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At least 199 records · Page 11Linked to original sources
PubMed2006

Plasminogen activator inhibitor-1 gene: selection of tagging single nucleotide polymorphisms and association with coronary heart disease.

OBJECTIVE: To explore the effect of plasminogen activator inhibitor-1 (PAI-1) gene variations on the risk of coronary heart disease (CHD) in Chinese Han population. METHODS AND RESULTS: We screened all exons and the promoter region of PAI-1 gene in 48 patients and identified 17 polymorphisms. Five tagging single nucleotide polymorphisms were selected and genotyped in 816 patients with CHD and 937 controls. In the total sample, no main effects of the loci or haplotypes reached statistical significance after adjusting environmental covariates. However, a strongly significant gene-smoking interaction was observed. Among nonsmokers, 2 polymorphisms located at promoter region (rs2227631 and rs1799889) showed significant association with CHD. The cases had higher frequency of rs2227631 A allele and rs1799889 4G allele than the controls (0.42 versus 0.33, P=0.001; 0.60 versus 0.52, P=0.002). Haplotype analyses confirmed the effects of the PAI-1 gene-smoking interaction on CHD risk. Compared with the most common haplotype G-5G-A-A-T (35.1%), the haplotype A-4G-A-A-C (32.7%) significantly increased the risk of CHD with adjusted odds ratio of 1.51 (95% CI, 1.12 to 2.05; P=0.008) in nonsmokers. CONCLUSIONS: This study identified a significant interaction between PAI-1 gene and smoking status. Both single locus and haplotype analyses indicated that rs2227631 A allele and rs1799889 4G allele increased the risk of CHD among nonsmokers in Chinese.

Adult↗
PubMed2003

Modified microplex vector enhances transfection of cells in culture while maintaining tumour-selective gene delivery in-vivo.

A non-commercial liposome (dimethyl dioctadecyl ammonium bromide:dioleoyl phosphatidylethanolamine) was compared with a commercial variety (Lipofectamine) for transfection of cultured rat adenocarcinoma cells and in an in-vivo kidney tumour model. Transfection of the cells in culture and in tumours in-vivo was variable with both types of liposomes. A high-dose microplex (lipoplex-microsphere) vector enhanced liposome-mediated transfection of cells in culture. When these high-dose microplexes were tested in-vivo, they were better than both microspherical and liposomal delivery modes in terms of transgene expression levels and the tumour-to-normal tissue ratio of gene delivery. Microplexes have been demonstrated to be capable of not only selective delivery of plasmids to solid tumours, but also of increasing transfection in cell culture, a finding that may be used in ex-vivo transfection studies. It is hypothesized that microspheres anchored the combination vector closer to the cultured cells, allowing attached liposomes to gain easier access into cells. In-vivo, microspheres permitted the microplexes to selectively deliver their genetic payload within the tumour tissue, from where the action of cationic liposomes on cellular membranes facilitated increased access of plasmids into the cytosol of target cells.

Cation Exchange Resins↗
PubMed2007

Identification of genes selectively regulated by IFNs in endothelial cells.

IFNs are highly pleiotropic cytokines also endowed with marked antiangiogenic activity. In this study, the mRNA expression profiles of endothelial cells (EC) exposed in vitro to IFN-alpha, IFN-beta, or IFN-gamma were determined. We found that in HUVEC as well as in other EC types 175 genes were up-regulated (>2-fold increase) by IFNs, including genes involved in the host response to RNA viruses, inflammation, and apoptosis. Interestingly, 41 genes showed a >5-fold higher induction by IFN-alpha in EC compared with human fibroblasts; among them, the gene encoding the angiostatic chemokine CXCL11 was selectively induced by IFN-alpha in EC along with other genes associated with angiogenesis regulation, including CXCL10, TRAIL, and guanylate-binding protein 1. These transcriptional changes were confirmed and extended by quantitative PCR analysis and ELISA; whereas IFN-alpha and IFN-beta exerted virtually identical effects on transcriptome modulation, a differential gene regulation by type I and type II IFN emerged, especially as far as quantitative aspects were concerned. In vivo, IFN-alpha-producing tumors overexpressed murine CXCL10 and CXCL11, guanylate-binding protein 1, and TRAIL, with evidence of CXCL11 production by tumor-associated EC. Overall, these findings improve our understanding of the antiangiogenic effects of IFNs by showing that these cytokines trigger an antiangiogenic transcriptional program in EC. Moreover, we suggest that quantitative differences in the magnitude of the transcriptional activation of IFN-responsive genes could form the basis for cell-specific transcriptional signatures.

