Adenosine 3',5'-cyclic monophosphate in Vibrio cholerae.
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The extracellular concentration of cyclic adenosine 3',5'-monophosphate (AMP) of three different strains of Vibrio cholerae growing in syncase medium were measured. Cyclic AMP secreted by V. cholerae 569B varied widely, with different carbon sources. Mutant 13, which produced little or no toxin, released half the amount of cyclic AMP as the wild type. The release of less cyclic AMP into the medium by mutant 13 may be accounted for by the lower activity of adenylate cyclase observed. High glucose (3%) in the culture medium reduced the concentration of cyclic AMP both in wild type and mutant 13. Reduction of cyclic AMP levels at high concentrations of glucose (3%) occurred without change of adenylate cyclase activity. The release of enterotoxin to the medium varied with carbon sources but was independent of conditions which reduced the cyclic AMP both within the cell and the medium. Neither adenylate cyclase activity nor toxin production was reduced by an increase concentration of glucose in wild-type V. cholerae, whereas cyclic AMP levels were reduced by sixfold. A lower activity of the adenylate cyclase was observed in a mutant of V. cholerae which produced no detectable toxin. Thus, a correlation exists between toxin production and adenylate cyclase activity in V. cholerae. (+info)
Human atherosclerotic plaque cells and leiomyoma cells. Comparison of in vitro growth characteristics.
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Cells derived from human atherosclerotic plaques and from arterial media were compared with cells obtained from human leiomyomata and myometrium with respect to growth behavior in long-term cell culture. None of numerous variations in culture media, including alterations of serum concentration and source, improved the rate of cell multiplication or in vitro longevity. Both uterine cell types, but neither arterial cell type, multiplied after tissue dissociation with enzymes (elastase, collagenase, hyaluronidase). The replicative life-span of each of eight samples of arterial plaque cells was equal to or less than that of the corresponding medial cells. A similar relationship was observed for eight paired sets of leiomyoma and myometrial cells. The results indicate that, under the conditions of culture in vitro, cells of a bona fide smooth muscle tumor have a finite replicative life-span and smooth muscle cells of atherosclerotic plaques behave in a similar manner. (+info)
Collagenolytic activity of some marine bacteria.
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Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources. (+info)
Collagen in the human lung. Quantitation of rates of synthesis and partial characterization of composition.
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The presence of collagen in lung is fundamental in normal lung structure and function. Methods have been developed to examine human fetal and adult lung collagen with respect to its composition and synthesis. The second trimester fetal lung has a large number of cells per unit lung mass (36.6 plus or minus 2.7 mug DNA/mg dry wt) and relatively small amounts of collagen (17.0 plus or minus 5.3 mug collagen/mg dry wt). The number of cells per unit lung mass in the adult lung (11.1 plus or minus 3.4 mug DNA/mg dry wt) is 30% of the number of cells in the fetal lung, but the adult has 11 times more collagen (196 plus or minus 25 mug collagen/mg dry wt). The composition of fetal lung collagen can be partially characterized by extraction with salt at neutral pH, acetic acid, or guanidine. The extracted chains, representing 10% of the total lung collagen, chromatograph as alpha1 and alpha2 chains, each with a mol wt of 100,000 and an animo acid composition characteristic for collagen but not specific for lung. Short-term explant cultures of fetal and adult lung synthesize alpha chains which can be isolated by ion-exchange chromatography. These chains, representing 30-40% of the total collagen synthesized by the explants, coelectrophorese with extracted collagen chains on acrylamide gels: they are destroyed by clostridial collagenase and they have a mol wt of 100,000. Although the composition of the collagen synthesized by these explants can be only partially characterized, the rate of synthesis of both collagen and noncollagen protein can be quantitated. In fetal lung, 4.0 plus or minus 1.2% of the amino acids incorporated into protein per hour are incorporated into collagen. In normal adult lung, this percentage (4.2 plus or minus 0.9%) is remarkably similar. These values are almost identical to the relative rate of collagen synthesis in rabbit lung in the same age range. This technology should be applicable to answer specific questions regarding collagen synthesis and degradation in human lung disease. (+info)
Sequence position of 3-hydroxyproline in basement membrane collagen. Isolation of glycyl-3-hydroxyprolyl-4-hydroxyproline from swine kidney.
