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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Abnormalities of tubular membrane structure and composition have been proposed as the primary defect in nephronophthisis (NEF). In order to characterize the protein composition of tubular cells in NEF, in vitro methods were developed to culture and propagate tubular cells obtained from biopsy fragments. Accordingly, microdissected cortical slices (1 x 3 mm) were first digested with collagenase and DNAse and then grown in RPMI medium supplemented with 10% NU serum and conditioned serum deriving from 3T3 cultures. At confluence, cultured cells from NEF showed characteristics which were typical of normal tubules, i.e. presence of cytokeratin and positivity for succinic dehydrogenase and alkaline phosphatase stainings, and presented no morphological alterations compared to cultured cells from normal tubular epithelium. Moreover, no difference was observed for fibronectin, collagen IV and laminin stains. Analysis by two-dimensional electrophoresis of cellular extracts revealed several changes in protein composition of NEF, the main one being the decrease in NEF cells of a polypeptide with a molecular weight of 120 kD and a pI of 4.8; this polypeptide was a constant finding in normal kidneys. These observations demonstrated that human tubular epithelial cells can be successfully cultured from very small biopsy fragments, which represents a new approach to the study of molecular disorders involving tubular cells in inherited disease. Cultured cells from NEF maintain the same morphological, immunological and cytochemical characteristics as normal tubular cells, but present a few alterations in polypeptide composition which may have pathogenetic relevance. A more careful analysis of these alterations is needed to define the molecular disorder(s) involving the tubule in NEF.
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PMID:Tubular epithelium culture from nephronophthisis-affected kidneys: a new approach to molecular disorders of tubular cells. 207 4

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.
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PMID:Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata. 208 49

The recent cloning of human androgen receptor (AR) cDNAs in this and other laboratories has provided valuable probes for investigating the structure and function of the AR at the molecular level. We now report the overexpression of a region of the human AR containing both the DNA- and hormone-binding domains in E. coli, which provides a means to produce large amounts of AR for analysis and use in functional studies. Under isopropyl-beta-D-thiogalactopyranoside induction, a tripartite protein, consisting of beta-galactosidase, a collagenase recognition site, and AR polypeptide, was produced in E. coli JM109 using pSS20 a as a vector. About 1 mg of the fused AR could be recovered per liter bacterial culture. The induced protein could readily be detected in a sodium dodecyl sulfate-polyacrylamide gel by Coomassie blue staining. Its identity was confirmed by Western blot analysis using antibodies to both beta-galactosidase and the AR. Scatchard analysis of the androgen-binding activity of the hybrid AR revealed high affinity binding to the synthetic androgen, Mibolerone (Kd, approximately 1.2 nM). Competition studies demonstrated the fusion protein's specificity for androgens. The hybrid receptor formed immune complexes with human anti-AR serum that sedimented at about 19S in 10-50% linear sucrose gradients containing 0.4 M KCl. Gel band shift assays revealed that the hybrid receptor protein forms specific complexes with a synthetic steroid response element derived from the mouse mammary tumor virus long terminal repeat region. These results demonstrate that the recombinant AR expressed in E. coli possesses many of the functional properties characteristic of DNA- and steroid-binding domains of the native AR.
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PMID:Overexpression of a partial human androgen receptor in E. coli: characterization of steroid binding, DNA binding, and immunological properties. 212 55

Procollagenase of human polymorphonuclear leucocytes was purified to homogeneity using a rapid and reproducible method. The purification procedure included affinity chromatography on zinc chelate Sepharose, ion exchange chromatography on Q-Sepharose fast flow, followed by affinity chromatography on orange Sepharose and finally a gel-permeation step on Sephacryl S-300. It was shown by SDS/PAGE, under reducing conditions, that the latent collagenase of human polymorphonuclear leucocytes consists of a single polypeptide chain with an apparent relative molecular mass of 85,000. Upon deglycosylation by endoglycosidase F digestion, the apparent relative molecular mass of the procollagenase was reduced to 53,000 which is similar to that of the fibroblast enzyme, and indicates a close relationship between both enzymes. Sequence data were determined by direct automated Edman degradation of the purified polymorphonuclear leucocyte procollagenase. The complete sequence of the propeptide region (residue 1-120) was thereby established. The proteolytic activation of the polymorphonuclear leucocyte procollagenase by various enzymes was investigated by determining the N-terminal sequences of the intermediate and final activated forms. Activation by chymotrypsin and cathepsin G led to the active form (Mr 64,000) by cleaving 79 N-terminal residues from the proenzyme. Trypsin activates in a two-step process. Cleavage of 48 N-terminal residues led to a still latent Mr 70,000 species. The final active form (Mr 65,000) was obtained by splitting off 20 additional N-terminal residues.
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PMID:Characterization and activation of procollagenase from human polymorphonuclear leucocytes. N-terminal sequence determination of the proenzyme and various proteolytically activated forms. 215 79

