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C Franzblau

Publications and source records attributed to C Franzblau.

At least 109 records · Page 6Linked to original sources
PubMed1982

Kinetics of reduction of the intersubunit disulfides of the carboxyl propeptide of type I procollagen.

The carboxyl propeptide produced by proteolysis during maturation of type I procollagen to collagen was purified to homogeneity from the medium of cultured chick embryo calvaria by a new method. The propeptide was identified as such by its amino acid composition and migration pattern through sodium dodecyl sulfate-polyacrylamide gels in the absence and presence of dithiothreitol. Reduction of the intersubunit disulfides, which covalently join the two C1 and one C2 polypeptides of the carboxyl propeptide, was studied by incubating the propeptide in the presence of dithiothreitol for various times under nondenaturing conditions at pH 8.2. The reduction process was characterized by the appearance of disulfide-linked dimers. The appearance of dimers correlated with the disappearance of the carboxyl propeptide. Monomers, retaining intrasubunit disulfides, appeared concomitant with dimer formation. Reduction of the intersubunit disulfides of the dimers followed; intrasubunit disulfides were retained. The rate of the first process, trimer to dimer plus monomer, was an order of magnitude larger than the rate for the second process, dimer to monomers. The dimeric intermediates were composed of approximately equivalent amounts of (C1)2 and (C1, C2). The kinetics of formation and reduction of (C1)2 and (C1, C2) could not be differentiated by the techniques used. The relative amounts of intermediates found were not those expected if quasi-equivalent intersubunit disulfides were reduced in a random fashion. A possible model for reduction of the intersubunit disulfides of the propeptide has been proposed, and implications for the intersubunit polypeptide surface contacts have been discussed.

Animals↗
PubMed1982

A gelatin-specific protease from hamster lung-derived cell cultures.

Gelatin-specific protease activity from hamster lung fibroblasts and their culture media is described. The fibroblasts were derived from hamster lung explant cultures. The gelatin-specific protease activity is latent and seen only after dialysis of either cells or media. The enzyme activity shares many properties of previously reported gelatinases. The activity is inhibited by EDTA, cysteine, and dithioerythritol, whereas it is not inhibited by p-chloromecuribenzoate, N-ethyl maleimide, or phenylmethylsulfonyl fluoride. Of all substrates tested, activity was observed only against gelatin and not against other substrates tested. It was inactive toward collagen, elastin, and methemoglobin. This enzyme may have a role in the digestion of collagen that has been previously cleaved by mammalian collagenase.

Animals↗
PubMed1982

A method to evaluate the biosynthesis of glycosaminoglycans by the aorta of Cynomolgus monkey.

The biosynthesis of the glycosaminoglycans (GAGs) was investigated in vitro in the aortic tissue of the Cynomolgus monkey incubated with [14C]glucosamine. With the use of a new micromethod, it was possible to quantify the glycosaminoglycans and their radioactive distribution in the aortic tissue and incubation medium. Labeled and nonlabeled chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid were measured. Since the sensitivity of this procedure is between 5 and 20 micrograms of GAGs, as little as 5 mg of dry defatted aortic tissue is sufficient for chemical and radioactive analyses.

Animals↗
PubMed1982

Role of alpha-macroglobulin-elastase complexes in the pathogenesis of elastase-induced emphysema in hamsters.

Radiolabeled, enzymatically active or chloromethyl ketone-inactivated porcine pancreatic elastase was endotracheally instilled into hamsters. Gel filtration of the bronchopulmonary lavage fluid revealed two major radioactive fractions: one, eluting at 780,000 daltons, corresponding to an alpha-macroglobulin-pancreatic elastase complex, and another, at 68,000 daltons, corresponding to an alpha-1-protease inhibitor-pancreatic elastase complex. Elastolytic activity was recovered in the bronchopulmonary lavage fluid up to 4 d after elastase instillation and was associated with the alpha-macroglobulin-pancreatic elastase complex. Small amounts of this complex were recovered 14 d after instillation. When less than 1% (1.5--1.7 micrograms) of the usual dose of elastase was instilled into hamsters, the major radioactive complex was alpha-1-protease inhibitor-pancreatic elastase complex, and little or no elastolytic activity was found in the lavage fluid. In contrast to the instillation of 220 micrograms of elastase, no disease or hemorrhagic reaction was detected with this low dose, and without hemorrhage only insignificant amounts of alpha-macroglobulin-pancreatic elastase complexes were recovered from the lungs. To study the interaction of circulating antiproteases with elastase, hamster plasma was allowed to interact directly with the radiolabeled elastase; alpha-macroglobulin bound much more of the elastase than alpha-1-protease inhibitor, confirming the findings in the lung lavage experiments. The hamster's susceptibility to pancreatic elastase-induced emphysema may depend on the preferential binding of elastase to alpha-macroglobulin, which protects the elastolytic potential, rather than to alpha-1-protease inhibitor, which inactivates elastase. We speculate that if even a fraction of the residual radioactivity found in the hamster lungs as long as 144 d after instillation of elastase represents enzymatically active alpha-macroglobulin-pancreatic elastase complex, this could serve as a source of persistent elastolytic activity, which might explain the progressive nature of the pulmonary lesion.