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The position of 3-hydroxyproline was investigated in the triplet sequences of peptides released by collagenase digestion of a collagen preparation from kidney cortex. Composition of the collagen preparation indicated that it was largely or wholly of basement membrane origin. 3-Hydroxyproline was detected in only one sequence, the tripeptide, glycyl-3-hydroxyprolyl-4-hydroxyproline, which accounted for a major fraction of the total 3-hydroxyproline obtained in the peptides released by collagenase. Preliminary data, based on sequencing the peptide mixture released by collagenase treatment, suggested that, in contrast, 4-hydroxyproline occurs predominantly if not exclusively in the Y position of Gly-X-Y triplet sequences in the collagen preparation studied. (+info)
The coagulation of insoluble and basic proteins from rat siminal vesicle secretion with vesiculase: influence of collagenase-like peptidase from rat testis.
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Proteins from the secretion of rat seminal vesicles were fractionated to yield (i) insoluble protein, (ii) basic proteins (soluble in the presence of urea) and (iii) neutral plus acidic proteins. The first two are necessary for the formation in vitro of a coagulum which is very similar to the copulatory plug. Collagenase-like peptidase from rat testis degrades both protein fractions and prevents coagulum formation. (+info)
Hormonal regulation of collagenolysis in uterine cervical fibroblasts. Modulation of synthesis of procollagenase, prostromelysin and tissue inhibitor of metalloproteinases (TIMP) by progesterone and oestradiol-17 beta.
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Rabbit uterine cervical fibroblasts produced a large amount of matrix metalloproteinases (MMPs) such as collagenase (MMP-1) and stromelysin (MMP-3) and a small relatively amount of tissue inhibitor of metalloproteinases (TIMP). When cells were treated with progesterone or oestradiol-17 beta, both steroids concurrently decreased the level of procollagenase and prostromelysin in the culture media and the steady-state levels of the respective mRNAs. On the other hand, the level of TIMP in the culture media and the steady-state level of its mRNA were simultaneously increased by these steroids. Similarly, the suppression of production of MMPs and the augmentation of TIMP production by both steroids were observed with interleukin 1 (IL-1)-treated cells, but the action of progesterone was more effective than that of oestradiol-17 beta in the IL-1-untreated and -treated cells. These results suggest that collagenolysis in uterine cervical fibroblasts is negatively regulated by steroid hormones via the acceleration of TIMP production and the suppression of synthesis of MMPs at the pretranslational level. (+info)
Distinction between Duchenne and other muscular dystrophies by ribosomal protein synthesis.
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Ribosome concentration, ribosome distribution on sucrose density gradients, and in-vitro ribosomal amino-acid incorporation (noncollagen and collagen synthesis) were studied in muscle biopsy samples obtained from 30 patients with Duchenne muscular dystrophy, seven patients with Becker muscular dystrophy, and 10 with facioscapulohumeral muscular dystrophy. Ribosome concentration was normal in Duchenne and facioscapulohumeral and decreased in Becker muscular dystrophy. Distribution of ribosomes in sucrose density gradients showed abnormalities (sharp monosomal peak and fewer polyribosomes) only in Duchenne muscular dystrophy and was normal in the other two types. In-vitro amino-acid incorporation of ribosomes in Duchenne muscular dystrophy revealed high collagen and low noncollagen synthesis of the heavy polyribosomes. This abnormality is controlled by an undetermined enzymatic factor belonging to the soluble enzyme fraction. Supplementation of the dystrophic heavy polyribosomes with normal soluble enzymes restored the synthesis of collagen to that of the controls. Heavy polyribosomes extracted from normals or from carriers produce proportionately more collagen in the presence of soluble enzyme fraction from Duchenne muscular dystrophy than in the presence of their homologous enzymes. In Becker muscular dystrophy, both noncollagen and collagen synthesis of the heavy polyribosomes were increased, under the influence of ribosomal factors. The different protein synthesis in Duchenne and Becker muscular dystrophies suggests that these conditions are non-allelic. In facioscapulohumeral muscular dystrophy the changes in protein synthesis occurred only in the early stage of the disease and consisted of increased noncollagen synthesis of the light polyribosomes, while the heavy polyribosomes had normal activity including collagen synthesis. This reaction was controlled by ribosomal factors. (+info)