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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of antigen retrieval (AR) technique on immunoreactivity in formalin-fixed, paraffin-embedded tissues is recognized. In focal reactive overgrowths of oral mucosa, we noted that patterns of immunoreactivity to a fibronectin polyclonal antibody were dependent on methods of AR. To establish these patterns we investigated eight pyogenic granulomas and eight fibroepithelial polyps. In the absence of AR no immunoreactivity was observed. After alpha-chymotrypsin AR a band of intense immunoreactivity was associated with vascular endothelial cells, with either minimal or no staining of connective tissue. After microwave AR immunoreactivity was observed in connective tissue, especially in the subepithelial region, but there was no specific vascular staining. After autoclave AR immunoreactivity was observed in connective tissue and also in epithelial nuclei. To determine if these results represented either changes induced by tissue fixation and processing or exposure of epitopes normally masked in vivo, we investigated frozen sections from two fibroepithelial polyps and one pyogenic granuloma. In frozen sections immunoreactivity was observed around vascular endothelial cells and within connective tissue, especially in the subepithelial region, but not in epithelia. This study provides indirect evidence that alpha-chymotrypsin and heat-mediated AR protocols expose different masked epitopes, and reinforces the need to undertake appropriate pilot studies for immunohistochemical investigation of archival tissue.
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PMID:Patterns of immunoreactivity to an anti-fibronectin polyclonal antibody in formalin-fixed, paraffin-embedded oral tissues are dependent on methods of antigen retrieval. 756 Aug 93

Vesicles made by Porphyromonas gingivalis possess several biological activities, including the ability to adhere to oral surfaces and to bacteria. In this study, a new and simple method was developed to measure the adherence capability of outer membrane vesicles from P. gingivalis. Vesicles were conjugated to fluorescent microspheres (0.7 micron) and added to wells of a Teflon-coated microscope slide previously covered with a variety of soluble ligands. After incubation and washes, the number of fluorescent microspheres per microscopic field were counted. Vesicle-coated microspheres attached best to gelatin (> 200 per field), whereas other compounds (such as fibronectin, fibrinogen, collagen and laminin) provided moderate attachment, and no attachment was observed to bovine serum albumin. Adherence to any of the tested ligands was not observed when fluorescent microspheres were conjugated to bovine serum albumin or lipopolysaccharides from P. gingivalis. The adherence of vesicle-coated microspheres to ligands was not significantly affected when the pH of the reaction mixture was between 4 and 10. None of the tested carbohydrates lowered the attachment capability of vesicle-coated microspheres to substrates. When vesicle-coated microspheres were treated with trypsin and chymotrypsin or heated, this resulted in a significant loss of attachment, suggesting a possible involvement of proteinaceous molecules in the process. The present study confirms that vesicles of P. gingivalis are capable of attachment to various molecules and indicate their potential role in colonization.
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PMID:Demonstration of adherence properties of Porphyromonas gingivalis outer membrane vesicles using a new microassay. 767 22