A novel polypeptide with a molecular weight of 17 kd (17k protein) was identified in bone marrow cell cultures. The synthesis of 17k protein is elevated in cell cultures maintained under Dexter conditions, which support myelopoiesis. The predominance of macrophages in the stromal layer of these cultures and the observation that a mouse myelomonocytic cell line P388D1 is capable of synthesizing large amounts of 17k protein led us to the study of its synthesis by bone marrow macrophages. Metabolic labeling with [14C]proline and partial amino acid analysis of 17k protein demonstrated that the polypeptide contains relatively high amounts of proline and is also sensitive to degradation with bacterial collagenase. However, no hydroxyproline is detectable in 17k protein, and it is extensively degraded with bacterial collagenase. However, no hydroxyproline is detectable in 17k protein, and it is extensively degraded by proteolysis with pepsin, using conditions under which collagen triple helices are resistant to degradation, suggesting that collagen-like structures are not contained in 17k protein. This polypeptide is found predominantly in the cellular layers of bone marrow macrophage cultures. Incorporation of [14C]proline into 17k protein is diminished by increasing concentrations of colony-stimulating factor 1 (CSF-1). The 17k protein may be involved in macrophage proliferation because its synthesis is inhibited by CSF-1, which is required for the maintenance of bone marrow macrophages in vitro.
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PMID:A 17-kd polypeptide, sensitive to bacterial collagenase, is synthesized by bone marrow macrophages. 216 40

The determined nucleotide sequence of the Klebsiella pneumoniae UNF5023 gene pulA comprises a single open reading frame coding for a 1090-residue precursor of the secreted protein pullulanase. The predicted sequence of this protein is highly homologous to that of pullulanase of Klebsiella aerogenes strain W70. However, the UNF5023 pullulanase lacks a collagen-like sequence present at the N-terminus of the mature W70 enzyme and differs further from the W70 pullulanase around residue 300 and at the C-terminus. Pullulanases with or without the collagen-like sequence could not be separated by gel electrophoresis under denaturing or non-denaturing conditions, and were unaffected by collagenase. A large central domain which is highly conserved in both UNF5023 and W70 polypeptides contains eight short sequences that are also found in amylases and iso-amylases. Linker mutations in the region of the UNF5023 pulA gene coding for this domain abolished catalytic activity without affecting transport of the polypeptide across the outer membrane. Hybrid proteins comprising at least the amino-terminal 656 residues of prepullulanase fused to alkaline phosphatase were partially localized to the cell surface, as judged by their accessibility to anti-pullulanase serum in immuno-fluorescence tests. On the basis of these results, we tentatively propose that secretion signals required for recognition and translocation across the outer membrane via the pullulanase-specific extension of the secretion pathway are located near the N-terminus of the pullulanase polypeptide.
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PMID:Molecular characterization of pulA and its product, pullulanase, a secreted enzyme of Klebsiella pneumoniae UNF5023. 218 Dec 42

Human and bovine glomerular basement membrane (GBM) preparations, representing the extracellular matrix of the renal filtration units, were found to contain type VI collagen. This protein was solubilized by guanidine and guanidine-dithiothreitol extractions and characterized after polyacrylamide gel electrophoretic resolution by immunoblotting with an antiserum directed against the alpha 1(VI) and alpha 2(VI) polypeptide chains and by its insensitivity to collagenase digestion in the nonreduced state. In contrast to GBM, which is the product of three distinct cells, type VI collagen could not be detected in extracts from calf lens capsule, an epithelial cell-derived basement membrane. Quantitation by radioimmunoassay of the type VI collagen content of GBM from 17 diabetic and 15 nondiabetic human subjects indicated a 2.8-fold higher level (P less than 0.001) in the diabetic preparations. Because in the glomerulus type VI collagen is considered on the basis of immunohistochemistry to be localized to the mesangium, we believe that measurement of this protein in GBM preparations can provide a valuable index of mesangial expansion in diabetic and other glomerulopathies.
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PMID:Occurrence of type VI collagen in extracellular matrix of renal glomeruli and its increase in diabetes. 221 58

