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)

Collagen VI is a microfibrillar component of the extracellular matrix that is predicted to have an important structural role in matrix organization and a biological function in mediating cell-matrix interactions. Secreted collagen VI molecules are composed of three distinct subunits, the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. To determine when, and in which tissues, collagen VI is likely to have a role in embryonic processes, we have analyzed the expression patterns of the three subunit chains during postimplantation mouse development by reverse transcriptase-PCR (RT-PCR), in situ hybridization, and immunofluorescence. No collagen VI protein could be detected in the mouse embryo until Day 11.5 of gestation, when low levels were localized within the mesoderm layer of the visceral yolk sac, the subepidermal matrix of branchial arches, and the vessel wall of the dorsal aorta. Levels of collagen VI mRNA and protein increased during the period from Days 12.5 to 14.5 in the visceral yolk sac, subepidermal mesenchyme, lung, gut, meninges, muscle, perichondrium, and vertebral column. The cartilage matrix of ribs and developing long bones was not stained with collagen VI antisera, but pericellular staining of chondrocytes was seen in both tissues. Low levels of collagen VI mRNA and protein were seen in the fetal liver except for the connective tissue of the liver capsule, which was highly stained. Collagen VI was first detected at significant levels in the developing heart on Day 14.5. These data demonstrate a tissue-specific onset of collagen VI synthesis and deposition in the extracellular matrix of developing mouse embryos at a much later stage of development than that reported for fibronectin or collagen I. Sensitive RT-PCR assays showed that alpha 1(VI) and alpha 2(VI) mRNAs were amplified from extracts of embryonic tissues as early as Day 7.5, while alpha 3(VI) mRNA was not detected until Day 10.5. Expression of the alpha 3(VI) gene immediately preceded the appearance of collagen VI protein in embryonic tissues. This correlation is consistent with the proposal that expression of alpha 3(VI) chains regulates the formation and secretion of collagen VI trimers and collagen VI matrix deposition during development.
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PMID:Deposition of collagen VI in the extracellular matrix during mouse embryogenesis correlates with expression of the alpha 3(VI) subunit gene. 880 34

Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).
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PMID:Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix. 1009 8

Collagen-induced arthritis (CIA) is an experimental model that mimics clinical and histological features of rheumatoid arthritis. In this disease, a crucial role in initiating the pathological changes has been assigned to T lymphocytes expressing the Th1 phenotype. Aiming at identifying type II collagen (CII)-specific T cells involved in CIA, T cell clones were generated in vitro from the lymph nodes (LN) of CII-immunized DBA / 1 mice. In three independent experiments, we repeatedly isolated CD4(+) Th1 clones recognizing the immunodominant epitope in the CB11 fragment of bovine CII and expressing a unique alpha betaTCR produced by the rearrangement of Valpha17/Jalpha20 and Vbeta10/Dbeta1.1/Jbeta2.5 gene segments. By reverse transcriptase-PCR, we demonstrated the presence of mRNA transcripts specific for the beta complementary-determining region 3 of this clonotype in the LN of the majority (73%) of mice with CIA whereas it was never detected in control animals. When transferred to CII-immunized DBA/1 mice, this recurrent Th1 clone augmented the incidence, aggravated significantly the clinical signs of CIA and greatly enhanced the anti-CII antibody response. Altogether, these results provide evidence that a CD4(+) Th1 clone belonging to the public arm of the response toward the immunodominant epitope of CII is involved in the cascade of events leading to CIA.
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PMID:A recurrent valpha17/vbeta10 TCR-expressing T cell clone is involved in the pathogenicity of collagen-induced arthritis in DBA/1 mice. 1055 19

Epidemiologic studies have indicated the association between tobacco smoking and skin aging, but the exact mechanism of tobacco smoke-induced premature skin aging is currently unknown. In this study, we investigated the alterations of collagen, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human fibroblasts treated with tobacco smoke extract. Human fibroblasts were exposed to different concentrations of water-soluble extract from tobacco smoke. Human fibroblasts irradiated with ultraviolet A1 (UVA1) were used as positive controls because the mechanism of UVA1-mediated MMP expression has been well characterized. The expression of MMP and TIMP was analyzed semiquantitatively following reverse transcriptase-polymerase chain reaction. Production of type I and type III collagens was detected by Western blotting and biosynthesis of new collagen was assessed by 3H-proline incorporation. Upon treatment with tobacco smoke extract or UVA1 irradiation, the expression of MMP-1 and MMP-3 mRNA was significantly increased in a dose-dependent manner. Maximum induction was observed with 25 microl/ml tobacco smoke extract. In contrast, the expression of TIMP-1 and TIMP-3 mRNA remained unchanged. Western blotting of the supernatant revealed that type I and type III collagens were decreased as compared with untreated controls. Collagen biosynthesis was significantly reduced by 40.1% following treatment with 25 microl/ml tobacco smoke extract. Sodium azide, L-ascorbic acid and Trolox (a water-soluble vitamin E) prevented both the UVA1- and the tobacco-induced alteration of MMP-1. These observations suggest that the imbalance of connective tissue matrix components might contribute to the molecular basis for premature skin aging in smokers. They also suggest that reactive oxygen species including singlet oxygen mediate this process.
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PMID:Alterations of extracellular matrix induced by tobacco smoke extract. 1083 12

