Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used two methods to detect specific transcription of the chicken alpha 2 (type I) collagen gene in cell-free extracts derived from Rous sarcoma virus-transformed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with 32P at the 5' end of the HindIII linker originally used to clone the collagen promoter region into PBR322. The probe distinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken alpha 2 (type I) collagen DNA template. Addition of either creatine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 mM was found to stimulate RNA polymerase II transcription by 5- to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primary extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell-free extracts.
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PMID:Transcription of the chicken alpha 2 (Type I) collagen gene by homologous cell-free extracts. 628 36

Chick embryos, chick embryo fibroblasts, and Rous sarcoma virus-transformed chick embryo fibroblasts contain a factor that preferentially blocks the accumulation of DNA-directed RNA polymerase II transcripts. The factor was detected by inhibition of transcription in a cell-free assay system utilizing partially purified RNA polymerase II from calf thymus, soluble factors from HeLa cells, and a purified DNA template. At low concentrations, it specifically prevents the accumulation of RNA polymerase II transcripts; at higher concentrations, it blocks the accumulation of other transcripts. The factor has been partially purified by sequential chromatography on BioRex 70, DNA-cellulose, Bio-Gel P-6, and HPX-87 from extracts of chicken embryos. The activity was resistant to treatment with trypsin, pronase, or micrococcal nuclease. A partial characterization of the molecule indicates that (i) it has an apparent molecular mass of about 200-300 daltons, (ii) it is stable at pH 2 and pH 12 and to heating at 100 degrees C, (iii) it is not extractable by ether or chloroform:methanol, (2:1, v/v), and (iv) it is labile to heating at 800 degrees C. These data suggest that it is a small, hydrolphilic compound probably organic in nature. The factor is active in a transcription assay utilizing either the Rous sarcoma virus Long Terminal Repeat promoter or the chick alpha 2 (Type I) collagen-promoter as DNA templates. The accumulation of promoter-specific transcripts is blocked in a cell-free assay utilizing either Rous sarcoma virus-chick embryo fibroblast extracts or HeLa S-100 factors and calf thymus RNA polymerase II. In the absence of S-100, the factor does not appreciably affect the accumulation of randomly initiated transcripts produced by calf thymus RNA polymerase II on a DNA template; this result indicates the factor interacts directly or indirectly with some component(s) of HeLa S-100 to prevent the accumulation of RNA.
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PMID:Chicken embryo extracts contain a factor that preferentially blocks the accumulation of RNA polymerase II transcripts in a cell-free system. 713 Jan 91

We previously isolated the human homeobox gene HOX4A (HOXD3) on chromosome 2 from a human genomic library and determined its nucleotide sequence. In the present study, expression of the HOX4A gene was investigated in human hematopoietic cell lines. Reverse transcriptase-mediated polymerase chain reaction analysis showed that the HOX4A gene was expressed in erythroleukemia HEL and K562 cells but not in promyelocytic leukemia HL-60 cells. To study the role of the HOX4A gene in erythropoiesis, expression vectors containing the HOX4A gene in the sense or antisense orientation were introduced into HEL cells. The sense transfectants overexpressing the HOX4A gene formed aggregates, which were composed of densely associated cells adhering to tissue-culture dishes, whereas the parental HEL cells and antisense transfectants adhered poorly to the dishes. Furthermore, the sense transfectants overexpressing the HOX4A gene attached more efficiently to fibronectin and collagen than did the antisense transfectants and parental HEL cells. Northern blot analysis showed that integrin beta 3 mRNA levels were significantly increased in the HEL cells overexpressing the HOX4A gene, whereas the integrin beta 1 and alpha IIb mRNA levels did not show a distinct correlation with HOX4A mRNA levels. Fluorescence-activated cell sorting analysis showed that the sense transfectants overexpressing the HOX4A gene expressed increased levels of integrin alpha IIb beta 3 (GP IIb-IIIa) complex as compared with the parental HEL cells and antisense transfectants. These results implicate the homeobox gene HOX4A in the regulation of cell adhesion processes.
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PMID:Overexpression of the HOX4A (HOXD3) homeobox gene in human erythroleukemia HEL cells results in altered adhesive properties. 774 39

