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)

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

Twenty distinct genetic types of collagen have been identified up to now. Their structure and function are not completely elucidated. We have chosen zebrafish as a model to bring information about the role of collagen during embryogenesis. In the present study, we isolated four overlapping DNA complementary to RNA clones covering the 4879 nucleotides of a zebrafish messenger RNA (mRNA) encoding a fibrillar procollagen chain. The comparison of its primary structure with known other vertebrate collagens allowed to conclude that it encodes collagen pro-alpha2(I) chain. The 5' untranslated region showed a typical stem-loop structure with three ATG codons which is found in mammals types I and III collagen chains (but not in type II), which are expressed in the same tissues. This suggests that the supposed regulatory role of the stem loop structure could be tissue specific. The comparison of the Gly-Gly doublets found along the helical domain of several species allowed to speculate that the Gly-Gly repeats could be a poikilotherm feature. Expression of pro-alpha2(I) was examined during zebrafish development by reverse transcriptase-polymerase chain reaction and in situ hybridization on whole embryo and tissue section. Col1a2 was expressed as early as stage10 h post fertilization (hpf) and two peaks of expression were observed at 20 and 48 hpf. alpha2 mRNAs, whose presence suggests a collagen synthesis, were detected principally in the superficial cell layers surrounding 20-72 hpf embryos which are characterized by an acellular collagen stratum. At 26-30 days, fibroblasts invade the dermis and take over from the epithelial cells to synthesize collagen. This suggests a fine regulation of collagen synthesis in these cells that remains to be elucidated. alpha2 mRNA were also detected in other tissues such as the tail fin primordium and the notochord primordium suggesting a participation of type I collagen in a pathway for notochord and tail formation.
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PMID:Structure and spatio temporal expression of the full length DNA complementary to RNA coding for alpha2 type I collagen of zebrafish. 1223 67

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

This study investigated the effects of mineral trioxide aggregate on cementoblast growth and osteocalcin production in tissue culture. For cellular morphology studies, cementoblasts on mineral trioxide aggregate, IRM, and amalgam were incubated for 48 h then fixed for scanning electron microscopic analysis. For gene expression on mineral trioxide aggregate and IRM, reverse transcriptase polymerase chain reaction was performed using primer sets for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein after 3 and 5 days. In vitro matrix protein expression was evaluated by confocal microscopy for the presence of osteocalcin on MTA after 7 and 12 days. Images were compared with controls to assess qualitative differences. Results suggest that mineral trioxide aggregate permits cementoblast attachment and growth and the production of mineralized matrix gene and protein expression. Our data indicates that mineral trioxide aggregate can be considered cementoconductive.
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PMID:Cementoblasts maintain expression of osteocalcin in the presence of mineral trioxide aggregate. 1281 26

Hepatic stellate cells (HSCs) were changed in their morphology, proliferative activity, and functions by culturing on type I collagen gel, as compared to the culture on polystyrene surface. HSCs have been found to produce extracellular matrix components and matrix metalloproteinases (MMPs). In this study, we have assessed the effects of several types of substrata on the expression of MMPs in HSC culture. MMP-1 expression was detectable in HSC culture on polystyrene surface and on type I collagen gel by immunofluorescence staining and reverse transcriptase-polymerase chain reaction (RT-PCR). The results from in situ zymography revealed the presence of interstitial collagenase activity around HSCs and along their cellular processes. Although proMMP-2 and proMMP-9 were detectable by gelatin zymography in the conditioned medium from both cultures using type I collagen gel and Matrigel as substratum, an active form of MMP-2 but not of MMP-9 was detected only in the culture using type I collagen as a substratum. Tissue inhibitor of metalloproteinase-2 expression was observed by RT-PCR in HSCs cultured on or in type I collagen gel, suggesting the suppression of MMP-2 activity detected in HSC culture using type I collagen. These results indicate a differential expression of MMP activity, hence the remodeling of extracellular matrix components is dependent on the substratum used for HSC culture. The HSC culture using several types of substrata appears to be a useful in vitro model to study the mechanism of extracellular matrix remodeling.
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PMID:Regulatory role of extracellular matrix components in expression of matrix metalloproteinases in cultured hepatic stellate cells. 1521 80

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.
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PMID:Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures. 1557 69

