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Biomedical subjects

D L Cochran

Publications and source records attributed to D L Cochran.

At least 109 records · Page 6Linked to original sources
PubMed1991

Bacteriologic monitoring of periodontal diseases: cultural, enzymatic, immunologic, and nucleic acid studies.

Evidence supporting the bacteriologic etiology of periodontal diseases has led investigators to monitor the microbiologic species associated with each disease. The type of study performed has evolved at different levels over the years and is predominantly influenced by available technologies. As the technology has advanced, so too have attempts at monitoring specific microorganisms. The ultimate goal in most research efforts on clinical bacteriologic monitoring of the periodontal diseases has been to obtain a method of measuring microorganisms in a specific, rapid, low-cost fashion. The method would be used on a daily basis at chairside in the dental operatory to direct treatment at a target microbial population, evaluate treatment, or predict future areas of attachment loss. It is suggested that future efforts try to incorporate microbiologic-host interactions that are likely to be important in the etiology and pathogenesis of the periodontal diseases.

Aggregatibacter actinomycetemcomitans↗
PubMed1990

The induction of specific metabolic alterations in mouse calvarial organ cultures by glycosaminoglycans.

Glycosaminoglycans specifically regulate the amount of calcium released from bone cultures; the mechanisms responsible for this regulation are not known. Media from glycosaminoglycan-stimulated bone organ cultures were analysed to determine (1) if specific calcium-releasing substances were selectively produced, and (2) if protein synthesis was differentially affected by glycosaminoglycans. Chondroitin sulphate B, hyaluronic acid and keratan sulphate at 100 micrograms/ml significantly increased prostaglandin release when compared with control cultures. In combination with suboptimal concentrations of PTH, chondroitin sulphate B, heparin and keratan sulphate significantly stimulated prostaglandin release. When indomethacin was included in the test assays, the stimulated prostaglandin release was abolished. Heparin-treated cultures released the greatest percentage of latent collagenase activity followed by hyaluronic acid-treated cultures. Organ cultures treated with heparin and PTH amount of active collagenase. Stimulation increased interleukin-1 above control levels but with no significant difference among the glycosaminoglycans except for keratan sulphate cultures with which had the greatest amount of interleukin-1. Collagen protein decreased between 48 and 72 h under both control and experimental conditions. Examination of the predominant [35S]-methionine labelled proteins revealed that prostaglandin E2 treatment resulted in a relative shift in labelling to higher molecular-weight proteins as time in culture increased (up to 144 h). After 48 h, when equal amounts of labelled protein were analysed, there was a predominance in labelling of a 200,000 Da protein in the prostaglandin-treated cultures. These findings demonstrate that modulation of calcium release by glycosaminoglycans results in the selective release of molecules capable of stimulating calcium release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Characterization of fibroblasts derived from human periodontal ligament and gingiva.

Growth characteristics and macromolecular synthesis of fibroblasts derived from human periodontal ligament (PDLF) and gingiva (GF) have been compared in cell culture. Cells were isolated from explants and plated at 500,000 cells/100 mm culture dish (day 0) with daily changes of culture medium. DNA histograms were obtained by flow microfluorimetric analysis to confirm the growth state of the cell cultures. Human PDLF cultures became confluent at day 6 as determined by cell number and cell cycle analysis while GF were confluent by day 4. Initially, DNA content of logarithmically growing cells was significantly greater in GF cultures; however, when confluent, DNA content and cell number was greater in PDLF cultures. Total protein content in GF was slightly greater than PDLF until day 7 but this difference was not significant. Analysis of collagen and noncollagen protein synthesis revealed a greater trend in noncollagen protein synthesis in the GF cultures compared to PDLF cultures. Analysis of glycosaminoglycans in the culture medium of GF and PDLF revealed similar distributions of components. In the cellular fraction, GF had greater amounts of hyaluronic acid and heparin and lesser amounts of chondroitin sulfates A and C than PDLF cultures. The results indicate that the growth characteristics of PDLF and GF, although similar in many respects, do exhibit specific differences in proliferative rates and macromolecular synthesis. The differences observed in these parameters may be important during in vivo events, such as guided tissue regeneration, where significant functional differences are observed between gingival connective tissue and periodontal ligament connective tissue.

Adult↗
PubMed1989

Protamine selectively inhibits collagen synthesis by human intestinal smooth muscle cells and other mesenchymal cells.

