the gastrointestinal tract. Its proliferative and survival effects are mediated by a G-protein coupled receptor, the NT1 receptor. NT1 receptor is not expressed in normal colon epithelial cells, but is over expressed in a number of cancer cells and tissues suggesting a link to the outgrowth of human colon cancer. Our results demonstrate that the upregulation of NT1 receptor occurring in colon cancer is the result of Wnt/APC signalling pathway activation. We first established the functionality of the Tcf response element within the NT1 receptor promoter. Consequently, we observed the activation of NT1 receptor gene by agents causing beta-catenin cytosolic accumulation, as well as a strong decline of endogenous receptor when wt-APC was restored. At the cellular level, the re-establishment of wt-APC phenotype resulted in the impaired functionality of NT1 receptor, like the breakdown in NT-induced intracellular calcium mobilization and the loss of NT pro-invasive effect. We corroborated the Wnt/APC signalling pathway on the NT1 receptor promoter activation with human colon carcinogenesis, and showed that NT1 receptor gene activation was perfectly correlated with nuclear or cytoplasmic beta-catenin localization while NT1 receptor was absent when beta-catenin was localized at the cell-cell junction in early adenomas of patients with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer and loss of heterozygosity tumours. In this report we establish a novel link in vitro between the Tcf/beta-catenin pathway and NT1 receptor promoter activation.

Characterization of arrangement and expression of the beta-2 microglobulin locus in the sandbar and nurse shark.

Science.gov (United States)

Chen, Hao; Kshirsagar, Sarika; Jensen, Ingvill; Lau, Kevin; Simonson, Caitlin; Schluter, Samuel F

2010-02-01

Beta 2 microglobulin (beta2m) is an essential subunit of major histocompatibility complex (MHC) type I molecules. In this report, beta2m cDNAs were identified and sequenced from sandbar shark spleen cDNA library. Sandbar shark beta2m gene encodes one amino acid less than most teleost beta2m genes, and 3 amino acids less than mammal beta2m genes. Although sandbar shark beta2m protein contains one beta sheet less than that of human in the predicted protein structure, the overall structure of beta2m proteins is conserved during evolution. Germline gene for the beta2m in sandbar and nurse shark is present as a single locus. It contains three exons and two introns. CpG sites are evenly distributed in the shark beta2m loci. Several DNA repeat elements were also identified in the shark beta2m loci. Sequence analysis suggests that the beta2m locus is not linked to the MHC I loci in the shark genome.

Defects in rhizobial cyclic glucan and lipopolysaccharide synthesis alter legume gene expression during nodule development

DEFF Research Database (Denmark)

D'Antuono, Alejandra L; Ott, Thomas; Krusell, Lene

2008-01-01

cDNA array technology was used to compare transcriptome profiles of Lotus japonicus roots inoculated with a Mesorhizobium loti wild-type and two mutant strains affected in cyclic beta(1-2) glucan synthesis (cgs) and in lipopolysaccharide synthesis (lpsbeta2). Expression of genes associated...... with the development of a fully functional nodule was significantly affected in plants inoculated with the cgs mutant. Array results also revealed that induction of marker genes for nodule development was delayed when plants were inoculated with the lpsbeta2 mutant. Quantitative real-time reverse......-transcriptase polymerase chain reaction was used to quantify gene expression of a subset of genes involved in plant defense response, redox metabolism, or genes that encode for nodulins. The majority of the genes analyzed in this study were more highly expressed in roots inoculated with the wild type compared with those...

DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells.

Science.gov (United States)

Singh, Kamaleshwar P; Treas, Justin; Tyagi, Tulika; Gao, Weimin

2012-03-01

Prolonged exposure to elevated levels of estrogen is a risk factor for breast cancer. Though increased cell growth and loss of DNA repair capacity is one of the proposed mechanisms for estrogen-induced cancers, the mechanism through which estrogen induces cell growth and decreases DNA repair capacity is not clear. DNA hypermethylation is known to inactivate DNA repair genes and apoptotic response in cancer cells. Therefore, the objective of this study was to determine the role of DNA hypermethylation in estrogen-induced cell growth and regulation of DNA repair genes expression in breast cancer cells. To achieve this objective, the estrogen-responsive MCF-7 cells either pretreated with 5-aza-2-deoxycytidine (5-aza-dC) or untreated (as control) were exposed to 17 beta-estradiol (E2), and its effect on cell growth and expression of DNA repair genes were measured. The result revealed that 5-aza-dC abrogates the E2-induced growth in MCF-7 cells. An increased expression of OGG1, MSH4, and MLH1 by 5-aza-dC treatment alone, suggest the DNA hypermethylation as a potential cause for decreased expression of these genes in MCF-7 cells. The decreased expression of ERCC1, XPC, OGG1, and MLH1 by E2 alone and its restoration by co-treatment with 5-aza-dC further suggest that E2 reduces the expression of these DNA repair genes potentially through promoter hypermethylation. Reactivation of mismatch repair (MMR) gene MLH1 and abrogation of E2-induced cell growth by 5-aza-dC treatment suggest that estrogen causes increased growth in breast cancer cells potentially through the inhibition of MMR-mediated apoptotic response. In summary, this study suggests that estrogen increases cell growth and decreases the DNA repair capacity in breast cancer cells, at least in part, through epigenetic mechanism. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Synthesis of 2'-deoxy-2'-[{sup 18}F]-fluoro-5-iodo-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]-FIAU) and micro-PET imaging of suicide gene expression in tumor-bearing nude mice

Energy Technology Data Exchange (ETDEWEB)

Alauddin, M.M.; Shahinian, A.; Park, R.; Tohme, M.; Fissekis, J.D.; Conti, P.S. [Univ. of Southern California, Los Angeles, CA (United States). PET Imaging Science Center

2004-07-01

Herpes simplex virus type-1 thymidine kinase (HSV1-tk) is being used as a suicide gene for gene therapy of cancer. An in vivo method to assess the HSV1-tk enzyme activity after gene transfer is desirable to monitor gene expression as an indicator of gene delivery. Imaging of the HSV1-tk reporter gene along with various reporter probes is of current interest. We originally developed [{sup 18}F]-FHPG and [{sup 18}F]-FHBG for PET imaging of HSV1-tk gene expression and demonstrated that [{sup 18}F]-FHBG is more useful than [{sup 18}F]-FHPG for this purpose. [{sup 124}I]-FIAU has been shown to be a potential PET imaging agent for HSV1-tk gene expression, and is superior to [{sup 18}F]-FHPG and [{sup 18}F]-FHBG. We also demonstrated that radiolabeled FMAU can be used as a marker for HSV-tk gene expression, and is superior to [{sup 18}F]-FHPG and [{sup 18}F]-FHBG. Earlier we reported a synthesis for 2'-deoxy-2'-[{sup 18}F]fluoro-5-methyl-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]-FMAU) and some other 5-substituted nucleosides. We have synthesized now [{sup 18}F]-FIAU, used the tracer for micro-PET imaging of suicide gene expression in tumor-bearing nude mice, and compared the results with earlier studies using [{sup 14}C]-FMAU. (orig.)

Nur77 coordinately regulates expression of genes linked to glucose metabolism in skeletal muscle.

Science.gov (United States)

Chao, Lily C; Zhang, Zidong; Pei, Liming; Saito, Tsugumichi; Tontonoz, Peter; Pilch, Paul F

2007-09-01

Innervation is important for normal metabolism in skeletal muscle, including insulin-sensitive glucose uptake. However, the transcription factors that transduce signals from the neuromuscular junction to the nucleus and affect changes in metabolic gene expression are not well defined. We demonstrate here that the orphan nuclear receptor Nur77 is a regulator of gene expression linked to glucose utilization in muscle. In vivo, Nur77 is preferentially expressed in glycolytic compared with oxidative muscle and is responsive to beta-adrenergic stimulation. Denervation of rat muscle compromises expression of Nur77 in parallel with that of numerous genes linked to glucose metabolism, including glucose transporter 4 and genes involved in glycolysis, glycogenolysis, and the glycerophosphate shuttle. Ectopic expression of Nur77, either in rat muscle or in C2C12 muscle cells, induces expression of a highly overlapping set of genes, including glucose transporter 4, muscle phosphofructokinase, and glycogen phosphorylase. Furthermore, selective knockdown of Nur77 in rat muscle by small hairpin RNA or genetic deletion of Nur77 in mice reduces the expression of a battery of genes involved in skeletal muscle glucose utilization in vivo. Finally, we show that Nur77 binds the promoter regions of multiple genes involved in glucose metabolism in muscle. These results identify Nur77 as a potential mediator of neuromuscular signaling in the control of metabolic gene expression.

Neighboring Genes Show Correlated Evolution in Gene Expression

Science.gov (United States)

Ghanbarian, Avazeh T.; Hurst, Laurence D.

2015-01-01

When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

Developmental expression of the alpha-skeletal actin gene

Directory of Open Access Journals (Sweden)

Vonk Freek J

2008-06-01

Full Text Available Abstract Background Actin is a cytoskeletal protein which exerts a broad range of functions in almost all eukaryotic cells. In higher vertebrates, six primary actin isoforms can be distinguished: alpha-skeletal, alpha-cardiac, alpha-smooth muscle, gamma-smooth muscle, beta-cytoplasmic and gamma-cytoplasmic isoactin. Expression of these actin isoforms during vertebrate development is highly regulated in a temporal and tissue-specific manner, but the mechanisms and the specific differences are currently not well understood. All members of the actin multigene family are highly conserved, suggesting that there is a high selective pressure on these proteins. Results We present here a model for the evolution of the genomic organization of alpha-skeletal actin and by molecular modeling, illustrate the structural differences of actin proteins of different phyla. We further describe and compare alpha-skeletal actin expression in two developmental stages of five vertebrate species (mouse, chicken, snake, salamander and fish. Our findings confirm that alpha-skeletal actin is expressed in skeletal muscle and in the heart of all five species. In addition, we identify many novel non-muscular expression domains including several in the central nervous system. Conclusion Our results show that the high sequence homology of alpha-skeletal actins is reflected by similarities of their 3 dimensional protein structures, as well as by conserved gene expression patterns during vertebrate development. Nonetheless, we find here important differences in 3D structures, in gene architectures and identify novel expression domains for this structural and functional important gene.

Differential gene expression profile in pig adipose tissue treated with/without clenbuterol

Directory of Open Access Journals (Sweden)

Deng Xue M

2007-11-01

Full Text Available Abstract Background Clenbuterol, a beta-agonist, can dramatically reduce pig adipose accumulation at high dosages. However, it has been banned in pig production because people who eat pig products treated with clenbuterol can be poisoned by the clenbuterol residues. To understand the molecular mechanism for this fat reduction, cDNA microarray, real-time PCR, two-dimensional electrophoresis and mass spectra were used to study the differential gene expression profiles of pig adipose tissues treated with/without clenbuterol. The objective of this research is to identify novel genes and physiological pathways that potentially facilitate clenbuterol induced reduction of adipose accumulation. Results Clenbuterol was found to improve the lean meat percentage about 10 percent (P Conclusion Pig fat accumulation was reduced dramatically with clenbuterol treatment. Histological sections and global evaluation of gene expression after administration of clenbuterol in pigs identified profound changes in adipose cells. With clenbuterol stimulation, adipose cell volumes decreased and their gene expression profile changed, which indicate some metabolism processes have been also altered. Although the biological functions of the differentially expressed genes are not completely known, higher expressions of these molecules in adipose tissue might contribute to the reduction of fat accumulation. Among these genes, five lipid metabolism related genes were of special interest for further study, including apoD and apoR. The apoR expression was increased at both the RNA and protein levels. The apoR may be one of the critical molecules through which clenbuterol reduces fat accumulation.

Gene Expression Commons: an open platform for absolute gene expression profiling.

Directory of Open Access Journals (Sweden)

Jun Seita

Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

PDGF-beta receptor expression and ventilatory acclimatization to hypoxia in the rat.

Science.gov (United States)

Alea, O A; Czapla, M A; Lasky, J A; Simakajornboon, N; Gozal, E; Gozal, D

2000-11-01

Activation of platelet-derived growth factor-beta (PDGF-beta) receptors in the nucleus of the solitary tract (nTS) modulates the late phase of the acute hypoxic ventilatory response (HVR) in the rat. We hypothesized that temporal changes in PDGF-beta receptor expression could underlie the ventilatory acclimatization to hypoxia (VAH). Normoxic ventilation was examined in adult Sprague-Dawley rats chronically exposed to 10% O(2), and at 0, 1, 2, 7, and 14 days, Northern and Western blots of the dorsocaudal brain stem were performed for assessment of PDGF-beta receptor expression. Although no significant changes in PDGF-beta receptor mRNA occurred over time, marked attenuation of PDGF-beta receptor protein became apparent after day 7 of hypoxic exposure. Such changes were significantly correlated with concomitant increases in normoxic ventilation, i.e., with VAH (r: -0.56, P < 0.005). In addition, long-term administration of PDGF-BB in the nTS via osmotic pumps loaded with either PDGF-BB (n = 8) or vehicle (Veh; n = 8) showed that although no significant changes in the magnitude of acute HVR occurred in Veh over time, the typical attenuation of HVR by PDGF-BB decreased over time. Furthermore, PDGF-BB microinjections did not attenuate HVR in acclimatized rats at 7 and 14 days of hypoxia (n = 10). We conclude that decreased expression of PDGF-beta receptors in the dorsocaudal brain stem correlates with the magnitude of VAH. We speculate that the decreased expression of PDGF-beta receptors is mediated via internalization and degradation of the receptor rather than by transcriptional regulation.

Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers

Directory of Open Access Journals (Sweden)

Pizzichini Daniele

2007-03-01

Full Text Available Abstract Background Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch and violaxanthin (in the beta-beta branch. None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold. Results In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species. Conclusion Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

TGF-beta1 modulates matrix metalloproteinase-13 expression in hepatic stellate cells by complex mechanisms involving p38MAPK, PI3-kinase, AKT, and p70S6k.

