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)

Colloidal gold is an electron-dense, lyophobic colloid that readily forms a stable electrostatic interaction with a variety of macromolecules. Monodispersed colloids ranging from 3-150 nm in diameter can be produced to provide the researcher with flexibility in selecting the optimally sized probe. Gold labeling of antibodies and lectins has been extensively used to study surface antigens and cell components. Recently, the use of gold labeling has been extended to study receptor-ligand binding, enzyme-substrate reactions, and transcellular pathways. Published applications include gold labeling of metabolites (low-density lipoproteins), enzymes (DNAase and RNAase, RNA polymerase, thrombin, collagenase, elastase), hormones (insulin, epidermal growth factor, glucagon), circulating plasma proteins (asialoglycoprotein, alpha 2-macroglobulin, factor VIII-von Willebrand factor), and endotoxins (tetanus toxin, cholera toxin). This broad spectrum of applications emphasizes the versatility and usefulness of colloidal gold as a probe in areas of cell biology related to receptors, endocytosis, transport, and functions of proteins.
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PMID:Colloidal gold: a pluripotent receptor probe. 635 33

A cDNA coding for a new human matrix metalloproteinase (MMP) has been cloned from a cDNA library derived from a breast tumor. The isolated cDNA contains an open reading frame coding for a polypeptide of 471 amino acids. The predicted protein sequence displays extensive similarity to the previously known MMPs and presents all the structural features characteristic of the members of this protein family, including the well conserved PRCGXPD motif, involved in the latency of the enzyme and the zinc-binding domain (HEXGHXXXXXHS). In addition, this novel human MMP contains in its amino acid sequence several residues specific to the collagenase subfamily (Tyr-214, Asp-235, and Gly-237) and lacks the 9-residue insertion present in the stromelysins. According to these structural characteristics, the MMP described herein has been tentatively called collagenase-3, since it represents the third member of this subfamily, composed at present of fibroblast and neutrophil collagenases. The collagenase-3 cDNA was expressed in a vaccinia virus system, and the recombinant protein was able to degrade fibrillar collagens, providing support to the hypothesis that the isolated cDNA codes for an authentic collagenase. Northern blot analysis of RNA from normal and pathological tissues demonstrated the existence in breast tumors of three different mRNA species, which seem to be the result of the utilization of different polyadenylation sites present in the 3'-noncoding region of the gene. By contrast, no collagenase-3 mRNA was detected either by Northern blot or RNA polymerase chain reaction analysis with RNA from other human tissues, including normal breast, mammary fibroadenomas, liver, placenta, ovary, uterus, prostate, and parotid gland. On the basis of the increased expression of collagenase-3 in breast carcinomas and the absence of detectable expression in normal tissues, a possible role for this metalloproteinase in the tumoral process is proposed.
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PMID:Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. 820

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

Collagenase-1 is invariantly expressed by migrating basal keratinocytes in all forms of human skin wounds, and its expression is induced by contact with native type I collagen. However, net differences in enzyme production between acute and chronic wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a dose-dependent manner. However, KGF was >100-fold more effective than bFGF at inhibiting collagenase-1 expression, suggesting that this differential signaling is transduced via an FGF receptor that binds these ligands with different affinities. Reverse transcriptase-polymerase chain reaction analysis of human keratinocyte mRNA for fibroblast growth factor receptors (FGFRs) revealed expression of only FGFR-2 IIIb, the KGF-specific receptor, which also binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF or KGF. FGFRs that bind bFGF with high affinity were not detected. Our results suggest that bFGF and KGF inhibit collagenase-1 expression through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix metalloproteinases in a tissue-specific manner.
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PMID:Cell type-specific inhibition of keratinocyte collagenase-1 expression by basic fibroblast growth factor and keratinocyte growth factor. A common receptor pathway. 921 49