Animals↗
PubMed1998

Developmental regulation of the cm2 muscarinic acetylcholine receptor gene: selective induction by a secreted factor produced by embryonic chick retinal cells.

The expression of the cm2 muscarinic acetylcholine receptor gene increases dramatically in chick retina during embryonic development in vivo. A similar developmental increase in cm2 expression occurs in embryonic chick retinal cells in culture. Conditioned medium from mature, but not young, retinal cultures contains a secreted factor that causes a selective increase in expression of cm2, but not cm3 or cm4, receptors. The secreted factor has been partially purified from serum-free medium, is protease-sensitive, and has a molecular weight >10 kDa. The cm2-inducing factor stimulates expression of a cm2 promoter/luciferase reporter gene, demonstrating that the increase in cm2 expression is attributable to increased gene transcription. Incubation of retinal cells with 14 identified neurotrophic and growth factors did not increase cm2 expression, suggesting that a novel developmentally regulated secreted factor mediates the subtype-specific induction of the cm2 receptor gene in retina.

Adrenal Gland Neoplasms↗
PubMed1995

Breast cancer selective gene expression and therapy mediated by recombinant adenoviruses containing the DF3/MUC1 promoter.

The high molecular weight mucin-like glycoprotein, DF3 (MUC1), is overexpressed in the majority of human breast cancers. Here we demonstrate that replication defective recombinant adenoviral vectors, containing the DF3 promoter (bp -725 to +31), can be used to express beta-galactosidase (Ad.DF3-betagal) and the herpes simplex virus thymidine kinase (HSV-tk) gene (Ad.Df3-tk) in DF3 positive breast carcinoma cell lines. In vivo experiments using breast tumor implants in nude mice injected with Ad.DF3-betagal demonstrated that expression of the beta-galactosidase gene is limited to DF3-positive breast cancer xenografts. Moreover, in an intraperitoneal breast cancer metastases model, we show that i.p. injection of Ad.DF3-tk followed by GCV treatment results in inhibition of tumor growth. These results demonstrate that utilization of the DF3 promoter in an adenoviral vector can confer selective expression of heterologous genes in breast cancer cells in vitro and in vivo.

Adenoviruses, Human↗
PubMed1984

Nucleotide sequence and expression in E. coli of a human interferon-alpha gene selected from a genomic library using synthetic oligonucleotides.

The complete nucleotide sequence of a human interferon-alpha gene is reported. The gene, designated IFN-alpha M1, was isolated from a human genomic library in phage lambda Charon 4A using synthetic oligonucleotides as hybridization probes. Based on a comparison of nucleotide sequence data obtained from this recombinant phage with published interferon-alpha gene sequences, a region of DNA capable of coding for a pre-interferon of 189 amino acids was identified. An AluI fragment containing the coding region for the mature interferon was inserted into the HincII site of the phage M13mp11, resulting in a fusion of portions of the IFN-alpha M1 and the beta-galactosidase genes. Antiviral activity was detected in extracts from E. coli infected with the recombinant M13 phage carrying the fused gene. The antiviral activity was completely neutralized by antibodies to human interferon-alpha.

Amino Acid Sequence↗
PubMed2000

[Use of PCR methods for detection of selected genes of Mycoplasma pneumoniae].

The aim of this study was standardization of PCR for the detection of gene encoding the P1 protein, 16S rRNA and elongation factor Tu of M. pneumoniae. A total of 13 strains of M. pneumoniae, 28 strains of other mycoplasmas and 14 strains of different bacteria causing respiratory tract infections were tested. In all of tested M. pneumoniae strains the presence of the sought genes was confirmed. The specificity of DNA was confirmed by the restriction endonuclease analysis with enzymes Hind III, Alu I and Hha I. With none of primers specific for the M. pneumoniae genes amplification of DNA from other bacteria was noted. The PCR method with the selected primers allowed to detect from 10(2) to 10(4) cfu M. pneumoniae/ml suspended in broth. The obtained results indicate that the PCR method can be used for detection of M. pneumoniae genes. A very good sensitivity and specificity predestine++ PCR as a potential quick diagnostic method for identification of M. pneumoniae in clinical specimens.

DNA, Bacterial↗
PubMed2002

TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila.