Science.gov (United States)

Lechuga, Carmen G; Hernández-Nazara, Zamira H; Domínguez Rosales, José-Alfredo; Morris, Elena R; Rincón, Ana Rosa; Rivas-Estilla, Ana María; Esteban-Gamboa, Andrés; Rojkind, Marcos

2004-11-01

Transforming growth factor-beta1 (TGF-beta1), the main cytokine involved in liver fibrogenesis, induces expression of the type I collagen genes in hepatic stellate cells by a transcriptional mechanism, which is hydrogen peroxide and de novo protein synthesis dependent. Our recent studies have revealed that expression of type I collagen and matrix metalloproteinase-13 (MMP-13) mRNAs in hepatic stellate cells is reciprocally modulated. Because TGF-beta1 induces a transient elevation of alpha1(I) collagen mRNA, we investigated whether this cytokine was able to induce the expression of MMP-13 mRNA during the downfall of the alpha1(I) collagen mRNA. In the present study, we report that TGF-beta1 induces a rapid decline in steady-state levels of MMP-13 mRNA at the time that it induces the expression of alpha1(I) collagen mRNA. This change in MMP-13 mRNA expression occurs within the first 6 h postcytokine administration and is accompanied by a twofold increase in gene transcription and a fivefold decrease in mRNA half-life. This is followed by increased expression of MMP-13 mRNA, which reaches maximal values by 48 h. Our results also show that this TGF-beta1-mediated effect is de novo protein synthesis-dependent and requires the activity of p38MAPK, phosphatidylinositol 3-kinase, AKT, and p70(S6k). Altogether, our data suggest that regulation of MMP-13 by TGF-beta1 is a complex process involving transcriptional and posttranscriptional mechanisms.

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

OpenAIRE

Ping, P; Gelzer-Bell, R; Roth, D A; Kiel, D; Insel, P A; Hammond, H K

1995-01-01

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling as...

Candidate Gene Study of TRAIL and TRAIL Receptors: Association with Response to Interferon Beta Therapy in Multiple Sclerosis Patients

Science.gov (United States)

Órpez-Zafra, Teresa; Pinto-Medel, María Jesús; Oliver-Martos, Begoña; Ortega-Pinazo, Jesús; Arnáiz, Carlos; Guijarro-Castro, Cristina; Varadé, Jezabel; Álvarez-Lafuente, Roberto; Urcelay, Elena; Sánchez-Jiménez, Francisca

2013-01-01

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10−4, pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS. PMID:23658636

Candidate gene study of TRAIL and TRAIL receptors: association with response to interferon beta therapy in multiple sclerosis patients.

Directory of Open Access Journals (Sweden)

Carlos López-Gómez

Full Text Available TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10(-4, pc = 0.048, OR = 0.30. This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A, a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

Characterization of the beta amyloid precursor protein-like gene in the central nervous system of the crab Chasmagnathus. Expression during memory consolidation

Directory of Open Access Journals (Sweden)

Fustiñana Maria

2010-09-01

Full Text Available Abstract Background Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information. Results Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl, showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels Conclusions cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation.

Alteration of gene expression by alcohol exposure at early neurulation.

Science.gov (United States)

Zhou, Feng C; Zhao, Qianqian; Liu, Yunlong; Goodlett, Charles R; Liang, Tiebing; McClintick, Jeanette N; Edenberg, Howard J; Li, Lang

2011-02-21

We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube

Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice

Directory of Open Access Journals (Sweden)

Liu Zhihui

2008-08-01

Full Text Available Abstract Background Interleukin 1 beta (IL-1β plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1β in disease processes and develop an in vivo screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1β gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models. Results In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1β mRNA and pro-IL-1β protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model. Conclusion Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1β gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug in vivo.

Synthesis of 2'-deoxy-2'-[{sup 18}F]-fluoro-5-ethyl-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]-FEAU) and micro-PET imaging of HSV-tk gene expression in tumor-bearing nude mice

Energy Technology Data Exchange (ETDEWEB)

Alauddin, M.M.; Shahinian, A.; Park, R.; Tohme, M.; Fissekis, J.D.; Conti, P.S. [Univ. of Southern California, Los Angeles, CA (United States). PET Imaging Science Center

2004-07-01

Herpes simplex virus type-1 thymidine kinase (HSV1-tk) is being used as a suicide gene for gene therapy of cancer. An in vivo method to assess the HSV1-tk enzyme activity after gene transfer is desirable to monitor gene expression as an indicator of gene delivery. Imaging of the HSV1-tk reporter gene along with various reporter probes is of current interest. We originally developed [{sup 18}F]-FHPG and [{sup 18}F]-FHBG for PET imaging of HSV1-tk gene expression and demonstrated that [{sup 18}F]-FHBG is more useful than [{sup 18}F]-FHPG for this purpose. [{sup 124}I]-FIAU has been shown to be a potential PET imaging agent for HSV1-tk gene expression, and is superior to [{sup 18}F]-FHPG and [{sup 18}F]-FHBG. We also demonstrated that radiolabeled FMAU can be used as a marker for HSV-tk gene expression, and is superior to [{sup 18}F]-FHPG and [{sup 18}F]-FHBG. Earlier we reported a synthesis for 2'-deoxy-2'-[{sup 18}F]fluoro-5-methyl-1-{beta}-D-arabinofuranosyluracil ([{sup 18}F]-FMAU) and some other 5-substituted nucleosides. We have synthesized now [{sup 18}F]-FEAU, used the tracer for micro-PET imaging of suicide gene expression in tumor-bearing nude mice, and compared the results with earlier studies using [{sup 14}C]-FMAU. (orig.)

The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of beta-carotene into astaxanthin and other xanthophylls.

Science.gov (United States)

Alvarez, Vanessa; Rodríguez-Sáiz, Marta; de la Fuente, Juan Luis; Gudiña, Eduardo J; Godio, Ramiro P; Martín, Juan F; Barredo, José Luis

2006-04-01

The conversion of beta-carotene into xanthophylls is a subject of great scientific and industrial interest. We cloned the crtS gene involved in astaxanthin biosynthesis from two astaxanthin producing strains of Xanthophyllomyces dendrorhous: VKPM Y2410, an astaxanthin overproducing strain, and the wild type ATCC 24203. In both cases, the ORF has a length of 3166 bp, including 17 introns, and codes for a protein of 62.6 kDa with similarity to cytochrome-P450 hydroxylases. crtS gene sequences from strains VKPM Y2410, ATCC 24203, ATCC 96594, and ATCC 96815 show several nucleotide changes, but none of them causes any amino acid substitution, except a G2268 insertion in the 13th exon of ATCC 96815 which causes a change in the reading frame. A G1470 --> A change in the 5' splicing region of intron 8 was also found in ATCC 96815. Both point mutations explain astaxanthin idiotrophy and beta-carotene accumulation in ATCC 96815. Mutants accumulating precursors of the astaxanthin biosynthetic pathway were selected from the parental strain VKPM Y2410 (red) showing different colors depending on the compound accumulated. Two of them were blocked in the biosynthesis of astaxanthin, M6 (orange; 1% astaxanthin, 71 times more beta-carotene) and M7 (orange; 1% astaxanthin, 58 times more beta-carotene, 135% canthaxanthin), whereas the rest produced lower levels of astaxanthin (5-66%) than the parental strain. When the crtS gene was expressed in M7, canthaxanthin accumulation disappeared and astaxanthin production was partially restored. Moreover, astaxanthin biosynthesis was restored when X. dendrorhous ATCC 96815 was transformed with the crtS gene. The crtS gene was heterologously expressed in Mucor circinelloides conferring to this fungus an improved capacity to synthesize beta-cryptoxanthin and zeaxanthin, two hydroxylated compounds from beta-carotene. These results show that the crtS gene is involved in the conversion of beta-carotene into xanthophylls, being potentially useful to

Increased expression of the auxiliary beta(2-subunit of ventricular L-type Ca(2+ channels leads to single-channel activity characteristic of heart failure.

Directory of Open Access Journals (Sweden)

Roger Hullin

2007-03-01

Full Text Available Increased activity of single ventricular L-type Ca(2+-channels (L-VDCC is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation.By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1 or beta(3 isoforms, beta(2a and beta(2b induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V1.2 also reveal increased single-channel activity and sarcolemmal beta(2 expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase", reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2 expression. Additional evidence for the cause-effect relationship between beta(2-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V1.2 and inducible beta(2 cardiac overexpression. Here in non-failing hearts induction of beta(2-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure.Our study presents evidence of the pathobiochemical relevance of beta(2-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

International Nuclear Information System (INIS)

Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

2008-01-01

Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

Directory of Open Access Journals (Sweden)

Radtke Arnold

2008-11-01

Full Text Available Abstract Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR. The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC is presented. Methods Six genes, beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hydroxymethyl-bilane synthase (HMBS, hypoxanthine phosphoribosyl-transferase 1 (HPRT1, succinate dehydrogenase complex, subunit A (SDHA and ubiquitin C (UBC, with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

Science.gov (United States)

Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

2008-01-01

Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Methods Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues

Analysis of gene expression in gecko digital adhesive pads indicates significant production of cysteine- and glycine-rich beta-keratins.

Science.gov (United States)

Hallahan, David L; Keiper-Hrynko, Natalie M; Shang, Tanya Q; Ganzke, Thaya S; Toni, Mattia; Dalla Valle, Luisa; Alibardi, Lorenzo

2009-01-15

Microscopic bristles (setae) present on digital pads permit the adhesion and climbing of geckos. Keratins of setae of the lizard Gekko gecko (Tokay gecko) were analyzed by the isolation of expressed mRNAs and by the generation of an EST library. Of the 510 sequences determined, 268 (52.9%) were unique. Of these, 14 appeared to encode alpha- and 111 beta-keratins. Within the beta-keratins, we identified five groups based on nucleotide sequence comparisons. Of these, one contained the bulk of beta-keratins, with 103 EST members. The mRNAs within this major group, together with two singlets, encoded cysteine-proline-serine-rich proteins of 10-14 kDa (Ge-cprp). One of the smaller groups of transcripts encoded slightly larger glycine-proline-serine-rich proteins, of 14-19 kDa (Ge-gprp). The remaining group consisted of smaller (9 kDa) serine-tyrosine-rich beta-keratins (Ge-strp). Thus three classes could be distinguished by amino acid sequence alignment. Exact matches for some of the peptide sequences obtained from setal proteins by ms/ms sequencing occur within several of these clones. Most of the beta-keratins were basic and contained a core-box region of two beta-strand sequences, with high homology to core-boxes present in avian scale and feather beta-keratins. Core-boxes are beta-folded regions that are likely responsible for polymerization into the beta-keratin filaments. The two deduced alpha-keratins of 52.7 kDa are both acidic, and contain the typical central rod region with some homology to mammalian and avian alpha-keratins, with variable N- and C-terminal regions. Basic beta-keratins and acidic alpha-keratins may interact electrostatically to form the resistant corneous material of setae. (c) 2008 Wiley-Liss, Inc.

Studies on gene expressions analyses for Arabidopsis thaliana plants stimulated by space flight condition

Science.gov (United States)

Lu, Jinying; Liu, Min; Pan, Yi; Li, Huasheng

We carried out whole-genome microarray to screen the transcript profile of Arabidopsis thaliana seedlings after three treatment: space microgravity condition( Seedlings grown in microgravity state of space flight of SIMBOX on Shenzhou-8), 1g centrifugal force in space(Seedlings grown in 1g centrifugal force state of space flight of SIMBOX on Shenzhou-8) and ground control. The result of microarray analysis is as followed: There were 368 genes significantly differentially expressed in space microgravity condition compared with that in 1g centrifuge space condition. Space radiation caused 246 genes significantly differentially expressed between seedlings in 1g centrifuge space condition and ground control. Space conditions (including microgravity and radiation) caused 621 genes significantly differentially expressed between seedlings in space microgravity condition and ground control. Microgravity and radiation as a single factor can cause plant gene expression change, but two factors synergism can produce some new effects on plant gene expression. The function of differential expression genes were analyst by bioinformatics, and we found the expression of genes related with stress were more different, such as the dehydration of protein (dehydrin Xero2) expression is up-regulated 57 times; low-temperature-induced protein expression is up-regulated in 49 times; heat shock protein expression is up-regulated 20 times; transcription factor DREB2A expression increase 25 times; protein phosphatase 2C expression is up-regulated 14 times; transcription factor NAM-like protein expression is up-regulated 13 times; cell wall metabolism related genes (xyloglucan, endo-1, 4-beta-D-glucanase) expression is down-regulated in 15 times. The results provide scientific data for the mechanism of space mutation.

Imaging gene expression in gene therapy

International Nuclear Information System (INIS)

Wiebe, Leonard I.

1997-01-01

Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on 'suicide gene therapy' of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k + ) has been use for 'suicide' in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k + gene expression where the H S V-1 t k + gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([ 18 F]F H P G; [ 18 F]-A C V), and pyrimidine- ([ 123 / 131 I]I V R F U; [ 124 / 131I ]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [ 123 / 131I ]I V R F U imaging with the H S V-1 t k + reporter gene will be presented

Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer’s disease model cell line

International Nuclear Information System (INIS)

Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee; Oh, Seikwan; Ahn, Jung-Hyuck

2011-01-01

Highlights: ► Genome-wide DNA methylation pattern in Alzheimer’s disease model cell line. ► Integrated analysis of CpG methylation and mRNA expression profiles. ► Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. ► The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer’s disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the −435, −295, and −271 CpG sites of CTIF, and at the −505 to −341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at −432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.

Astrocytic expression of the Alzheimer's disease beta-secretase (BACE1) is stimulus-dependent

DEFF Research Database (Denmark)

Hartlage-Rübsamen, Maike; Zeitschel, Ulrike; Apelt, Jenny

2003-01-01

The beta-site APP-cleaving enzyme (BACE1) is a prerequisite for the generation of beta-amyloid peptides, which give rise to cerebrovascular and parenchymal beta-amyloid deposits in the brain of Alzheimer's disease patients. BACE1 is neuronally expressed in the brains of humans and experimental...... paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1-immunoreactive astrocytes in proximity to beta-amyloid plaques in the brains of aged Tg2576 mice and Alzheimer's disease patients....

Prediction of graft-versus-host disease in humans by donor gene-expression profiling.

