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D Holderbaum

Publications and source records attributed to D Holderbaum.

26 records · Page 2Linked to original sources
PubMed1984

A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium.

Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans or other products of smooth muscle cell metabolism.

Animals↗
PubMed1984

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro.

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.

Animals↗
PubMed1982

Platelet stimulation by antifibronectin antibodies requires the Fc region of antibody.

Anti-human fibronectin antibodies produced in a goat or in rabbits stimulate the release of serotonin from washed or gelatin/Sepharose-treated human platelets in a dose-dependent manner. This finding led us to propose that fibronectin on the platelet plasma membrane might serve as a collagen receptor on these cells [Bensusan, H. B., Koh, T. L., Henry, K. G., Murray, B. A. & Culp, L. A. (1978) Proc. Natl. Acad. Sci. USA 75, 5864-5868]. To determine whether direct interaction of the antibody with platelet membrane fibronectin was responsible for this stimulation, we prepared proteolytic fragments of the antifibronectin antibodies. Purified F(ab')2 fragments had a greatly diminished ability to elicit degranulation, and rabbit F(ab')2 fragments were totally ineffective in this regard. Preimmune IgG from these sources was capable of inhibiting antibody-induced serotonin release in a dose-dependent manner. Purified antibody Fc fragments also inhibited this stimulation in doses stoichiometrically equivalent to the inhibition seen with preimmune IgG. These results suggest that (i) platelet stimulation elicited by anti-human fibronectin antibody is mediated by the platelet Fc receptor and therefore is likely to be dependent on antigen-antibody complex formation and (ii) fibronectin on the platelet surface may not be the primary receptor for platelet adherence to collagen.

Antigen-Antibody Complex↗
PubMed1980

Aortic collagen, elastin and non-fibrous protein synthesis in rabbits red cholesterol and peanut oil.

Alteration of the fatty acid composition of atherogenic test diets has been a widely recognized method for influencing the character and severity of atherosclerotic lesions. The addition of peanut oil or coconut oil to cholesterol-supplemented diets has been shown to produce lesions of a fibrous nature in several species. In the present study, addition of 8% peanut oil to a 2% cholesterol diet accelerated the formation of atherosclerotic lesions which were more fibrous after only 90 days than those previously seen in rabbits even after 6 months on a diet supplemented with cholesterol alone. Collagen, elastin and non-fibrous protein synthesis were all increased over control values, as previously seen in aortas from rabbits given cholesterol supplementation alone. However, the addition of peanut oil to the 2% cholesterol diet produced a preferential increase in the rate of aortic collagen synthesis per unit dry, defatted weight compared with the increases seen in elastin, non-fibrous protein or total protein synthesis. Collagen deposition in proliferative intimal plaques was evident by histological examination. These focal accumulations, however, did not result in significant increases in either total collagen content of the whole descending thoracic aorta or in collagen concentration expressed per unit of dry, defatted weight. These data suggest that, while a portion of the increased synthetic rates may be a direct result of aortic hyperplasia, the proportionally greater increase in collagen synthesis in these lesions is attributable to the addition of peanut oil to the atherogenic diet. Although the lesions produced in this experiment lacked the overt fibrosis seen in man and in some forms of experimentally induced atherosclerosis, the relative synthetic rates of collagen, elastin and nonfibrous protein described here suggest that even a small preferential increase in collagen synthesis compared with non-collagen protein synthesis may gradually lead to a more fibrous lesion.

Animals↗
PubMed1980

Inhibition of collagen and non-collagen protein synthesis in cultured aortic smooth muscle cells by hyperlipoproteinemic serum.

The response of rabbit arterial tissue to prolonged extreme hyperlipoproteinemia is the development of a state of enhanced in vitro protein synthesis where severe atherosclerosis is present. In order to examine this stimulation, arterial cells were grown in cell culture and exposed to hyperlipemic serum as a part of the growth medium. Rather than observing an overall increase in protein synthesis as seen in atherosclerotic tissue, the cells exposed to hyperlipemic serum produced less collagen and non-collagen protein per cell than counterparts exposed to normolipemic homologous serum. We conclude that exposure to hyperlipemic serum for six days in vitro causes enhanced cellular proliferation, as seen by others, and a general perturbation of protein synthesis which may be related to cell population density.

Animals↗
PubMed1977

Stimulation of aortic protein synthesis in experimental rabbit atherosclerosis.

Collagen, elastin and non-fibrous protein synthesis were measured in the aortas of male New Zealand white rabbits fed a diet containing 2% cholesterol for 140 or 180 days. At these time periods increases in aortic cholesterol and cholesteryl esters were evident. The atherosclerotic lesions induced were predominantly of the foam cell type although some areas of early fibrous lesion formation were noted. These changes in lipid concentration and arterial morphology were accompanied by a significant increase in collagen synthesis as determined by the formation of [14C]hydroxyproline. This increase, however, was not confined specifically to collagen since both elastin and non-collagenous proteins were also being synthesized at a higher rate. The two-fold increase in the rates of both fibrous and non-fibrous protein synthesis may in part be a consequence of marked intimal hyperplasia necessitating a general increase in protein synthesis.

Animals↗
PubMed1975

Effects of hyperlipoproteinemic serum and exogenous proline concentration on collagen synthesis by isolated rabbit aortas.

Collagen synthesis was measured in segments of normal rabbit aorta, incubated in vitro, by monitoring the formation of peptidyl-14C-hydroxyproline from [U-14C]-L-proline added to the incubation medium. The effect of hyperlipoproteinemic rabbit serum on the rate of collagen synthesis was compared with the effect of normal rabbit serum. No differences in the rates of synthesis were detected between the two batches of serum, despite a 60-fold difference in serum cholesterol concentration. Increases in free proline concentration in the incubation medium resulted in changes in proline flux between medium and tissue pools of free proline, but medium proline concentration had no effect on the rate of collagen synthesis.

Animals↗