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)

Normal human adult articular chondrocytes were used to determine how the chondrocyte phenotype is modulated by culture conditions following long-term culture. We report here for the first time that human articular chondrocytes have a lifespan in the range of 34-37 population doublings. While chondrocytes cultured as monolayers displayed a fibroblastoid morphology and grew faster, those cultured as suspensions over agarose adopted a round morphology and formed clusters of cells reminiscent of chondrocyte differentiation in intact cartilage, with little or no DNA synthesis. These morphologies were independent of the age of the culture. Despite, these morphological differences, however, chondrocytes expressed markers at mRNA and protein levels characteristic of cartilage: namely, types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein, but not syndecan, under both culture conditions. However, they also expressed type I collagen alpha 1(I) and alpha 2(I) chains. It has been suggested that expression of collagen alpha 1(I) by chondrocytes cultured as monolayers is a marker of the loss of the chondrocyte phenotype. However, we show here, using reverse transcriptase/polymerase chain reaction, that normal fresh intact human articular cartilage expresses collagen alpha 1(I). The data show that following long-term culture human articular chondrocytes retain their differentiated characteristics and that cell shape does not correlate with the expression of the chondrocyte phenotype. It is proposed that loss of the chondrocyte phenotype is marked by the loss of one or more cartilage-specific molecules rather than by the appearance of non-cartilage-specific molecules.
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PMID:Expression of cartilage-specific molecules is retained on long-term culture of human articular chondrocytes. 765 19

A competitive reverse transcriptase-PCR (RT-PCR) assay has been developed for the quantification of particular mRNA species in human articular cartilage. Competitor RNA species were synthesized that differed from the amplified target sequence only by the central insertion of an EcoRI restriction site. By using known amounts of synthetic target and competitor RNA, it was shown that competitor RNA molecules designed in this way are reverse-transcribed and amplified with equal efficiency to the target of interest. Furthermore quantification could be performed during the plateau phase of the PCR, which was necessary when using ethidium bromide fluorescence as a detection system. The inhibition of aggrecan and link-protein mRNA expression by interleukin 1 or tumour necrosis factor in monolayers of human articular chondrocytes quantified by this competitive RT-PCR method compared favourably with Northern hybridization studies. The main advantage of this technique is that it can be used to quantify levels of mRNA with RNA extracted directly from 100 mg wet weight of human articular cartilage. Age-related changes in aggrecan and link-protein mRNA were therefore quantified in human articular cartilage directly after dissection from the joint. The concentration of link-protein mRNA was higher in immature cartilage than in mature cartilage when expressed relative to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA, but no age-related changes were observed in aggrecan mRNA expression. The ratio of aggrecan to link-protein mRNA was higher in mature cartilage than in immature tissue. These age-related differences in the molecular stoichiometry of aggrecan and link-protein mRNA might have implications with respect to the regulation of the formation and the stability of the proteoglycan aggregates in cartilage.
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PMID:Quantification of aggrecan and link-protein mRNA in human articular cartilage of different ages by competitive reverse transcriptase-PCR. 891 86

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.
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PMID:Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage. 952 44

A clonal cell line, CS-OKB, was derived from a human chondrosarcoma and characterized by cytogenetic study, immunocytochemical staining, and reverse transcriptase polymerase chain reaction (RT-PCR). Chromosomal abnormalities characteristic of malignant cartilaginous neoplasms were identified. CS-OKB cells were intensely stained with anti-type II collagen and anti-keratan sulphate antibodies. RT-PCR indicated that CS-OKB transcribes cartilage-specific genes such as type II, X procollagen, and aggrecan. This human chondrosarcoma cell line is stable and expresses well-differentiated chondrocyte-specific genes. It synthesizes well-differentiated chondrocyte-specific molecules in uncoated plastic dishes. CS-OKB may be useful for studies of human chondrocytes and in characterizing human chondrosarcomas.
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PMID:Characterization of a newly established human chondrosarcoma cell line, CS-OKB. 967 94