Collagen synthesis is a major function of human intestinal smooth muscle (HISM) cells and contributes to intestinal fibrosis in chronic inflammatory bowel disease. As an extension of previous in vitro studies of the role of heparin in regulating HISM cell proliferation and collagen synthesis, the effect of protamine sulfate was studied. Protamine decreased collagen production by 50% in confluent and proliferating cultures. This effect was concentration-dependent and was selective for collagen in that neither noncollagen production nor DNA accumulation in the culture plates was affected. Other human mesenchymal cells which produce collagen, such as dermal fibroblasts and aortic smooth muscle cells, responded to protamine in a similar fashion. Protamine has a strong cationic charge and is rich in lysine and arginine. To determine which of these properties was important in decreasing collagen production, the effect of protamine was compared to that of other polyionic compounds. Poly-L-lysine decreased collagen production to a lesser degree than protamine. Poly-L-arginine was toxic to the cells. Poly-L-glutamic acid, which has an opposite charge to protamine, had no effect. These findings suggest that both the number and the arrangement of lysyl residues, in addition to positive charge, are important. Binding assays demonstrated that protamine did not inhibit collagen production by binding to ascorbate in the culture medium. Electrophoretic separation and chromatography of collagen types expressed following protamine treatment showed that the ratio of type I to type III collagen remained 2:1. This observation suggests that suppression of collagen production is not specific to a particular collagen type. The selective inhibition of collagen production by protamine provides an important tool to study the regulation of collagen production in human cells and may also provide potential therapy of fibrotic disorders.

Ascorbic Acid↗
PubMed1988

Heparin modulates the secretion of a major excreted protein-like molecule by vascular smooth muscle cells.

Previous work from our laboratory has shown that heparin specifically induces the release of a pair of proteins of approximately 35,000 and 37,000 Da into the culture medium of vascular smooth muscle cells (SMC). In this report, we demonstrate that the previously identified 37,000-Da smooth muscle protein is composed of two protein species with very similar molecular weights based on migration patterns in SDS-polyacrylamide gels. The larger molecular weight species in this doublet has a similar molecular weight and shares antigenic determinants with major excreted protein (MEP), a lysosomal proteinase previously shown to be secreted by normal and transformed fibroblasts and epidermal cells. Antisera to MEP precipitated the higher molecular weight band from the doublet; preimmune serum was not reactive with the smooth muscle protein. Exposure of smooth muscle cells to heparin resulted in decreased amounts of immunoprecipitable protein released into the medium. Thus, it now appears that three proteins in the 35,000-38,000 molecular weight range are modulated by heparin, and that the largest of the heparin-modulated vascular SMC proteins has a similar molecular weight and is immunologically related to MEP. The release of MEP-like protein from SMC is decreased by heparin, while the remaining two heparin-modulated proteins are increased in the presence of heparin.

Animals↗
PubMed1988

Modulation of bone resorption by glycosaminoglycans: effects of parathyroid hormone and interleukin-1.

Regulation of calcium release from mouse calvariae by glycosaminoglycans and known modulators of bone resorption has been examined. Anticoagulant and nonanticoagulant fragments of heparin were combined with parathyroid hormone (PTH) and interleukin-1, beta (IL1), and calcium release from radiolabeled bone was determined. Three heparin fragments, dextran and dextran sulfate, when tested alone, released similar amounts of calcium as did native heparin alone. Suboptimal concentrations of PTH combined with heparin fragments of varying amounts of anticoagulant activity released similar amounts of calcium as did native heparin. When dextran or dextran sulfate was combined with PTH, the amount of calcium released was significantly greater than dextran or dextran sulfate alone but not from PTH when tested alone. Heparin, in combination with 0.1 mu/ml IL1, stimulated significant calcium release compared to heparin but not to IL1 tested alone. There was no significant difference in calcium release when hyaluronic acid in combination with 0.1 U/ml IL1 was compared to the amount of calcium released by either agent alone. Two of the heparin fragments combined with 0.1 U/ml IL1 significantly inhibited calcium release compared to IL1 alone. In combination with 1.0 U/ml of IL1, heparin and hyaluronic acid inhibited calcium release compared to IL1 alone but this inhibition was not significant. The heparin fragments, combined with 1.0 U/ml IL1, had decreasing inhibitory activity as the degree of anticoagulant activity decreased. A comparison of glycosaminoglycan-modulated calcium release in mouse calvariae to that release in mouse radii/ulnae revealed consistently greater percentages of calcium release from calvariae.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Heparin induces specific protein release from human intestinal smooth muscle cells.