Animals↗
PubMed1982

Influence of asbestos fibers on collagen and prostaglandin production in fibroblast and macrophage co-cultures.

The interaction of fibroblasts, macrophages, and crocidolite asbestos fibers was studied in cell culture. We determined the effects of co-cultivation and asbestos fibers on collagen, total protein, and PG production. The co-cultivation of guinea pig alveolar macrophages and fetal lung fibroblasts resulted in a 155% increase in protein and a 31% increase in collagen production above fibroblast controls. The collagen was derived exclusively from the fibroblasts. Although total protein production was derived predominantly from the fibroblasts, the macrophages in co-culture also contributed to the protein levels. The addition of asbestos fibers to fibroblast cultures resulted in a decrease in collagen and total protein production. The addition of asbestos fibers to fibroblast and macrophage co-cultures prevented the enhancement of collagen production and limited the increase in protein production above fibroblast controls. PGE2, PGI2, and TXA2 were measured in macrophage and fibroblast cultures. Very low, almost undetectable PG production was observed under basal conditions by either cell type alone or in co-culture. Bradykinin induced release of these PGs in fibroblast but not macrophage cultures. This release was enhanced in co-cultures. Asbestos fibers, when added to the co-cultures caused a significant increase in the release of PGs, particularly PGE2. PGE2 is known to inhibit collagen and total protein production by fibroblasts. The increased release of PGs in asbestos-treated co-cultures may have contributed to the described asbestos suppression of collagen production

Animals↗
PubMed1981

Partial purification and characterization of a gelatin-specific protease from the culture media of human pulmonary alveolar macrophages.

A gelatin-specific protease from the culture media of human pulmonary alveolar macrophages has been partial purified by gel filtration and characterized. The macrophages were obtained by bronchopulmonary lavage from the lungs of disease-free smoking volunteers. The gelatin-specific protease initially requires trypsin activation. After chromatographing the culture media on a Sephadex G-200 column, trypsin is no longer required for activation. The gelatin-specific protease reported here shares many properties of previously reported gelatinases. It is inhibited by EDTA, cysteine, dithiothreitol and serum. It is unaffected by other protease inhibitors: phenylmethylsulfonyl fluoride, tosyllysine chloromethyl ketone and p-chloromercuribenzoate. Of all substrates tested activity was observed only with gelatin. It was inactive toward collagen, elastin and methemoglobin. This enzyme may have a role in the digestion of collagen which has been cleaved by a mammalian collagenase.

Animals↗
PubMed1981

Measurement of medium lysyl oxidase activity in aorta smooth muscle cells. Effects of multiple medium changes and inhibition of protein synthesis.

Cultures of rabbit aortic smooth muscle (RSM) cells are a valuable model system for studying production and metabolism of connective tissue components. This report describes various assay procedures for lysyl oxidase, the enzyme responsible for deaminating lysine residues to give aldehyde cross-link precursors, in culture medium from these cells. Studies of the medium enzyme from second-passage RSM cells indicate that approximately 40% of the total enzyme activity in the flask of cells is in the medium. The medium enzyme levels are replenished quite rapidly following refeeding, and enzyme levels in the medium appear to be feedback controlled. The mechanism for this control is unknown at present. Multiple refeeding experiments in which the medium was changed every 2-4 h for up to 40 h indicate that these cells are cap]able of producing large amounts of enzyme and are capable of altering enzyme production and secretion quite rapidly in response to changes in their environment. Protein synthesis inhibitor studies with cycloheximide suggest that the major portion of the enzyme released into the medium following refeeding is newly synthesized although a pool of latent enzyme is also present. As in intact tissue, extraction of the enzyme from the cell layer requires strong denaturing reagents such as 4 M urea. These results suggest that the production of lysyl oxidase is closely regulated and is very responsive to changes in the external environment of the cells. This cell culture system appears to be an excellent one to study the production of lysyl oxidase and its role in connective tissue fibrillogenesis.

Amino Acid Oxidoreductases↗
PubMed1981

Elastin biosynthesis and cross-link formation in rabbit aortic smooth muscle cell cultures.

Rabbit aortic smooth muscle cells in culture produce insoluble elastin which can be purified by treatment with hot alkali. These cells, when maintained in the same flask for long periods of time, continue to accumulate elastin. Both desmosine and isodesmosine, cross-links unique to insoluble elastin, have also been found to increase with time in culture. The results from pulse-chase studies with radiolabeled proline and lysine confirm these observations. All the data indicate that the appearance of the desmosines in the elastin in these cell cultures is a relatively slow process, while the lysine-derived aldehydes appear quite rapidly.

Animals↗
PubMed1981

Effects of 17 beta-estradiol on the biosynthesis of collagen in cultured bovine aortic smooth muscle cells.