The sensitivity to serine proteinases of cellular proteins involved in cell-matrix adhesion was investigated using C32 melanoma cells. Cells dissociated from monolayers by the metal chelator ethylenediaminetetraacetic acid were incubated with proteolytic enzymes, and then attachment was quantified by standard cell adhesion assays. The effect of proteinases was found to depend on the presence of Ca2+ in the incubations. Incubation with 100 nM trypsin or chymotrypsin for 1-2 h (37 degrees C) in the absence of Ca2+ reduced cell attachment to vitronectin (Vn), fibrinogen (Fb), laminin, and fibronectin by approximately 80, 80, 40, and 30%, respectively. Viability studies indicated that such treatment with proteinases was not cytotoxic. Inclusion of 0.1 nM CaCl2 in the incubations prevented the loss in attachment to all substrata. In the case of Fb, proteinase treatment in the presence of Ca2+ had an additional effect; it improved cell attachment to this substratum by about 50%. C32 cells have been shown to express the integrin alpha v beta 3 (Vn receptor) which mediates attachment to Vn and Fb in a GRGDS-sensitive manner. Attachment of C32 cells to Vn and Fb prior to proteinase treatment and after proteinase treatment in the presence of Ca2+ was 90% inhibited by the addition of GRGDS peptide to the attachment assays. These results suggest that the adhesion observed both before and after proteinase treatment was mediated by this integrin. Analysis of the Vn receptor from proteinase-treated cells by immunoblotting of cell extracts and by SDS gel electrophoresis of immunoprecipitated receptor revealed no detectable change in either the alpha v or beta 3 subunit that correlated with loss in attachment. Similarly proteinase treatment in the presence of Ca2+ did not produce detectable alterations in the subunits which might correlate with the improved attachment to Fb. Consistent with these results, an enzyme-linked immunoassay to quantify cell surface receptors revealed little difference in the amount of Vn receptor on cells treated with proteinase in the presence or absence of Ca2+. Degradation of the alpha v subunit was demonstrated, however, at proteinase concentrations higher than those required to affect cell attachment. Thus, treatment of cells with serine proteinases can affect integrin-mediated attachment to matrix proteins in a manner moderated by Ca2+, but the alterations in attachment do not appear to be accompanied by detectable proteolytic modification of the integrin.
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PMID:Modification of integrin-mediated cell attachment to substrata by serine proteinases in the presence and absence of divalent cations. 768 80

The domains of laminin utilized by cells from human squamous-cell carcinoma (SCC) to promote adhesion were investigated. The ability of cultured SCC cells to adhere to surfaces adsorbed with laminin, laminin fragments, or laminin peptides was examined in a direct, solid-phase adhesion assay. The cells adhered in a concentration-dependent and saturable manner to laminin and E3 and E8 fragments of elastase-digested laminin. These results suggest that SCC cells adhere to at least two distinct sites within the carboxy terminal long arm of laminin. In contrast, SCC cells adhered poorly to the 440-kDa chymotrypsin-resistant fragment of laminin, and the E1' and E4 elastase-digested fragments of laminin, suggesting that the short arms, including the cross-region, of laminin does not contain binding sites for these cells. Synthetic peptides GD-2 and -6, comprised of amino acid sequences derived from the E3 fragment, promoted the adhesion of SCC cells in a concentration-dependent and saturable manner. The specific interaction of SCC cells with GD-2 was demonstrated by competition assays in which soluble GD-2 and anti-peptide GD-2 IgG inhibited cell adhesion to GD-2. The anti-peptide GD-2 IgG partially inhibited the adhesion of SCC cells to the E3 fragment and intact laminin, but not to fibronectin. These results suggest that SCC cells recognize the sequence of GD-2 within laminin. The role of integrins in mediating the adhesion of SCC cells to laminin and GD-2 was then investigated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Promotion of human oral squamous cell carcinoma adhesion in vitro by the carboxy-terminal globular domain of laminin. 769 5

An extracellular proteinase has been partially purified from culture filtrates of Trichophyton rubrum by ultrafiltration, isoelectric focusing and gel filtration chromatography. The enzyme has a non-reduced molecular weight of 235,000 by substrate SDS-PAGE. It has a pH optimum of 8.5 using azocasein and azoalbumin as substrates and a pI of 3.6-3.8. The metalloproteinase inhibitors EDTA and 1,10-phenanthroline, together with the chymotrypsin inhibitor chymostatin, strongly inhibited its activity. The serine proteinase inhibitors phenylmethanesulphonyl fluoride and diisopropylfluorophosphate showed weak inhibitory activity. The proteinase exhibited broad substrate activity against azocoll, azoalbumin, azocasein, laminin and fibronectin. It exhibited weak activity against elastin and keratin. Observations on the occurrence of this proteinase together with previously described lower molecular weight proteinases suggests that the former is the first to appear in minimal medium cultures. Freeze/thaw cycling of the partially purified 235,000 M(r) proteinase was found to generate low molecular weight proteinases, particularly at 53,000, 27,000 and 25,000 M(r), indicating that the latter may originate from the larger molecule.
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PMID:Partial purification and characterization of a 235,000M(r) extracellular proteinase from Trichophyton rubrum. 784 25