The mechanical function and the collagen phenotype of the disc are complex, each a hybrid of elements of ligament and cartilage. In detail, the collagen properties are unique. Collagens I and II provide the bulk of the tissue fabric interwoven in opposing radial concentration gradients. From analysis of isolated cross-linked peptides, some degree of commingling of these major fibrillar collagens occurs down to the molecular level. Collagens V, VI, IX, XI, XII and XIV all contribute to the matrix. Collagen IX is the short molecular form that lacks a non-collagenous (NC)4 domain, not the long form found in most hyaline cartilages. Protein sequence and reverse transcriptase-PCR analysis confirmed this was the result of expression from the alternative transcription start site, not proteolysis of the long form. In view of recent reports that common single nucleotide polymorphisms in COL9A2 and COL9A3 are linked to chronic sciatica associated with disc pathology, the specific interactions and role of collagen IX in disc tissue are important to define.
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PMID:Collagen polymorphisms of the intervertebral disc. 1244 Sep 30

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/APO-2L) is a member of the tumor necrosis factor family that induces apoptosis in a variety of transformed cell lines and in normal human hepatocytes and brain cells. Soluble TRAIL at high concentrations was found to induce apoptotic death in normal human lung fibroblasts, whereas at low concentrations it was found to stimulate collagen production by these cells. Collagen alpha2(I) mRNA expression was assessed by semiquantitative reverse transcriptase/polymerase chain reaction; total soluble collagen was measured in culture supernatants by the Sircol assay. Both alpha2(I) collagen mRNA level and total soluble collagen secretion were increased upon TRAIL stimulation, with peak response (> 4-fold increase in mRNA level) at 1 ng/ml TRAIL. Analysis of the transcriptional response in TRAIL-stimulated fibroblasts, using DNA microarray hybridization, revealed an augmented expression of a number of genes involved in tissue remodeling, including those related to the transforming growth factor-beta (TGF-beta) pathway. DNA microarray results for the increase in TGF-beta1 mRNA level were confirmed by Northern blot analysis and by measurements of total active TGF-beta1 in culture supernatants. In addition, pan-specific TGF-beta antibody was shown to inhibit TRAIL-stimulated collagen mRNA and protein expression. These data suggest that TRAIL can enhance extracellular matrix synthesis in fibroblasts by triggering TGF-beta production that acts in an autocrine manner.
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PMID:Tumor necrosis factor-related apoptosis-inducing ligand enhances collagen production by human lung fibroblasts. 1254 Apr 90

Collagen is the primary structural component in connective tissue. The poor mechanical properties of most cell-seeded cartilage grafts used for cartilage repair can be attributed to the low level of collagen synthesized compared with native cartilage. In this study, the synthesis and assembly of collagen by chondrocytes in hydrogels were investigated, with particular attention paid to the role of cross-link formation in this process. Primary bovine chondrocytes were seeded in alginate and collagen synthesis was assessed in the presence and absence of beta-aminopropronitrile (BAPN), a potent inhibitor of the enzyme lysyl oxidase and collagen cross-link formation. Cultures on days 21, 35, and 49 were evaluated by stereology, biochemistry, and real-time reverse transcriptase-polymerase chain reaction. All measures of collagen synthesis (except hydroxyproline) significantly increased in the presence of 0.25 mM BAPN. By 35 days of culture, the average collagen fibril diameter was 62 +/- 10 nm in control cultures and 109 +/- 20 nm with BAPN supplementation. The collagen volume density increased from 5 +/- 3% in control cultures to 17 +/- 1% in the presence of BAPN. Likewise, the expression of cartilage-specific collagens (type II and XI) and aggrecan increased significantly as a result of BAPN culture. These findings demonstrate the prominent role of collagen cross-linking in collagen fibrillogenesis and suggest approaches by which collagen synthesis and assembly could be controlled in tissue-engineered constructs.
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PMID:Collagen fibrillogenesis by chondrocytes in alginate. 1254 43