Certain regions of coronary and other arteries undergo cyclic flexure due to attachment to the heart or crossing of joints. Such motion gives rise to fluctuations in transmural stress and luminal shear stress. It is well known that cyclic variation of these biomechanical forces influences many aspects of vascular cell biology including gene expression. The purpose of this work was to investigate the hypothesis that cyclic flexure of arterial segments influences their gene expression. Bilateral porcine femoral arteries were obtained fresh from the abattoir. One vessel was mounted in an ex vivo perfusion system and subjected to an intraluminal pressure of 60 mmHg and flow of 50 ml/min to serve as a control. The other vessel was mounted in a second perfusion system with similar hemodynamic conditions, but also subjected to controlled cyclic bending consistent with that found in coronary arteries in vivo. Reverse transcriptase-polymerase chain reaction analysis demonstrated that E-selectin and matrix metalloproteinase-1 (MMP-1) were consistently and significantly downregulated in the specimens subjected to 4 h of cyclic bending as compared to the control (n = 8, p < 0.05). Our results show that cyclic flexure of arterial segments in vitro may influence their gene expression. Further investigation should follow this novel observation and focus on other known mediators to more carefully elucidate the consequence of cyclic flexure on arterial pathobiology.
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PMID:Gene expression is altered in perfused arterial segments exposed to cyclic flexure ex vivo. 1037 28

Extracellular matrix-destructive enzymes, like matrix metalloproteinases (MMP), have been recognized in the process of inflammation and tissue remodeling and repair. The affected tissues often contain markedly increased numbers of mast cells. Although mast cells are capable of activating latent collagenase and proMMP, it has so far been unknown whether human mast cells themselves produce and secrete MMP9. In this study, MMP9 production by cord blood-derived cultured human mast cells and HMC-1 human mast cells was examined by reverse-transcriptase PCR, gelatin zymography and Western blot analysis using an antibody against MMP9. Cultured mast cells and HMC-1 cells treated with phorbol 12-myristate 13-acetate were shown to express MMP9 mRNA, and the cultured conditioned media from these cells showed gelatinolytic activity, identical with MMP9. Immunohistochemical examination was performed to detect MMP9 in tissue mast cells; mast cells localized in the skin, lung and synovial tissue showed strongly positive reactions for MMP9. Thus, these findings indicate that human mast cells can produce MMP9, which might contribute to extracellular matrix degradation and absorption in the process of allergic and nonallergic responses.
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PMID:Human mast cells produce matrix metalloproteinase 9. 1045 79

Changes in masticatory muscle structure and function are either developmental, as seen in anomalies of facial form, or adaptive, as seen during procedures such as orthognathic surgery and functional-appliance orthodontic therapy. Remodelling of muscle extracellular matrix is pivotal in these processes. This turnover is mediated via members of the family of enzymes known as matrix metalloproteinases (MMP) and inhibited by the tissue inhibitors of metalloproteinases (TIMP). The aim here was to investigate the in vivo pattern of expression and distribution of MMPs and TIMPs in masseter muscle of humans with both normal and abnormal facial forms. Masseter muscle biopsies were taken from 10 patients, four with long-face syndrome and six normal controls as confirmed by cephalometry. Immunohistochemical techniques were used to show the pattern and distribution of MMPs and TIMP proteins in the muscle. Zymography of tissue extracts was used to determine the presence of MMP activity. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the presence of MMP and TIMP-2 mRNA. MMP-1 was expressed around the individual muscle fibres, especially in those fibre surfaces in contact with the interstices of the connective tissue and around blood vessels. MMP-9 staining was less intense and was expressed in the interstices of the connective tissue and around blood vessels. Zymography of protein extracts confirmed that MMP-9 activity was present. MMP-2 and MMP-3 were not expressed in the samples, although MMP-2 mRNA could be detected by RT-PCR and its activity could be detected by zymography. Intense TIMP-1 staining was present around each muscle fibre, in the interstices of the connective tissue and surrounding blood vessels; TIMP-2 mRNA could be detected in all samples. These staining patterns were seen in all biopsies examined and were irrespective of the facial form of the donor. These findings provide evidence that the mechanisms required for matrix remodelling are present in the human masseter muscle.
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PMID:Identification of matrix metalloproteinases and their tissue inhibitors type 1 and 2 in human masseter muscle. 1077 72