To determine the relationship between pancreatic secretory capacity and nutritional status in celiac patients, we studied 52 patients with celiac disease (24 males, 28 females; age range 6-36 months) and 30 healthy control subjects (14 males, 16 females; age range 6-42 months). A secretin-cerulein test was performed on all patients, and levels of serum albumin and plasma fibronectin were assayed. In addition, weight/height ratios were calculated in the celiacs, who were then divided into three groups on this basis, as follows: celiacs with weight/height ratio < or = 3rd percentile; those with weight/height ratio between the 4th and 10th percentiles; and those with weight/height ratio > 10th percentile. There was no significant difference in the duodenal output of chymotrypsin, phospholipase and lipase between these groups. When the total celiac group was compared to control subjects, only lipase levels were significantly lower (P < 0.009). However, subnormal values in one or more pancreatic enzymes were observed in 15/52 celiacs (29%). A residual enzyme activity < 10% of normal secretory capacity, was also found in 4/52 patients. There was no correlation between the output of the various pancreatic enzymes and levels of albumin, fibronectin, and weight/height ratios in the patients. Furthermore, there was no difference in weight/height ratios and levels of albumin and fibronectin between the celiac subjects with pancreatic deficiency and those with normal pancreatic function. We conclude that a mild/moderate pancreatic insufficiency is quite frequent in celiacs, but that it may be completely independent of nutritional status; further studies are therefore required to shed light on its pathogenesis.
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PMID:Pancreatic insufficiency in celiac disease is not dependent on nutritional status. 792 48

Clusters of nicotinic acetylcholine receptor (AChR) in cultured rat myotubes are organized into rectilinear arrays of receptor-rich and receptor-poor domains. Extracellular matrix (ECM) molecules, including fibronectin, heparan sulfate proteoglycan, laminin, and type IV collagen, codistribute with AChR in these clusters. We have examined the stability of this association. We disrupted the AChR clusters in intact myotubes with sodium azide, an energy metabolism inhibitor, and with culture medium free of Ca2+. We also altered or extracted proteins from detergent-isolated AChR clusters by treating with buffers of low ionic strength or alkaline pH or with insoluble chymotrypsin. Each of these treatments dispersed AChR clusters and, simultaneously, caused fibronectin, heparan sulfate proteoglycan, laminin, and type IV collagen to disperse from AChR-rich strips of membrane. Control experiments indicated that insoluble chymotrypsin had no direct effect on the ECM at AChR clusters. It did, however, remove spectrin and the receptor-associated 58-kDa protein from the cytoplasmic surface of receptor clusters. Thus, the ECM at AChR clusters is disrupted by an agent acting at the cytoplasmic surface of the membrane. We discuss the possibility that both AChR and ECM are bound to a common membrane skeleton and the implications this may have for synaptogenesis.
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PMID:Evidence for transmembrane anchoring of extracellular matrix at acetylcholine receptor clusters. 850 May 52

The mechanism of human bladder cancer cell invasion is not clear, but it appears that extracellular matrix components, such as fibronectin, may be involved. To investigate the role of fibronectin in tumor cell invasion and progression, we used an in vitro invasion assay to define the motility stimulating fragment of fibronectin for invasive human bladder cancer T24 cells. Using a modified Boyden chamber assay and purified fragments of fibronectin, we demonstrated that both the 120 kDa chymotrypsin generated fragment of fibronectin (containing the cell attachment RGD motif and additional sequences towards the carboxyl-terminal heparin binding domain), as well as the trypsin generated 60 kDa fragment of fibronectin (containing the carboxyl-terminal heparin binding domain and additional sequences towards the cell attachment RGD motif), were able to stimulate the migration of invasive human bladder cancer T24 cells. Control fragments containing only the amino-terminal gelatin binding region of fibronectin did not stimulate the motility of the human bladder cancer T24 cells. To determine the molecular mechanism in which these fragments may stimulate the migration of the T24 cells, we assayed for intracellular signal transduction pathway protein kinase C (PKC). We demonstrated that both the 120 kDa and the 60 kDa fragments were able to stimulate the activation of protein kinase C. Non-motility stimulating fragments of fibronectin were not able to activate protein kinase C. We conclude that the PKC signal transduction pathway may be involved in matrix mediated motility, and suggest that the inhibition of such pathway(s) may alter the malignant phenotype of human bladder cancer.
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PMID:Specific sequences of fibronectin activate the protein kinase C signal transduction pathway in invasive bladder cancer. 862 Apr 37