The purpose of this study was to determine whether or not membrane-bound and soluble forms of IL-4 receptors are expressed by isolated subsets of murine lung fibroblasts and to evaluate the potential functional consequences of IL-4 receptor triggering. Recent studies demonstrate that IL-4-synthesizing Th2 cells and mast cells are present in increased numbers in the lung during inflammation and fibrosis, suggesting that IL-4 may play a regulatory role in these events. We hypothesize that pulmonary fibroblasts and subsets thereof are intimately involved in this inflammatory response and that IL-4 is an active player in stimulating fibroblast collagen synthesis and hyperproliferation, creating a fibrotic environment in the lung. The fibroblast subsets used in these experiments differ not only in surface expression of the thymocyte-1 (Thy-1) Ag, but also in function and morphology. We now report the novel finding that IL-4 receptors are present at discordant levels on Thy-1+ and Thy-1- lung fibroblasts. IL-4R level and affinity were analyzed using a monoclonal anti-IL-4R Ab and equilibrium binding analysis with 125I-labeled IL-4. Reverse transcriptase PCR demonstrated the presence of mRNA for membrane-bound and soluble IL-4R. Lung fibroblast subsets secrete soluble IL-4R protein at dramatically different levels, as detected by an ELISA. Thy-1+ and Thy-1- lung fibroblasts were treated with IL-4 to determine whether this cytokine was profibrotic. Thy-1+ fibroblasts responded to IL-4 by proliferating and up-regulating collagen production. In contrast, Thy-1- fibroblasts proliferate to a lesser degree than Thy-1+ fibroblasts and were not stimulated to secrete increased levels of collagen. Overall, these results suggest that elevated levels of IL-4 at a site of injury could result in the development of fibrosis by enhancing fibroblast subset proliferation and collagen synthesis.
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PMID:Subsets of murine lung fibroblasts express membrane-bound and soluble IL-4 receptors. Role of IL-4 in enhancing fibroblast proliferation and collagen synthesis. 790 5

Dermal fibroblasts from a Chinese Ehlers-Danlos syndrome type VII patient synthesized approximately equal amounts of normal pro-alpha 2(I) chains of type I procollagen and abnormal ones with electrophoretic mobility of pN alpha 2(I) chains, in which the amino-propeptide (N-propeptide) was retained. Reverse-transcriptase PCR analysis of the proband's RNA showed outsplicing of the 54 base exon 6 in half of the pro-alpha 2(I) mRNAs. Exon 6 encodes 18 amino acids of the N-telopeptide which contains the procollagen N-proteinase cleavage site and a cross-link precursor lysine. Loss of these sequences would result in failure to cleave the amino-propeptide of pro-alpha 2(I) and the accumulation of pN-alpha 2(I) chains. Nucleotide sequencing analyses of the proband's COL1A2 gene showed the presence of a T to C transition at position +2 of intron 6 in one allele and the proband is heterozygous for the defect. This mutation which destroyed the consensus GT dinucleotide at the 5' splice donor site of the intron is responsible for the loss of exon 6 by exon skipping. Electron microscopic analysis of the patient's dermis showed the presence of abnormal collagen I fibrils of irregular diameter and circularity. This mutation in COL1A2 in an EDS VII patient is the first reported case in the Chinese population and is identical to one reported for another EDS-VII (Libyan) patient. The occurrence of an identical mutation in two probands of different ethnic origin is direct evidence that the mutant genotype is the cause of the EDS VII phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Further evidence that the failure to cleave the aminopropeptide of type I procollagen is the cause of Ehlers-Danlos syndrome type VII. 808 89

Angiotensin II (Ang II), a vasoactive octapeptide, has been implicated in cardiac growth and the development of hypertrophy and fibrosis secondary in hypertensive disease. These consequences of Ang II imply an effect on the function and morphology of cardiac interstitial cells (fibroblasts). The present investigation was designed to (1) determine whether neonatal heart fibroblasts (NHFs) possess functional Ang II receptors on their plasma membrane and (2) examine the effects of Ang II on NHFs in vitro using three- and two-dimensional (3D and 2D, respectively) cultures. Several analytic techniques were used to test the specific questions of the present study. Since cardiac fibroblast phenotype can be influenced by culture conditions, both 2D and 3D cultures were used in the present investigations. Reverse-transcriptase polymerase chain reaction and radioligand binding analysis were used to test for the presence of Ang II receptors on NHFs. Both revealed that NHFs in 2D culture possess Ang II receptor mRNA and Ang II receptors. When isolated NHFs were cultured in 3D collagen gels and treated with Ang II, gel contraction was stimulated by NHFs. This effect was attenuated by the specific Ang II receptor antagonist [Sar1,Ala8]Ang II. Ang II-stimulated gel contraction was completely inhibited by extracellular matrix receptor (beta 1-integrin) antibodies (P < .05), supporting previous studies indicating that collagen gel contraction is mediated via the integrins. Immunofluorescent staining was used to test the localization of cell-surface integrins. A more intense staining pattern for beta 1-integrin in Ang II-treated versus control cells was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Integrin-mediated collagen gel contraction by cardiac fibroblasts. Effects of angiotensin II. 829 68