Human intestinal smooth muscle cells have recently been identified as the major cell type responsible for stricture formation in Crohn's disease. Heparin, a sulfated glycosaminoglycan, has been shown to be a key modulator of vascular smooth muscle cell growth both in vivo and in vitro and to affect the release of proteins from these cells. Heparin has also been shown to affect the growth of human intestinal smooth muscle cells. In this report we demonstrate that heparin, in addition to its effects on proliferation, also has very specific effects on proteins released by these cells in vitro. Examination of the culture medium proteins of heparin-treated human intestinal cells revealed an increase in three proteins of molecular weight between 150-250 kd, an increase in a 37 kd protein and a decrease in synthesis of lower molecular weight (less than 20 kd) proteins. In substrate-attached material a transient effect on a 48 kd protein was observed. No effects on intracellular labeled proteins could be demonstrated. The 35S-methionine labeled protein profile of human intestinal smooth muscle cells exposed to heparin is similar to that observed in rat vascular smooth muscle cells yet distinct differences do exist. Extracellular processing does not account for the released proteins nor is de novo protein synthesis required suggesting that altered intracellular protein processing is the mechanism for the heparin-induced protein pattern. The release of specific proteins following exposure to heparin may reflect a significant influence of this glycosaminoglycan on the metabolism of smooth muscle cells in general and particularly in the human intestine.

Cells, Cultured↗
PubMed1987

Glycosaminoglycan stimulation of calcium release from mouse calvariae. Specificity for hyaluronic acid and dermatan sulfate.

Heparin in combination with suboptimal concentrations of parathyroid hormone (PTH) has been shown to stimulate calcium release from bone organ cultures. The mechanism of action of heparin, however, is not known. One possible mechanism relates to the highly sulfated structure of heparin. We have compared heparin to other glycosaminoglycans to stimulate calcium release from mouse calvarial organ cultures in the presence and absence of suboptimal concentrations of parathyroid hormone. The exogenous addition of heparin to bone cultures demonstrated only slight effects on calcium release at 5.0, 10, and 100 micrograms/ml. The addition of hyaluronic acid to the calvarial cultures caused a significant release of calcium at 10 and 100 micrograms/ml compared to 5 micrograms/ml hyaluronic acid. Dermatan sulfate was equally as effective as hyaluronic acid at 100 micrograms/ml but not at 10 micrograms/ml. A comparison of heparin- and hyaluronic acid-stimulated release demonstrated a significantly greater amount of calcium release with hyaluronic acid 100 micrograms/ml. At 5.0 and 10 micrograms/ml, there was no difference between heparin and hyaluronic acid in the amount of calcium released into the culture medium. When heparin was added to the organ cultures with suboptimal concentrations of PTH, there was a significant enhancement of calcium release observed with 10 and 100 micrograms/ml heparin compared to heparin addition alone. When hyaluronic acid was added with suboptimal concentrations of PTH, no significant enhancement of calcium release was observed with 100 micrograms/ml hyaluronic acid. Dermatan sulfate, chondroitin sulfates A and C, and keratan sulfate, in combination with PTH, stimulated significant calcium release compared to the glycosaminoglycan added alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Effect of heparin on vascular smooth muscle cells. I. Cell metabolism.

Previous work from our laboratory has shown that heparin inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. The mechanism of action of this glycosaminoglycan is unknown. In this communication, we have examined the antiproliferative effect of heparin on smooth muscle and other cell types, and have investigated several aspects of heparin on smooth muscle cell metabolism. Smooth muscle and closely related cell types from several species, including human, were much more sensitive to heparin than any other cell type tested, including primary and established cell lines, normal and transformed cell pairs, fibroblasts, epithelial, and endothelial cells. Flow microfluorimetric analysis of cell cycle distribution indicated that heparin blocked either the G0----S transition or a very early S-phase event in smooth muscle cells. Heparin rapidly inhibited DNA and RNA synthesis, but did not affect the rate of protein synthesis. The decrease in nucleic acid synthesis could be accounted for by an inhibition of thymidine and uridine uptake. Interestingly, heparin did not block amino acid or glucose transport. Although no change in the overall rate of protein synthesis was observed in the presence of heparin, we noted at least two changes in the synthesis of specific proteins by smooth muscle cells: two 35,000-dalton proteins which appeared in the culture medium of heparin-treated cells, and the transient disappearance of a 48,000-dalton protein in the substrate attached material of smooth muscle cells exposed to heparin. The role of the observed changes in smooth muscle cell metabolism is yet to be determined, but they may provide valuable clues to the molecular mechanisms controlling the antiproliferative activity of heparin.

Animals↗
PubMed1985

Effect of heparin on vascular smooth muscle cells. II. Specific protein synthesis.