Directory of Open Access Journals (Sweden)

Chantal Baron

2007-01-01

Full Text Available BACKGROUND: Graft-versus-host disease (GVHD results from recognition of host antigens by donor T cells following allogeneic hematopoietic cell transplantation (AHCT. Notably, histoincompatibility between donor and recipient is necessary but not sufficient to elicit GVHD. Therefore, we tested the hypothesis that some donors may be "stronger alloresponders" than others, and consequently more likely to elicit GVHD. METHODS AND FINDINGS: To this end, we measured the gene-expression profiles of CD4(+ and CD8(+ T cells from 50 AHCT donors with microarrays. We report that pre-AHCT gene-expression profiling segregates donors whose recipient suffered from GVHD or not. Using quantitative PCR, established statistical tests, and analysis of multiple independent training-test datasets, we found that for chronic GVHD the "dangerous donor" trait (occurrence of GVHD in the recipient is under polygenic control and is shaped by the activity of genes that regulate transforming growth factor-beta signaling and cell proliferation. CONCLUSIONS: These findings strongly suggest that the donor gene-expression profile has a dominant influence on the occurrence of GVHD in the recipient. The ability to discriminate strong and weak alloresponders using gene-expression profiling could pave the way to personalized transplantation medicine.

Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

OpenAIRE

Ezer, Daphne; Moignard, Victoria; G?ttgens, Berthold; Adryan, Boris

2016-01-01

Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete ...

Imaging gene expression in gene therapy

Energy Technology Data Exchange (ETDEWEB)

Wiebe, Leonard I. [Alberta Univ., Edmonton (Canada). Noujaim Institute for Pharmaceutical Oncology Research

1997-12-31

Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on `suicide gene therapy` of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k{sup +}) has been use for `suicide` in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k{sup +} gene expression where the H S V-1 t k{sup +} gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([{sup 18} F]F H P G; [{sup 18} F]-A C V), and pyrimidine- ([{sup 123}/{sup 131} I]I V R F U; [{sup 124}/{sup 131I}]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [{sup 123}/{sup 131I}]I V R F U imaging with the H S V-1 t k{sup +} reporter gene will be presented

Effects of early weaning and social isolation on the expression of glucocorticoid and mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase 1 and 2 mRNAs in the frontal cortex and hippocampus of piglets.

Science.gov (United States)

Poletto, R; Steibel, J P; Siegford, J M; Zanella, A J

2006-01-05

Pigs weaned at young ages show more abnormal and aggressive behaviors and cognitive deficits compared to later weaned pigs. We investigated the effects of age, weaning and/or social isolation on the expression of genes regulating glucocorticoid response [glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11beta-hydroxysteroid dehydrogenases 1 and 2 (11beta-HSD1 and 11beta-HSD2)] in the frontal cortex and hippocampus. Early- (EW; n = 6) and conventionally-weaned (CW; n = 6) piglets were weaned at 10 and 21 days after birth, respectively. Non-weaned (NW) piglets of both ages (NW; n = 6/group) remained with their dams. Immediately before euthanasia, half of CW, EW and NW animals were socially isolated for 15 min at 12 (EW, NW) and 23 (CW, NW) days of age. Differences in amounts of 11beta-HSD1, 11beta-HSD2, GR and MR mRNA were determined by quantitative real-time RT-PCR and data subjected to multivariate linear mixed model analysis. When compared with NW piglets at 12 days of age, the hippocampi of EW piglets showed decreased gene expression (P Social isolation decreased gene expression (P social isolation affected frontal cortex regardless of age. These results may be correlated with behavioral and cognitive changes reported in EW piglets.

Expression of Estrogen Alpha and Beta Receptors in Prostate ...

African Journals Online (AJOL)

Expression of Estrogen Alpha and Beta Receptors in Prostate Cancer and Hyperplasia: Immunohistochemical Analysis. ... Additionally, ER-α was not expressed in either luminal or basal cells in any of the 35 BPH cases. However ... Key Words: ER-α, ER-β, prostate, hyperplasia, premalignant, cancer, immunohistochemistry ...

Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

DEFF Research Database (Denmark)

Zhao, Heng; Stet, René J M; Skjødt, Karsten

2008-01-01

Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable ...... MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association...... antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently......, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC...

TGF-beta3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells.

Science.gov (United States)

Nakamura, Shin-ichi; Kawai, Takayuki; Kamakura, Takashi; Ookura, Tetsuya

2010-01-01

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

Science.gov (United States)

Chen, Xi'en; Zhang, Yalin

2015-03-10

NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. Copyright © 2015 Elsevier B.V. All rights reserved.

Analysis of T cell receptor alpha beta variability in lymphocytes infiltrating melanoma primary tumours and metastatic lesions

DEFF Research Database (Denmark)

Schøller, J; thor Straten, P; Jakobsen, Annette Birck

1994-01-01

The T cell receptor (TCR) alpha beta variable (V) gene family usage of tumour-infiltrating lymphocytes (TIL) in four different primary human malignant melanomas and their corresponding metastatic lesions was characterized using a recently developed method based on the reverse-transcription-couple......The T cell receptor (TCR) alpha beta variable (V) gene family usage of tumour-infiltrating lymphocytes (TIL) in four different primary human malignant melanomas and their corresponding metastatic lesions was characterized using a recently developed method based on the reverse...... usage of the TCR V gene families V alpha 4, V alpha 5, V alpha 22 and V beta 8, whereas the V beta 3 gene family appeared to be expressed together with HLA-A1. Other highly expressed V gene families, apparently not restricted to either HLA-A1 or -A2, were V alpha 1 (expressed in three of four primary...... tumours) and V alpha 21 (expressed in two of four tumours). We found no evidence suggesting any correlations between the haplotypes HLA-A1 and -A2 and preferential V gene family expression in the metastatic lesions, and the only common feature was V alpha 8, which was found to be highly expressed in two...

A gene encoding the major beta tubulin of the mitotic spindle in Physarum polycephalum plasmodia

Energy Technology Data Exchange (ETDEWEB)

Burland, T.G.; Paul, E.C.A.; Oetliker, M.; Dove, W.F.

1988-03-01

The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. The authors identified a ..beta..-tubulin cDNA clone, ..beta..105, which is shown to correspond to the transcript of the betC ..beta..-tubulin locus and to encode ..beta..2 tubulin, the ..beta.. tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that ..beta..2 tubulin is only 83% identical to the two ..beta.. tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum ..beta..2 tubulin and the ..beta.. tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum ..beta..2 tubulin is no more similar to, for example, Aspergillus ..beta.. tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged ..beta.. tubulin as well as one or more ..beta.. tubulins that conform more closely to a consensus ..beta..-tubulin sequence. The authors suggest that ..beta..-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among ..beta.. tubulins could have resulted through neutral drift. For example, exclusive use of Physarum ..beta..2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the ..beta.. tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of ..beta.. tubulins may allow positive selection to operate more freely to refine ..beta..-tubulin function.

Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

Energy Technology Data Exchange (ETDEWEB)

Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F. (Hagedorn Research Laboratory, Gentofte (Denmark))

1988-09-01

The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression.

Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo

International Nuclear Information System (INIS)

Madsen, O.D.; Andersen, L.C.; Michelsen, B.; Owerbach, D.; Larsson, L.I.; Lernmark, A.; Steiner, D.F.

1988-01-01

The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. The authors propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression

Global regulator SATB1 recruits beta-catenin and regulates T(H2 differentiation in Wnt-dependent manner.

Directory of Open Access Journals (Sweden)

Dimple Notani

2010-01-01

Full Text Available In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs to activate target genes. Wnt/beta -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1, the T lineage-enriched chromatin organizer and global regulator, interacts with beta-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon beta-catenin signalling. GATA-3 is a T helper type 2 (T(H2 specific transcription factor that regulates production of T(H2 cytokines and functions as T(H2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4(+ T cells, suggesting that SATB1 influences T(H2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1, an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature T(H2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H2 cells in a Wnt-dependent manner. SATB1 coordinates T(H2 lineage commitment by reprogramming gene expression. The SATB1:beta-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1

In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression.

Science.gov (United States)

Carlin, Sean; Pugachev, Andrei; Sun, Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C Clifton; Humm, John L

2009-10-01

To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer (18)F-fluoromisonidazole ((18)F-FMISO). Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe (124)I-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil ((124)I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between (124)I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe (18)F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with (124)I-FIAU (3 h before sacrifice) and (18)F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between (18)F-FMISO and (124)I-FIAU on a pixel-by-pixel basis was performed. Correlation coefficients between (124)I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between (18)F-FMISO and (124)I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

Directory of Open Access Journals (Sweden)

Holger A Russ

Full Text Available Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT. Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2 using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug

Expression of SCM-1alpha/lymphotactin and SCM-1beta in natural killer cells is upregulated by IL-2 and IL-12.

Science.gov (United States)

Hennemann, B; Tam, Y K; Tonn, T; Klingemann, H G

1999-07-01

Recruitment of lymphocytes is an important feature of the host immune response against pathogens. However, the mechanisms by which lymphocytes are attracted are not yet fully understood. Recently, the cDNA of a lymphocyte-specific chemokine, lymphotactin (Lptn), was isolated from murine and human T cells and was also found to be expressed in murine NK cells and human NK cell clones. This study investigated the influence of interleukin (IL)-2 and IL-12 on the expression of Lptn, also known as SCM (single cysteine motif)-1alpha, and SCM-1beta, a 97% homolog of Lptn, in freshly isolated human NK cells and the human NK cell line NK-92. Northern blot analysis and RT-PCR confirmed that nonactivated human NK cells expressed both genes at low level. After activation with IL-2 or IL-12, the expression of both Lptn and SCM-1beta was upregulated within hours. NK-92 cells maintained in medium supplemented with IL-2 constitutively expressed SCM-1 mRNA. However, after 24 h of IL-2 starvation and subsequent culturing at various IL-2 concentrations, the expression of Lptn/SCM-1alpha was upregulated in a dose-dependent manner, whereas the expression of SCM-1beta remained consistently high. These observations indicate that NK cells, in addition to T lymphocytes, express Lptn/SCM-1alpha and SCM-1beta after cytokine activation. The upregulation of these chemokines in NK cells on activation likely acts to increase the number of effector cells reaching the site of an immune response such as inflammation.

The murine homeobox gene Msx-3 shows highly restricted expression in the developing neural tube.

Science.gov (United States)

Shimeld, S M; McKay, I J; Sharpe, P T

1996-04-01

The mouse homeobox-genes Msx-1 and Msx-2 are expressed in several areas of the developing embryo, including the neural tube, neural crest, facial processes and limb buds. Here we report the characterisation of a third mouse Msx gene, which we designate Msx-3. The embryonic expression of Msx-3 was found to differ from that of Msx-1 and -2 in that it was confined to the dorsal neural tube. In embryos with 5-8 somites a segmental pattern of expression was observed in the hindbrain, with rhombomeres 3 and 5 lacking Msx-3 while other rhombomeres expressed Msx-3. This pattern was transient, however, such that in embryos with 18 or more somites expression was continuous throughout the dorsal hindbrain and anterior dorsal spinal cord. Differentiation of dorsal cell types in the neural tube can be induced by addition of members of the Tgf-beta family. Additionally, Msx-1 and -2 have been shown to be activated by addition of the Tgf-beta family member Bmp-4. To determine if Bmp-4 could activate Msx-3, we incubated embryonic hindbrain explants with exogenous Bmp-4. The dorsal expression of Msx-3 was seen to expand into more ventral regions of the neurectoderm in Bmp-4-treated cultures, implying that Bmp-4 may be able to mimic an in vivo signal that induces Msx-3.

Chromosome-encoded narrow-spectrum Ambler class A beta-lactamase GIL-1 from Citrobacter gillenii.

Science.gov (United States)

Naas, Thierry; Aubert, Daniel; Ozcan, Ayla; Nordmann, Patrice

2007-04-01

A novel beta-lactamase gene was cloned from the whole-cell DNA of an enterobacterial Citrobacter gillenii reference strain that displayed a weak narrow-spectrum beta-lactam-resistant phenotype and was expressed in Escherichia coli. It encoded a clavulanic acid-inhibited Ambler class A beta-lactamase, GIL-1, with a pI value of 7.5 and a molecular mass of ca. 29 kDa. GIL-1 had the highest percent amino acid sequence identity with TEM-1 and SHV-1, 77%, and 67%, respectively, and only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (C. koseri), CdiA1 (C. diversus), and SED-1 (C. sedlaki), respectively. The substrate profile of the purified GIL-1 was similar to that of beta-lactamases TEM-1 and SHV-1. The blaGIL-1 gene was chromosomally located, as revealed by I-CeuI experiments, and was constitutively expressed at a low level in C. gillenii. No gene homologous to the regulatory ampR genes of chromosomal class C beta-lactamases was found upstream of the blaGIL-1 gene, which fits the noninducibility of beta-lactamase expression in C. gillenii. Rapid amplification of DNA 5' ends analysis of the promoter region revealed putative promoter sequences that diverge from what has been identified as the consensus sequence in E. coli. The blaGIL-1 gene was part of a 5.5-kb DNA fragment bracketed by a 9-bp duplication and inserted between the d-lactate dehydrogenase gene and the ydbH genes; this DNA fragment was absent in other Citrobacter species. This work further illustrates the heterogeneity of beta-lactamases in Citrobacter spp., which may indicate that the variability of Citrobacter species is greater than expected.

Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase β-subunit gene family

International Nuclear Information System (INIS)

Pestov, Nikolay B.; Zhao, Hao; Basrur, Venkatesha; Modyanov, Nikolai N.

2011-01-01

Highlights: → Structural properties of BetaM and Na,K-ATPase β-subunits are sharply different. → BetaM protein is concentrated in nuclear membrane of skeletal myocytes. → BetaM does not associate with a Na,K-ATPase α-subunit in skeletal muscle. → Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. → BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a β-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase β-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass

Chromatin loops, gene positioning, and gene expression

NARCIS (Netherlands)

Holwerda, S.; de Laat, W.