Peripheral lymphoid tissues contain a fibroblastic cell type referred to as stromal cells or reticulum cells which interact with lymphocytes as part of the lymphoid microenvironment. After isolation from human tonsils and expansion in vitro we analyzed the surface phenotype, extracellular matrix components, cytoskeletal products, cytokine production, binding and functional interaction with B lymphocytes of in vitro cultured stromal cells (HTSC) both in resting condition and after activation with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our results show that HTSC do not express specific myeloid, lymphoid, endothelial or epithelial markers. HTSC express CD54 (ICAM-1), CD49a (VLA-1), CD49b (VLA-2), CD49c (VLA-3), CD49e (VLA-5), CD49f (VLA-6), CD29, CD51, CD44 and produce vinculin, beta-tubulin, alpha-actin, vimentin, fibronectin, laminin and collagen types I, III and IV. Activation of HTSC up-regulated CD54 (ICAM-1) and induced HLA-DR and CD106 (VCAM-1). HTSC constitutively produce interleukin (IL)-6 which is enhanced upon activation with TNF-alpha. IL-8 and granulocyte/macrophage colony-stimulating factor are detected only in the supernatants of activated HTSC. Reverse transcriptase polymerase chain reaction analysis revealed that HTSC display mRNA for IL-1 alpha, leukemia inhibitory factor and IL-7. The adhesion of tonsillar B lymphocytes to activated HTSC is mediated by CD11a/CD18 and CD54. Furthermore, HTSC can induce maximal proliferation of IL-2-activated B lymphocytes cocultured in direct cell-cell contact with HTSC. These results clearly distinguish in vitro cultured HTSC from common fibroblasts and other non-lymphoid elements present in the lymphoid parenchyma, such as follicular dendritic cells, and show that HTSC actively participate in the lymphoid microenvironment. In vitro cultures of HTSC could therefore be a useful model system for detailed analysis of the interactions between stromal cells and lymphocytes under physiological and pathological conditions.
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PMID:In vitro cultured stromal cells from human tonsils display a distinct phenotype and induce B cell adhesion and proliferation. 856 62

Recently, a new human collagenase, collagenase-3 has been identified. Since collagen changes are of particular importance in cartilage degeneration, we investigated if collagenase-3 plays a role in osteoarthritis (OA). Reverse transcriptase-PCR analysis revealed that in articular tissues collagenase-3 was expressed by the chondrocytes but not by the synoviocytes. Northern blot analysis of the chondrocyte mRNA revealed the presence of two major gene transcripts of 3.0 and 2.5 kb, and a third one of 2.2 kb was occasionally present. Compared to normal, OA showed a significantly higher (3.0 kb, P < or = 0.05; 2.5 kb, P < or = 0.03) level of collagenase-3 mRNA expression. Collagenase-3 had a higher catalytic velocity tate (about fivefold) than collagenase-1 on type II collagen. With the use of two specific antibodies, we showed that human chondrocytes had the ability to produce collagenase-3 as a proenzyme and as a glycosylated doublet. The chondrocyte collagenase-3 protein is produced in a significantly higher (P < or = 0.04) level in OA (approximately 9.5-fold) than in normal. The synthesis and expression of this new collagenase could also be modulated by two proinflammatory cytokines, IL-1 beta and TNF-alpha, in a time- and dose-dependent manner. This study provides novel and interesting data on collagenase-3 expression and synthesis in human cartilage cells and suggest its involvement in human OA cartilage patho-physiology.
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PMID:The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis. 862 89

Conglutinin is a bovine serum protein which was first described as a vertebrate lectin. This protein belongs to the family of C-type lectins. These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD). Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization. Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties. In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined. A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique. The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained. SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa. Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin. The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity. These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.
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PMID:Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus. 864 31

Since recent studies demonstrated that platelet-derived growth factor (PDGF) induces vascular smooth muscle cell (SMC) proliferation by stimulating polyamine synthesis, we examined whether the transcellular transport of L-ornithine, the cationic amino acid precursor of polyamines, could regulate the mitogenic response of PDGF. Treatment of SMC with PDGF stimulated DNA and putrescine synthesis, and this was enhanced further by increasing the extracellular concentration of L-ornithine. The potentiating effect of L-ornithine was reversed by the competitive inhibitor of cationic amino acid transport, methyl-L-arginine, or by preventing putrescine formation with alpha-difluoromethylornithine. Cationic amino acid uptake by SMC was Na+-independent and was mediated by both a high and low affinity carrier system. Treatment of SMC with PDGF initially (0-2 h) decreased basic amino acid transport, while longer exposures (6-24 h) progressively increased uptake. Kinetic studies indicated that PDGF-induced inhibition was associated with a decrease in affinity for cationic amino acids, while the stimulation was mediated by an increase in transport capacity. Endogenous PDGF released by collagen-activated platelets likewise up-regulated cationic amino acid transport in SMC. Reverse transcriptase-polymerase chain reaction detected the presence of mRNA encoding two distinct cationic amino acid transporter (CAT) proteins, CAT-1 and CAT-2B. Treatment of SMC with PDGF strongly induced the expression CAT-2B mRNA and modestly elevated the level of CAT-1 mRNA. These results demonstrate that PDGF-induced polyamine synthesis and SMC mitogenesis are dependent on the transcellular transport of L-ornithine. The capacity of PDGF to up-regulate the transport of L-ornithine by inducing the expression of the genes for CAT-1 and CAT-2B may modulate its mitogenic effect by providing SMC with the necessary intracellular precursor for polyamine biosynthesis.
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PMID:Platelet-derived growth factor regulates vascular smooth muscle cell proliferation by inducing cationic amino acid transporter gene expression. 866 68


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