The effect of 17 beta-estradiol (E2) on the biosynthesis of collagen in cultured bovine aortic smooth muscle cells was explored. Cells treated with various concentrations of the hormone for 14 days following subcultivation were subjected to growth studies. The cultures were also evaluated for [14C]hydroxyproline formation, the presence of collagenase-susceptible protein, prolyl hydroxylase activity, and procollagen types. There were no effect of E2 on the growth of these cells. At 10(-8) M E2, the hydroxylation of proline when compared to control cultures was reduced by 25-30%; however, little difference in extractable prolyl hydroxylase activity or total [14C]proline incorporation into protein was observed. The effect on collagen synthesis appears to be dose dependent over concentrations of E2 ranging from 10(-6) to 10(-12) M when measured by collagenase susceptibility. Procollagen typing on diethylaminoethylcellulose displayed reduced amounts of procollagen type I and type III fractions as well as other collagenous components. More importantly, however, the ratio of these two procollagen types were also altered. Similar results were obtained from the medium or cell layer. It is concluded that aortic smooth muscle cells cultured in the presence of 17 beta-estradiol display a decreased production of collagen in addition to altering the ratio of type I to type III procollagen fractions produced.

Animals↗
PubMed1981

Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils.

We have examined the effect of the myeloperoxidase-hydrogen peroxide-halide system and of activated human neutrophils on the ability of serum alpha 1-protease inhibitor (alpha 1-PI) to bind and inhibit porcine pancreatic elastase. Exposure to the isolated myeloperoxidase system resulted in nearly complete inactivation of alpha 1-PI. Inactivation was rapid (10 to 20 s); required active myeloperoxidase, micromolar concentrations of H2O2 (or glucose oxidase as a peroxide generator), and a halide cofactor (Cl- or I-); and was blocked by azide, cyanide, and catalase. Intact neutrophils similarly inactivated alpha 1-PI over the course of 5 to 10 min. Inactivation required the neutrophils, a halide (Cl-), and a phorbol ester to activate secretory and metabolic activity. It was inhibited by azide, cyanide, and catalase, but not by superoxide dismutase. Neutrophils with absent myeloperoxidase or impaired oxidative metabolism (chronic granulomatous disease) failed to inactivate alpha 1-PI, and these defects were specifically corrected by the addition of myeloperoxidase or H2O2, respectively. Thus, stimulated neutrophils secrete myeloperoxidase and H2O2 which combine with a halide to inactivate alpha 1-PI. We suggest that leukocyte-derived oxidants, especially the myeloperoxidase system, may contribute to proteolytic tissue injury, for example in elastase-induced pulmonary emphysema, by oxidative inactivation of protective antiproteases.

Alpha-Globulins↗
PubMed1981

Lysine-derived cross-links in the egg shell membrane.

Egg shell membrane protein contains significant quantities of the lysine-derived aldehyde, allysine, and its aldol condensation product. NaB3H4 reduction followed by alkaline hydrolysis of purified protein revealed that there were six residues/1000 of both allysine and the reduced aldol while only traces of desmosine and isodesmosine were detected. The amino acid composition of the membrane protein did not resemble that of mammalian elastin.

2-Aminoadipic Acid↗
PubMed1981

Ultrastructural changes in hamster lung four hours to twenty-four days after exposure to elastase.

A single endotracheal instillation of elastase initiates a series of changes in animal lungs that results in a condition resembling human panlobular emphysema. An ultrastructural examination of this series of changes was conducted on the lungs of male golden hamsters exposed to 3H-methylated pancreatic elastase and sacrificed at intervals between 4 hour and 24 days after exposure to enzyme. Lung tissue between 4 and 48 hours showed evidence of hemorrhage and progressive degradation of elastic fibers. Very little indication of epithelial cell damage accompanied these changes. Four days after exposure to elastase, synthesis of new elastic fibers began with the appearance of small clumps of microfibrils in close association with interstitial cells, fibroblasts, and smooth muscle cells. There was also evidence of alterations in alveolar type II cells at this time. Small fibrillar elastic fibers continued to be present in the lung through twenty-four days and may represent a slow repair process or may indicate a structural difference in elastic fibers synthesized after exposure to elastase. Evidence of the continued degradation of elastic fiber could be found up to 16 days after exposure to elastase, revealing that repair processes were occurring in some areas of the lung while destructive process still predominated in other areas.

Animals↗
PubMed1981

Ultrastructural studies of a polygonal myofilament network in cultured rat aortic smooth muscle cells.

Cultured rat aortic smooth muscle cells (SMC) were examined by electron microscopy and found to contain polygonal networks of 75 A degrees thin myofilament bundles. The cells also had large bundles of longitudinally aligned thin myofilaments with periodically spaced dense bodies. Abundant plasmalemmal vesicles were present at the cell periphery, and the cells were connected by desmosomes. Intercellular spaces contained sparse amounts of elastic fibers, a material generally present in SMC cultures. Analyses of amino acids by automated column-chromatography showed that isodesmosine and desmosine, two amino acid residues unique for elastin, were present. Accordingly, it was concluded that polygonal networks, previously detected solely in cultured nonmuscle cells, were present in SMC. Other findings suggest 1) a change in myofilament arrangement takes place during cell migration, and 2) rat aortic SMC grown in tissue culture flasks is an important experimental tool in the study of cell motility since such myofilament rearrangements were observed to occur up to fourteen days in first passage.

Animals↗