2012-01-01

Technological developments and intense research over the last years have led to a better understanding of the 3D structure of the genome and its influence on genome function inside the cell nucleus. We will summarize topological studies performed on four model gene loci: the alpha- and beta-globin

Mechanical loading prevents the stimulating effect of IL-1{beta} on osteocyte-modulated osteoclastogenesis

Energy Technology Data Exchange (ETDEWEB)

Kulkarni, Rishikesh N.; Bakker, Astrid D.; Everts, Vincent [Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Amsterdam (Netherlands); Klein-Nulend, Jenneke, E-mail: j.kleinnulend@acta.nl [Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Amsterdam (Netherlands)

2012-03-30

Highlights: Black-Right-Pointing-Pointer Osteocyte incubation with IL-1{beta} stimulated osteocyte-modulated osteoclastogenesis. Black-Right-Pointing-Pointer Conditioned medium from IL-1{beta}-treated osteocytes increased osteoclastogenesis. Black-Right-Pointing-Pointer IL-1{beta} upregulated RANKL and downregulated OPG gene expression by osteocytes. Black-Right-Pointing-Pointer CYR61 is upregulated in mechanically stimulated osteocytes. Black-Right-Pointing-Pointer Mechanical loading of osteocytes may abolish IL-1{beta}-induced osteoclastogenesis. -- Abstract: Inflammatory diseases such as rheumatoid arthritis are often accompanied by higher plasma and synovial fluid levels of interleukin-1{beta} (IL-1{beta}), and by increased bone resorption. Since osteocytes are known to regulate bone resorption in response to changes in mechanical stimuli, we investigated whether IL-1{beta} affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. MLO-Y4 osteocytes were pre-incubated with IL-1{beta} (0.1-1 ng/ml) for 24 h. Cells were either or not subjected to mechanical loading by 1 h pulsating fluid flow (PFF; 0.7 {+-} 0.3 Pa, 5 Hz) in the presence of IL-1{beta} (0.1-1 ng/ml). Conditioned medium was collected after 1 h PFF or static cultures. Subsequently mouse bone marrow cells were seeded on top of the IL-1{beta}-treated osteocytes to determine osteoclastogenesis. Conditioned medium from mechanically loaded or static IL-1{beta}-treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Gene expression of cysteine-rich protein 61 (CYR61/CCN1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) by osteocytes was determined immediately after PFF. Incubation of osteocytes with IL-1{beta}, as well as conditioned medium from static IL-1{beta}-treated osteocytes increased the formation of osteoclasts. However, conditioned medium from mechanically loaded IL

Changes of laminin beta 2 chain expression in congenital muscular dystrophy

DEFF Research Database (Denmark)

Cohn, R D; Herrmann, R; Wewer, U M

1997-01-01

We studied the distribution of laminin beta 2 chain in the skeletal muscle basement membrane of 16 patients with congenital muscular dystrophy (CMD) by immunohistochemistry. A dramatic reduction in the laminin beta 2 staining was observed in four patients with classical merosin-negative CMD....... A moderate reduction of laminin beta 2 labelling was observed in four patients with partial merosin deficiency and two patients with merosin-positive CMD. Two patients with merosin-positive CMD had no apparent changes in the expression of laminin beta 2. In three patients and one fetus diagnosed as Walker...

Repair of full-thickness articular cartilage defects by cultured mesenchymal stem cells transfected with the transforming growth factor {beta}{sub 1} gene

Energy Technology Data Exchange (ETDEWEB)

Guo Xiaodong [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Zheng Qixin [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Yang Shuhua [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Shao Zengwu [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Yuan Quan [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Pan Zhengqi [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Tang Shuo [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Liu Kai [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Quan Daping [Institute of Polymer Science, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275 (China)

2006-12-15

Articular cartilage repair remains a clinical and scientific challenge with increasing interest focused on the combined techniques of gene transfer and tissue engineering. Transforming growth factor beta 1 (TGF-{beta}{sub 1}) is a multifunctional molecule that plays a central role in promotion of cartilage repair, and inhibition of inflammatory and alloreactive immune response. Cell mediated gene therapy can allow a sustained expression of TGF-{beta}{sub 1} that may circumvent difficulties associated with growth factor delivery. The objective of this study was to investigate whether TGF-{beta}{sub 1} gene modified mesenchymal stem cells (MSCs) could enhance the repair of full-thickness articular cartilage defects in allogeneic rabbits. The pcDNA{sub 3}-TGF-{beta}{sub 1} gene transfected MSCs were seeded onto biodegradable poly-L-lysine coated polylactide (PLA) biomimetic scaffolds in vitro and allografted into full-thickness articular cartilage defects in 18 New Zealand rabbits. The pcDNA{sub 3} gene transfected MSCs/biomimetic scaffold composites and the cell-free scaffolds were taken as control groups I and II, respectively. The follow-up times were 2, 4, 12 and 24 weeks. Macroscopical, histological and ultrastructural studies were performed. In vitro SEM studies found that abundant cartilaginous matrices were generated and completely covered the interconnected pores of the scaffolds two weeks post-seeding in the experimental groups. In vivo, the quality of regenerated tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 24 weeks post-implantation. Joint repair in the experimental groups was better than that of either control group I or II, with respect to: (1) synthesis of hyaline cartilage specific extracellular matrix at the upper portion of the defect; (2) reconstitution of the subchondral bone at the lower portion of the defect and (3) inhibition of

Deletion of Nhlh2 results in a defective torpor response and reduced Beta adrenergic receptor expression in adipose tissue.

Directory of Open Access Journals (Sweden)

Umesh D Wankhade

2010-08-01

Full Text Available Mice with a targeted deletion of the basic helix-loop-helix transcription factor, Nescient Helix-Loop-Helix 2 (Nhlh2, display adult-onset obesity with significant increases in their fat depots, abnormal responses to cold exposure, and reduced spontaneous physical activity levels. These phenotypes, accompanied by the hypothalamic expression of Nhlh2, make the Nhlh2 knockout (N2KO mouse a useful model to study the role of central nervous system (CNS control on peripheral tissue such as adipose tissue.Differences in body temperature and serum analysis of leptin were performed in fasted and ad lib fed wild-type (WT and N2KO mice. Histological analysis of white (WAT and brown adipose tissue (BAT was performed. Gene and protein level expression of inflammatory and metabolic markers were compared between the two genotypes.We report significant differences in serum leptin levels and body temperature in N2KO mice compared with WT mice exposed to a 24-hour fast, suggestive of a defect in both white (WAT and brown adipose tissue (BAT function. As compared to WT mice, N2KO mice showed increased serum IL-6 protein and WAT IL-6 mRNA levels. This was accompanied by slight elevations of mRNA for several macrophage markers, including expression of macrophage specific protein F4/80 in adipose, suggestive of macrophage infiltration of WAT in the mutant animals. The mRNAs for beta3-adrenergic receptors (beta3-AR, beta2-AR and uncoupling proteins were significantly reduced in WAT and BAT from N2KO mice compared with WT mice.These studies implicate Nhlh2 in the central control of WAT and BAT function, with lack of Nhlh2 leading to adipose inflammation and altered gene expression, impaired leptin response to fasting, all suggestive of a deficient torpor response in mutant animals.

Gene expression changes in rat prostate after activation or blocking of the androgen and estrogen receptor

DEFF Research Database (Denmark)

Nellemann, Christine Lydia; Dalgaard, Majken; Holst, Bjørn

2005-01-01

responsive genes (complement C3, ER alpha, ER beta, AR, TRPM-2, PBPC3, ODC, and IGF-1 mRNA) was analyzed in rat ventral prostate by real time RT-PCR. Administration of estradiol benzoate (EB) to castrated testosterone-treated rats had no effect on reproductive organ weights or gene expression levels...... reversed by ICI 182780, and affected TRPM-2, PBP C3, ODC, IGF-1, AR, and ERa mRNA levels. AR expression in the prostate seemed to be under regulation of both estrogens and androgens, as ICI 182780 inhibited the testosterone-induced AR expression, and flutamide inhibited the EB-induced AR expression...... administration abolished the effects of EB. First choice of gene expression profiles in the Hershberger assay to study androgenic or anti-androgenic effects would be the traditional, TRPNI-2 and PBP C3, supplemented with the new complement C3....

Common changes in global gene expression induced by RNA polymerase inhibitors in Shigella flexneri.

Directory of Open Access Journals (Sweden)

Hua Fu

Full Text Available Characterization of expression profile of organisms in response to antimicrobials provides important information on the potential mechanism of action of the drugs. The special expression signature can be used to predict whether other drugs act on the same target. Here, the common response of Shigella flexneri to two inhibitors of RNA polymerase was examined using gene expression profiling. Consistent with similar effects of the two drugs, the gene expression profiles indicated that responses of the bacteria to these drugs were roughly the same, with 225 genes affected commonly. Of them, 88 were induced and 137 were repressed. Real-time PCR was performed for selected genes to verify the microarray results. Analysis of the expression data revealed that more than 30% of the plasmid-encoded genes on the array were up-regulated by the antibiotics including virF regulon, other virulence-related genes, and genes responsible for plasmid replication, maintenance, and transfer. In addition, some chromosome-encoded genes involved in virulence and genes acquired from horizontal transfer were also significantly up-regulated. However, the expression of genes encoding the beta-subunit of RNA polymerase was increased moderately. The repressed genes include those that code for products associated with the ribosome, citrate cycle, glycolysis, thiamine biosynthesis, purine metabolism, fructose metabolism, mannose metabolism, and cold shock proteins. This study demonstrates that the two antibiotics induce rapid cessation of RNA synthesis resulting in inhibition of translation components. It also indicates that the production of virulence factors involved in intercellular dissemination, tissue invasion and inflammatory destruction may be enhanced through derepressing horizontal transfer genes by the drugs.

Gene expression profiling in the stress control brain region hypothalamic paraventricular nucleus reveals a novel gene network including Amyloid beta Precursor Protein

Directory of Open Access Journals (Sweden)

Deussing Jan M

2010-10-01

Full Text Available Abstract Background The pivotal role of stress in the precipitation of psychiatric diseases such as depression is generally accepted. This study aims at the identification of genes that are directly or indirectly responding to stress. Inbred mouse strains that had been evidenced to differ in their stress response as well as in their response to antidepressant treatment were chosen for RNA profiling after stress exposure. Gene expression and regulation was determined by microarray analyses and further evaluated by bioinformatics tools including pathway and cluster analyses. Results Forced swimming as acute stressor was applied to C57BL/6J and DBA/2J mice and resulted in sets of regulated genes in the paraventricular nucleus of the hypothalamus (PVN, 4 h or 8 h after stress. Although the expression changes between the mouse strains were quite different, they unfolded in phases over time in both strains. Our search for connections between the regulated genes resulted in potential novel signalling pathways in stress. In particular, Guanine nucleotide binding protein, alpha inhibiting 2 (GNAi2 and Amyloid β (A4 precursor protein (APP were detected as stress-regulated genes, and together with other genes, seem to be integrated into stress-responsive pathways and gene networks in the PVN. Conclusions This search for stress-regulated genes in the PVN revealed its impact on interesting genes (GNAi2 and APP and a novel gene network. In particular the expression of APP in the PVN that is governing stress hormone balance, is of great interest. The reported neuroprotective role of this molecule in the CNS supports the idea that a short acute stress can elicit positive adaptational effects in the brain.

Expression stability and selection of optimal reference genes for gene expression normalization in early life stage rainbow trout exposed to cadmium and copper.

Science.gov (United States)

Shekh, Kamran; Tang, Song; Niyogi, Som; Hecker, Markus

2017-09-01

Gene expression analysis represents a powerful approach to characterize the specific mechanisms by which contaminants interact with organisms. One of the key considerations when conducting gene expression analyses using quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the selection of appropriate reference genes, which is often overlooked. Specifically, to reach meaningful conclusions when using relative quantification approaches, expression levels of reference genes must be highly stable and cannot vary as a function of experimental conditions. However, to date, information on the stability of commonly used reference genes across developmental stages, tissues and after exposure to contaminants such as metals is lacking for many vertebrate species including teleost fish. Therefore, in this study, we assessed the stability of expression of 8 reference gene candidates in the gills and skin of three different early life-stages of rainbow trout after acute exposure (24h) to two metals, cadmium (Cd) and copper (Cu) using qPCR. Candidate housekeeping genes were: beta actin (b-actin), DNA directed RNA polymerase II subunit I (DRP2), elongation factor-1 alpha (EF1a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PD), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein L8 (RPL8), and 18S ribosomal RNA (18S). Four algorithms, geNorm, NormFinder, BestKeeper, and the comparative ΔCt method were employed to systematically evaluate the expression stability of these candidate genes under control and exposed conditions as well as across three different life-stages. Finally, stability of genes was ranked by taking geometric means of the ranks established by the different methods. Stability of reference genes was ranked in the following order (from lower to higher stability): HPRT

An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

OpenAIRE

Itzhaki, H; Maxson, J M; Woodson, W R

1994-01-01

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define ...

Altered choroid plexus gene expression in major depressive disorder

Directory of Open Access Journals (Sweden)

Cortney Ann Turner

2014-04-01

Full Text Available Given the emergent interest in biomarkers for mood disorders, we assessed gene expression in the choroid plexus, the region that produces cerebrospinal fluid (CSF, in individuals with major depressive disorder (MDD. Genes that are expressed in the choroid plexus (CP can be secreted into the CSF and may be potential biomarker candidates. Given that we have previously shown that fibroblast growth factor family members are differentially expressed in post-mortem brain of subjects with MDD and the CP is a known source of growth factors in the brain, we posed the question whether growth factor dysregulation would be found in the CP of subjects with MDD. We performed laser capture microscopy of the choroid plexus at the level of the hippocampus in subjects with MDD and psychiatrically normal controls. We then extracted, amplified, labeled and hybridized the cRNA to Illumina BeadChips to assess gene expression. In controls, the most highly abundant known transcript was transthyretin. Moreover, half of the 14 most highly expressed transcripts in controls encode ribosomal proteins. Using BeadStudio software, we identified 169 transcripts differentially expressed (p< 0.05 between control and MDD samples. Using pathway analysis we noted that the top network altered in subjects with MDD included multiple members of the transforming growth factor-beta (TGFβ pathway. Quantitative real-time PCR (qRT-PCR confirmed downregulation of several transcripts that interact with the extracellular matrix in subjects with MDD. These results suggest that there may be an altered cytoskeleton in the choroid plexus in MDD subjects that may lead to a disrupted blood-CSF-brain barrier.

Stunned Silence: Gene Expression Programs in Human Cells Infected with Monkeypox or Vaccinia Virus

Science.gov (United States)

Rubins, Kathleen H.; Hensley, Lisa E.; Relman, David A.; Brown, Patrick O.

2011-01-01

Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection. PMID:21267444

Rapid evolution of Beta-keratin genes contribute to phenotypic differences that distinguish turtles and birds from other reptiles.

Science.gov (United States)

Li, Yang I; Kong, Lesheng; Ponting, Chris P; Haerty, Wilfried

2013-01-01

Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles.

Differential Gene Expression in Colon Tissue Associated With Diet, Lifestyle, and Related Oxidative Stress.