Molecular biology techniques help to study details of testicular cell interactions, which play an important role in the control of spermatogenesis and secretory function of the male gonad. This paper presents an example of application of such techniques, with evaluation of their usability, in the study on expression of some cytokines involved in cellular interactions in the rat testis. Testicular cells were isolated by digestion with collagenase followed by percoll gradient centrifugation or by the adherence technique (macrophages). Then RNA was isolated from cells using Chomczynski-Sacchi method and analysed by electrophoresis and spectrophotometry. RNA was reversely transcribed using oligo-dT15 primer and AMV (Avian Myeloblastosis Virus) transcriptase. cDNA was amplified by PCR with primers for rat IL-1alpha, IL-1beta, IL-6 and Tbr (Thermus brockianus) polymerase. RT-PCR products were analysed by electrophoresis. Chomczynski-Sacchi method proved to be effective for isolation of good quality RNA even from small number of cells. RT-PCR revealed the presence of mRNA for studied cytokines in isolated cell. The results indicate that the applied techniques are useful in studies on the function of male gonad and indicate the need for use of semi-quantitative methods for evaluation of cytokine expression.
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PMID:An example of application of molecular techniques to the study on the local regulatory system controlling the function of the rat male gonad. 1137 83

Collagenase-2 (matrix metalloproteinase-8 or MMP-8) is synthesized mainly by polymorphonuclear neutrophils and plays a crucial role in inflammatory periodontal tissue destruction. We tested the effect of interleukin(IL)-1beta, a proinflammatory cytokine, on collagenase-2 gene expression in cultured human gingival fibroblasts and also compared this effect with IL-1beta-induced changes in collagenase-1 and -3 gene expression. By a combination of reverse transcription-polymerase chain reaction and Southern analysis, IL-1beta was found to dose-dependently induce gene expression for collagenase-1, -2, and -3 in gingival fibroblasts. Although collagenase-2 mRNA was the least abundant among the three collagenase mRNAs tested in the cultured fibroblast system, addition of 1 ng/ml IL-1beta significantly increased collagenase-2 gene transcription within 6 h, and maximal stimulation was maintained for 12 to 48 h. Significant mRNA induction was observed with as little as 0.1 ng/ml IL-1beta. IL-1beta was also found to increase the stability of collagenase-2 mRNAs after transcription arrest was induced by an RNA polymerase inhibitor. Stimulation of collagenase-2 mRNA expression by IL-1beta was prevented by pretreatment with cycloheximide, an inhibitor of protein synthesis. These results indicate that IL-1beta increased mRNA expression for collagenases including collagenase-2 in gingival fibroblasts. The findings also suggest that enhancement of collagenase-2 mRNA expression by IL-1beta involves both protein synthesis and suppression of mRNA degradation.
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PMID:Induction of collagenase-2 (matrix metalloproteinase-8) gene expression by interleukin-1beta in human gingival fibroblasts. 1145 13

Previous in vitro data on type I collagen self-assembly into fibrils suggested that the amino acid 776-796 region of the alpha1(I) chain is crucial for fibril formation because it serves as the recognition site for the telopeptide of a docking collagen monomer. We used a natural collagen mutation with a deletion of amino acids 766-801 to confirm the importance of this region for collagen fibril formation. The proband has type III osteogenesis imperfecta and is heterozygous for a COL1A1 IVS 41 A(+4) --> C substitution. The intronic mutation causes splicing of exon 41, confirmed by sequencing of normal and shorter reverse transcriptase-PCR products. Reverse transcriptase-PCR using RNA from proband dermal fibroblasts and clonal cell lines showed the mutant cDNA was about 15% of total alpha1(I) cDNA. The mutant transcript is translated; structurally abnormal alpha chains are demonstrated in the cell layer of proband fibroblasts by SDS-urea-PAGE. The proportion of mutant chains in the secreted procollagen was determined to be 10% by resistance to digestion with MMP-1, since chains lacking exon 41 are missing the vertebral collagenase cleavage site. Secreted proband collagen was used for analysis of kinetics of binding of alpha1(I) C-telopeptide using an optical biosensor. Telopeptide had slower association and faster dissociation from proband than from normal collagen. Purified proband pC-collagen was used to study fibril formation. The presence of the mutant molecules decreases the rate of fibril formation. The fibrils formed in the presence of 10-15% mutant molecules have strikingly increased length compared with normal collagen, but are well organized, as demonstrated by D-periodicity. These results suggest that some collagen molecules containing the mutant chain are incorporated into fibrils and that the absence of the telopeptide binding region from even a small portion of the monomers interferes with fibril growth. Both abnormal fibrils and slower remodeling may contribute to the severe phenotype.in
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PMID:Procollagen with skipping of alpha 1(I) exon 41 has lower binding affinity for alpha 1(I) C-telopeptide, impaired in vitro fibrillogenesis, and altered fibril morphology. 1170 4


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