Reza I. Bashey

Identification of collagen α1(I) trimer in embryonic chick tendons and calvaria

Abstract Collagen with a molecular composition [ α 1 (I)] 3 has been identified in acetic acid extracts from lathyritic chick embryo tendons and calvaria. These molecules characteristically have greater solubility than Type I collagen at neutral pH and contain increased amounts of hydroxylysine residues. It is suggested that these molecules represent a separate gene product of embryonic cells which may be important in the process of maturation and development.

Collagenous abnormalities in the heart of the tight-skin mouse

The tight-skin (TSK) mouse, a possible animal model for scleroderma, has multiple abnormalities including increased dermal thickness, cardiomegaly, emphysematous lungs, and an enlarged skeleton. Previous investigations have demonstrated an increased collagen and glycosaminoglycan (GAG) content in the skin and lungs of these mice. The present correlative investigation of the biochemical and ultrastructural properties of the heart in the TSK mouse also revealed an increased presence of collagen. Analysis of collagen types in the TSK heart showed there was a shift in the ratio of type I: type III: type V from the normal values. Over 90% of the collagen was type I, while both types III and V were decreased in this organ. The ultrastructural examination of the left ventricle demonstrated extensive accumulations of perivascular and intercellular edema fluid, foci of myocytolysis, and areas of moderately increased collagen deposits within interstitial sites. These findings suggest that an increased collagen deposition (type 1) may be a contributing factor to cardiac enlargement in the TSK mouse.

Biosynthesis of a low molecular weight collagen by rabbit growth plate cartilage organ cultures.

Analysis of [14C]-proline labeled proteins synthesized by organ cultures of rabbit growth plate cartilage slices revealed that this tissue synthesized a low molecular weight collagen in addition to Type II, 1 alpha, 2 alpha, and 3 alpha collagens. This collagen appears to be a distinct molecular species on the basis of electrophoretic mobility, hydroxyproline content and cyanogen bromide peptide mapping. In subsequent studies, a non-disulfide-bonded, higher molecular weight form of the same molecule was identified.

Increased sensitivity of scleroderma fibroblasts in culture to stimulation of protein and collagen synthesis by serum

Abstract Serum effects on 14 C-proline incorporation and 14 C-hydroxyproline synthesis by normal and scleroderma fibroblasts in culture were studied. Serum resulted in 97% and 212% increases in 14 C-proline incorporation in two lines of scleroderma fibroblasts while the increase in normal fibroblasts was only 53%. Effects on collagen synthesis were more pronounced. Addition of serum resulted in 124% and 445% increments in 14 C-hydroxyproline synthesis in the scleroderma fibroblasts but only a 43% increment in the normal fibroblasts. The results indicate that cultured scleroderma fibroblasts have increased sensitivity to biosynthetic stimulation by serum and this mechanism may be of pathogenetic importance in the excessive collagen accumulation characteristic of the disease.

Identification of two new collagen α-chains in extracts of lathyritic chick embryo tendons

Abstract Two new collagen polypeptide chains have been identified in extracts of lathyritic embryonic chick tendons. The electrophoretic migration of these polypeptides in sodium dodecyl sulfate-polyacrylamide gels indicates that they have about 20% greater apparent molecular weights than α 1 and α 2 chains of Type I collagen. These chains are not held by disulfide bonds since reduction does not affect their electrophoretic behavior. Further, they do not represent incompletely cleaved procollagen since their apparent molecular size remains greater than that of Type I collagen polypeptides after limited proteolytic digestion. Because the ratio of these polypeptides in the purified extracts is not 2:1 it appears that they are components of two separate tropocollagen molecules.

Biochemical Characterization of Collagens and of a Non-Collagenous Protein Synthesized by Guinea Pig Lung Fibroblasts in Culture

Employing various radioactive amino acids, protein biosynthesis by guinea pig lung fibroblasts has been studied in monolayer culture. The cells were shown to synthesize and secrete several collagenous and noncollagenous proteins. The biosynthesized macromolecules have been characterized employing molecular sieve and ion exchange chromatography (DEAE-cellulose column), SDS-polyacrylamide gel electrophoresis, and enzymatic digestion. It was found that the guinea pig lung fibroblasts synthesized mainly type I procollagen molecules which appeared in the media in various stages of cleavage. The intact procollagen molecules and the various processed products were identified by their electrophoretic migration in SDS-polyacrylamide slab gels and further characterized by 1) elution position on SDS-agarose columns under reducing conditions; 2) hydroxyproline content; 3) change in elution position on SDS-agarose after pepsin digestion; 4) chromatographic separation on DEAE-cellulose columns and electrophoretic mobility of the various peaks; and 5) susceptibility to collagenase digestion. Slab gel electrophoresis under non-reducing conditions of aliquots of culture media after limited pepsin digestion indicated the presence of disulfide-bonded type III collagen alpha-chains. In addition, the lung fibroblasts synthesized a non-collagenous protein composed of two disulfide-bonded chains. The individual chains appeared to have a molecular weight of approximately 220,000 when examined under reducing conditions. This protein has been identified as fibronectin based on its molecular weight, its resistance to collagenase attack, its susceptibility to protease digestion and its precipitation with specific antisera to fibronectin.

Isolation and partial characterization of proteoglycans from sheep lung parenchyma.

Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE-cellulose in urea and eluted with increasing concentrations of NaCl. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaCl and further purified by Sepharose Cl-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminoglycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single-chain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.