Directory of Open Access Journals (Sweden)

Martha L Slattery

Full Text Available Several diet and lifestyle factors may impact health by influencing oxidative stress levels. We hypothesize that level of cigarette smoking, alcohol, anti-inflammatory drugs, and diet alter gene expression. We analyzed RNA-seq data from 144 colon cancer patients who had information on recent cigarette smoking, recent alcohol consumption, diet, and recent aspirin/non-steroidal anti-inflammatory use. Using a false discovery rate of 0.1, we evaluated gene differential expression between high and low levels of exposure using DESeq2. Ingenuity Pathway Analysis (IPA was used to determine networks associated with de-regulated genes in our data. We identified 46 deregulated genes associated with recent cigarette use; these genes enriched causal networks regulated by TEK and MAP2K3. Different differentially expressed genes were associated with type of alcohol intake; five genes were associated with total alcohol, six were associated with beer intake, six were associated with wine intake, and four were associated with liquor consumption. Recent use of aspirin and/or ibuprofen was associated with differential expression of TMC06, ST8SIA4, and STEAP3 while a summary oxidative balance score (OBS was associated with SYCP3, HDX, and NRG4 (all up-regulated with greater oxidative balance. Of the dietary antioxidants and carotenoids evaluated only intake of beta carotene (1 gene, Lutein/Zeaxanthine (5 genes, and Vitamin E (4 genes were associated with differential gene expression. There were similarities in biological function of de-regulated genes associated with various dietary and lifestyle factors. Our data support the hypothesis that diet and lifestyle factors associated with oxidative stress can alter gene expression. However genes altered were unique to type of alcohol and type of antioxidant. Because of potential differences in associations observed between platforms these findings need replication in other populations.

Gene expression markers of age-related inflammation in two human cohorts.

Science.gov (United States)

Pilling, Luke C; Joehanes, Roby; Melzer, David; Harries, Lorna W; Henley, William; Dupuis, Josée; Lin, Honghuang; Mitchell, Marcus; Hernandez, Dena; Ying, Sai-Xia; Lunetta, Kathryn L; Benjamin, Emelia J; Singleton, Andrew; Levy, Daniel; Munson, Peter; Murabito, Joanne M; Ferrucci, Luigi

2015-10-01

Chronically elevated circulating inflammatory markers are common in older persons but mechanisms are unclear. Many blood transcripts (>800 genes) are associated with interleukin-6 protein levels (IL6) independent of age. We aimed to identify gene transcripts statistically mediating, as drivers or responders, the increasing levels of IL6 protein in blood at older ages. Blood derived in-vivo RNA from the Framingham Heart Study (FHS, n=2422, ages 40-92 yrs) and InCHIANTI study (n=694, ages 30-104 yrs), with Affymetrix and Illumina expression arrays respectively (>17,000 genes tested), were tested for statistical mediation of the age-IL6 association using resampling techniques, adjusted for confounders and multiple testing. In FHS, IL6 expression was not associated with IL6 protein levels in blood. 102 genes (0.6% of 17,324 expressed) statistically mediated the age-IL6 association of which 25 replicated in InCHIANTI (including 5 of the 10 largest effect genes). The largest effect gene (SLC4A10, coding for NCBE, a sodium bicarbonate transporter) mediated 19% (adjusted CI 8.9 to 34.1%) and replicated by PCR in InCHIANTI (n=194, 35.6% mediated, p=0.01). Other replicated mediators included PRF1 (perforin, a cytolytic protein in cytotoxic T lymphocytes and NK cells) and IL1B (Interleukin 1 beta): few other cytokines were significant mediators. This transcriptome-wide study on human blood identified a small distinct set of genes that statistically mediate the age-IL6 association. Findings are robust across two cohorts and different expression technologies. Raised IL6 levels may not derive from circulating white cells in age related inflammation. Published by Elsevier Inc.

Angiotensin II increases CTGF expression via MAPKs/TGF-{beta}1/TRAF6 pathway in atrial fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Gu, Jun [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University School of medicine, Shanghai (China); Liu, Xu, E-mail: xkliuxu@yahoo.cn [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University School of medicine, Shanghai (China); Wang, Quan-xing, E-mail: shmywqx@126.com [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Tan, Hong-wei [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University School of medicine, Shanghai (China); Guo, Meng [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Jiang, Wei-feng; Zhou, Li [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University School of medicine, Shanghai (China)

2012-10-01

The activation of transforming growth factor-{beta}1(TGF-{beta}1)/Smad signaling pathway and increased expression of connective tissue growth factor (CTGF) induced by angiotensin II (AngII) have been proposed as a mechanism for atrial fibrosis. However, whether TGF{beta}1/non-Smad signaling pathways involved in AngII-induced fibrogenetic factor expression remained unknown. Recently tumor necrosis factor receptor associated factor 6 (TRAF6)/TGF{beta}-associated kinase 1 (TAK1) has been shown to be crucial for the activation of TGF-{beta}1/non-Smad signaling pathways. In the present study, we explored the role of TGF-{beta}1/TRAF6 pathway in AngII-induced CTGF expression in cultured adult atrial fibroblasts. AngII (1 {mu}M) provoked the activation of P38 mitogen activated protein kinase (P38 MAPK), extracellular signal-regulated kinase 1/2(ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). AngII (1 {mu}M) also promoted TGF{beta}1, TRAF6, CTGF expression and TAK1 phosphorylation, which were suppressed by angiotensin type I receptor antagonist (Losartan) as well as p38 MAPK inhibitor (SB202190), ERK1/2 inhibitor (PD98059) and JNK inhibitor (SP600125). Meanwhile, both TGF{beta}1 antibody and TRAF6 siRNA decreased the stimulatory effect of AngII on TRAF6, CTGF expression and TAK1 phosphorylation, which also attenuated AngII-induced atrial fibroblasts proliferation. In summary, the MAPKs/TGF{beta}1/TRAF6 pathway is an important signaling pathway in AngII-induced CTGF expression, and inhibition of TRAF6 may therefore represent a new target for reversing Ang II-induced atrial fibrosis. -- Highlights: Black-Right-Pointing-Pointer MAPKs/TGF{beta}1/TRAF6 participates in AngII-induced CTGF expression in atrial fibroblasts. Black-Right-Pointing-Pointer TGF{beta}1/TRAF6 participates in AngII-induced atrial fibroblasts proliferation. Black-Right-Pointing-Pointer TRAF6 may represent a new target for reversing Ang II-induced atrial fibrosis.

Transcriptome-wide identification of preferentially expressed genes in the hypothalamus and pituitary gland

Directory of Open Access Journals (Sweden)

Jonny eSt-Amand

2012-01-01

Full Text Available To identify preferentially expressed genes in the central endocrine organs of the hypothalamus and pituitary gland, we generated transcriptome-wide mRNA profiles of the mouse hypothalamus, pituitary gland and parietal cortex using serial analysis of gene expression (SAGE. Total counts of SAGE tags for the hypothalamus, pituitary gland and parietal cortex were 165824, 126688 and 161045 tags, respectively. This represented 59244, 45151 and 55131 distinct tags, respectively. Comparison of these mRNA profiles revealed that 22 mRNA species, including three potential novel transcripts, were preferentially expressed in the hypothalamus. In addition to well-known hypothalamic transcripts, such as hypocretin, several genes involved in hormone function, intracellular transduction, metabolism, protein transport, steroidogenesis, extracellular matrix and brain disease were identified as preferentially expressed hypothalamic transcripts. In the pituitary gland, 106 mRNA species, including 60 potential novel transcripts, were preferentially expressed. In addition to well-known pituitary genes, such as growth hormone and thyroid stimulating hormone beta, a number of genes classified to function in transport, amino acid metabolism, intracellular transduction, cell adhesion, disulfide bond formation, stress response, transcription, protein synthesis and turnover, cell differentiation, the cell cycle and in the cytoskeleton and extracellular matrix were also preferentially expressed. In conclusion, the current study identified not only well-known hypothalamic and pituitary transcripts but also a number of new candidates likely to be involved in endocrine homeostatic systems regulated by the hypothalamus and pituitary gland.

Transcriptome-wide identification of preferentially expressed genes in the hypothalamus and pituitary gland.

Science.gov (United States)

St-Amand, Jonny; Yoshioka, Mayumi; Tanaka, Keitaro; Nishida, Yuichiro

2011-01-01

To identify preferentially expressed genes in the central endocrine organs of the hypothalamus and pituitary gland, we generated transcriptome-wide mRNA profiles of the hypothalamus, pituitary gland, and parietal cortex in male mice (12-15 weeks old) using serial analysis of gene expression (SAGE). Total counts of SAGE tags for the hypothalamus, pituitary gland, and parietal cortex were 165824, 126688, and 161045 tags, respectively. This represented 59244, 45151, and 55131 distinct tags, respectively. Comparison of these mRNA profiles revealed that 22 mRNA species, including three potential novel transcripts, were preferentially expressed in the hypothalamus. In addition to well-known hypothalamic transcripts, such as hypocretin, several genes involved in hormone function, intracellular transduction, metabolism, protein transport, steroidogenesis, extracellular matrix, and brain disease were identified as preferentially expressed hypothalamic transcripts. In the pituitary gland, 106 mRNA species, including 60 potential novel transcripts, were preferentially expressed. In addition to well-known pituitary genes, such as growth hormone and thyroid stimulating hormone beta, a number of genes classified to function in transport, amino acid metabolism, intracellular transduction, cell adhesion, disulfide bond formation, stress response, transcription, protein synthesis, and turnover, cell differentiation, the cell cycle, and in the cytoskeleton and extracellular matrix were also preferentially expressed. In conclusion, the current study identified not only well-known hypothalamic and pituitary transcripts but also a number of new candidates likely to be involved in endocrine homeostatic systems regulated by the hypothalamus and pituitary gland.

Mural granulosa cell gene expression associated with oocyte developmental competence

Directory of Open Access Journals (Sweden)

Jiang Jin-Yi

2010-03-01

Full Text Available Abstract Background Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. Methods Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC. Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. Results The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox and nerve growth factor receptor associated protein 1 (Ngfrap1, which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2, which is involved in the regulation of extracellular matrix organization and biogenesis. Conclusions The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and

Identification of distal regulatory regions in the human alpha IIb gene locus necessary for consistent, high-level megakaryocyte expression.

Science.gov (United States)

Thornton, Michael A; Zhang, Chunyan; Kowalska, Maria A; Poncz, Mortimer

2002-11-15

The alphaIIb/beta3-integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of alphaIIb, and studies of the alphaIIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the alphaIIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) alphaIIb loci revealed high levels of conservation at intergenic regions both 5' and 3' to the alphaIIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive (HS) sites that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the h(alpha)IIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue specificity, but that a region 2.5 to 7.1 kb upstream of the h(alpha)IIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold and led to levels of h(alpha)IIb mRNA that were about 30% of the native m(alpha)IIb mRNA level. These constructs also resulted in detectable h(alpha)IIb/m(beta)3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the alphaIIb gene for tissue specificity, but also characterizes the distal organization of the alphaIIb gene locus and provides an initial localization of 2 important regulatory regions needed for the expression of the alphaIIb gene at high levels during megakaryopoiesis.

Gene expression demonstrates an immunological capacity of the human endolymphatic sac

DEFF Research Database (Denmark)

Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

2015-01-01

OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the innate immune system in the human endolymphatic sac. It is hypothesized that the endolymphatic sac has a significant immunological function in the human inner ear...... was obtained. Multiple key elements of both the cellular and humoral innate immune system were expressed, including Toll-like receptors 4 and 7, as well as beta-defensin and lactoferrin. CONCLUSIONS: The present data provides the first direct evidence of an immunological capacity of the human endolymphatic sac...... immunological entity of the inner ear. LEVEL OF EVIDENCE: N/A....

Expression of genes related to the hypothalamic-pituitary-adrenal axis in murine fetal lungs in late gestation

Directory of Open Access Journals (Sweden)

Côté Mélissa

2010-11-01

Full Text Available Abstract Background Lung maturation is modulated by several factors, including glucocorticoids. Expression of hypothalamic-pituitary-adrenal (HPA axis-related components, with proposed or described local regulatory systems analogous to the HPA axis, was reported in peripheral tissues. Here, HPA axis-related genes were studied in the mouse developing lung during a period overlapping the surge of surfactant production. Methods Expression of genes encoding for corticotropin-releasing hormone (CRH, CRH receptors (CRHR 1 and 2beta, CRH-binding protein, proopiomelanocortin (POMC, melanocortin receptor 2 (MC2R, and glucocorticoid receptor was quantified by real-time PCR and localized by in situ hydridization in fetal lungs at gestational days (GD 15.5, 16.5, and 17.5, and was also quantified in primary mesenchymal- and epithelial cell-enriched cultures. In addition, the capability of CRH and adrenocorticotropic hormone (ACTH to stimulate pulmonary expression of enzymes involved in the adrenal pathway of glucocorticoid synthesis was addressed, as well as the glucocorticoid production by fetal lung explants. Results We report that all the studied genes are expressed in fetal lungs according to different patterns. On GD 15.5, Mc2r showed peaks in expression in samples that have previously presented high mRNA levels for glucocorticoid synthesizing enzymes, including 11beta-hydroxylase (Cyp11b1. Crhr1 mRNA co-localized with Pomc mRNA in cells surrounding the proximal epithelium on GD 15.5 and 16.5. A transition in expression sites toward distal epithelial cells was observed between GD 15.5 and 17.5 for all the studied genes. CRH or ACTH stimulation of genes involved in the adrenal pathway of glucocorticoid synthesis was not observed in lung explants on GD 15.5, whereas CRH significantly increased expression of 21-hydroxylase (Cyp21a1 on GD 17.5. A deoxycorticosterone production by fetal lung explants was observed. Conclusions Temporal and spatial

IL-1beta signals through the EGF receptor and activates Egr-1 through MMP-ADAM.

Directory of Open Access Journals (Sweden)

Estella Sanchez-Guerrero

Full Text Available The immediate-early gene Egr-1 controls the inducible expression of many genes implicated in the pathogenesis of a range of vascular disorders, yet our understanding of the mechanisms controlling the rapid expression of this prototypic zinc finger transcription factor is poor. Here we show that Egr-1 expression induced by IL-1beta is dependent on metalloproteinases (MMP and a disintegrin and a metalloproteinase (ADAM. Pharmacologic MMP/ADAM inhibitors and siRNA knockdown prevent IL-1beta induction of Egr-1. Further, IL-1beta activates Egr-1 via the epidermal growth factor receptor (EGFR. This is blocked by EGFR tyrosine kinase inhibition and EGFR knockdown. IL-1beta induction of Egr-1 expression is reduced in murine embryonic fibroblasts (mEFs deficient in ADAM17 despite unbiased expression of EGFR and IL-1RI in ADAM17-deficient and wild-type mEFs. Finally, we show that IL-1beta-inducible wound repair after mechanical injury requires both EGFR and MMP/ADAM. This study reports for the first time that Egr-1 induction by IL-1beta involves EGFR and MMP/ADAM-dependent EGFR phosphorylation.

