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Biomedical subjects

C C Clark

Publications and source records attributed to C C Clark.

At least 55 records · Page 3Linked to original sources
PubMed1988

Underhydroxylated minor cartilage collagen precursors cannot form stable triple helices.

Matrix-free cells from chick-embryo sterna were incubated with various concentrations of 2,2'-bipyridyl, an iron chelator that inhibits prolyl hydroxylase and lysyl hydroxylase. At concentrations in the region of 0.1 mM, significant effects on cartilage collagen hydroxylation and secretion were observed. When the underhydroxylated collagens were subsequently digested with chymotrypsin or chymotrypsin plus trypsin at 4 degrees C for 15 min, the minor cartilage collagen precursors (namely types IX and XI) were extensively degraded; type II procollagen was only partially susceptible and was converted into underhydroxylated collagen. The results demonstrate that there were significant differences in triple-helix stability among cartilage collagens such that the underhydroxylated minor collagen precursors were unable to attain a native structure under conditions where type II procollagen was successful.

2,2'-Dipyridyl↗
PubMed1987

Modulation of heparan sulfate biosynthesis. Effects of 6-diazo-5-oxo-L-norleucine and low glutamine on the synthesis of heparan sulfate proteoglycan by human colon carcinoma cells.

We have investigated the biosynthesis of heparan sulfate proteoglycan in human colon carcinoma cells cultured in either the absence of L-glutamine or the presence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue. Following a 24-h incubation with 100 micrograms/ml DON (0.58 mM) or without L-glutamine, the incorporation of [35S]sulfate was maximally inhibited to about 50%, whereas the incorporation of [3H]leucine or [3H]serine and their specific activity were not significantly affected. Several lines of evidence indicate that the inhibition of [35S]sulfate incorporation was mediated by a reduction in the intracellular pool of UDP-N-acetylhexosamine: the intracellular hexosamine levels were reduced by about 50%; taking into account the changes in specific activity, the incorporation of [3H]glucosamine was also significantly inhibited; and exogenous D-glucosamine (2.3 mM) was capable of substantially reversing the inhibitory effects of DON. This decrease in endogenous sugar supply resulted in the generation of an underglycosylated, lower buoyant density proteoglycan which contained significantly fewer heparan sulfate chains of otherwise normal size and sulfation and even fewer O-linked oligosaccharides. These biochemical changes were corroborated ultrastructurally by the appearance of smaller ruthenium red-stained proteoglycans on the surface of the cells. DON also caused a marked inhibition of cell proliferation and profound morphological changes, both of which were reversible upon culturing in DON-free medium. These results demonstrate that perturbations in glutamine metabolism have profound effects on the structure of heparan sulfate proteoglycan and on the phenotype of human colon carcinoma cells and indicate that DON treatment could be useful for studying post-translational modifications of proteoglycans in various cell systems.

Azo Compounds↗
PubMed1987

The effects of 6-diazo-5-oxo-L-norleucine, a glutamine analogue, on the structure of the major cartilage proteoglycan synthesized by cultured chondrocytes.

Incubation of embryonic chick chondrocytes with 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue, led to a dose-dependent inhibition of [35S]sulfate incorporation into proteoglycan. In the absence of exogenous L-glutamine, a maximal inhibition of 50-60% was achieved with DON concentrations greater than or equal to 1 microgram/ml (6 microM); the ED50 was approximately 0.2 microM. This inhibitory effect could be partially restored by the addition of 100-fold molar excess of either exogenous L-glutamine or M-glucosamine. The quantitative changes were due neither to inhibition of protein core synthesis nor to undersulfation of glycosaminoglycan chains. Rather, the proteoglycan synthesized in the presence of DON contained substantially fewer (approximately 50% of control) and smaller (10-15% of control, on the average) chondroitin sulfate chains as well as a paucity of keratan sulfate chains. The result of these structural changes was a proteoglycan with significantly lower molecular weight, buoyant density, and anionic charge. In spite of these modifications, the altered proteoglycan synthesized in the presence of DON was secreted normally and retained the ability to interact with exogenous hyaluronic acid and link proteins. The results of our experiments also indicate that DON substantially diminished the pool of hexosamine precursors required for glycosaminoglycan synthesis. We conclude that this decrease was responsible for the molecular alterations described above; and these, in turn, can account for the morphological changes previously seen in cartilage matrix synthesized in the presence of DON.

