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)

We studied the effects of megestrol acetate (MA) and ketoconazole (KC) on protein synthesis of epithelial and stromal cells of human prostate. Patients with benign prostatic hypertrophy (BPH) were treated with MA (160 mg/day) plus KC (1,200 mg/day) for 7 days. Prostate tissues obtained from transurethral resection (TURP) were separated into epithelial and stromal cells with 0.5% collagenase. The separated cells were incubated with L-35S-methionine in methionine-free MEM at 37 degrees C for 3 hr. The protein synthesis in both epithelial and stromal cells was significantly inhibited in the group treated with MA and KC when compared to a control group (p less than 0.05). The proteins incorporated with L-35S-methionine were analyzed by SDS-PAGE and autoradiography. The molecular weights of the epithelial and stromal proteins inhibited ranged between 35-55K.
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PMID:Effects of androgen blockade with ketoconazole and megestrol acetate on human prostatic protein patterns. 242 23

To evaluate the role of small amounts of DHT in prostate tissue as a stimulus to epithelial cell growth (protein synthesis) we studied tissue from patients given various androgen-blocking drugs prior to transurethral resection of the prostate (TURP) and measured epithelial protein synthesis and DHT in the tissue specimens. We also studied the effects on stromal cell protein synthesis of an antiestrogen, tamoxifen. Test drugs prior to TURP included Megace 160 mg per day, Megace 160 mg per day plus Tamoxifen 40 mg per day, Megace 160 mg a day plus ketoconazole 1200 mg per day and tamoxifen 40 mg/day. The tissue was processed immediately and epithelial and stromal cells separated by digestion of tissue with 0.5% collagenase. After separation, epithelial cells were labeled with either [3H]leucine or L-[35S]methionine. Stromal cells were labelled with [3H]proline. DHT was measured in whole prostate tissue. Megace alone and Megace plus tamoxifen significantly decreased both [3H]leucine incorporation into protein and tissue concentration of DHT; Megace plus ketoconazole significantly decreased L-[35S]methionine incorporation into protein and DHT. Tamoxifen significantly decreased stromal protein synthesis. When the data correlating DHT with epithelial protein synthesis using both labeling techniques were combined, the curves were parallel and a strong correlation was noted between DHT and protein synthesis over a wide range of values (P less than 0.001). These results suggest that in hormone-dependent prostate cancer even small amounts of prostate DHT such as may occur from adrenal androgens following castration may significantly stimulate growth of the tumor epithelial cells. Since tamoxifen decreased stromal protein synthesis, estrogen is likely a significant growth stimulus to the increased stromal mass characteristic of benign prostatic hypertrophy.
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PMID:Effect of antiandrogen and/or antiestrogen blockade on human prostate epithelial and stromal cell protein synthesis. 243 6

Granulomas are chronic, usually focal, tissue-destructive inflammatory reactions that usually form around slowly degradable, poorly soluble substances. They are dynamic lesions, regulated by complex immune mechanisms. Tachykinins are a family of neuropeptides characterized by the common C-terminal amino acid sequence -Phe-X-Gly-Leu-Met-NH2. One such tachykinin, substance P, has been reported to modulate immunologic responses. In this investigation, granulomas were examined for substance P. Granulomas were isolated from the livers of mice infected with murine schistosomiasis, and substance P was extracted. Immunoreactive substance P was detected by RIA. The authenticity of the molecule was confirmed by elution profile on HPLC. Immunoreactive substance P, identified by immunostaining, localized to eosinophils derived from collagenase-dispersed granulomas. Granulomas were then probed for expression of the gene for substance P (preprotachykinin). Preprotachykinin mRNA was localized to granuloma eosinophils by in situ oligonucleotide hybridization. It is concluded that substance P is present within the granuloma as a result of preprotachykinin production by eosinophils.
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PMID:Eosinophils from granulomas in murine schistosomiasis mansoni produce substance P. 245 38

Synthesis and secretion of basement membrane proteins by 3T3-L1 adipocytes were studied by metabolic labeling of the cells with [35S]methionine. Enhanced synthesis and secretion of many polypeptides of high molecular weight were observed by stimulating the adipose conversion of 3T3-L1 fibroblasts with dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Among these polypeptides, alpha 1(IV) and alpha 2(IV) chains of collagen were identified based on specific immunoprecipitation and digestion with bacterial collagenase. Synthesis and secretion of alpha 1(IV) and alpha 2(IV) chains were negligible in the fibroblasts, but remarkably enhanced in adipocytes. Based on specific immunoprecipitation of a sulfated polypeptide of 150 kDa, enhanced (6-fold) synthesis and secretion of entactin were also demonstrated. Immunoprecipitation with anti-laminin antiserum showed synthesis of three polypeptides with sizes corresponding to B subunits but failed to demonstrate synthesis of the A subunit. Synthesis of these laminin-related polypeptides remained constant during the conversion. Nonreducing sodium dodecyl sulfate electrophoresis showed intracellular assembly of three laminin-related polypeptides into binary and ternary complexes in a similar sequence of AB1B2 formation via B1B2 in embryonal carcinoma F9 (Morita, A., Sugimoto, E., and Kitagawa, Y. (1985) Biochem. J. 229, 259-264). The ternary complex of laminin in 3T3-L1 cells had a size significantly smaller than AB1B2 complex in F9 cells. In this complex, a novel subunit appears to take the place of the A subunit. Thus, a novel laminin complex is produced by 3T3-L1 cells.
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PMID:Enhanced synthesis and secretion of type IV collagen and entactin during adipose conversion of 3T3-L1 cells and production of unorthodox laminin complex. 246 Apr 44

