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Biomedical subjects

R L Church

Publications and source records attributed to R L Church.

At least 55 records · Page 3Linked to original sources
PubMed1979

Biochemical characterization of procollagen-collagen synthesized by rabbit corneal endothelial cells in culture.

Collagen synthesis was studied in monolayer cultures of rabbit corneal endothelial cells by following [14C]proline and [3H]glucosamine or [3H]fucose incorporation into a fraction enriched for collagen and its precursor molecules. Sodium dodecyl sulfate gel electrophoresis of this fraction showed that it consisted of a high-molecular-weight (greater than 300,000 daltons) polypeptide. This component was collagenase sensitive and, in the presence of dithiothreitol, gave rise to two polypeptides of the apparent molecular weights of 200,000 and 160,000 daltons. Pepsin digestion of this material destroyed all the high-molecular-weight material and gave rise to a single collagenase-sensitive component of an apparent molecular weight of 115,000 daltons. This 115,000 dalton material is similar to previously observed basement membrane collagens, and the 160,000 and 200,000 dalton components are probably precursor chains of basement membrane collagen. The very-high-molecular-weight material (greater than 300,000 daltons) may represent a disulfide-linked complex of these precursor chains. DEAE-cellulose column chromatography confirmed the presence of a single procollagen species distinct from the collagen fraction. Amino acid analysis of collagen and procollagen fractions showed a decreased hydroxyproline value as compared with previously reported basement membrane collagens or collagen precursors.

Amino Acids↗
PubMed1978

Partial characterization of human collagen and procollagen secreted by human corneal stromal fibroblasts in cell culture.

Human corneal stromal fibroblasts were established into cell culture. Fractions highly enriched for collagen and procollagen were purified from the medium and cell layer. Analysis using polyacrylamide gel electrophoresis and DEAE cellulose chromatography indicated the presence of a heterogeneous population of procollagen molecules ranging in molecular weights from 200,000 to 120,000 daltons along with alpha1 and alpha2 collagen chains. Cyanogen bromide peptides of a corneal peptide fraction migrated concomitantly with type I standard cyanogen bromide peptides.

Cells, Cultured↗
PubMed1977

Procollagen and collagen produced by a teratocarcinoma-derived cell line, TSD4: evidence for a new molecular form of collagen.

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of theta chains (150,000 daltons), pro alpha chains (130,000 daltons), and alpha chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (alpha1)3, as did collagen isolated from the cell layer. Each alpha1 chain appears to be slightly larger than alpha1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type alpha1(X)3, although similar to that of the alpha1 chain of type I collagen, [alpha1(1)]2alpha2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [alpha1(I)]3.

Amino Acids↗
PubMed1977

Genetics of the connective tissue proteins: assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids.

Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [(3)H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17.

Animals↗
PubMed1975

Structure of cultured fibroblasts from dermatosparaxic calves.

Clonal cell lines from the dermis of a dermatosparaxic calf were grown in tissue culture. After fixation in a mixture of glutaraldehyde and osmium, they were prepared for electron microscopy. Most cells contained a well-developed Golgi region, lysosomes, mitochondria, and dilated cisternae of rough endoplasmic reticulum. They also contained numerous, large bundles of intracellular fialments, many lipid droplets and extensive arrays of vesicles. Cultures accumulated substantial amounts of extracellular fibrillar material. The fibrils were loosely packed and indistinctly cross-banded. Bundles of intracellular filaments were commonly parallel in adjacent cells and also parallel to extracellular fibrils. These cytoplasmic features may result from the inability of the secreted collagen to form normal fibrils.

Animals↗
PubMed1975

Immunological properties of procollagens obtained from the culture medium of dermatosparactic cells.

