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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated type 11 collagen matrices with and without CS. Type II collagen matrices were prepared using purified, pepsin-treated, type II collagen. Techniques applied to prepare type I collagen matrices were found unsuitable for type II collagen. Crosslinking of collagen and covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. Porous matrices were prepared by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature, collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In contrast to type I collagen, type II collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix. Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse transcriptase PCR for collagen types I, II and X. No major differences were observed with respect to the presence or absence of CS in the matrices.
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PMID:Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering. 1210 90

The hallmark feature of abdominal aortic aneurysm (AAA) is the progressive degeneration of aortic wall. Matrix proteoglycans (PGs) play important roles in the development of vascular diseases and the function of the tissue. In this study, we examined the concentration, expression and localization of the small extracellular matrix PG biglycan and decorin. The concentration of small PGs present in normal and aneurysmal aortas was determined by biochemical methods following extraction of the tissues with guanidine hydrochloride and treatment with collagenase/elastase, isolation by ion-exchange and gel chromatographies and identification by Western blotting. The levels of mRNA encoding for biglycan and decorin were evaluated in corresponding tissue samples by reverse transcriptase polymerase chain reaction (RT-PCR). Distribution of extracellular matrix macromolecules was examined using Movat's pentachrome staining and localization of biglycan and decorin by immunohistochemistry. Both normal and aneurysmal aortas contained almost equal amounts of decorin (1.13+/-0.08 and 1.22+/-0.10 mg uronic acid per g of dry defatted (dd) tissue, respectively). Furthermore, the expression of decorin was almost constant in both tissues. In normal specimens decorin accounts for 22% of total PGs, whereas in AAA ones for 60%, due to the significant loss of other matrix PGs. In contrast, the concentration of biglycan was markedly decreased in aneurysmal aortas (57%, 0.478+/-0.04 mg uronic acid per g of dd tissue) in comparison to normal ones (1.12+/-0.10 mg uronic acid per g of dd tissue). Biglycan accounts for 22% of total PGs in normal aortas and 25% of total in aneurysmal tissue. A similar decrease (60%) in the amounts of mRNA encoding for biglycan was observed in the AAA. Immunohistochemical study showed that all aortic layers of AAA were characterized by a significant loss of elastin, biglycan and other PGs/GAGs and replacement of these molecules with collagen fibrils and decorin. The obtained data suggest that the altered matrix architecture of aorta, i.e. the differential expression of biglycan and localization of decorin may well be crucial parameters accounting for the functional degeneration of the tissue and the development of aneurysmal dilatation.
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PMID:Decreased biglycan expression and differential decorin localization in human abdominal aortic aneurysms. 1241 72

Articular cartilage degeneration in osteoarthritis (OA) involves type II collagen degradation and chondrocyte differentiation (hypertrophy). Because these changes resemble growth plate remodeling, we hypothesized that collagen degradation may be inhibitable by growth factors known to suppress growth plate hypertrophy, namely transforming growth factor (TGF)-beta2, fibroblast growth factor (FGF)-2, and insulin. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with TGF-beta2, FGF-2, and insulin in combination (growth factors) or individually. In cultured explants from five OA patients, collagenase-mediated type II collagen cleavage was significantly down-regulated by combined growth factors as measured by enzyme-linked immunosorbent assay. Individually, FGF-2 and insulin failed to inhibit collagen cleavage in some OA explants whereas TGF-beta2 reduced collagen cleavage in these 5 explants and in 19 additional explants. Moreover, TGF-beta2 effectively suppressed cleavage at low concentrations. Together or individually these growth factors did not inhibit glycosaminoglycan (primarily aggrecan) degradation while TGF-beta2 occasionally did. Semiquantitative reverse transcriptase-polymerase chain reaction of articular cartilage from six OA patients revealed that TGF-beta2 suppressed expression of matrix metalloproteinase-13 and matrix metalloproteinase-9, early (PTHrP) and late (COL10A1) differentiation-related genes, and proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha). In contrast, TGF-beta2 up-regulated PGES-1 expression and prostaglandin E(2) release. These observations show that TGF-beta2 can suppress collagen resorption and chondrocyte differentiation in OA cartilage and that this may be mediated by prostaglandin E(2). Therefore TGF-beta2 could provide therapeutic control of type II collagen degeneration in OA.
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PMID:Transforming growth factor-beta2 suppresses collagen cleavage in cultured human osteoarthritic cartilage, reduces expression of genes associated with chondrocyte hypertrophy and degradation, and increases prostaglandin E(2) production. 1640 16