This study describes 11 monoclonal antibodies (Mabs) against human fibroblast collagenase that (i) inhibit the specific catalytic activity of the enzyme and/or (ii) react with one or more forms of the enzyme on Western blots. Each of the Mabs specifically immunoprecipitated the Mr 57,000/52,000 procollagenase from [35S]methionine-labeled culture medium. Five Mabs, designated VI-3, VI-4, 2C5, 4A2, and 7C2, inhibited the activity of fibroblast-type collagenase against soluble monomeric collagen and against reconstituted collagen fibrils but did not inhibit the genetically distinct human PMN leukocyte collagenase. The interstitial collagenase produced by human mucosal keratinocytes (SCC-25) was also inhibited, whereas the corresponding enzyme from rat was not. Assignment of epitopes to structural domains within the molecule based on immunoperoxidase staining of Western blots of collagenase and its autocatalytic fragments revealed that 9 of 11 epitopes, including those recognized by 4 inhibitory Mabs, were clustered in a 169-residue domain, which constitutes the NH2-terminal part of the Mr 46,000/42,000 active enzyme. One Mab (X-2a) specifically recognized the Mr 57,000/52,000 zymogen species and failed to react with the active Mr 46,000/42,000 form. The inhibitory Mab VI-3 was used for immunoaffinity purification of procollagenase from culture media with a recovery better than 80% and a yield of approximately 1.4 mg of enzyme/L of medium.
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PMID:Monoclonal antibodies to human fibroblast procollagenase. Inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH2-terminal end of the activated enzyme. 246 32

Polymorphonuclear leukocytes (PMNs) recovered from 4-h lapine peritoneal exudates contained factors which provoked the synthesis of collagenase, gelatinase, caseinase, and prostaglandin E2 by lapine articular chondrocytes. Rapid secretion of these factors occurred after exposing the polymorphs to phorbol myristate acetate or formyl-Met-Leu-Phe. Fractionation of polymorph lysates by HPLC size exclusion chromatography provided a molecular weight of approximately 14,000 for the active principle. Examination of the most active fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining, confirmed the presence of a single band with this apparent molecular weight. Isoelectric focusing of this fraction revealed the presence of four distinct bands with the pI values 6.90, 7.05, 7.45, and 7.55. This fraction tested positive in a bioassay for interleukin-1. We were unable to activate chondrocytes by exposure to extracts of human PMNs from either peripheral blood or inflammatory synovial fluid.
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PMID:Chondrocyte activation by a putative interleukin-1 derived from lapine polymorphonuclear leukocytes. 253 51

The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts (Flaherty, M., and Chojkier, M. (1986) J. Biol. Chem. 261, 12060-12065). In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+ (Thomas, A.P., Marks, J.S., Coll, K.E., and Williamson, J. R. (1983) J. Biol. Chem. 258, 5716-5725). However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with [5-3H]proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic [8-arg]vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with [35S]methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific [32P]cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment. Vasopressin did not affect collagen production in quiescent cultures of mouse Swiss 3T3, human myofibroblast or rat smooth muscle cells; and hepatocyte collagen production was unaffected by treatment with glucagon or dibutyryl cAMP. Thus, accelerated Ca2+ fluxes induced by vasopressin are associated with decreased production of hepatocyte collagen and albumin in primary cultures that simulate quiescent adult rat liver.
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PMID:Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes. 254 14

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.
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PMID:Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem. 259 13

A high molecular weight extracellular protein has been purified from cell culture medium of Ewing's sarcoma cell lines, by high performance liquid chromatography and electroelution from SDS-PAGE electrophoresis. This protein has an apparent molecular mass of about 500,000 Da on SDS-PAGE. Immunoprecipitation studies with several extracellular matrix glycoproteins (laminin, fibronectin) specific antisera indicate it is a separate protein. Reduction of disulphide bonds with 2-ME or DTT fails to significantly alter its migration on SDS-PAGE gels, other than a slight apparent increase in molecular mass, indicating an apparent single polypeptide chain structure. The slightly greater mobility observed in unreduced gels suggests one or more regions of intrachain disulfide bonding. It is sensitive to pepsin and trypsin, but resistant to bacterial collagenase indicating that it does not contain collagenous domains. Metabolic labelling with 3H-proline, 3H-leucine, and 35S-methionine indicate that this protein is proline-poor, but leucine, and especially methionine, rich. Sodium 35S-sulfate incorporation is totally negative and treatment with glycosaminoglycan degrading enzymes has no effect on the mobility of the protein on gels, unlike typical proteoglycans. This protein appears by rotary shadowing electron microscopy as a long, thin, filamentous molecule at least 500 nm (0.5 um) in length. The tissue localization and function are unknown at this time, but are under active investigation.
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PMID:A novel 500,000 Da, linear, single chain extracellular protein synthesized by several childhood tumors. 263 60

An enzymic method for recovering primordial follicles from the pig ovary consists of incubating cortical slices for 2 h with 0.025% collagenase 1A. An average of 185,000 or 419,000 primordial follicles per ovary were recovered from ovaries collected in Cambridge and Kansas, respectively. Following a discontinuous Percoll gradient, primordial follicles can be separated from contaminating somatic cells by mouth pipette or a micromanipulator to collect 100-1500 follicles but for large scale recovery of approximately 30,000 follicles flow cytometry is recommended. Two types of primordial follicles can be distinguished by electron microscopy: peripheral clusters of small oocytes with an incomplete investment of pregranulosa cells and a deeper region of individual oocytes surrounded by a complete layer of pregranulosa cells. The viability of the purified primordial follicles is attested by their ability to synthesize proteins for at least 12 h after incubation with [35S]methionine. Moreover, the primordial follicles showed several polypeptide bands in common with mature oocytes especially with Mr of 60,000-90,000 but with considerable differences from somatic cells.
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PMID:Isolation and preliminary characterization of pig primordial follicles. 268 42


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