The alternatively spliced type III connecting segment (IIICS) of fibronectin (Fn) contains an amino acid sequence, CS-1, which is recognized by the integrin receptor, alpha 4 beta 1. Plasma Fn inhibits alpha 4 beta 1-dependent binding of lymphocytes and monocytes to CS-1 containing Fn derivatives poorly, suggesting limited exposure of the CS-1 sequence in Fn. To test the availability of CS-1 in plasma Fn, an antibody was raised to the synthetic peptide CS-1. The CS-1 sequence was found to be minimally exposed in plasma Fn; and immobilization of Fn, a model of matrix deposition, caused only a modest increase in its exposure. Digestion of Fn with selected proteases, however, induced substantial expression of the CS-1 sequence. The acid protease cathepsin D generated fragments of 31-33.5 kDa from the COOH-terminal heparin-binding domain of Fn which possessed high immunoreactivity with anti-CS-1. Digestion of Fn with cathepsin B also resulted in the exposure of CS-1 sequence in a 140 kDa fragment. Although the digestion of Fn with neutral proteases (neutrophil elastase, cathepsin G, chymotrypsin, trypsin) generated fragments from the COOH-terminal heparin-binding domain of similar molecular weight as with cathepsin D, the exposure of CS-1 did not occur. Exposure of the CS-1 region by the cathepsins was supported by cell adhesion experiments; digestion of Fn with cathepsins D and B transformed inert plasma Fn to an effective inhibitor of adhesion of lymphoblastoid B and T cells (Ramos, Jurkat, Molt-4) to an immobilized CS-1 conjugate. These results suggest that exposure of the CS-1 sequence in plasma Fn by proteolysis with cathepsins D and B, enzymes implicated in several pathological processes, may serve a regulatory function in cell adhesion. The adhesive function of the CS-1 region in intact Fn appears to be suppressed by the native conformation of the molecule.
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PMID:Proteolysis regulates exposure of the IIICS-1 adhesive sequence in plasma fibronectin. 871 84

The binding properties of the COOH-terminal hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelatinase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components. Recombinant C domain (rC domain) (Gly417-Cys631) was expressed in Escherichia coli, and the purified protein, identified using two antipeptide antibodies, was determined by electrospray mass spectrometry to have a mass of 25,925 Da, within 0.1 Da of that predicted. As assessed by microwell substrate binding assays and by column affinity chromatography, the matrix proteins laminin, denatured type I collagen, elastin, SPARC (secreted protein that is acidic and rich in cysteine), tenascin, and MatrigelTM were not bound by the rC domain. Unlike the hemopexin-like domains of collagenase and stromelysin, the rC domain also did not bind native type I collagen. Nor were native or denatured types II, IV, V, and X collagen, or the NC1 domain of type VII collagen bound. However, binding to heparin and fibronectin (Kd, 1.1 x 10(-6) M) could be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively. Using nonoverlapping chymotrypsin-generated fragments of fibronectin, binding sites for the rC domain were found on both the 40-kDa heparin binding and the 120-kDa cell binding fibronectin domains (Kd values, approximately 4-6 x 10(-7) M). The Ca2+ ion, but not the potential structural Zn2+ ion, were found to be essential for maintaining the binding properties of the protein. The apo-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca2+ ion chelator 1, 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, but not the Zn2+ ion chelator 1,10-phenanthroline, eluted the holo form of the rC domain from both heparin-Sepharose and fibronectin. Inductive coupled plasma mass spectrometry also did not detect a Zn2+ ion in the rC domain. In contrast, reduction with 65 mM dithiothreitol did not interfere with heparin binding, further emphasizing the crucial structural role played by the Ca2+ ion. Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca2+ ions are important in maintaining the structure and function of the domain.
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PMID:The hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2) requires Ca2+ for fibronectin and heparin binding. Binding properties of recombinant gelatinase A C domain to extracellular matrix and basement membrane components. 905 49


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