A gentle method to isolate glomeruli simply by cutting renal cortices without forced sieving was devised in our previous study of primary podocyte culture. Yields of glomeruli isolated by this method, however, were too small to perform subculture or biological assays. In the present study, we tried an isolation method with magnetic beads and collagenase to increase the yields. Rat kidneys were perfused with magnetic particles. Renal cortices were digested with collagenase and filtered. Utilizing magnetic particles trapped within glomeruli, glomeruli were collected by attractive power of a magnet and cultured. The number of glomeruli isolated from one adult rat was more than 20,000 and the purity was more than 97%. About half of them were attached to culture dishes and exhibited cellular outgrowths, which were identified as podocytes by their distinct staining for podocyte markers. After 3 days of primary culture, the cellular outgrowths were subcultured. Approximately 60 podocytes were obtained per attached glomerulus. Their significant expression of podocytes markers was demonstrated by immunostaining and quantitative reverse transcriptase-polymerase chain reaction. The isolation method with magnetic beads and collagenase provides a number of glomeruli suitable for primary podocyte culture.
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PMID:An improved method for primary culture of rat podocytes. 1662 47

Tissue engineering of articular cartilage usually requires the isolation and culture of chondrocytes. Previous studies have suggested that enzymatic isolation may alter the metabolic activity and growth rate of chondrocytes. This study examined the effects of 4 common isolation protocols on chondrocyte gene expression, morphology, and total cell yield immediately following the digest (t = 0) and after 2 culture periods (24 h and 1 week). Cartilage explants were digested using 1 of 4 protocols: (1) 6-h collagenase digest, (2) 22-h collagenase digest, (3) 45-min trypsin digest followed by a 3-h collagenase digest, or (4) 1.5-h pronase digest followed by a 3-h collagenase digest. Gene expression levels for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, type II collagen, aggrecan, superficial zone protein, matrix metalloproteinase- 1, and tissue inhibitor of metalloproteinase-1 were measured at t = 0 h, 24 h, and 1 week using quantitative reverse transcriptase-polymerase chain reaction. In this study, cell yield was greatest for the 22-h collagenase and pronase-collagenase digests. However, the data indicate that a 6-h collagenase digest has the fewest gene expression changes compared to native cells. For tissue engineering, data from this study suggest that when cell yield is critical, a 22-h collagenase digest is preferable, but when obtaining cells closest to native chondrocytes is more desired, the 6-h collagenase digest is more beneficial.
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PMID:The effects of isolation on chondrocyte gene expression. 1699 90

Incisional hernias represent one of the most common complications of laparotomies. Previous investigations have suggested that a disorder in collagen fiber structure and production level may be an important pathologic cause of abdominal wall hernias. We hypothesized that a cross-examination of multiple extracellular matrix biomarkers might identify underlying defects contributing to the development of hernias. We examined two patient populations: patients with incisional hernias (presenting for hernia repair) and patients with no hernia after previous laparotomy (undergoing a second laparotomy). Patients with previous wound infections, open abdomens, or on steroids were excluded. Fascia samples were obtained from all patients at the time of their second operation and they were studied. Western blots and reverse transcriptase-polymerase chain reaction were used to determine the ratio of type I, III, and IV collagens, as well as matrix metalloproteinase 1 (MMP1) and MMP2 in both groups. Values of P < 0.05 were considered statistically significant. At the protein level, collagen I/III ratio was slightly decreased in patients with incisional hernias compared with those with no hernia, whereas it was significantly decreased at the mRNA transcript level (0.49 vs 1.03, P < 0.01, respectively). The MMP-1 mRNA transcripts were not different in incisional hernia (IH) versus nonincisional hernia, but the MMP-2 level was significantly increased in patients with IH. Reduced collagen I/III and MMP-1/MMP-2 ratios in IH might be consequence of the biological activities between key elements participating in the development of IH after laparotomies. The potential role of MMP-2-specific inhibitors in preventing IH is of significance for future studies.
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PMID:Role of biomarkers in incisional hernias. 1765 92