Brevican is a member of the aggrecan/versican family of proteoglycans. In contrast to the other family members, brevican occurs both as soluble isoforms secreted into the extracellular space and membrane-bound isoforms which are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety. Expression of both variants, which are encoded by two differentially processed transcripts from the same gene, is confined to the nervous system. In the current study, we have used in situ hybridization to examine the cellular sites of synthesis for both mRNAs during postnatal development of the rat brain. Whereas the 3.6-kb transcript encoding secreted brevican displays a widespread distribution in grey matter structures, including cerebellar and cerebral cortex, hippocampus and thalamic nuclei with silver grains accumulating over neuronal cell bodies, the smaller transcript (3.3 kb) encoding GPI-anchored isoforms appears to be largely confined to white matter tracts and diffusely distributed glial cells. This expression pattern is further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments with RNA from different glial cell cultures, and by biochemical data demonstrating that the crude membrane fraction from isolated optic nerve contains high amounts of phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive brevican immunoreactivity. During ontogenetic development, both brevican transcripts are generally up-regulated. However, the expression of glypiated brevican is delayed by about 1 week, compared with the expression of the secreted isoform. This late appearance of GPI-linked brevican, its predominant expression in glial cells and its tight association with brain myelin fractions suggest a functional role in neuroglia.
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PMID:Transcripts for secreted and GPI-anchored brevican are differentially distributed in rat brain. 975 Nov 35

Multipotential mesenchymal stem cells capable of chondro-osseous induction contribute to the endochondral callus of healing fractured bone. Microvascular pericytes serving the role of multipotential mesenchymal stem cells are considered osteoprogenitors because they express type I collagen, alkaline phosphatase enzyme activity, osteocalcin immunoreactivity, and bone sialoprotein mRNA. Previous electron microscopic studies indicate that this cell type has a contribution to the fracture callus. Limited data suggest that pericytes may also assume a chondrogenic phenotype. We undertook in vitro studies to understand how the chondro-osseous phenotype of the pericyte might be regulated. Using Northern analysis and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that cultured pericytes produce aggrecan and type II collagen mRNA indicating their chondrogenic potential. Aggrecan message is elevated by BMP-2 as analyzed by both Northern hybridization and RT-PCR. This finding suggests a regulatory role for this morphogen on this phenotype in pericytes. RT-PCR amplified versican product was also associated with pericyte cultures but was not affected by BMP-2. Our data strongly support a chondrogenic role for the pericyte and that the phenotype is regulated at least in part by BMP.
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PMID:Microvascular pericytes express aggrecan message which is regulated by BMP-2. 1069 96

Brain-enriched hyaluronan binding (BEHAB)/brevican is a brain-specific extracellular matrix protein containing a cleavage site between Glu(395)-Ser(396), which bears remarkable homology to the "aggrecanase" site in the cartilage proteoglycan aggrecan. Expression of BEHAB/brevican is dramatically increased in human gliomas, notoriously invasive tumors. Recently, we showed that the rat 9L gliosarcoma cell line, which does not express BEHAB/brevican and forms non-invasive tumors when grown as intracranial grafts, can form invasive tumors when transfected with a 5' cDNA fragment of BEHAB/brevican, but not when transfected with the full-length cDNA. In marked contrast, the highly invasive CNS-1 glioma cell line expresses and cleaves BEHAB/brevican protein when grown as an intracranial graft. These results suggest that both synthesis and cleavage of BEHAB/brevican protein may play a role in the invasiveness of gliomas. We report here, using an antibody developed to the neoepitope created by BEHAB/brevican cleavage at the Glu(395)-Ser(396) site, that the CNS-1 cells are able to cleave the protein in vitro. We characterized the CNS-1-derived cleavage activity by assaying its ability to cleave BEHAB/brevican proteoglycan, and determined that the enzyme is a constitutively expressed, secreted activity. Using a variety of protease inhibitors, reverse transcriptase-polymerase chain reaction, and specific antibodies, we determined that this activity is likely to be a member of the ADAMTS family of metalloproteinases, specifically ADAMTS4. These results suggest a novel function for ADAMTS family members in BEHAB/brevican cleavage and glioma and indicate that inhibition of ADAMTS in glioma may provide a novel therapeutic strategy.
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PMID:Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member. 1080 87