Hydroxyapatite/soluble calcium phosphate composites (HAp/SCaP) are novel HAp-based materials with enhanced solubility that have been developed by annealing HAp in a vacuum. This study compared the effects of HAp and HAp/SCaP on osteoblast proliferation, differentiation, and mineralization using an MC3T3-E1 cell culture system. MC3T3-E1 cells were cultured on HAp or HAp/SCaP, and the number of attached cells and their morphology were examined. The influence of the extract from HAp/SCaP on osteoblast differentiation was determined by the measurement of alkaline phosphatase activity and reverse transcriptase-polymerase chain reaction analysis of the expression of osteoblastic markers. In addition, mineralization was evaluated by the staining of calcium deposits with Alizarin red. Attachment of a greater number of cells exhibiting no degeneration in their morphology was observed on HAp/SCaP compared with HAp after incubation for 7 days or more. Culturing cells with the extract from HAp/SCaP resulted in promotion of alkaline phosphatase activity, the expression of type I collagen, and bone-like tissue formation. The results of the present study indicate that HAp/SCaP shows greater ability in osteogenesis than HAp by increasing collagen synthesis and calcification of the extracellular matrix.
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PMID:Comparison of osteoblast responses to hydroxyapatite and hydroxyapatite/soluble calcium phosphate composites. 1562 83

We have generated transgenic mice expressing a kinase-deficient type II transforming growth factor-beta (TGFbeta) receptor selectively on fibroblasts (TbetaRIIDeltak-fib). These mice develop dermal and pulmonary fibrosis. In the present study we explore activation of TGFbeta signaling pathways in this strain and examine the profibrotic properties of explanted transgenic fibroblasts including myofibroblast differentiation and abnormal metalloproteinase production. Gene expression profiles of littermate wild type or transgenic fibroblasts were compared using high-density gene arrays and validated by Taqman reverse transcriptase-PCR, Northern and Western blotting. Using a specific inhibitor (SD-208) we demonstrate that the abnormal phenotype of these cells is dependent upon TbetaRI kinase (ALK5) activity, and that transgenic fibroblasts show enhanced expression and activation of TGFbeta together with increased levels of wild type TbetaRII. Moreover, we confirm that transgene expression is itself regulated by TGFbeta and that expression at low levels facilitates signaling, whereas high level expression is inhibitory. For a subset of TGFbeta responsive genes basal up-regulation is normalized or suppressed by exogenous recombinant TGFbeta1 at time points coincident with increased transgene expression. These findings explain the profound refractoriness of TbetaRIIDeltak-fib fibroblasts to exogenous TGFbeta1, despite their activated phenotype. Thus, transgenic fibroblasts recapitulate many hallmark biochemical properties of fibrotic cells, including high level CTGF (CCN2) expression and type I collagen overproduction, altered MMP production, and myofibroblast differentiation. These cells also show an enhanced ability to contract collagen gel matrices. Our study demonstrates that altered high affinity TGFbeta receptor function may lead to ligand-dependent activation of downstream signaling, and provides further evidence of a pivotal role for sustained TGFbeta overactivity in fibrosis.
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PMID:Activation of key profibrotic mechanisms in transgenic fibroblasts expressing kinase-deficient type II Transforming growth factor-{beta} receptor (T{beta}RII{delta}k). 1570 53

Transforming growth factor-beta (TGF-beta) plays a critical role in the progression of renal fibrosis. The activity of TGF-beta is tightly controlled by various mechanisms, among which antagonizing Smad-mediated gene transcription by co-repressors represents one of the important components. We investigated the expression, degradation, and ubiquitination of Smad transcriptional co-repressors SnoN (ski-related novel gene N) and Ski (Sloan-Kettering Institute proto-oncogene) in renal fibrogenesis. We also studied the involvement of Smad-ubiquitination regulatory factor 2 (Smurf2) in ubiquitination of SnoN protein. The kidneys of mice with unilateral ureteral obstruction (UUO) and those of sham-operated mice were used. Renal lesions and the expression of TGF-beta1, type I collagen, SnoN, Ski, and Smurf2 were examined by immunohistochemistry, Western blot, and/or real-time reverse transcriptase-polymerase chain reaction. Degradation and ubiquitination of SnoN/Ski proteins were also investigated. The obstructed kidneys of UUO mice showed progressive tubulointerstitial fibrosis, high expression levels of TGF-beta1, type I collagen, SnoN and Ski mRNAs, and low levels of SnoN and Ski proteins. Both degradation and ubiquitination of SnoN/Ski proteins were markedly increased in the obstructed kidneys, in which Smurf2 expression was increased. Smurf2 immunodepletion in extracts of obstructed kidneys resulted in reduced ubiquitination of SnoN. Our results suggest that the reduction of SnoN/Ski proteins resulting from increased ubiquitin-dependent degradation is involved in the progression of tubulointerstitial fibrosis.
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PMID:Ubiquitin-dependent degradation of SnoN and Ski is increased in renal fibrosis induced by obstructive injury. 1668 90

The use of periosteum-derived progenitor cells (PCs) combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional (3D) poly-lactic-co-glycolic acid (PLGA) fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type I collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules of neo-bone tissue formation and the presence of matrix mineralization were confirmed by positive von Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.
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PMID:Osteogenic potential of human periosteum-derived progenitor cells in PLGA scaffold using allogeneic serum. 1697 24


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