Heparin suppresses the proliferation of vascular smooth muscle cells both in vivo and in vitro. The mechanism of action of the antiproliferative activity of heparin is not known. We have detected differences in the synthesis of specific proteins when vascular smooth muscle cells are exposed to heparin and report here that many characteristics of these protein alterations parallel the properties of the antiproliferative activity. The induction into the culture medium of a pair of proteins of approximately 35,000 dalton mw in heparin-treated smooth muscle cell cultures and the antiproliferative effect of heparin share the following characteristics: 1) the effect is reversible, 2) the effect is specific for smooth muscle cells, 3) anticoagulant and non-anticoagulant heparin are equally effective, 4) the effect is lost with time in culture and, 5) heparin is the most potent glycosaminoglycan in producing the effect. Furthermore, heparin causes a transient suppression of a 48,000 dalton substrate-attached protein, whereas chondroitin sulfate A and C and dermatan sulfate had much less effect. Dextran sulfate was almost as effective as heparin in suppressing the synthesis of the substrate-attached protein. These proteins appear to be noncollagenous and the induced synthesis of the 35,000 dalton proteins is inhibited by actinomycin D. Although a direct relationship between these specific protein changes and the antiproliferative effect of heparin has not been proven, these protein alterations may play a crucial role in the effect of heparin on smooth muscle cell growth.

Carbohydrate Metabolism↗
PubMed1982

Oligomeric structure of the major nuclear envelope protein lamin B.

Lamins A and B, the two most abundant proteins in the nuclear envelope fraction, can each be converted into disulfide cross-linked homotypic oligomers by oxidation of intrinsic disulfide bonds. The cross-linked polymers of lamin A include dimers, trimers, tetramers, and larger polymers. Those that enter acrylamide gels migrate as expected for their molecular weights. Lamin B has yielded predominantly two oligomers and the number of monomers in these oligomers has been controversial. In the present study, it is demonstrated that migration of the lamin B oligomers is sensitive to the electrophoretic conditions; thus, the oligomers have appeared to be two forms of dimer or two forms of trimer, depending upon conditions. In the present study, a third, smaller oligomer of lamin B has been detected. Because there are three oligomers of lamin B, it is possible that these are a dimer, a trimer, and a tetramer. Support for this model has been obtained by analysis of cross-linked fragments from a mild trypsin digestion of cross-linked lamina proteins. This procedure yields 62,000-, 46,000-, and 30,000-dalton fragments of lamin B. Each fragment also occurs as three homotypic oligomers. The largest oligomer, which appears to be a tetramer, is obtained in high yield under brief, mild cross-linking conditions in several cell types. These results suggest that lamin B occurs as a tetramer in the nuclear envelope.

Animals↗
PubMed1979

Comparison of the major polypeptides of the erythrocyte nuclear envelope.

The three most abundant nonhistone polypeptides (molecular weights 75,000, 71,000 and 61,000) of the avian erythrocyte nucleus have previously been isolated in the nuclear envelope fraction. They have been separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and peptide-mapped after limited enzymatic digestion. Three enzymes -- chymotrypsin, papain and Staphylococcus aureus protease -- were used. Results obtained with each enzyme indicate strong similarities between the three nuclear envelope polypeptides. The amino acid compositions of the two most abundant polypeptides (P75 and P71) have been determined and found to be similar. Further, they readily yield large fragments upon brief alkaline hydrolysis. For both P75, and P71 the degree and the pattern of alkaline fragmentation are almost identical. A 61,000-dalton polypeptide which appears to be P61 is obtained from P75 and P71 by mild acid hydrolysis. These results establish the close chemical similarity of these predominant polypeptides in the erythrocyte nucleus and suggest that they serve related functions.

Amino Acids↗
PubMed1978

In vitro oxidation of intrinsic sulfhydryl groups yields polymers of the two predominant polypeptides in the nuclear envelope fraction.

The two quantitatively predominant nuclear nonhistone polypeptides of the avian erythrocyte comprise a significant portion of the nuclear envelope protein. Each of these two polypeptides can be converted almost completely to homogeneous polymeric species via oxidation of intrinsic sulfhydryl groups by omicron-phenanthroline cupric ion complex. Major products are two dimeric species arising from each of the approximately 77 000-dalton pair. The larger of the two polypeptides also yields oligomers greater than dimers. These observations extend out earlier solubility and polypeptide cross-linking evidence which indicated that these polypeptides were involved in an oligomeric, proteinaceous nuclear structure. Ultramicroscopic studies from other laboratories indicate that such a structure may function as a nuclear skeleton. The architectural details of the structure now appear to be accessible at the molecular level.

Animals↗