The search for biocompatible materials that can support the growth and phenotypic expression of osteoblasts and chondrocytes is a major challenge in the application of tissue engineering techniques for the repair of bone and cartilage defects. Chitosan, a copolymer of glucosamine and N-acetylglucosamine, may provide an answer to this search. Chitosan is the deacetylated product of chitin, a ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates. Little is known about the utility of chitosan in propagating human osteoblasts and chondrocytes. In this study, we test the hypothesis that chitosan promotes the survival and function of osteoblasts and chondrocytes. Chitosan (4%, w/v in 2% HAc) was coated onto plastic coverslips that had been fitted into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan-coated coverslips at 1 x 10(5)/cells per well. Cultures were incubated at 37 degrees C, 5% CO(2) for a period of 7 days. Cell viability was assessed at that time using a fluorescent molecular probe. The phenotypic expression of osteoblasts and chondrocytes was analyzed by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Osteoblasts and chondrocytes appeared spherical and refractile on chitosan-coated coverslips. In contrast, greater than 90% of cells on plastic coverslips were elongated and spindle shaped after 7 days of culture. Similar to cells propagated on uncoated control wells, greater than 90% of human osteoblasts and chondrocytes propagated on chitosan remained viable. Human osteoblasts propagated on chitosan films continued to express collagen type I whereas chondrocytes expressed collagen type II and aggrecan, as shown by reverse transcriptase-polymerase chain reaction analysis and immunostaining. The present in vitro work demonstrates the biocompatibility of chitosan as a substrate for the growth and continued function of human osteoblasts and chondrocytes. Chitosan may have potential use as a tissue engineering tool for the repair of osseous and chondral defects.
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PMID:Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. 1088 Jan 6

Hyaluronan (HA) is a component of cartilage matrix with known effects on chondrocytes. We tested the effects of adding HA to 3-dimensional (3-D) collagen. sponges on chondrocyte function in vitro. Bovine articular chondrocytes isolated by collagenase digestion were injected into either collagen or HA/collagen scaffolds comprising different amounts of HA (2, 5, 10, and 14% w/w). Expression of aggrecan and type II collagen genes was measured by gene-specific quantitative competitive reverse transcriptase-polymerase chain reactions, and the extracellular matrix was estimated by histomorphometrical analyses. After 7-day culture, the chondrocytes in 2% (w/w) HA sponges expressed fourfold more mRNA transcripts for type II collagen (p = 0.002) and twofold more mRNA transcripts for aggrecan (p = 0.022) than in control collagen sponges. Furthermore, there was 45% more extracellular matrix in 2% (w/w) HA sponges and 43% less matrix in the 10% (w/w) HA sponges compared with plain collagen sponges (p > 0.05). In sum, a small amount of HA in 3-D collagen scaffolds enhanced chondrogenesis, but a greater amount was inhibitory. This 3-D system represents a novel tool to identify mechanisms by which extracellular matrix molecules influence chondrocyte function. Further, these results show the potential for modifying scaffolds to improve production of engineered cartilage for in vivo applications.
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PMID:Effects of hyaluronan on engineered articular cartilage extracellular matrix gene expression in 3-dimensional collagen scaffolds. 1142 90

Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate molecular and structural changes occurring in four articular cartilage (AC) regions from the knees of anterior cruciate ligament (ACL)-transected rabbits at 3 and 8 weeks post-surgery. Rabbit AC from the lateral and medial femoral condyles (LFC and MFC) as well as from the medial and lateral tibial plateau (MTP and LTP) were processed for histology and for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules (collagen II, aggrecan. biglycan, decorin, fibromodulin, MMP-1, -3, -13, and TIMP-1). While the most severe histological changes were observed in the MTP starting as early as 3 weeks post-ACL transection based on Mankin scores, histological examination demonstrated a progression of osteoarthritic changes in the MFC from 3 to 8 weeks post-surgery. In contrast, very few changes were observed within both the LFC and LTP, and these changes did not worsen with increasing time after surgery. The water content increased significantly in the MFC at 8 weeks post-ACL transection and at both 3 and 8 weeks post-ACL transection in the MTP. Significant decreases in DNA content were observed for the MFC, LTP and MTP at 8 weeks post-ACL transection. Total RNA yields from the MFC and MTP were significantly elevated at 8 weeks post-ACL transection, while in the lateral compartment total RNA was unchanged following ACL transection. Analysis of mRNA levels for a subset of matrix molecules, proteinases and proteinase inhibitors, by RT-PCR demonstrated significant region-specific changes at the mRNA level following ACL transection. These results show that following ACL transection, complex molecular, as well as structural changes occur early in cartilage and that the observed changes are both region-specific and time-dependent.
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PMID:Assessment of specific mRNA levels in cartilage regions in a lapine model of osteoarthritis. 1203 28


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