1. Ecdysis of infective Haemonchus contortus larvae is effected by the enzymatic degradation of a specialized region of the second molt cuticle containing a biochemically unique polypeptide (mol. wt = 160,000). 2. The 160,000 mol. wt polypeptide and related polypeptides are synthesized at approximately 6 days of larval development. Antigenically similar polypeptides occur in other ruminant trichostrongyles. 3. Cuticle polypeptides digested during ecdysis differ from second molt cuticle collagens in amino acid composition and collagenase sensitivity. However, some antigenic homology between the 160,000 mol. wt polypeptide and cuticle collagens suggests structurally similar regions.
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PMID:Biochemical characterization of cuticle polypeptides from the infective larvae of Haemonchus contortus. 236 68

Recent reports suggesting that the actions of certain neuroenteric peptides may be mediated in part by the secretion of histamine and other mast cell contents could have important implications for gastrointestinal motility and secretion. However, evidence for a mast cell-hormonal interaction is based on studies using peritoneal or cutaneous mast cells. Because intestinal mucosal mast cells (MMC) differ functionally from peritoneal mast cells (PMC), we compared the effects of several neurotransmitters and intestinal hormones on histamine secretion from two mast cell types in the rat. MMC hyperplasia was induced in rats by infection with the nematode Nippostrongylus brasiliensis, and MMC were isolated from the small intestine by collagenase digestion. Substance P, somatostatin, vasoactive intestinal polypeptide (VIP), neurotensin, and bradykinin had a potent secretagogue effect on (10(-7) to 10(-4)M) PMC which was temperature-, energy-, and calcium-dependent. In contrast to PMC, MMC released significant amounts of histamine only when challenged with substance P. Acetylcholine, bombesin, motilin, and pentagastrin had no secretory effect on either PMC or MMC. The differences between PMC and MMC in responsiveness to peptides could not be attributed to the MMC isolation procedure because PMC treated similarly or mixed with MMC suspensions retained their responsiveness to these stimuli. Our results extend the concept of neurocrine control of mast cell function, but indicate that mast cells from different sites have distinct profiles of responsiveness to regulatory peptides.
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PMID:Mast cell heterogeneity: effects of neuroenteric peptides on histamine release. 240 46

Rabbit uterine collagenase was purified from the medium of involuting uterus (1-2 days postpartum) in culture using ammonium sulfate fractionation, DEAE-cellulose, heparin-affinity, and high performance liquid chromatography. The enzyme was purified more than 1600 fold. Hybridoma cell-lines producing monoclonal antibodies were prepared by fusing the spleen cells of mice immunized with the purified enzyme with mouse myeloma cells (Sp2/O-Ag14). The hybridoma cells were selected with HAT medium, cloned, and screened by ELISA. Antibody-producing ascites were prepared by injecting hybridoma cell-lines into the peritoneal cavities of mice. Western-blot analysis indicated that the antibodies recognized a polypeptide having a molecular weight of 52,000. The IgG isolated from the ascites inhibited the enzyme. Indirect immunofluorescent staining demonstrated that polymorphonuclear leukocytes (PMNs) in the superficial layer of alkali-burned corneas contained collagenase, whereas stromal cells and PMNs within the stroma were not stained by the antibodies. Our results suggest that collagenases produced by rabbit PMNs are different from those produced by fibroblasts from cornea. We hypothesize that PMNs in alkali-burned corneas secrete all or most of their collagenases by degranulation at the anterior surface of the cornea, and then continue to migrate into the deeper portion of the stroma.
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PMID:Development of monoclonal antibodies recognizing collagenase from rabbit PMN; the presence of this enzyme in ulcerating corneas. 243 Jul 58


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