Rabbit antisera were produced against purified calf dermatosparatic procollagen and against the purified procollagens obtained from the culture medium of calf dermatosparatic cells. These antisera and their derived gamma-globulins were characterized by immunoprecipitation, double immunodiffusion and immunoelectrophoresis. Antiserum directed against dermatosparatic procollagen cross-reacted with the two different forms of procollagen obtained from the culture medium of dermatosparatic calf cells. Antiserum directed against onw of these procollagens, namely (pro alpha1)2 pro alpha2, cross reacted with dermatosparatic procollagen and also cross-reacted with the other procollagen,(pro alpha1)3. Antiserum directed against procollagen (pro alpha1)3 cross-reacted with dermatosparatic procollagen and with the procollagen (pro alpha1)2 pro alpha2. None of the antisera reacted with authentic calf skin collagen, or with the collagen extracted from the cell layer of the dermatosparatic calf cells in culture. Reduction andalkylation of the procollagens abolished the antigen-antibody reactions, while prior digestion of the antigens with bacterial collagenase did not eliminate the immunological reaction. Antigenic determinants in the cell culture procollagens were found at the COOH-terminal non-collagen peptide as well as at the NH2-terminal non-collagen peptide.

Animals↗
PubMed1974

Procollagen: intermediate forms containing several types of peptide chains and non-collagen peptide extensions at NH2 and COOH ends.

A population of procollagen molecules has been isolated from the culture medium of a clonal line of calf dermatosparactic cells and shown to have the amino-acid composition, physical properties, and molecular structure consistent with collagen precursors. Although this procollagen population shares immunologic determinants with the procollagen obtained from dermatosparactic skin, it differs from the latter in aminoacid composition, in subunit properties, and by its content of both amino and carboxyl terminal non-collagen peptide appendages. We propose that the cell culture procollagen contains earlier biosynthetic forms of dermatosparactic procollagen.

Carbon Radioisotopes↗
PubMed1973

Collagen and procollagen production by a clonal line of Schwann cells.

A well-characterized line of rat Schwann cells has been examined for its ability to produce collagen. About 14% of the [(3)H]proline in the proteins that were secreted into the culture medium by the cells was hydroxylated, while only 1% of the labeled proteins in the cell layer contained [(3)H]hydroxyproline. Three sizes of procollagen polypeptides, of molecular weights about 105,000, 120,000, and 155,000, were present in the medium, as well as tropocollagen molecules that contained the usual alpha1 and alpha2 chains. Subsequently, the Schwann cells ceased producing the smaller collagenous polypeptides, although the total [(3)H]hydroxyproline content of the medium was unchanged. The [(3)H]hydroxyproline was almost entirely accounted for by the polypeptide of 155,000 daltons; this peptide was rapidly digested by collagenase or pepsin or chymotrypsin. The destruction by pepsin and chymotrypsin indicates that the large polypeptide, in contrast to procollagen and tropocollagen, is not in the collagenous (helical) conformation. Possibly, this substance is a very early form of procollagen that does not fold into the collagen conformation. The data show that cells of neuroectodermal origin can synthesize collagen, and also suggest that Schwann cells may be responsible for a large proportion of the collagen seen in peripheral neurinomas in vivo.

Animals↗
PubMed1971

Collagen biosynthesis: synthesis and secretion of a high molecular weight collagen precursor (procollagen).

Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular-sieve chromatography on 8% agarose demonstrate the existence of a very high molecular weight (500,000-600,000), proline-rich protein in cultured 3T6 fibroblasts that appears to be the precursor molecule (procollagen) of collagen. The kinetics of [(3)H]-proline uptake indicate that this precursor is synthesized at a different rate than are other cell proteins and is secreted apparently unchanged into the medium, where it undergoes modification; it then precipitates around the cells as collagen fibrils that contain the characteristic tropocollagen polypeptide chains. The solubility of this precursor in hot 5% Cl(3)CCOOH, its hydroxyproline to proline ratio, and its sensitivity to highly-purified bacterial collagenase all indicate that this molecule is of collagenous nature, but that it has considerable regions of noncollagen peptide (about half of the molecule is collagenase sensitive, and it has half of the normal amount of hydroxylated proline residues). These results support the concept of a procollagen molecule, of molecular weight about 500,000-600,000, that contains large regions of noncollagen peptides, which might allow the collagen alpha-chain regions to associate in register and that also can provide the correct chain composition for tropocollagen. Upon secretion of the procollagen molecule, the intercollagen-peptide regions are cleaved and the finished tropocollagen molecule then polymerizes into typical intercellular fibrils.

Acrylamides↗