Vibrio parahaemolyticus, a causative agent of wound infections as well as food poisoning, harbors two collagenase genes: vppC and prtV. When cultivated at 26 degrees C in gelatin broth supplemented with 3.0% NaCl, significant collagenolytic activity was detected in the culture supernatant at the early stationary phase. Native polyacrylamide gel electrophoresis analysis revealed a 90-kDa protein, and N-terminal amino acid sequencing showed that this protein was VppC, generated through truncation of 72 N-terminal amino acid residues. Additionally, significant expression of only vppC was observed by reverse transcriptase PCR. By contrast, a vppC-negative mutant constructed through single crossover homologous recombination secreted a 50-kDa-collagenolytic enzyme; however, this enzyme was a serine protease that was reported previously. These results suggest that VppC is a primary extracellular collagenase produced by V. parahaemolyticus.
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PMID:Differential gene expression and extracellular secretion of the collagenolytic enzymes by the pathogen Vibrio parahaemolyticus. 1842 26

Sinusoidal endothelial liver cells (SECs) have a key role in the pathophysiology of chronic liver disease. Leptin is an important profibrogenic and proinflammatory cytokine whose expression in sinusoidal endothelial liver has not been documented. The authors studied the potential of rat SECs to express the leptin and leptin receptor genes. Two cell lines of rat SECs were generated from a male rat liver by pronase-collagenase perfusion and dilution cloning. They were characterized according to morphology, ploidy, von Willebrand antigen immunoreactivity, CD31 transcription, matrix metalloproteinase secretion, and pseudocapillary formation. Expression of the leptin and leptin receptor genes was studied using qualitative reverse transcriptase-polymerase chain reaction. Both cell lines fulfilled the accepted criteria for consideration as being derived from the liver sinusoidal endothelium. Confluent monolayers of both cell lines transcribed leptin and leptin receptor genes. This work demonstrated that SECs can transcribe the leptin gene in vitro, cotranscribing with the leptin receptor gene. Leptin production and signaling at this level could be of paramount importance in liver physiopathology; further studies of this issue are warranted because it represents a potential intervention point during chronic liver diseases.
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PMID:Identification of leptin gene expression in sinusoidal endothelial rat liver cells. 1856 52

Mesenchymal stem cells (MSCs) have the capacity to differentiate into several cell lineages, some of which can generate bone, cartilage, or adipose tissue. The presence of MSCs in the synovial membrane was recently reported. Data from comparative studies of MSCs derived from various mesenchymal tissues suggest that MSCs from synovial membranes have a superior chondrogenesis capacity. Previous chondrogenic differentiation studies have used the total population of MSCs, including cells with several MSC markers, such as CD44, CD90, CD105, or CD73. However the chondrogenic capacity of an individual population of MSCs has not been examined. Our aim was to study the chondrogenic capacity of the cellular MSC subset, CD105(+), derived from synovial membrane tissues of patients with osteoarthritis (OA) and normal donors. The tissues were digested with a cocktail of collagenase/dispase and the isolated MSCs were seeded into plates. The subpopulation of CD105(+)-MSCs was separated using a magnetic separator. The MSCs were then differentiated towards chondrocyte-like cells using a specific medium to promote spheroid formation. Spheroids were collected after 14, 28, and 46 days in chondrogenic medium and stained with hematoxylin, eosin, Safranin O or Alcian blue to evaluate the extracellular matrix. Immunohistochemistry was performed to study collagen types I (COLI) and II (COLII) and aggrecan expression. Phenotypic characterization of the isolated CD105(+)-MSCs shows that these cells are also positive for CD90 and CD44, but negatives for CD34 and CD45. In addition, this cellular subset expressed Sox-9. Spheroids appeared after 7 days in culture in the presence of chondrogenic medium. Our studies show no differences between MSCs obtained from OA and normal synovial membranes during chondrogenesis. The morphological analysis of spheroids revealed characteristics typical of chondrocyte cells. The intensity of Safranin O, Alcian blue and aggrecan staining was positive and constant throughout the culture period. However, the intensity of COL2 staining was higher at 28 days (84.29 +/- 0.1 U) than at 46 days (61.28 +/- 01 U), while COL1 staining was not detected in any samples analyzed. These results were confirmed by reverse transcriptase-polymerase chain reaction assays. We conclude that the cellular subset of CD105(+)-MSCs has chondrogenic capacity. The study also show the similar chondrogenic capacity of CD105(+)-MSCs cultured from normal and OA synovial membranes.
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PMID:Differentiation of synovial CD-105(+) human mesenchymal stem cells into chondrocyte-like cells through spheroid formation. 1954 99


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