Animals↗
PubMed1987

Release of corrosion products by F-75 cobalt base alloy in the rat. III: Effects of a carbon surface coating.

A pyrolytic carbon coating was applied to F-75 chromium-cobalt-molybdenum alloy in an effort to reduce the release of corrosion products in vivo. After intramuscular implantation in the rat, a complex pattern of serum and urine concentration elevations of chromium, cobalt, and nickel was seen. The carbon-coated implants released more chromium and cobalt than uncoated controls, as seen by significantly elevated metal concentrations in serum and urine. Animals receiving carbon-coated implants showed a high rate of recurrent implant site inflammation. Neoplastic infiltration of 24 animals with coated implants, but not in any of the 16 animals which received either uncoated F-75 microsphere or poly(ethylene) particulate implants.

Alloys↗
PubMed1987

Chondroitin sulfate proteoglycan is a constituent of the basement membrane in the rat embryo parietal yolk sac.

In addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reichert's membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20-30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal antibodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40-100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reichert's membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.

Animals↗
PubMed1987

Surface distribution of flash-evoked and pattern reversal-evoked potentials in hooded rats.

Simultaneous recording from 21 electrode sites in a 4 X 4 mm area over the posterior cortex was used to determine the surface distribution of all major peaks which constitute flash-evoked potentials (FEPs) and pattern reversal evoked-potentials (PREPs) in hooded rats. Topographical maps were constructed with respect to Bregma and midline reference points. The data indicate that not all of the peaks which constitute either evoked potential have their greatest amplitude within the classically defined primary visual cortex. Further, since the FEPs were produced by uniform stimulation, the data suggest that surface regions of the rat visual cortex differ in ways other than simply the portion of the visual field from which information is received.

Animals↗
PubMed1987

The specificity of electron impact mass spectroscopy for the identification of N-ethyl-1-phenylcyclohexylamine (PCE).

The electron impact mass spectrum of N-ethyl-1-phenylcyclohexylamine (PCE) was studied using both deuterium-labeled compounds and structurally related analogs. The deuterium-labeled compounds used were d2 . PCE with two deuterium atoms on the methylene carbon of the N-ethyl group, d3 . PCE with three deuterium atoms on the methyl carbon of the N-ethyl group, d4 . PCE with four deuterium atoms on the beta carbons of the cyclohexyl rings, and d5 . PCE with five deuterium atoms on the phenyl ring. Structurally related compounds used included the N,N-dimethyl, N-propyl, and cyclopentyl analogs. The identities of some major fragments and possible pathways leading to their formation are shown. Electron impact mass spectroscopy is shown to be a definitive test for the identification of PCE.

Cyclohexylamines↗
PubMed1986

Biosynthesis of proteoglycans by rat embryo parietal yolk sacs in organ culture.

The embryonic rat parietal yolk sac has been previously shown to synthesize a number of basement membrane glycoconjugates including type IV procollagen, laminin, and entactin. In this study, parietal yolk sacs were isolated from 14.5-day rat embryos and incubated in organ culture for 4-7 h with [35S]sulfate, [3H] glucosamine, and/or 3H-labeled amino acids, and the newly synthesized proteoglycans were characterized. The major [35S]sulfate-labeled macromolecule represented approximately 90% of the medium and 80% of the tissue radioactivity. It also represented nearly 80% of the total [3H]glucosamine-labeled glycosaminoglycans. After purification by sequential ion-exchange chromatography and isopycnic CsCI density gradient ultracentrifugation, size-exclusion high-performance liquid chromatography showed a single species with an estimated Mr of 8-9 X 10(5). The intact proteoglycan did not form aggregates in the presence of exogenous hyaluronic acid or cartilage aggregates. Alkaline borohydride treatment released glycosaminoglycan chains with Mr of 2.0 X 10(4) which were susceptible to chondroitinase AC II and chondroitinase ABC digestion. Analysis by high-performance liquid chromatography of the disaccharides generated by chondroitinase ABC digestion revealed that chondroitin 6-sulfate was the predominant isomer. The uronic acid content of the glycosaminoglycans was 92% glucuronic acid and 8% iduronic acid, and the hexosamine content was 96% galactosamine and 4% glucosamine. No significant amounts of N- or O-linked oligosaccharides were detected. Deglycosylation of the proteoglycan with chondroitinase ABC in the presence of protease inhibitors revealed a protein core with an estimated Mr of 1.25-1.35 X 10(5). These results indicated that the major proteoglycan synthesized by the 14.5-day rat embryo parietal yolk sac is a high-density chondroitin sulfate containing small amounts of copolymeric dermatan sulfate. Hyaluronic acid and minor amounts of heparan sulfate proteoglycan were also detected.

