Beta-adrenergic receptor binding in crude porcine adipose tissue plasma membranes. (25/881)

Methods have been detailed to prepare a crude membrane fraction from isolated porcine adipose tissue cells. Adipocytes were obtained after incubation of 5 g of adipose tissue slices with 4,500 units of a selected lot of collagenase in a total volume of 15 mL at 37 degrees C for 90 min. There was no bovine serum albumin present during cell isolation because albumin did not enhance cell yield or yield of lipolytic activity. Isolated cells were lysed by exposure to hypotonic conditions in the presence of 7.5 mM ethylene glycol tetraacetic acid (EGTA) and .8 mM phenylmethylsulfonyl fluoride (PMSF). A 30,000 x g centrifugal pellet was used as the crude membrane preparation. Binding of tritiated dihydroalprenolol (DHA), a beta-adrenergic antagonist, was measured in the presence of 7.5 mM EGTA and .2 mM PMSF, because these protease inhibitors improved specific binding by approximately 50% to greater than 150 fmol/mg of protein and decreased non-specific binding to less than 10% at 2.5 nM DHA.  (+info)

Heparin inhibits collagenase gene expression mediated by phorbol ester-responsive element in primate arterial smooth muscle cells. (26/881)

Heparin is a potent inhibitor of arterial smooth muscle cell (SMC) migration and proliferation in vivo and in vitro. We propose that heparin affects these SMC functions by interfering with either the expression or the activity of secreted proteases required for cell movement. We have reported that heparin selectively inhibits the expression of tissue-type plasminogen activator in SMCs during mitogenesis. In this study we show that the gene expression of another kind of protease, interstitial collagenase, is induced by fetal bovine serum and is also suppressed by heparin. The inhibitory effect on the induced collagenase mRNA is specific to heparin-like molecules and does not depend on the anticoagulant activity of heparin. The induction of the collagenase gene depends on the protein kinase C pathway, since it can be induced by phorbol esters such as phorbol 12-myristate 13-acetate and blocked by inhibitors such as H-7 and staurosporine. In transient transfection assays with chloramphenicol acetyltransferase constructs containing the phorbol ester-responsive element introduced into baboon SMCs, heparin inhibits transcription induced by serum or phorbol 12-myristate 13-acetate. These results support the conclusion that, in primate SMCs, interstitial collagenase gene transcription mediated by the phorbol ester-responsive element is blocked by heparin.  (+info)

Advanced glycosylation end products in the mesenteric artery. (27/881)

We measured advanced glycosylation end products in the mesenteric artery of 37 patients (ages 29-82 years), 34 of whom were nondiabetic. Samples of arterial tissue were obtained during bowel resectioning. Advanced glycosylation end products were measured as collagen-linked fluorescence (excitation wavelength 370 nm, emission wavelength 440 nm) after collagenase digestion of tissue samples. Mean fluorescence of the arterial samples was 15 U/mg (range 5.3-27). Collagen fluorescence correlated with patients' age (r = 0.57; P less than 0.001). No difference in the collagen-linked fluorescence was observed between men and women (P = 0.63), hypertensive and normotensive patients (P = 0.44), smokers and nonsmokers (P = -0.52), and patients with and without symptomatic coronary heart disease (P = 0.7). This study demonstrates, for the first time, the relationship between collagen-linked fluorescence and patients' age in human arterial tissue ex vivo.  (+info)

MAP kinase is constitutively activated in gip2 and src transformed rat 1a fibroblasts. (28/881)

