Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00790 (PGA)
 2,475 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated a large noncollagenous glycoprotein, laminin, from a mouse tumor that produces basement membrane. The protein consists of at least two polypeptide chains (Mr = 220,000 and Mr = 440,000) joined to each other by disulfide bonds. Laminin and type IV collagen are major constituents of the tumor. Laminin is distinctly different from fibronectin, another component of basement membranes, in amino acid composition and immunological reactivity. Pepsin digestion of laminin releases a large, cystine-rich fragment which retains most of the antigenicity of the original protein. Immunological studies using purified antibody against laminin show that it is produced by a variety of cultured cells. In addition, these antibodies react with the basement membranes of normal tissues, suggesting that this protein or an immunologically related protein is a constituent of the basement membranes of these tissues.
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PMID:Laminin--a glycoprotein from basement membranes. 11 18

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.
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PMID:Collagen synthesis by bovine aortic endothelial cells in culture. 39 Dec 67

We have shown that thrombospondin (tsp), like fibronectin (fn) and von Willebrand factor (vWf), exhibits a rapid, specific and saturable binding to collagen type I fibres (from bovine tendon). The level of binding at saturation is very similar to that of vWf. As with fn and vWf, the interaction is ionic in character and appears to occur by a polyvalent mechanism since there is little inhibition of interaction by monomeric collagen. The conformation of tsp, like that of fn and vWf, is important since denaturation causes reduced complexing. Furthermore, conformational changes in tsp due to the presence of Ca++ can modulate the amount of complex formed under physiological conditions. Tsp, like vWf but in contrast to fn, shows little affinity for denatured fibres emphasizing the importance of collagen conformation. Pepsin digestion suggests an important role for collagen telopeptides; vWf- and fn-binding sites are located more within the collagen triple helix. Comparison of the effect on binding after leucine aminopeptidase or carboxypeptidase digestion suggests involvement of the N- rather than C-terminal telopeptides. No evidence was found for a role for fn, the proteoglycan PG2 or collagen type V, which could be present in type I fibres, in mediating the interaction between tsp and the fibres. VWf did not inhibit the interaction of tsp, but fn did slightly when tested in large excess. This suggests separate binding sites in collagen for all three ligands since fn and vWf are also known to bind independently of each other.
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PMID:Characterization of thrombospondin binding to collagen (type I) fibres: role of collagen telopeptides. 187 Apr 47

Treatment of cultured human skin fibroblasts with increasing doses of gamma-interferon produces a distinct reduction of steady-state levels of the alpha 3 chain of collagen VI mRNA by about 60% but not of the alpha 1 and alpha 2 chain mRNAs. A similar decrease was also observed for collagen I and III mRNA while fibronectin mRNA remained at the same level. The decrease in alpha 3(VI) mRNA is accompanied by a reduced synthesis of collagen VI and by a reduced deposition of both collagen VI and fibronectin in urea-insoluble form in the cell matrix. No other gamma-interferon effects were observed for fibronectin biosynthesis. Immunoprecipitation of metabolically labeled collagen VI demonstrated a strongly reduced synthesis (by 65-80%) of intracellular alpha 3(VI) chains with no decrease found for alpha 1(VI) and alpha 2(VI) chains. All three chains were, however, found to be reduced in the culture medium. Pepsin treatment of immunoprecipitated collagen VI showed similar chain ratios for material in the culture medium obtained in the absence or presence of gamma-interferon. It indicates that correctly assembled heterotrimers of the composition [alpha 1(VI) alpha 2(VI) alpha 3(VI)] are formed and secreted also in the absence of an equivalent alpha 3(VI) chain synthesis but at a reduced rate. The data support previous predictions from sequence analyses [Chu et al. (1988) J. Biol. Chem. 263, 18,601-18,606] that collagen VI molecules composed of all three constituent chains are more stable than other assembly alternatives.
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PMID:Down-regulation of alpha 3(VI) chain expression by gamma-interferon decreases synthesis and deposition of collagen type VI. 250 96

The major collagenous component secreted into the medium of cultured HT-1080 tumor cells was identified as type IV procollagen by specific antibodies and characteristic ratios of incorporated labeled 3-hydroxyproline and 4-hydroxyproline. The disulfide-bonded molecules consisted of two subunits, pro-alpha 1(IV) and pro-alpha 2(IV) chains with apparent molecular weights of 180 000 and 165 000. No conversion of the procollagen to collagen or to procollagen intermediates was detected in the cell cultures. The two subunits apparently represent different gene products, since enzymatic digestion of the separated chains produced quite different peptide maps. Pepsin degraded native type IV procollagen successively into several fragments, some still disulfide-linked, giving rise to a complex set of polypeptide chains (Mr = 30 000-140 000). This agrees with similar diverse patterns produced by pepsin from authentic type IV collagens. The ratio between the pro-alpha 1(IV) and pro-alpha 2(IV) chains varied in several experiments between 1.3 and 1.8, suggesting that the two chains belong to different triple-helical molecules. The cells also produced distinct amounts of fibronectin (subunit Mr = 230 000) and of the basement membrane glycoprotein laminin. The latter showed three subunits with Mr = 220 000, 210 000, and 400 000. A further disulfide-bonded, non-collagenous polypeptide (Mr = 160 000) was detected but not yet identified. Immunofluorescence demonstrated these proteins within the cells but not in a pericellular matrix. The production of basement membrane components by HT-1080 cells and lack of interstitial collagens disagree with the original classification of the cell line as a fibrosarcoma.
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PMID:Biosynthesis of two subunits of type IV procollagen and of other basement membrane proteins by a human tumor cell line. 625 Aug 36