Expression of the benign HEXA mutations, Arg247Trp and Arg249Trp, associated with beta-hexosaminidase A pseudodeficiency

Energy Technology Data Exchange (ETDEWEB)

Cao, Z.; Petroulakis, E.; Salo, T. [Univ. of Manitoba (Canada)] [and others

1994-09-01

{beta}-Hexosaminidase (Hex A) is a heterodimer of {alpha} and {beta} subunits encoded by the HEXA and HEXB genes, respectively. Mutations in the HEXA gene typically cause Tay-Sachs disease or less severe forms of G{sub M2} gangliosidosis. However, two benign mutations (Arg247Trp and Arg249Trp) in the {alpha}-subunit of Hex A account for Hex A deficiency in {approximately}36% of non-Jewish enzyme-defined Tay-Sachs disease carriers. These mutations do not result in any apparent clinical phenotype in individuals who are genetic compounds with a second disease-causing mutation. We expressed the {alpha}-subunit harboring each of the benign mutations separately to study activity toward the synthetic substrate, 4-MUGS, for comparison to activity from enzymes containing mutations associated with other forms of G{sub M2} gangliosidosis. The C739T (Arg247Trp;benign), C745T (Arg 249Trp; benign), G805A (Gly269Ser; adult-onset), G749A (Gly250Asp; juvenile), and C508T (Arg170Trp; infantile) mutations were introduced into the {alpha}-subunit cDNA. These were transfected alone, or with the {beta}-subunit cDNA, to generate Hex S ({alpha}{alpha}) or Hex A ({alpha}{beta}), respectively. The activities were monitored using 4-MUGS, and the levels of {alpha}-subunit protein were assessed by Western blotting. Repeated experiments show that the benign mutations produce approximately 35% of normal Hex S and 40% of normal Hex A activity. This level is much higher than that of Hex A harbouring the Gly169Ser adult-onset mutation (12%). A sequential decrease in expressed Hex A activity is observed as mutations associated with more severe phenotypes are expressed. The benign mutations also result in lower levels of mature {alpha}-subunit protein compared to normal, and slightly reduced levels of {alpha}-subunit precursor protein. The Hex A deficiency resulting from benign mutations is not as great as that associated with disease-causing mutations.

Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

Energy Technology Data Exchange (ETDEWEB)

Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae [Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Sang Chul, E-mail: scpark@snu.ac.kr [Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

2010-01-01

One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin {alpha}, karyopherin {beta}, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

Expression of interleukin-1 beta in rat dorsal root ganglia

NARCIS (Netherlands)

Copray, JCVM; Mantingh, [No Value; Brouwer, N; Biber, K; Kust, BM; Liem, RSB; Huitinga, [No Value; Tilders, FJH; Van Dam, AM; Boddeke, HWGM

2001-01-01

The expression of interleukin-lp was examined in dorsal root ganglion (DRG) neurons from adult rats using non-radioactive in Situ hybridization and immunocytochemistry. At all spinal levels, approximately 70% of the DRG neurons appeared to express IL-1 beta mRNA: about 80% of these DRG neurons

Murrayafoline A attenuates the Wnt/{beta}-catenin pathway by promoting the degradation of intracellular {beta}-catenin proteins

Energy Technology Data Exchange (ETDEWEB)

Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Lee, Gye Won [Department of Pharmaceutical Engineering, Konyang University, Nonsan 320-711 (Korea, Republic of); Yun, Mi-Young [Department of Beauty Health Care, Daejeon University, Daejeon 305-764 (Korea, Republic of); Cuong, Nguyen Manh [Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi (Viet Nam); Shin, Jae-Gook [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Song, Gyu-Yong, E-mail: gysong@cnu.ac.kr [College of Pharmacy, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Oh, Sangtaek, E-mail: ohsa@inje.ac.kr [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)

2010-01-01

Molecular lesions in Wnt/{beta}-catenin signaling and subsequent up-regulation of {beta}-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3{beta} (GSK-3{beta}), and promoted the degradation of intracellular {beta}-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known {beta}-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

Glucocorticoid effects on the programming of AT1b angiotensin receptor gene methylation and expression in the rat.

Directory of Open Access Journals (Sweden)

Irina Bogdarina

2010-02-01

Full Text Available Adverse events in pregnancy may 'programme' offspring for the later development of cardiovascular disease and hypertension. Previously, using a rodent model of programmed hypertension we have demonstrated the role of the renin-angiotensin system in this process. More recently we showed that a maternal low protein diet resulted in undermethylation of the At1b angiotensin receptor promoter and the early overexpression of this gene in the adrenal of offspring. Here, we investigate the hypothesis that maternal glucocorticoid modulates this effect on fetal DNA methylation and gene expression. We investigated whether treatment of rat dams with the 11beta-hydroxylase inhibitor metyrapone, could prevent the epigenetic and gene expression changes we observed. Offspring of mothers subjected to a low protein diet in pregnancy showed reduced adrenal Agtr1b methylation and increased adrenal gene expression as we observed previously. Treatment of mothers with metyrapone for the first 14 days of pregnancy reversed these changes and prevented the appearance of hypertension in the offspring at 4 weeks of age. As a control for non-specific effects of programmed hypertension we studied offspring of mothers treated with dexamethasone from day 15 of pregnancy and showed that, whilst they had raised blood pressure, they failed to show any evidence of Agtr1b methylation or increase in gene expression. We conclude that maternal glucocorticoid in early pregnancy may induce changes in methylation and expression of the Agtr1b gene as these are clearly reversed by an 11 beta-hydroxylase inhibitor. However in later pregnancy a converse effect with dexamethasone could not be demonstrated and this may reflect either an alternative mechanism of this glucocorticoid or a stage-specific influence.

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

Science.gov (United States)

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Cytokine gene expression profiles in chicken spleen and intestinal tissues during Ascaridia galli infection

DEFF Research Database (Denmark)

Pleidrup, Janne A.; Norup, Liselotte R.; Dalgaard, Tina S.

2014-01-01

In the poultry production industry, chickens with access to outdoor areas are exposed to a wide range of parasites e.g. the helminth Ascaridia galli. By real-time quantitative RTPCR, the relative gene expression of the T helper 1 (Th1) cytokine IFN-gamma, the T helper 2 (Th2) cytokine IL-13...... expression of jejunal IFN-gamma and IL-13 was observed. Finally, at the expected period of an adaptive immune response (days 14-21) a general decreased expression of IFN-gamma and TGF-beta 4 in spleen and IFN-gamma in jejunum was followed by a decreased expression of IFN-gamma and IL-10 at day 21 in caecal...

Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

Science.gov (United States)

Ghaye, Aurélie P; Bergemann, David; Tarifeño-Saldivia, Estefania; Flasse, Lydie C; Von Berg, Virginie; Peers, Bernard; Voz, Marianne L; Manfroid, Isabelle

2015-09-02

In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In

GENE EXPRESSION DYNAMICS IN PATIENTS WITH SEVERE THERAPY-RESISTANT ASTHMA DURING TREATMENT PERIOD

Directory of Open Access Journals (Sweden)

Ye. S. Kulikov

2014-01-01

Full Text Available Introduction: The leading mechanisms and causes of severe therapy resistant asthma are poorly understood. The aim of this study was to define global patterns of gene expression in adults with severe therapy-resistant asthma in dynamic during treatment period.Methods: Performed 24-week prospective interventional study in parallel groups. Severe asthma patients was aposterior divided at therapy sensitive and resistant patients according to ATS criteria. Global transcriptome profile was characterized using the Affymetrix HuGene ST1.0 chip. Cluster analysis was performed.Results and conclusion: According to our data several mechanisms of therapy resistance may be considered: increased levels of nitric oxide and beta2-agonists nitration, dysregulation of endogenous steroids secretion and involvement in the pathogenesis of Staphylococcus aureus. Absence of suppression of gene expression KEGG-pathway “asthma" may reflect the low efficiency or long period of anti-inflammatory therapy effect realization.

Housekeeping gene expression during fetal brain development in the rat-validation by semi-quantitative RT-PCR.

Science.gov (United States)

Al-Bader, Maie Dawoud; Al-Sarraf, Hameed Ali

2005-04-21

Mammalian gene expression is usually carried out at the level of mRNA where the amount of mRNA of interest is measured under different conditions such as growth and development. It is therefore important to use a "housekeeping gene", that does not change in relative abundance during the experimental conditions, as a standard or internal control. However, recent data suggest that expression of some housekeeping genes may vary with the extent of cell proliferation, differentiation and under various experimental conditions. In this study, the expression of various housekeeping genes (18S rRNA [18S], glyceraldehydes-3-phosphate dehydrogenase [G3PDH], beta-glucuronidase [BGLU], histone H4 [HH4], ribosomal protein L19 [RPL19] and cyclophilin [CY]) was investigated during fetal rat brain development using semi-quantitative RT-PCR at 16, 19 and 21 days gestation. It was found that all genes studied, with exception to G3PDH, did not show any change in their expression levels during development. G3PDH, on the other hand, showed increased expression with development. These results suggest that the choice of a housekeeping gene is critical to the interpretation of experimental results and should be modified according to the nature of the study.

Prebiotic and Synbiotic Modifications of Beta Oxidation and Lipogenic Gene Expression after Experimental Hypercholesterolemia in Rat Liver

Directory of Open Access Journals (Sweden)

Claudia C. Alves

2017-10-01

Full Text Available Background and aims: Non-alcoholic fatty liver disease (NAFLD is characterized by the presence of fat in hepatocytes because of decreased β-oxidation and increased lipogenesis. Prebiotics, probiotics, and synbiotic have modulatory effects on intestinal microbiota and may influence the gut-liver axis. Our aim was to evaluate the effects of prebiotic, probiotics, and synbiotic on liver histopathology and gene expression related to β-oxidation and lipogenesis after hypercholesterolemia.Methods: Wistar male adult rats (n = 40 were submitted to hypercholesterolemic conditions (HPC (60 days. On Day 30 of HPC, rats were subdivided in 5 groups: negative control (NC: without HPC + Gv (distilled water; positive control (PC: with HPC + Gv (distilled water; prebiotic (PRE: HPC + Gv with prebiotic (Fiber FOS®; probiotic (PRO: HPC + Gv with probiotic strains Gv (Probiatop®; and synbiotic (SYN: HPC + Gv with synbiotic (Simbioflora®. All rats were sacrificed on Day 30 post-treatment. Blood was collected to verify total serum cholesterol, and liver tissue was sampled to verify histopathological changes and gene expression. Gene expression related to ß-oxidation (PPAR-α and CPT-1 and lipogenesis (SREBP-1c, FAS and ME was evaluated in liver tissue using RT-qPCR.Results: PC had higher cholesterol levels when compared to NC. PRE and SYN rats had lower cholesterol levels than PC. PC rats showed more histopathological changes than NC rats; PRE and SYN rats showed fewer alterations than PC rats. PPAR-α was expressed at higher levels in SYN and PC rats compared with PRE and PRO rats. CPT-1 expression was similar in all groups. SREBP-1c was expressed at higher levels in PC rats compared with NC rats; levels were lower in SYN rats compared with PRO rats; levels were lower in PRE rats compared with PC and PRO rats. FAS was expressed at lower levels in PRE rats compared with SYN rats. ME expression was lower in PC rats compared with NC rats.Conclusion: Prebiotic and

Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

International Nuclear Information System (INIS)

Krutmann, J.; Koeck, A.S.; Schauer, E.; Parlow, F.; Moeller, A.K.; Kapp, A.; Foerster, E.S.; Schoepf, E.L.; Luger, T.A.

1990-01-01

Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC

Characterization of a large deletion in the {beta}-globin gene cluster in a newborn with hemoglobin FE

Energy Technology Data Exchange (ETDEWEB)

Louie, E.; Dietz, L.; Shafer, F. [Children`s Hosptial, Oakland, CA (United States)] [and others

1994-09-01

A sample on a newborn with hemoglobin FE screen results was obtained to investigate whether E/E or B/{beta}{degrees} thalassemia was present using polymerase chain reaction (PCR) methodology. The newborn appeared homozygous for the hemoglobin E mutation in our initial study, but the parents` genotypes did not support this diagnosis. The father is homozygous for the absence of the hemoglobin E mutation (non E/non E) and the mother is heterozygous (E/non E) for this mutation. The limitation of PCR analysis is an assumption that the amplification of the two {beta}-globin alleles is equivalent. A large deletion on one {beta}-globin gene, which would produce E/{beta}{degrees} thalassemia, would be missed if it included part or the entire region subjected to amplification. The family results were consistent with either non-paternity, sample mix-up or such a deletion of the {beta}-globin gene in the father and child. To rule out the possibility of non-paternity, two polymorphic loci (HLA on chromosome 6 and a VNTR system of chromosome 17) that are outside of the {beta}-globin gene were analyzed and show that inheritance is consistent and the likelihood of a sample mix-up is then reduced. We therefore believe there is a gene deletion in this family. At the present time, analyses of the RFLPs that are 5{prime} of the {beta}-globin gene cluster show that the polymorphisms most distal from the 5{prime} {beta}-globin gene are not being inherited as expected. These results support our interpretation that a deletion exists in the father and was inherited by the child. The father`s clinical picture of possible HPFH (the father has 12% hemoglobin F) also supports the interpretation of a deletion in this family. Deletions of the {beta}-globin gene within this ethnic group are rare. Currently, Southern blots on the family are being probed to determine the extent of the putative deletion.

The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition.

Science.gov (United States)

Schneider, P M; Witzel-Schlömp, K; Rittner, C; Zhang, L

2001-02-01

Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.

FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data

DEFF Research Database (Denmark)

Manijak, Mieszko P.; Nielsen, Henrik Bjørn

2011-01-01

circumvented by instead matching gene expression signatures to signatures of other experiments. FINDINGS: To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700...... Arabidopsis microarray experiments. CONCLUSIONS: Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/....

Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

DEFF Research Database (Denmark)

Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S

2008-01-01

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...

Expression of beta-catenin, COX-2 and iNOS in colorectal cancer: relevance of COX-2 adn iNOS inhibitors for treatment in Malaysia.

Science.gov (United States)

Hong, Seok Kwan; Gul, Yunus A; Ithnin, Hairuszah; Talib, Arni; Seow, Heng Fong

2004-01-01

Promising new pharmacological agents and gene therapy targeting cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) could modulate treatment of colorectal cancer in the future. The aim of this study was to elucidate the expression fo beta-catenin and teh presence of COX-2 and iNOS in colorectal cancer specimens in Malaysia. This is a useful prelude to future studies investigating interventions directed towards COX-2 adn iNOS. A cross-section study using retrospective data over a 2-year period (1999-2000) involved 101 archival, formalin-fixed, paraffin-embedded tissue samples of colorectal cancers that were surgically resected in a tertiary referral. COX-2 production was detected in adjacent normal tissue in 34 sample (33.7%) and in tumour tissue in 60 samples (59.4%). More tumours expressed iNOS (82/101, 81.2%) than COX-2. No iNOS expression was detected in adjacent normal tissue. Intense beta-catenin immunoreactivity at the cell-to-cell border. Poorly differentiated tumours had significantly lower total beta-catenin (p = 0.009) and COX-2 scores (p = 0.031). No significant relationships were established between pathological stage and beta-catenin, COX-2 and iNOS scores. the accumulation of beta-catenin does not seem to be sufficient to activate pathways that lead to increased COX-2 and iNOS expression. A high proportion of colorectal cancers were found to express COX-2 and a significant number produced iNOS, suggesting that their inhibitors may be potentially useful as chemotherapeutic agents in the management of colorectal cancer.

Digital gene expression analysis of gene expression differences within Brassica diploids and allopolyploids.

Science.gov (United States)

Jiang, Jinjin; Wang, Yue; Zhu, Bao; Fang, Tingting; Fang, Yujie; Wang, Youping

2015-01-27

Brassica includes many successfully cultivated crop species of polyploid origin, either by ancestral genome triplication or by hybridization between two diploid progenitors, displaying complex repetitive sequences and transposons. The U's triangle, which consists of three diploids and three amphidiploids, is optimal for the analysis of complicated genomes after polyploidization. Next-generation sequencing enables the transcriptome profiling of polyploids on a global scale. We examined the gene expression patterns of three diploids (Brassica rapa, B. nigra, and B. oleracea) and three amphidiploids (B. napus, B. juncea, and B. carinata) via digital gene expression analysis. In total, the libraries generated between 5.7 and 6.1 million raw reads, and the clean tags of each library were mapped to 18547-21995 genes of B. rapa genome. The unambiguous tag-mapped genes in the libraries were compared. Moreover, the majority of differentially expressed genes (DEGs) were explored among diploids as well as between diploids and amphidiploids. Gene ontological analysis was performed to functionally categorize these DEGs into different classes. The Kyoto Encyclopedia of Genes and Genomes analysis was performed to assign these DEGs into approximately 120 pathways, among which the metabolic pathway, biosynthesis of secondary metabolites, and peroxisomal pathway were enriched. The non-additive genes in Brassica amphidiploids were analyzed, and the results indicated that orthologous genes in polyploids are frequently expressed in a non-additive pattern. Methyltransferase genes showed differential expression pattern in Brassica species. Our results provided an understanding of the transcriptome complexity of natural Brassica species. The gene expression changes in diploids and allopolyploids may help elucidate the morphological and physiological differences among Brassica species.

Gene expression profiling in limb-girdle muscular dystrophy 2A.

Directory of Open Access Journals (Sweden)

Amets Sáenz

Full Text Available Limb-girdle muscular dystrophy type 2A (LGMD2A is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3. Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens, cell adhesion (fibronectin, muscle development (myosins and melusin and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB is upregulated in LGMD2A muscle samples, it could be hypothesized that beta-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1. Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies.

CISH has no non-redundant functions in glucose homeostasis or beta cell proliferation during pregnancy in mice.

Science.gov (United States)

Jiao, Yang; Rieck, Sebastian; Le Lay, John; Kaestner, Klaus H

2013-11-01

Increased beta cell proliferation during pregnancy is mediated by the Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signalling pathway in response to increased lactogen levels. Activation of the pathway leads to transcriptional upregulation of Cish (encoding cytokine-inducible SH2 domain-containing protein), a member of the suppressor of cytokine signalling (SOCS) family of genes, forming a negative-feedback loop. Here, we examined whether conditional gene ablation of Cish in the pancreas improves beta cell proliferation and beta cell function during pregnancy in mice. We derived mice with a novel, conditional loxP allele for Cish. Pancreas-specific ablation of Cish was achieved by crossing Cish (loxP/loxP) mice with Pdx1-Cre (Early) mice. Beta cell proliferation was quantified by BrdU labelling. Glucose homeostasis was examined with glucose tolerance tests and determination of plasma insulin levels. The expression of other Socs genes and target genes of p-STAT5 related to beta cell function and beta cell proliferation was determined by quantitative PCR. There was no difference in beta cell proliferation or glucose homeostasis between the Cish mutant group and the control group. The p-STAT5 protein level was the same in Cish mutant and control mice. Socs2 gene expression was higher in Cish mutant than control mice at pregnancy day 9.5. The expression of other Socs genes was the same between control and mutant mice. Our results show that CISH has no non-redundant functions in beta cell proliferation or glucose homeostasis during pregnancy in mice. Socs2 might compensate for the loss of Cish during pregnancy.

Stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus.

Directory of Open Access Journals (Sweden)

Kathleen H Rubins

2011-01-01

Full Text Available Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV, an emerging human pathogen, and Vaccinia virus (VAC, a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA, or poly (I-C was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai.

Science.gov (United States)

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-11-18

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%-3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai

Directory of Open Access Journals (Sweden)

Mi-Jin Choi

2015-11-01

Full Text Available The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%–3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3, vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

Energy Technology Data Exchange (ETDEWEB)

Hong, Wei, E-mail: hongwei@tijmu.edu.cn [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Li, Jinru; Wang, Bo [College of Basic Medicine, Tianjin Medical University, 300070 Tianjin (China); Chen, Linfeng [Department of Medical Oncology, Harvard Medical School, Dana Farber Cancer Institute, Boston, 02115 MA (United States); Niu, Wenyan; Yao, Zhi [Department of Immunology, Tianjin Medical University, 300070 Tianjin (China); Baniahmad, Aria, E-mail: aban@mti.uni-jena.de [Institute for Human Genetics, Jena University Hospital, 07740 Jena (Germany)

2011-12-02

Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by which Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.

The expression of myosin genes in developing skeletal muscle in the mouse embryo

International Nuclear Information System (INIS)

Lyons, G.E.; Ontell, M.; Cox, R.; Sassoon, D.; Buckingham, M.

1990-01-01

Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and acculumation

Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

Science.gov (United States)

Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

2014-05-01

Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Increased expression of alpha- and beta-globin mRNAs at the pituitary following exposure to estrogen during the critical period of neonatal sex differentiation in the rat

DEFF Research Database (Denmark)

Leffers, H; Navarro, V M; Nielsen, John E

2006-01-01

Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element in the neuroe......Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element......, we screened for differentially expressed genes at the pituitary and hypothalamus of rats after neonatal exposure to estradiol benzoate. Our analyses identified persistent up-regulation of alpha- and beta-globin mRNAs at the pituitary following neonatal estrogenization. This finding was confirmed...... by combination of RT-PCR analyses and in situ hybridization. Induction of alpha- and beta-globin mRNA expression at the pituitary by neonatal exposure to estrogen was demonstrated as dose-dependent and it was persistently detected up to puberty. In contrast, durable up-regulation of alpha- and beta-globin genes...

In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs.

Science.gov (United States)

Chung, Jin Young; Choi, Jung Hoon; Shin, Il Seob; Choi, Eun Wha; Hwang, Cheol Yong; Lee, Sang Koo; Youn, Hwa Young

2008-12-01

Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

Gonadotropin-releasing hormone regulates expression of the DNA damage repair gene, Fanconi anemia A, in pituitary gonadotroph cells.

Science.gov (United States)

Larder, Rachel; Chang, Lynda; Clinton, Michael; Brown, Pamela

2004-09-01

Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse L beta T2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of L beta T2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature alpha T3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA.

Advanced Glycation End-Products affect transcription factors regulating insulin gene expression

International Nuclear Information System (INIS)

Puddu, A.; Storace, D.; Odetti, P.; Viviani, G.L.

2010-01-01

Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic β-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation prevents FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.

Molecular characterization of cDNAs encoding G protein alpha and beta subunits and study of their temporal and spatial expression patterns in Nicotiana plumbaginifolia Viv.

Science.gov (United States)

Kaydamov, C; Tewes, A; Adler, K; Manteuffel, R

2000-04-25

We have isolated cDNA sequences encoding alpha and beta subunits of potential G proteins from a cDNA library prepared from somatic embryos of Nicotiana plumbaginifolia Viv. at early developmental stages. The predicted NPGPA1 and NPGPB1 gene products are 75-98% identical to the known respective plant alpha and beta subunits. Southern hybridizations indicate that NPGPA1 is probably a single-copy gene, whereas at least two copies of NPGPB1 exist in the N. plumbaginifolia genome. Northern analyses reveal that both NPGPA1 and NPGPB1 mRNA are expressed in all embryogenic stages and plant tissues examined and their expression is obviously regulated by the plant hormone auxin. Immunohistological localization of NPGPalpha1 and NPGPbeta1 preferentially on plasma and endoplasmic reticulum membranes and their immunochemical detection exclusively in microsomal cell fractions implicate membrane association of both proteins. The temporal and spatial expression patterns of NPGPA1 and NPGPB1 show conformity as well as differences. This could account for not only cooperative, but also individual activities of both subunits during embryogenesis and plant development.

Modulation of hepatocyte growth factor gene expression by estrogen in mouse ovary.

Science.gov (United States)

Liu, Y; Lin, L; Zarnegar, R

1994-09-01

Hepatocyte growth factor (HGF) is expressed in a variety of tissues and cell types under normal conditions and in response to various stimuli such as tissue injury. In the present study, we demonstrate that the transcription of the HGF gene is stimulated by estrogen in mouse ovary. A single injection of 17 beta-estradiol results in a dramatic and transient elevation of the levels of mouse HGF mRNA. Sequence analysis has found that two putative estrogen responsive elements (ERE) reside at -872 in the 5'-flanking region and at +511 in the first intron, respectively, of the mouse HGF gene. To test whether these ERE elements are responsible for estrogen induction of HGF gene expression, chimeric plasmids containing variable regions of the 5'-flanking sequence of HGF gene and the coding region for chloramphenicol acetyltransferase (CAT) gene were transiently transfected into both human endometrial carcinoma RL 95-2 cells and mouse fibroblast NIH 3T3 cells to assess hormone responsiveness. Transfection results indicate that the ERE elements of the mouse HGF gene can confer estrogen action to either homologous or heterologous promoters. Nuclear protein extracts either from RL95-2 cells transfected with the estrogen receptor expression vector or from mouse liver bound in vitro to ERE elements specifically, as shown by band shift assay. Therefore, our results demonstrate that the HGF gene is transcriptionally regulated by estrogen in mouse ovary; and such regulation is mediated via a direct interaction of the estrogen receptor complex with cis-acting ERE elements identified in the mouse HGF gene.

Perinatal exposure to diesel exhaust affects gene expression in mouse cerebrum

Energy Technology Data Exchange (ETDEWEB)

Tsukue, Naomi [Tokyo University of Science, Department of Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Noda, Chiba (Japan); Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Kawaguchi, Saitama (Japan); Japan Automobile Research Institute, Health Effects Research Group, Energy and Environment Research Division, Tsukuba, Ibaraki (Japan); Watanabe, Manabu; Kumamoto, Takayuki; Takeda, Ken [Tokyo University of Science, Department of Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Noda, Chiba (Japan); Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Kawaguchi, Saitama (Japan); Takano, Hirohisa [Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Kawaguchi, Saitama (Japan); National Institute for Environmental Studies, Pathophysiology Research Team, Tsukuba, Ibaraki (Japan)

2009-11-15

Many environmental toxins alter reproductive function and affect the central nervous system (CNS). Gonadal steroid hormones cause differentiation of neurons and affect brain function and behavior during the perinatal period, and the CNS is thought to be particularly susceptible to toxic insult during this period. It was, therefore, hypothesized that inhalation of diesel exhaust (DE) during the fetal or suckling period would disrupt the sexual differentiation of brain function in mice, and the effects of exposure to DE during the perinatal period on sexual differentiation related gene expression of the brain were investigated. In the fetal period exposure group, pregnant ICR mice were exposed to DE from 1.5 days post-coitum (dpc) until 16 dpc. In the neonatal period exposure group, dams and their offspring were exposed to DE from the day of birth [postnatal day (PND)-0] until PND-16. Then, the cerebrums of males and females at PND-2, -5, and -16 from both groups were analyzed for expression level of mRNA encoding stress-related proteins [cytochrome P450 1A1 (CYP1A1), heme oxygenase-1 (HO-1)] and steroid hormone receptors [estrogen receptor alpha (ER alpha), estrogen receptor beta (ER beta), androgen receptor (AR)]. Expression levels of ER alpha and ER beta mRNA were increased in the cerebrum of newborns in the DE exposure groups as well as mRNA for CYP1A1 and HO-1. Results indicate that perinatal exposure to DE during the critical period of sexual differentiation of the brain may affect endocrine function. (orig.)

Large scale gene expression meta-analysis reveals tissue-specific, sex-biased gene expression in humans

Directory of Open Access Journals (Sweden)

Benjamin Mayne

2016-10-01

Full Text Available The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analysed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes, followed by the heart (375 genes, kidney (224 genes, colon (218 genes and thyroid (163 genes. More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases.

Steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation

Directory of Open Access Journals (Sweden)

Gisele Negro Lima

2015-02-01

Full Text Available OBJECTIVE: To analyze steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation. METHODS: Thirty-two virgin adult female rats were randomized to two groups as follows: the control group GI received vehicle and the experimental group GII received melatonin supplementation (10 µg/night per animal for 60 consecutive days. After the treatment, animals were anesthetized and the collected ovaries were immediately placed in liquid nitrogen for complementary deoxyribonucleic acid microarray analyses. A GeneChip¯ Kit Rat Genome 230 2.0 Affymetrix Array was used for gene analysis and the experiment was repeated three times for each group. The results were normalized with the GeneChip¯ Operating Software program and confirmed through analysis with the secondary deoxyribonucleic acid-Chip Analyzer (dChip software. The data were confirmed by real-time reverse transcription polymerase chain reaction analysis. Genes related to ovarian function were further confirmed by immunohistochemistry. RESULTS: We found the upregulation of the type 9 adenylate cyclase and inhibin beta B genes and the downregulation of the cyclic adenosine monophosphate response element modulator and cytochrome P450 family 17a1 genes in the ovarian tissue of GII compared to those of the control group. CONCLUSION: Our data suggest that melatonin supplementation decreases gene expression of cyclic adenosine monophosphate, which changes ovarian steroidogenesis.

CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

Science.gov (United States)

Christenson, L K; Johnson, P F; McAllister, J M; Strauss, J F

1999-09-10

Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

RFLP for the human retinoic acid receptor gene RAR-. beta

Energy Technology Data Exchange (ETDEWEB)

Datson, N A; Oostra, B A [Erasmus Univ., Rotterdam (Netherlands); van der Saag, P T [Netherlands Institute for Developmental Biology, Utrecht (Netherlands)

1989-11-11

1.4 kb Mae I fragment containing the entire RAR-{beta} ORF was cloned into the Sma I site of pTZ18U, yielding the plasmid pCOD20. Msp I digestion of genomic DNA and hybridization with the pCOD20 probe detects a two allele polymorphism with allelic fragments of 8.1 and 7.7 kb. The human RAR-{beta} gene has been localized to the p24 band of chromosome 3. Co-dominant segregation of the alleles was observed in 4 Caucasian families.

Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

Energy Technology Data Exchange (ETDEWEB)

Sopjani, Mentor [Department of Physiology, University of Tuebingen (Germany); Department of Chemistry, University of Prishtina, Kosovo (Country Unknown); Alesutan, Ioana; Wilmes, Jan [Department of Physiology, University of Tuebingen (Germany); Dermaku-Sopjani, Miribane [Department of Physiology, University of Tuebingen (Germany); Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Lam, Rebecca S. [Department of Physiology, University of Tuebingen (Germany); Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main (Germany); Koutsouki, Evgenia [Department of Physiology, University of Tuebingen (Germany); Jakupi, Muharrem [Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Foeller, Michael [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

2010-11-19

Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

The Impact of Oxytocin Gene Knockout on Sexual Behavior and Gene Expression Related to Neuroendocrine Systems in the Brain of Female Mice.

Science.gov (United States)

Zimmermann-Peruzatto, Josi Maria; Lazzari, Virgínia Meneghini; Agnes, Grasiela; Becker, Roberta Oriques; de Moura, Ana Carolina; Guedes, Renata Padilha; Lucion, Aldo Bolten; Almeida, Silvana; Giovenardi, Márcia

2017-07-01

Social relations are built and maintained from the interaction among individuals. The oxytocin (OT), vasopressin (VP), estrogen, dopamine, and their receptors are involved in the modulation of sexual behavior in females. This study aimed to analyze the impact of OT gene knockout (OTKO) on sexual behavior and the gene expression of oxytocin (OTR), estrogen alpha (ERα), estrogen beta (ERβ), vasopressin (V 1a R), and dopamine (D 2 R) receptors in the olfactory bulb (OB), prefrontal cortex (PFC), hippocampus (HPC), and hypothalamus (HPT), as well as in the synthesis of VP in the HPT of female mice. Wild-type (WT) littermates were used for comparisons. The C DNAs were synthesized by polymerase chain reaction and the gene expression was calculated with the 2 -ΔΔCt formula. Our results showed that the absence of OT caused an increase in the frequency and duration of non-receptive postures and a decrease in receptive postures in the OTKO. OTKO females showed a significant decrease in the gene expression of OTR in the HPC, V 1a R in the HPT, and ERα and ERβ in the PFC. There was no significant difference in the gene expression of D 2 R of OTKO. However, OTKO showed an increased gene expression of V 1a R in the HPC. There is no significant difference in VP mRNA synthesis in the HPT between OTKO and WT. Our findings demonstrate that the absence of OT leads to significant changes in the expression of the studied genes (OTR, ERα, ERβ, V 1a R), and these changes may contribute to the decreased sexual behavior observed in OTKO females.

Differential Gene Expression and Aging

Directory of Open Access Journals (Sweden)

Laurent Seroude

2002-01-01

Full Text Available It has been established that an intricate program of gene expression controls progression through the different stages in development. The equally complex biological phenomenon known as aging is genetically determined and environmentally modulated. This review focuses on the genetic component of aging, with a special emphasis on differential gene expression. At least two genetic pathways regulating organism longevity act by modifying gene expression. Many genes are also subjected to age-dependent transcriptional regulation. Some age-related gene expression changes are prevented by caloric restriction, the most robust intervention that slows down the aging process. Manipulating the expression of some age-regulated genes can extend an organism's life span. Remarkably, the activity of many transcription regulatory elements is linked to physiological age as opposed to chronological age, indicating that orderly and tightly controlled regulatory pathways are active during aging.

Efficacy of Selenium Supplement on Gene Expression of Inflammatory Cytokines and Vascular Endothelial Growth Factor in Gestational Diabetes

Directory of Open Access Journals (Sweden)

Mehri Jamilian

2018-01-01

Full Text Available Abstract Background: Selenium supplement has multiple important effects, including anti-inflammatory effect. The aim of this study was to assess the effects of selenium supplement on gene expression of inflammatory cytokines and vascular endothelial growth factor in gestational diabetes. Materials and Methods: This randomized double blind placebo control trial was performed on 40 patients suffering from GDM aged 18–40 years old. Participants were randomly divided into interventional group receiving 200mg/day selenium supplements (n=20 and control group receiving placebo (n=20 for 6 weeks. Primary outcome was gene expression of inflammatory cytokines and VEGF which were assessed in lymphocyte of GDM patients by RT-PCR method. Results: After 6 weeks intervention, in comparison with the control group, interventional group showed down regulation of gene expression of tumor necrosis factor alpha (TNF–α (p=0.02 and transforming growth factor beta (TGF–β (p=0.01 and up-regulation of gene expression of vascular endothelial (VEGF (p = 0.03 in lymphocytes of GDM. There was not any significant change following intervention with selenium regarding gene expression of interleukin IL-1 β and IL-8 in lymphocytes of GDM patients. Conclusion: 6 weeks supplementation with selenium in patients with GDM can cause down regulated gene expression of TNF-α and TGF–β, and up regulated gene expression of VEGF. Selenium supplement had not any effect on gene expression of IL-1 β and IL-8.

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

Science.gov (United States)

Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

1992-01-01

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

Polycistronic gene expression in Aspergillus niger.

Science.gov (United States)

Schuetze, Tabea; Meyer, Vera

2017-09-25

Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell

Directory of Open Access Journals (Sweden)

Jing Dan

2017-03-01

Full Text Available AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2, transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future. METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in

Hippocampal gene expression patterns in oxytocin male knockout mice are related to impaired social interaction.

Science.gov (United States)

Lazzari, Virginia Meneghini; Zimmermann-Peruzatto, Josi Maria; Agnes, Grasiela; Becker, Roberta Oriques; de Moura, Ana Carolina; Almeida, Silvana; Guedes, Renata Padilha; Giovenardi, Marcia

2017-11-02

Social interaction between animals is crucial for the survival and life in groups. It is well demonstrated that oxytocin (OT) and vasopressin (AVP) play critical roles in the regulation of social behaviors in mammals, however, other neurotransmitters and hormones are involved in the brain circuitry related to these behaviors. The present study aimed to investigate the gene expression of neurotransmitter receptors in the brain of OT knockout (OTKO) male mice. In this study, we evaluated the expression levels of the OT receptor (Oxtr), AVP receptors 1a and 1b (Avpr1a; Avpr1b), dopamine receptor 2 (Drd2), and the estrogen receptors alpha and beta (Esr1; Esr2) genes in the hippocampus (HPC), olfactory bulb (OB), hypothalamus (HPT) and prefrontal cortex (PFC). AVP gene (Avp) expression was analyzed in the HPT. Gene expression results were discussed regarding to social interaction and sexual behavior findings. Additionally, we analyzed the influence of OT absence on the Avp mRNA expression levels in the HPT. RNA extraction and cDNAs synthesis followed by quantitative polymerase chain reaction were performed for gene expression determination. Results were calculated with the 2 -ΔΔCt method. Our main finding was that HPC is more susceptible to gene expression changes due to the lack of OT. OTKOs exhibited decreased expression of Drd2 and Avpr1b, but increased expression of Oxtr in the HPC. In the PFC, Esr2 was increased. In the HPT, there was a reduced Avp expression in the OTKO group. No differences were detected in the OB and HPT. Despite these changes in gene expression, sexual behavior was not affected. However, OTKO showed higher social investigation and lower aggressive performance than wild-type mice. Our data highlight the importance of OT for proper gene expression of neurotransmitter receptors related to the regulation of social interaction in male mice. Copyright © 2017. Published by Elsevier B.V.

Bayesian inference of the number of factors in gene-expression analysis: application to human virus challenge studies

Directory of Open Access Journals (Sweden)

Hero Alfred

2010-11-01

Full Text Available Abstract Background Nonparametric Bayesian techniques have been developed recently to extend the sophistication of factor models, allowing one to infer the number of appropriate factors from the observed data. We consider such techniques for sparse factor analysis, with application to gene-expression data from three virus challenge studies. Particular attention is placed on employing the Beta Process (BP, the Indian Buffet Process (IBP, and related sparseness-promoting techniques to infer a proper number of factors. The posterior density function on the model parameters is computed using Gibbs sampling and variational Bayesian (VB analysis. Results Time-evolving gene-expression data are considered for respiratory syncytial virus (RSV, Rhino virus, and influenza, using blood samples from healthy human subjects. These data were acquired in three challenge studies, each executed after receiving institutional review board (IRB approval from Duke University. Comparisons are made between several alternative means of per-forming nonparametric factor analysis on these data, with comparisons as well to sparse-PCA and Penalized Matrix Decomposition (PMD, closely related non-Bayesian approaches. Conclusions Applying the Beta Process to the factor scores, or to the singular values of a pseudo-SVD construction, the proposed algorithms infer the number of factors in gene-expression data. For real data the "true" number of factors is unknown; in our simulations we consider a range of noise variances, and the proposed Bayesian models inferred the number of factors accurately relative to other methods in the literature, such as sparse-PCA and PMD. We have also identified a "pan-viral" factor of importance for each of the three viruses considered in this study. We have identified a set of genes associated with this pan-viral factor, of interest for early detection of such viruses based upon the host response, as quantified via gene-expression data.

Bayesian inference of the number of factors in gene-expression analysis: application to human virus challenge studies.

Science.gov (United States)

Chen, Bo; Chen, Minhua; Paisley, John; Zaas, Aimee; Woods, Christopher; Ginsburg, Geoffrey S; Hero, Alfred; Lucas, Joseph; Dunson, David; Carin, Lawrence

2010-11-09

Nonparametric Bayesian techniques have been developed recently to extend the sophistication of factor models, allowing one to infer the number of appropriate factors from the observed data. We consider such techniques for sparse factor analysis, with application to gene-expression data from three virus challenge studies. Particular attention is placed on employing the Beta Process (BP), the Indian Buffet Process (IBP), and related sparseness-promoting techniques to infer a proper number of factors. The posterior density function on the model parameters is computed using Gibbs sampling and variational Bayesian (VB) analysis. Time-evolving gene-expression data are considered for respiratory syncytial virus (RSV), Rhino virus, and influenza, using blood samples from healthy human subjects. These data were acquired in three challenge studies, each executed after receiving institutional review board (IRB) approval from Duke University. Comparisons are made between several alternative means of per-forming nonparametric factor analysis on these data, with comparisons as well to sparse-PCA and Penalized Matrix Decomposition (PMD), closely related non-Bayesian approaches. Applying the Beta Process to the factor scores, or to the singular values of a pseudo-SVD construction, the proposed algorithms infer the number of factors in gene-expression data. For real data the "true" number of factors is unknown; in our simulations we consider a range of noise variances, and the proposed Bayesian models inferred the number of factors accurately relative to other methods in the literature, such as sparse-PCA and PMD. We have also identified a "pan-viral" factor of importance for each of the three viruses considered in this study. We have identified a set of genes associated with this pan-viral factor, of interest for early detection of such viruses based upon the host response, as quantified via gene-expression data.

Alteration in gene expression profile and oncogenicity of esophageal squamous cell carcinoma by RIZ1 upregulation.

Science.gov (United States)

Dong, Shang-Wen; Li, Dong; Xu, Cong; Sun, Pei; Wang, Yuan-Guo; Zhang, Peng

2013-10-07

To investigate the effect of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) upregulation in gene expression profile and oncogenicity of human esophageal squamous cell carcinoma (ESCC) cell line TE13. TE13 cells were transfected with pcDNA3.1(+)/RIZ1 and pcDNA3.1(+). Changes in gene expression profile were screened and the microarray results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Nude mice were inoculated with TE13 cells to establish ESCC xenografts. After two weeks, the inoculated mice were randomly divided into three groups. Tumors were injected with normal saline, transfection reagent pcDNA3.1(+) and transfection reagent pcDNA3.1(+)/RIZ1, respectively. Tumor development was quantified, and changes in gene expression of RIZ1 transfected tumors were detected by RT-PCR and Western blotting. DNA microarray data showed that RIZ1 transfection induced widespread changes in gene expression profile of cell line TE13, with 960 genes upregulated and 1163 downregulated. Treatment of tumor xenografts with RIZ1 recombinant plasmid significantly inhibited tumor growth, decreased tumor size, and increased expression of RIZ1 mRNA compared to control groups. The changes in gene expression profile were also observed in vivo after RIZ1 transfection. Most of the differentially expressed genes were associated with cell development, supervision of viral replication, lymphocyte costimulatory and immune system development in esophageal cells. RIZ1 gene may be involved in multiple cancer pathways, such as cytokine receptor interaction and transforming growth factor beta signaling. The development and progression of esophageal cancer are related to the inactivation of RIZ1. Virus infection may also be an important factor.

Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

Science.gov (United States)

Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

2008-01-01

The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792