Specific identification of collagens and their fragments by clostridial and anti-collagenase antibody.

A method specific for identification of collagens irrespective of type, species, or tissue origin, and of their derived fragments of molecular weight more than 10,000, is described. The method is based on the low-temperature affinity between clostridial collagenase and almost all types of collagens as well as on the affinity between collagenase and its antibodies. Various collagens or fragments derived from them by treatment with CNBr were separated by SDS-PAGE and immobilized onto a nitrocellulose membrane by a slot-blot technique or electrotransfer.

Biochemistry and molecular biology of MNSs blood group antigens.

This chapter has reviewed the nature of antigens of the MNSs blood group system. The structures of the proteins and the molecular features and organization of glycophorin genes were described, emphasizing their domain arrangement and the extensive sequence homology that indicates that their common and variant alleles belong to a single gene family. Methods currently used to examine these antigens are immunoblotting and DNA typing.

Properties of heart fibroblasts of adult rats in culture.

In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate.

Collagen accumulation in heart ventricles as a function of growth and aging.

Increase in resting tension of left ventricular papillary muscle with age has been attributed to the amount of collagen present. We therefore studied the total amount and structure of myocardial collagen as a function of age in the hearts of male Fischer 344 rats. Using amino acid analysis and quantification of hydroxyproline, we showed that collagen accumulates in relation to ventricular protein after 3 months of age and continues in that mode with increased age of the animal, levelling off at 22 months.

Localization of types I, III and IV collagen mRNAs in rat heart cells by in situ hybridization.

Previous studies investigating the cellular origins of several collagens in young adult rat hearts (Eghbali et al., 1988) demonstrated that the mRNAs for types I and III collagen occurred in non-myocyte cells, mostly fibroblasts, whereas the mRNA for type IV collagen was observed in both myocytes and non-myocyte cells. In the present study, cellular localization of collagen mRNAs has been achieved by in situ hybridization in rat heart tissue and in isolated heart cells.

Visualization of collagenase-sensitive acetylcholinesterase in isolated cardiomyocytes and in heart tissue.

Previous studies have indicated that the asymmetric form of acetylcholinesterase (collagen-tailed) is localized in the basal lamina of the neuromuscular junction of skeletal muscle. The present study shows localization of the asymmetric acetylcholinesterase in the heart of the rat. Antiserum to 14 + 18 S acetylcholinesterase of the electric eel was raised in rabbits.

Collagen chain mRNAs in isolated heart cells from young and adult rats.

Collagen is the predominant component of the extracellular matrix of the heart, where it is organized in a hierarchy of structures. To establish the cellular origin of the various collagen types, type I-procollagen alpha 2 chain and types III and IV collagen mRNAs were examined in preparations of myocytes and non-myocyte heart cells freshly isolated from rats 1 to 6 months old. The cardiomyocytes appeared morphologically intact and functionally competent.

Morphology, composition, and function of struts between cardiac myocytes of rat and hamster.

The morphology, composition, and function of struts that interconnect the lateral surfaces of cardiomyocytes were examined in the hearts of rats and hamsters. Methods included brightfield and fluorescent light microscopy, secondary and backscatter scanning electron microscopy, and transmission electron microscopy in conjunction with silver stain, cationic dye, and antibody to type-I collagen. These studies reveal a twisted, beaded appearance and a complex substructure of collagen fibrils embedded in a ground substance that has a positive reaction with cationic dye.

Enzyme-antibody histochemistry. A method for detection of collagens collectively.

Different types of distinct molecular forms of collagen are components of the extracellular matrix in most tissues. The common types can usually be detected by immunohistochemical methods but others may escape detection for lack of specific antisera. However, all these collagens are substrates for the collagenase of Clostridium histolyticum.

Periodate oxidation products of hydroxylysine in the synthesis of 5-substituted prolines.

The amino acid hydroxylysine was subjected to oxidation by sodium metaperiodate under various conditions. It was found that in acid and high temperature, the initial oxidation product alpha-aminoglutaric gamma-semialdehyde was converted to glutamic acid with a yield of 60%. The use of alkaline conditions of oxidation favored the cyclization of alpha-aminoglutaric gamma-semialdehyde to form delta 1-pyrroline 5-carboxylic acid.

Phosphorylation of hydroxylysine residues in collagen synthesized by cultured aortic smooth muscle cells.

O5-Phosphohydroxylysine was chemically synthesized and techniques were established for its identification by combined use of cation-exchange chromatography, thin-layer electrophoresis at pH 1.9 and 3.5, and thin-layer chromatography.

The nonenzymatic glycosylation of collagen.

Calf skin acid-soluble collagen, containing about 34 residues of lysine plus hydroxylysine per 100,000 dalton polypeptide chain, was treated with [14C]glucose in the presence or absence of NaCNBH3. In 144 h, under the conditions employed, the presence of NaCNBH3 increased the extent of glycosylation from 8 to 15% of the total residues of lysine plus hydroxylysine. The extent of glycosylation was estimated, using acid hydrolysates of the protein, by isolation and determination of reduced adducts (1-lysinohexitols) employing a system of paper chromatography followed by chromatography on an amino acid analyzer.

Proline trapping in granulomas, the site of collagen biosynthesis in murine schistosomiasis.

Proline, a critical substrate for collagen synthesis, is increased in liver undergoing fibrosis. In mice with schistosomiasis, the incorporation of proline into collagen occurs within liver granulomas. To study the interaction of liver cells and granulomas in the development of fibrosis, we assayed the enzymes that catalyze the formation and degradation of proline in isolated granulomas and liver.

Liver collagenase in murine schistosomiasis.

Mice infected with Schistosoma mansoni represent a model for study of the most prevalent form of hepatic fibrosis in humans. In the present study, collagenase activity was measured in relation to collagen synthesis and accumulation in the livers of mice 6-11 wk after after infection. Total and latent collagenase and elastase activities and collagen synthesis were maximal 8 wk after infection and decreased thereafter, whereas collagen content progressively increased to the 11th wk.

Glucocorticoid receptors in WI-38 fibroblasts: characterization and changes with population doubling in culture.

A high-affinity dexamethasone binding macromolecule was identified in WI-38 human fetal lung fibroblasts. High specificity of binding for glucocorticoids was shown by competition studies in which binding of dexamethasone was inhibited by cortisol and corticosterone but not by testosterone or 17 beta-estradiol. WI-38 cells exposed to [3H]dexamethasone at 30 degrees C were able to transfer the 3H-labeled steroid-receptor complex to the nuclear materal.

The characterization of human uterine smooth muscle cells in culture.

Primary cultures initiated from normal human uterine endometrium after total enzymatic dissociation contained epithelioid cells and smooth muscle cells. The smooth muscle cells were subsequently isolated by differential trypsinization and grown in culture for 36 +/- 4 generations. Ultrastructural examination of log and post-confluent cultures of cells at low and high population doubling levels revealed characteristics similar to those of published reports on other smooth muscle cells studied in vivo and in vitro.

Specific cleavage of reduced and S-carboxamidomethylated neurophysin II by the collagenase of Clostridium histolyticum.

Purified collagenase of Clostridium histolyticum was shown to cleave reduced and S-carboxamidomethylated bovine neurophysin between Cys-13 and Gly-14. The scission resulted in formation of two separable fragments: a smaller peptide arising from residues 1 through 13, and a larger peptide comprising the remainder of the residues of the protein. By dansylation procedures, the smaller peptide was shown to have amino-terminal alanine as expected from the sequence of neurophysin II, and the larger peptide had amino-terminal glycine as anticipated.