Animals↗
PubMed1985

Isolation and partial characterization of precursors to minor cartilage collagens.

Suspension cultures of cartilage cells were prepared from 17-day chick embryo sterna and radiolabeled with [14C]-proline under conditions which sought to minimize proteolytic conversion of procollagen to collagen. Collagenous proteins were isolated from the culture medium and cell fraction, were purified in their native state by (NH4)2SO4 precipitation and DEAE-cellulose chromatography, and were characterized by protease susceptibility, SDS-gel-filtration and SDS-polyacrylamide gel electrophoresis. Qualitatively, the precursor components present in the medium were similar to those in the cell extract; quantitatively, it appeared that the minor cartilage collagen precursor components derived from 1 alpha, 2 alpha, 3 alpha and type IX collagens were more prevalent in the cell extract. SDS-PAGE of unreduced samples showed that precursors to both of these collagens migrated as distinct high-molecular-weight aggregates. After chymotrypsin digestion, unreduced type IX collagen migrated as two disulfide-bonded aggregates--a large one (Mr approximately 210K) and a small one (Mr approximately 43K); whereas 1 alpha, 2 alpha, 3 alpha chains migrated identically whether reduced or unreduced. Reduction of undigested type IX aggregate yielded two components of Mr approximately 97K and 78K; whereas reduction of the chymotrypsin resistant 210K and 43 K aggregates gave a single component of Mr approximately 61K and a component which migrated at the dye front, respectively. The molecular origin of these components was confirmed by differential NaCl precipitation. It was concluded that this culture system synthesized precursors to 1 alpha, 2 alpha, 3 alpha and type IX collagens in addition to type II; type X collagen was not detected even though the 17-day sternum contained a population of cells morphologically similar to hypertrophic chondrocytes. The precursor chains to 1 alpha, 2 alpha, 3 alpha collagen had an apparent Mr greater than pro-alpha (II) and could be isolated as a disulfide-bonded aggregate(s); the precursor chains to type IX collagen had an apparent Mr less than pro alpha (II) and could also be isolated as a disulfide-bonded aggregate. All of the cartilage collagen precursors had protease-susceptible regions, but those in type IX appeared to be more sensitive to pepsin than to chymotrypsin.

Animals↗
PubMed1983

Deposition of fibronectin and laminin in the basement membrane of the rat parietal yolk sac: immunohistochemical and biosynthetic studies.

Rat parietal yolk sacs (PYS) at gestational ages 7.5, 9.5, 11.5, 13.5, 14.5, and 16.5 d were reacted with antibodies against laminin or plasma fibronectin. At all times studied, laminin consistently gave a positive reaction with Reichert's membrane and with the cytoplasm of PYS cells. In contrast, fibronectin gave a negative reaction with Reichert's membrane at day 7.5, was weakly positive at day 9.5, and from then on was increasingly positive with maximum reactivity at 14.5 d. By electron microscopic immunohistochemistry, antilaminin reacted strongly with 14.5-d Reichert's membrane and with the contents of the rough endoplasmic reticulum RER cisternae of the PYS cells. Antifibronectin had some spotty reactivity with Reichert's membrane, but the cytoplasm of the PYS cells was negative. The contents of the vitelline vessels and the interface between trophoblast and Reichert's membrane were strongly positive. Metabolic labeling of PYS cells in organ culture clearly demonstrated the presence of laminin, type IV procollagen, and entactin both in the medium and in tissues, but fibronectin was absent. No component in the medium bound to gelatin-Sepharose columns. These studies demonstrate that PYS cells, which actively synthesize and secrete basement membrane components, do not synthesize any detectable fibronectin. Furthermore, the anti-fibronectin staining pattern in the vitelline vessels and trophoblast-Reichert's membrane interface strongly suggests that the fibronectin present in Reichert's membrane is derived from the maternal circulation and is merely "trapped" in the membrane.

Animals↗
PubMed1982

Location and identification of the collagen found in the 14.5-d rat embryo visceral yolk sac.