Drosophila TATA-box-binding protein (TBP)-related factor 2 (TRF2) is a member of a family of TBP-related factors present in metazoan organisms. Recent evidence suggests that TRF2s are required for proper embryonic development and differentiation. However, true target promoters and the mechanisms by which TRF2 operates to control transcription remain elusive. Here we report the antibody affinity purification of a Drosophila TRF2-containing complex that contains components of the nucleosome remodelling factor (NURF) chromatin remodelling complex as well as the DNA replication-related element (DRE)-binding factor DREF. This latter finding led us to potential target genes containing TRF2-responsive promoters. We have used a combination of in vitro and in vivo assays to show that the DREF-containing TRF2 complex directs core promoter recognition of the proliferating cell nuclear antigen (PCNA) gene. We also identified additional TRF2-responsive target genes involved in DNA replication and cell proliferation. These data suggest that TRF2 functions as a core promoter-selectivity factor responsible for coordinating transcription of a subset of genes in Drosophila.

Animals↗
PubMed2000

Tumor-selective gene transduction and cell killing with an oncotropic autonomous parvovirus-based vector.

A recombinant MVMp of the fibrotropic strain of minute virus of mice (MVMp) expressing the chloramphenicol acetyltransferase reporter gene was used to infect a series of biologically relevant cultured cells, normal or tumor-derived, including normal melanocytes versus melanoma cells, normal mammary epithelial cells versus breast adenocarcinoma cells, and normal neurons or astrocytes versus glioma cells. As a reference cell system we used normal human fibroblasts versus the SV40-transformed fibroblast cell line NB324K. After infection, we observed good expression of the reporter gene in the different tumor cell types, but only poor expression if any in the corresponding normal cells. We also constructed a recombinant MVMp expressing the green fluorescent protein reporter gene and assessed by flow cytometry the efficiency of gene transduction into the different target cells. At a multiplicity of infection of 30, we observed substantial transduction of the gene into most of the tumor cell types tested, but only marginal transduction into normal cells under the same experimental conditions. Finally, we demonstrated that a recombinant MVMp expressing the herpes simplex virus thymidine kinase gene can, in vitro, cause efficient killing of most tumor cell types in the presence of ganciclovir, whilst affecting normal proliferating cells only marginally if at all. However, in the same experimental condition, breast tumor cells appeared to be resistant to GCV-mediated cytotoxicity, possibly because these cells are not susceptible to the bystander effect. Our data suggest that MVMp-based vectors could prove useful as selective vehicles for anticancer gene therapy, particularly for in vivo delivery of cytotoxic effector genes into tumor cells.

Adenocarcinoma↗
PubMed2004

Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern.

The development of in vitro methods for regulatory embryotoxicity testing is challenging since the understanding of chemical effects on the mammalian development is still poor. The aim of the project is to identify marker genes during in vitro cell differentiation of murine embryonic stem cells, in order to predict chemical effects on cell differentiation of specific target tissues. The present study is focusing on the expression pattern by using semi-quantitative reverse transcriptase (RT)-PCR of key genes involved in cardiomyocytes development; i.e. Oct-4, Brachyury, Nkx2.5 and alpha myosin heavy chain (alpha-MHC). Two reference chemicals with well-known in vivo data have been analysed by using this approach: retinoic acid and lithium chloride. Retinoic acid has been selected as a teratogen affecting several target tissues, whereas lithium chloride has been described to affect the development of the cardiovascular system. We demonstrate that retinoic acid already affects in the early stage of germ layer formation, which was demonstrated by a change of Oct-4 and Brachyury gene expression. As we expected, the expression of cardiac specific genes (Nkx2.5, alpha-MHC) has been also modified. In contrary, the Oct-4 and Brachyury expression was not changed by lithium treatment. In this case, we observed a modification in the normal gene expression pattern, for alpha-MHC and Nkx2.5, demonstrating that lithium chloride affects the later stage of heart development. These data suggest that the inclusion of selective target organ genes in an established embryotoxicity test allows to predict effects of chemicals and drugs to the heart development.

Animals↗
PubMed1989

Characterization of the methyltransferases in the yeast phosphatidylethanolamine methylation pathway by selective gene disruption.