The development of high myopia is associated with reduced scleral collagen accumulation, scleral thinning, and loss of scleral tissue, in both humans and animal models. Reduced collagen fibril diameter is also observed in the sclera of eyes with high myopia. The present study investigated aspects of scleral collagen synthesis and degradation, in a mammalian model of high myopia, to elucidate the factors underlying scleral changes. General synthesis and degradation of scleral collagen was investigated in monocularly deprived tree shrews, through the in vivo administration of [(3)H]proline and subsequent assay of scleral tissue for [(3)H]collagen. In addition, PCR enriched cDNA, produced from tree shrew scleral mRNA, was used to synthesize probes for hybridization to custom gene arrays consisting of partial sequences for 11 collagen subtypes. Finally, real-time reverse transcriptase-PCR was employed to investigate collagen type I, III, and V mRNA expression in the sclera of myopic, contralateral control, and normal tree shrew eyes. Scleral [(3)H]proline incorporation was reduced at the posterior pole of myopic eyes following 5 days of monocular deprivation (-36 +/- 4%), whereas [(3)H]proline content was similar in treated and control eyes before myopia induction (-1 +/- 8%) but was reduced in myopic eyes following 5 (-8 +/- 2%), 12 (-15 +/- 4%), and 24 (-10 +/- 4%) days of myopia induction. The majority of the collagens investigated were found to be expressed in the sclera, with 11 subtypes being identified. Collagen type I mRNA expression was reduced in the sclera of myopic eyes (-20 +/- 7%), however, collagen type III (+2 +/- 9%) and type V (-1 +/- 6%) expression was unchanged relative to control, resulting in a net increase in the ratio of expression of collagen type III/type I and collagen type V/type I (22 and 25%, respectively). These results show that reduced scleral collagen accumulation in myopic eyes is a result of both decreased collagen synthesis and accelerated collagen degradation. Furthermore, changes in collagen synthesis are driven by reduced type I collagen production. Short term increases in the ratio of newly synthesized collagen type III/type I and type V/type I are likely to be important in the increasing frequency of small diameter scleral collagen fibrils observed in high myopia and may be important in the subsequent development of posterior staphyloma in humans with pathological myopia.
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PMID:Collagen gene expression and the altered accumulation of scleral collagen during the development of high myopia. 1260 41

Three-dimensional porous scaffolds made from blend polyesters of polyhydroxyalkanoates (PHA) were evaluated with respect to their effects on collagen II production. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to assay collagen II mRNA for evaluation of the ability for the blend scaffolds to induce collagen II production. It was found that scaffold of poly-3-hydroxybutyrate (PHB) blended with a copolyester consisting of 3-hydoxybutyrate-co3-hydroxyhexanate (PHBHHx) in a radio of 1:2 had strong ability to induce collagen II production compared with other ratios of PHB/PHBHHx and poly-L-lactic acid (PLA). Collagen II expression level remained approx. 1.5-times higher on the above-mentioned blend compared with that of PLA throughout four weeks cultivation. At the same time, the PHB/PHBHHx blend (1:2) exhibited a similar tendency as PLA to maintain the collagen 11 expression level over the entire culture period. The results showed that the PHB/PHBHHx blend (1:2) reported to improve mechanical properties compared with PHB or PHBHHx improved biocompatibility in comparison with PHB, PHBHHx, PLA and other blending systems consisting of PHB/PHBHHX in ratios of 2:1 and 1:1, respectively.
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PMID:Induced production of rabbit articular cartilage-derived chondrocyte collagen II on polyhydroxyalkanoate blends. 1290 31

We have evaluated a biomaterial to serve as a scaffold for the propagation and amplification of chondrocytes that promotes the original cellular phenotype of these cells. The goal of the present study was to investigate the use of thermally reversible polymer gels poly(NiPAAm-co-AAc), as a biocompatible supporting scaffold for the propagation of chondrocytic cells. The polymer gels at temperatures above its lower critical solution temperature whereas liquefying at temperatures below its lower critical solution temperature of 34.5 degrees C. Hence, the polymer, in its gelled form, has the ability to hold cells in situ, forming a matrix similar to the natural cellular environment or the extracellular matrix that comprises cartilage. We tested the hypothesis that the polymer gel promotes cell viability and function. Human osteoblast-like cells, nasal chondrocytes, and articular chondrocytes (1 x 10(5)/150 microL) were resuspended in enriched Dulbecco's minimal essential media and were plated onto control (without gel) and gel containing 24-well plates. The plates were reincubated at 37 degrees C, 5% CO(2) for the time point of interest. Additional media was added to the plates and exchanged as needed. After cell culture, cells were retrieved, enumerated, and cell viability was determined. Other aliquots of the cells were stained for morphological analysis whereas expression of chondrocyte markers including collagen type II and aggrecan were determined using reverse transcriptase-polymerase chain reaction. The polymer gel was not cytotoxic because the cell number retrieved from three-dimensional culture gel was found to be one to two times higher than that retrieved from monolayer culture. Chondrocytes propagated in the thermo-reversible polymers expressed enhanced or maintained expression of collagen type II and aggrecan. Collagen type I expression was decreased or unaltered. The N-isopropylacrylamide and acrylic acid copolymer gel has potential use as a cell culture substrate and as a cell delivery vehicle.
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PMID:Thermally reversible polymer gel for chondrocyte culture. 1462 18


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