The collagens associated with 14.5-d rat visceral yolk sacs were localized and identified by a variety of procedures. Morphological examination showed that both the visceral epithelium and mesothelium rested upon thin basement membranes, whereas the majority of the extracellular matrix consisted of a stroma containing occasional cells and abundant banded fibrils. Immunohistochemistry at the electron microscope level showed that the basement membranes specifically cross-reacted with antibodies directed against mouse basement membrane components, whereas the stroma specifically cross-reacted with antibodies directed against rat type I collagen. Extractions of acellular visceral yolk sacs and subsequent analyses showed that type I collagen components were prevalent. Furthermore, in vitro biosynthetic studies showed only the presence of type I procollagen components (or their conversion products) and alpha-fetoprotein. These findings, taken together with our previous studies on the 14.5-d rat parietal yolk sac, provide us with protein markers for studying the origin of cells in rat parietovisceral yolk sac carcinomas.

Animals↗
PubMed1982

Partial characterization of collagenous and noncollagenous basement membrane proteins synthesized by the 14.5-day rat embryo parietal yolk sac in vitro.

Parietal yolk sacs isolated from 14.5-day rat embryos and incubated in vitro with either [14C]proline, [3H]mannose or 3H-labeled amino acid mixture synthesized and secreted basement membrane collagenous and noncollagenous glycoprotein components with relative molecular weights of 350,000 (350K), 220,000 (220K), 185,000 (185K), 175,000 (175K) and 150,000 (150K). The 185K and 175K components appeared to be similar to the pro-alpha 1 (IV) and pro-alpha 2(IV) chains, respectively, which have been isolated from other sources. These components were completely susceptible to bacterial collagenase, but were only partially susceptible to alpha-chymotrypsin digestion. The 350K and 220K components appeared to be similar to subunits of laminin (or PYS A and PYS B, respectively) which have been characterized by others, while the 150K component may be similar to entactin (or PYS C). These components were completely resistant to bacterial collagenase and completely susceptible to alpha-chymotrypsin digestion. In addition, the basement membrane of the parietal yolk sac (Reichert's membrane) stained intensely with antibodies directed against either rat laminin or mouse basement membrane procollagen. The results of these experiments suggest that the 14.5-day rat embryo parietal yolk sac is a useful system for studying the structure, biosynthesis and deposition of basement membrane components.

Animals↗
PubMed1981

Fibronectin: its relationship to basement membranes. I. Light microscopic studies.

Fibronectin is a large molecular weight glycoprotein which has been shown to be associated with cell surfaces, extracellular fluids, and connective tissues. Its possible relationship with basement membranes remains controversial. To define this relationship, the distribution of this antigen was evaluated by light microscopic immunoperoxidase techniques in kidney, skin, skeletal muscle, gastrointestinal tract and parietal yolk sac carcinoma. In addition, antibodies against basement membrane and interstitial collagen were used as controls of the specificity of this reaction. Any possible cross-reactivity between plasma fibronectin and basement membrane was examined by immunodiffusion, immunoelectrophoresis and ELISA techniques. The results indicate that antibodies to plasma fibronectin do not co-localize with antibodies to basement membrane constituents. Furthermore, by immunodiffusion or ELISA, there was no cross-reactivity between plasma fibronectin and anti-basement membrane antibody, nor between basement membrane and anti-plasma fibronectin antibody. We conclude that fibronectin is probably not part of the basement membranes studied.

Animals↗
PubMed1981

Fibronectin: its relationship to basement membranes. II. Ultrastructural studies in rat kidney.

Fibronectin, basement membrane and type I collagen antigens have been localized in normal rat kidney by electron immunohistochemical methods. Immunoreactive fibronectin was found in the interstitial connective tissue matrix and on collagen fibers, while tubular, endothelial and smooth muscle basement membranes throughout the kidney were consistently negative. In the glomerulus immunoreactive fibronectin was abundant in the mesangial matrix. The peripheral glomerular basement membrane was occasionally reactive in a spotty, irregular manner. These findings suggest that fibronectin antigens are probably not a constituent of basement membranes. It is proposed that some fibronectin antigen may be trapped in the glomerular filter, and that normal glomerular cleansing mechanisms would transport this trapped fibronectin toward the mesangial areas where it would be eventually processed.

Animals↗