PEM1 and PEM2 are structural genes for the yeast phosphatidylethanolamine methylation pathway which mediates the three-step methylation of phosphatidylethanolamine to phosphatidylcholine. Selective disruption of each locus in the yeast genome was performed using the in-vitro-inactivated gene with insertion of yeast LEU2 or HIS3. Complementation test and spore analysis indicated that the disruptants were allelic with our previous mutants that were isolated by chemical mutagenesis and used for the cloning of PEM1 and PEM2. The methyltransferase activities of the disruptants were assayed using their membrane fractions. When the PEM1 locus was disrupted, the activity for the first methylation was greatly decreased but was still detectable, while the activities for the second and third methylations were well retained. The remaining three activities exhibited nearly identical pH optima and apparent Km values for S-adenosyl-L-methionine. The disruptant incorporated radioactivity from L-[methyl-14C]Met into phosphatidylcholine at a low but measurable rate and required choline for optimal growth. When choline was omitted from the culture medium, the phosphatidylcholine content of the cells significantly decreased, but was restored by the addition of N-monomethylethanolamine or choline. When the PEM2 locus was disrupted, the activities for the second and third methylations were totally lost, but that for the first methylation remained. This activity could be distinguished from those remaining in the pem1 disruptant by its different pH optimum and apparent Km for S-adenosyl-L-methionine. When incubated with [methyl-14C]Met, the pem2 disruptant accumulated the radioactivity in phosphatidylmonomethylethanolamine. This disruptant also required choline for optimal growth. In the absence of choline, it accumulated phosphatidylmonomethylethanolamine with a concomitant decrease in phosphatidylcholine and phosphatidylethanolamine. When both loci were disrupted, all phospholipid-methylating activities were lost and cells absolutely required choline for growth. The flux through the pathway became negligible. Thus, the PEM1-encoded methyltransferase was strictly specific to the first step while the PEM2-encoded methyltransferase exhibited a somewhat broader specificity with a preference for the second and third steps of the pathway. These two enzymes accounted for all the activities in the yeast phosphatidylethanolamine methylation pathway.

Cloning, Molecular↗
PubMed2005

RelA repression of RelB activity induces selective gene activation downstream of TNF receptors.

TNF-alpha is a potent proinflammatory cytokine that regulates immune and inflammatory responses and programmed cell death. TNF-alpha stimulation causes nuclear translocation of several NF-kappaB dimers, including RelA/p50 and RelB/p50. However, contrary to RelA, RelB entering the nucleus in response to TNF-alpha cannot bind to DNA in mouse embryonic fibroblasts, strongly suggesting that RelB DNA-binding activity is modulated by additional nuclear mechanisms. Here, we demonstrate that TNF-alpha promotes the association of RelA with RelB in the nucleus and that TNF-alpha-induced RelA/RelB heterodimers do not bind to kappaB sites. Remarkably, we show that RelA serine-276, the phosphorylation of which is induced by TNF receptor ligation, is crucial for RelA/RelB complex formation and subsequent inhibition of RelB DNA binding. In the absence of RelA phosphorylation on serine-276, TNF-alpha stimulation leads to a strong increase in the expression of endogenous NF-kappaB-responsive genes, such as Bcl-xL, whose transcriptional up-regulation is mainly controlled by RelB. Our findings demonstrate that RelA has a major regulatory role serving to dampen RelB activity in response to TNF-alpha and define a previously unrecognized mechanism that represents an essential step leading to selective NF-kappaB target gene expression.

Animals↗
PubMed1997

Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium.

Vascular endothelium is an important transducer and integrator of both humoral and biomechanical stimuli within the cardiovascular system. Utilizing a differential display approach, we have identified two genes, Smad6 and Smad7, encoding members of the MAD-related family of molecules, selectively induced in cultured human vascular endothelial cells by steady laminar shear stress, a physiologic fluid mechanical stimulus. MAD-related proteins are a recently identified family of intracellular proteins that are thought to be essential components in the signaling pathways of the serine/threonine kinase receptors of the transforming growth factor beta superfamily. Smad6 and Smad7 possess unique structural features (compared with previously described MADs), and they can physically interact with each other, and, in the case of Smad6, with other known human MAD species, in endothelial cells. Transient expression of Smad6 or Smad7 in vascular endothelial cells inhibits the activation of a transfected reporter gene in response to both TGF-beta and fluid mechanical stimulation. Both Smad6 and Smad7 exhibit a selective pattern of expression in human vascular endothelium in vivo as detected by immunohistochemistry and in situ hybridization. Thus, Smad6 and Smad7 constitute a novel class of MAD-related proteins, termed vascular MADs, that are induced by fluid mechanical forces and can modulate gene expression in response to both humoral and biomechanical stimulation in vascular endothelium.