Rat 1a fibroblasts transformed by the Gi2 oncogene, gip2, exhibit a constitutively elevated mitogen-activated protein (MAP) kinase activity that correlates with enhanced tyrosine phosphorylation of the p42 MAP kinase polypeptide. The MAP kinase activity in gip2 transformed cells is 50-60% of the pertussis toxin-sensitive, thrombin-stimulated activity observed in wild-type Rat 1a cells. A similar activation of MAP kinase is observed in src but not ras or raf transformed Rat 1a cells, indicating that the persistent MAP kinase activity results from the action of the specific oncoprotein and is not the consequence of cellular transformation. The enhanced transactivation function of c-Jun characteristic of the transformed phenotype, measured using a collagenase promoter-CAT reporter gene, is observed in gip2, src, ras, and raf transformed Rat 1a cells. The regulatory networks controlled by the four transforming oncogenes therefore alter the activity of specific transcription factors, but only gip2 and src constitutively activate MAP kinase. The findings demonstrate that the catalytic activity of growth factor-regulated cytoplasmic kinases are selectively and stably activated as a consequence of specific oncogene expression.  (+info)

Bovine retained placenta: effects of collagenase and hyaluronidase on detachment of placenta. (29/881)

A significant percentage of cows (11%) fail to release the placenta within 12 h postpartum. Failure of collagen breakdown seems to be related to the retention of placentas. Sections of placentomes incubated with bacterial collagenase caused an increase in placentome proteolysis (6.6-fold) and placentome collagenolysis (94-fold) within 4 h in a dose-related fashion (r = 0.94). Injections of collagenase (825 U/cc) into the placentomes, via umbilical vessels, decreased the cotyledon-caruncle binding force (determined by manometry) to 30 +/- 5 mm Hg from 97 +/- 2 mm Hg, and increased proteolysis by 42% within 8 h (r = -0.95). Hyaluronidase at various concentrations (400-8 250 U/cc) and at various incubation times (up to 8 h) was not effective. Hyaluronidase (825 U/cc) and collagenase (825 U/cc) were not synergistic in loosening cotyledon-caruncle attachment. A single 15-min collagenase pulse, given prior to perfusion with collagenase-free blood, was as effective in loosening cotyledon attachment as was a sustained 2-h perfusion of blood with collagenase added. It was concluded that collagenase caused collagenolysis and loosening of cotyledon from caruncle, but collagenolysis and cotyledon-caruncle separation were not facilitated by the presence of hyaluronidase.  (+info)

The role of the C-terminal domain in collagenase and stromelysin specificity. (30/881)

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.  (+info)

Specificity of the anticollagenase action of tetracyclines: relevance to their anti-inflammatory potential. (31/881)

The concentrations of doxycycline and 4-de-dimethylaminotetracycline required to inhibit 50% of collagenase activity were found to be 15 to 30 microM for human neutrophil and gingival crevicular fluid collagenases. Fibroblast collagenase was relatively resistant to inhibition by tetracyclines; the 50% inhibitory concentrations of doxycycline and 4-de-dimethylaminotetracycline were 280 and 510 microM, respectively.  (+info)

Tumor necrosis factor-alpha stimulates the biosynthesis of matrix metalloproteinases and plasminogen activator in cultured human chorionic cells. (32/881)

To investigate the role of tumor necrosis factor-alpha (TNF alpha) in advanced collagenolysis and degradation of connective tissue components in preterm parturition, the effects of human recombinant TNF alpha (hrTNF alpha) on the production of matrix metalloproteinase 1 (MMP-1)/tissue collagenase, MMP-3/stromelysin, tissue inhibitor of metalloproteinases (TIMP), urokinase type-plasminogen activator (uPa) and prostaglandin (PG) E2 in human chorionic cells were examined in vitro. Human chorionic cells, but not amniotic cells, were found to respond to macrophage-conditioned medium (contains mainly interleukin 1) to produce MMP-1 and MMP-3. This indicated that the chorionic cell is one of the MMP-producing cells of fetal membranes. When confluent chorionic cells were treated with hrTNF alpha, the production of MMP-1 and MMP-3 as well as of uPa and PGE2 was greatly increased in a dose-dependent manner. In contrast, the production of TIMP was suppressed by hrTNF alpha. These results suggested that TNF alpha may participate in destruction of collagen and other connective tissue matrix components of fetal membranes and in promotion of uterine contractility in preterm parturition with intraamniotic infection.  (+info)