The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed.
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PMID:Restoration of antigenicity of tissue antigens, cell-bound immunoglobulins and immune deposits in paraffin-embedded tissue. The influence of fixation and proteolytic enzymatic digestion. 643 48

Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains 1 (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed KD = 60 nM for variant C and KD > 1 microM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.
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PMID:Structural characterization of two variants of fibulin-1 that differ in nidogen affinity. 784 16

Biodegradable polymers such as poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA) and PGA coated with PLLA are being employed for cell transplantation and for in vivo regeneration of vascular tissue. Ingrowth and organization of fibrovascular tissue inside polymer scaffolds lead to the occlusion of the regenerated blood vessel. In order to provide regulatory mechanisms to control the development of an inner capsule, endothelialization of these materials is necessary. To achieve this, we employed a novel ammonia plasma technique to surface modified PLLA substrates. Human endothelial cell (HUVEC) and rabbit microvascular endothelial cell (RbMVEC) growth was studied on modified PLLA and control PLLA. Our studies show that modified PLLA and fibronectin (Fn)-coated modified PLLA exhibited statistically significant improvement in HUVEC and RbMVEC growth (P<0.001) when compared to PLLA and Fn-coated PLLA. Therefore, ammonia plasma treatment gives us the unique capability of modifying prosthetic biomaterials of various constructs with the eventual transplantation of mammalian cells to be used in tissue engineering or as biological implants.
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PMID:Enhanced growth of animal and human endothelial cells on biodegradable polymers. 1056 62

Morphology and compliance of tissue-engineered blood vessels (TEBV) are dependent on the culture period and production of extracellular matrix (ECM) components in order to increase the strength of the developing tissue. The aim of the present study was to evaluate the potential of TEBVs to produce an ECM similar to native arteries and veins. Human smooth muscle cells (SMC) were seeded onto the poly(glycolic acid) (PGA) scaffold and placed in bioreactors filled with DMEM supplemented with growth factors. After 6 weeks, the vessels were harvested from the bioreactors and seeded with human endothelial cells at the lumen for another 3 days. Then, the TEBVs were harvested for RNA and protein isolation for further RT-PCR and Western blot. TEBVs had a similar macroscopic appearance to that of native vessels with no visible evidence of the original PGA. Histological and immunohistochemical analyses indicated the presence of high cell density and development of a highly organized structure of ECM. After 6 weeks of culture, there were significantly lower gene expression of SMC-specific markers, such as alpha-actin, caldesmon, and vimentin, and proteoglycans, such as biglycan, decorin, and versican, and other ECM components, such as collagen I and elastin, in TEBVs, with and without pulsatile conditions, compared to that of native arteries. Gene expression of fibronectin was significantly lower in TEBVs grown during pulsatile conditions compared to that of native arteries. No difference was observed in TEBVs grown during non-pulsatile conditions. The presence of alpha-actin, collagen I, decorin, and fibronectin at protein level was demonstrated in TEBVs with and without pulsatile conditions after 6 weeks and in native veins and arteries as well. How this deviation translates into mechanical properties remains to be explored.
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PMID:Production of extracellular matrix components in tissue-engineered blood vessels. 1667 96

While the brain vasculature can be imaged with many methods, immunohistochemistry has distinct advantages due to its simplicity and applicability to archival tissue. However, immunohistochemical staining of the murine brain vasculature in aldehyde fixed tissue has proven elusive and inconsistent using current protocols. Here we investigated whether antigen retrieval methods could improve vascular staining in the adult mouse brain. We found that pepsin digestion prior to immunostaining unmasked widespread collagen IV staining of the cerebrovasculature in the adult mouse brain. Pepsin treatment also unmasked widespread vascular staining with laminin, but only marginally improved isolectin B4 staining and did not enhance vascular staining with fibronectin, perlecan or CD146. Collagen IV immunoperoxidase staining was easily combined with cresyl violet counterstaining making it suitable for stereological analyses of both vascular and neuronal parameters in the same tissue section. This method should be widely applicable for labeling the brain vasculature of the mouse in aldehyde fixed tissue from both normal and pathological states.
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PMID:Pepsin pretreatment allows collagen IV immunostaining of blood vessels in adult mouse brain. 1740 41


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