Amino Acid Sequence↗
PubMed2006

Molecular analysis of constitutively expressed erm(C) genes selected in vitro in the presence of the non-inducers pirlimycin, spiramycin and tylosin.

OBJECTIVES: As known from other lincosamides and 16-membered macrolides, the antimicrobial agents pirlimycin, spiramycin and tylosin, which are frequently used for the therapy of bovine mastitis, cannot induce the expression of the resistance gene erm(C). The aim of this study was to confirm the ability of these three non-inducers to select for constitutively expressed erm(C) mutants in Staphylococcus aureus. METHODS: A S. aureus strain carrying an inducibly expressed erm(C) gene was incubated on agar plates containing inhibitory concentrations of each of the three antimicrobial agents. The erm(C) regulatory region of mutants obtained after overnight incubation was amplified by PCR; selected amplicons were sequenced and compared with the wild-type sequence. RESULTS: Mutants developed in the presence of each of the three antimicrobial agents. Constitutive expression of erm(C) was due to variations in the erm(C) regulatory region. A total of 10 different types of deletions ranging in size between 16 and 121 bp as well as 20 different types of duplications ranging between 24 and 602 bp were detected. The frequencies by which sequence alterations occurred as well as the types of alterations detected varied with regard to the antimicrobial agents used for selection. CONCLUSIONS: All sequence alterations observed explained constitutive erm(C) gene expression by functional inactivation of translational attenuation. In order to prevent the development of constitutively resistant isolates under therapy, the results of this study support the recommendation not to use lincosamides or 16-membered macrolides for the control of staphylococcal infections caused by strains harbouring inducibly expressed erm(C) genes.

Animals↗
PubMed2001

Gene-selective developmental roles of general transcription factors.

Innumerable transcription factors integrate cellular and intercellular signals to generate a profile of expressed genes that is characteristic of the biochemical and cellular properties of the cell. This profile of expressed genes changes dynamically along with the developmental stage and differentiation state of the cell. The biochemical machinery upon which transcription factors integrate their signals is referred to as the general transcription machinery. However, this machinery is not of universal composition, and variants of the general transcription factors play specific roles in embryonic development, reflecting the constraints and requirements of developmental gene regulation.

Animals↗
PubMed1986

A universal oligonucleotide probe for acetylcholine receptor genes. Selection and sequencing of cDNA clones for the mouse muscle beta subunit.

A region of 25 nucleotides is highly conserved in genes coding for the alpha, beta, gamma, and delta subunits of the nicotinic acetylcholine receptor (AChR) of human, mouse, calf, chicken, and Torpedo. Based on this observation, a 2-fold degenerate oligonucleotide was synthesized and used as a probe to screen a cDNA library made from a mouse myogenic cell line. Clones coding for the beta, gamma, and delta subunits were identified by the probe. The protein sequence deduced from the beta subunit clones codes for a precursor polypeptide of 501 amino acids with a calculated molecular weight of 56,930 daltons, which includes a signal peptide of 23 amino acids. The protein sequence and structural features of the beta subunits of mouse, calf, and Torpedo are conserved. A clone coding for the mouse gamma subunit was isolated, and its identity was confirmed by alignment of its sequence to previously published cDNA sequences for the mouse and calf gamma subunits. The clone contained approximately 200 nucleotides more at its 3' end untranslated region than a mouse gamma clone recently described. Northern blot analysis, utilizing as probes these beta and gamma subunit cDNAs and previously characterized alpha and delta subunit cDNAs, shows that the steady-state levels of the four AChR mRNAs increase coordinately during terminal differentiation of cultured C2 and C2i mouse myoblasts. The increase in mRNA levels can account for the rise of cell surface receptors during myogenesis and suggests that the muscle AChR genes may be regulated during development by a common mechanism. Utilization of this oligonucleotide probe should prove useful for screening a variety of libraries made from different species and tissues which are known to express AChRs.

Amino Acid Sequence↗
PubMed1979

[The "hitch-hiking" effect of a selected gene and gamete combination (linkage disequilibrium): the example of 2 closely linked loci in the swine, Hal (halothane sensitivity) and PHI (phosphohexose isomerase)].

The observation of two lines of pigs selected differently for four generations confirms a recent theoretical work (Thomson, 1977) showing that a possible source of linkage disequilibrium may be the hitch-hiking effect of a selected locus (Hal) on another closely linked neutral locus (PHI).

Alleles↗