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C K Silbert

Publications and source records attributed to C K Silbert.

18 recordsLinked to original sources
PubMed1991

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

Aggrecans↗
PubMed1990

Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe.

We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.

Animals↗
PubMed1989

Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine and [35S]-sulfate for varying periods of time. Incorporation of 3H into macromolecules appearing in the medium was linear after approximately 45 min, and incorporation of 35S was linear after approximately 30 min. The amounts of 35S-proteoglycan formed by each of the cultures during 5-h incubations were compared and were found to be fairly similar for the six lines, varying from 0.08 to 0.14 nmol sulfate/microgram DNA. Isolated 3H,35S-glycosaminoglycans were then treated with chondroitin ABC lyase to characterize the location and degree of sulfation. Results indicated a considerable variation in completeness of chondroitin/dermatan sulfation and in proportions of 6-sulfation to 4-sulfation among the various lines. However these variations did not seem to be related to whether the cells were from normals or diabetics. 3H,35S-Labeled disaccharides were isolated and ratios of 3H to 35S determined in order to calculate the [3H]glucosamine dilution by endogenous glucosamine derived from glucose or other sources during the period of incubation. Dilutions varied widely from 160- to 635-fold among the different cell lines, but the variations did not seem to be related to whether the cells were from normals or diabetics.

Adult↗
PubMed1988

Sulphation by cultured cells. Cysteine, cysteinesulphinic acid and sulphite as sources for proteoglycan sulphate.

Bovine aortic smooth-muscle cells, bovine aortic endothelial cells, and IMR-90 human embryonic lung fibroblasts were tested to determine their ability to use cysteine or cysteine metabolites as a source of sulphate (SO4). Cells were incubated in SO4-depleted medium containing [3H]glucosamine plus 0.2 mM-cystine, 0.3 mM-cysteinesulphinic acid or 0.3 mM-sulphite (SO3). The [3H]chondroitin sulphate produced by the different cells was found to vary considerably in degree of sulphation under these conditions. One line of smooth-muscle cells utilized cysteine effectively as a SO4 source and thus produced chondroitin sulphate which was highly sulphated. IMR-90 fibroblasts produced partly sulphated chondroitin sulphate under these conditions, while another smooth-muscle cell line could not utilize cysteine, but could utilize cysteinesulphinic acid as a partial SO4 source. In contrast with the above cells, endothelial cells could not use cysteine or cysteinesulphinic acid as a source of SO4 and produced chondroitin with almost no SO4. All of the cells were able to utilize SO3. Incubation of the cells in the SO4-depleted medium containing [35S]cysteine confirmed that only the first line of smooth-muscle cells could convert significant amounts of [35S]cysteine to 35SO4. Furthermore, the addition of 0.4 mM inorganic SO4 did not inhibit the production of SO4 from cysteine by these cells.

Cells, Cultured↗
PubMed1986

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin.

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.

Adult↗
PubMed1986

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium.

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.

Animals↗
PubMed1986

Effects of heparan sulfate removal on attachment and reattachment of fibroblasts and endothelial cells.

Human skin fibroblasts and calf aorta endothelial cells were grown as tissue culture monolayers in the presence of [35S]sulfate in order to label the glycosaminoglycan portions of proteoglycans for investigation of their role in cell attachment. The [35S]glycosaminoglycans were then selectively removed from the cell monolayers by the addition of various glycosaminoglycan-degrading enzymes. As previously described, in contrast to trypsin treatment none of these enzymes removed any cells from the culture plates. Incubation with a preparation from Flavobacterium heparinum left only small stubs of [35S]glycosaminoglycans on the cell monolayers, indicating that all the cell-surface proteoheparan [35S]sulfate and proteochondroitin [35S]sulfate was accessible to this enzyme preparation. The treatment did not change the amount or time of incubation with trypsin necessary for release of the cells from the monolayers. Thus, cell attachment was not weakened by removal of heparan sulfate or chondroitin sulfate. In contrast, neither fibroblasts nor endothelial cells in suspension would reattach in the presence of the F. heparinum preparation while reattachment occurred readily in the presence of chondroitin ABC lyase. This provides evidence that heparan sulfate, but not chondroitin sulfate, is involved in the process of cell attachment even though neither is necessary for maintaining attachment.

Animals↗
PubMed1986

Relationship of fat distribution to glucose tolerance. Results of computed tomography in male participants of the Normative Aging Study.

Computed tomography (CT) scanning was used to assess the relationship of glucose tolerance to fat distribution in men. Three cross sections [chest (including upper arms), abdomen, and thigh] were scanned in 41 men randomly selected from the Normative Aging Study, a longitudinal study of aging. Greater amounts of fat in the upper body and greater ratios of upper-body fat to lower-body fat were significantly correlated with higher 2-h serum glucose levels after adjustment for age and body mass index. In particular, intra-abdominal fat, a feature uniquely measured by CT, was a significant correlate of 2-h glucose. Largely parallel results were obtained when we compared a sample of male diabetic subjects (N = 8) with the male normal subjects from our random sample. This investigation demonstrates that body fat distribution, adjusted for overall degree of obesity, is a significant correlate of glucose tolerance even in a sample unselected for extremes of physique.

Abdomen↗
PubMed1979

Studies of cultured human fibroblasts in diabetes mellitus: changes in heparan sulfate.

The incorporation of [35S]sulfate into glycosaminoglycans was studied in cultures of normal and diabetic skin fibroblasts. Heparan sulfate was determined by column chromatography after enzymatic degradation of chondroitin sulfates and dermatan sulfate by chondroitinase ABE. Cultured skin fibroblasts from both insulin-dependent and noninsulin-dependent diabetics were found to have increased proportions of heparan sulfate in the media relative to the other sulfated glycosaminoglycans.

Adult↗
PubMed1976

Tumor hypoglycemia: deficient splanchnic glucose output and deficient glucagon secretion.

Fasting hypoglycemia occurred in a patient with a histologically benign mesothelioma; the serum insulin was low (2-4 muU./ml.), as was the glucose utilization rate. Splanchnic glucose output was markedly decreased on direct measurement (21 mg./min.; normal: 108-180 mg./min.). Splanchnic uptake of gluconeogenic substrates plasma glucagon was low normal during hypoglycemia and responded poorly to oral and intravenous alanine. The nonsuppressible insulin-like (NSILA-s) and somatomedin-like activities of the serum were not elevated, and the tumor did not release insulin-like activity on incubation nor did it contain somatostatin. The marked decrease in splanchnic glucose output was the principal cause of hypoglycemia, was associated with an apparent decrease in glycogenolysis, and was at least partly due to deficient glucagon secretion. The relationship of the tumor to these defects is unclear. The tumor may have secreted an unknown insulin-like material affecting primarily the liver and/or pancreatic alpha cell. The approach used here may serve as a paradigm for the analysis of hypoglycemia not caused by excessive insulin.

Glucagon↗
PubMed1975

The effect of glucose on the growth hormone response to glucagon and propranolol-glucagon in normal subjects.

UNLABELLED: The mean (+/- SE) peak level of serum growth hormone (GH) after intramuscular injection of glucagon in ten normal adult men was 15.1 +/- 2.1 ng/ml; glucose infusion suppressed the mean peak GH to 9.6 +/- 3.7 ug/ml (p less than 0.05). Pretreatment of eight of these subjects with propranolol caused a modest increase in the mean peak GH after glucagon (19.4 +/- 2.8 ng/ml) but did not improve the mean peak GH after glucagon when glucose was infused (8.7 +/- 2.8 ng/ml). Individual analysis of the peak GH showed that glucose infusion did not uniformly suppress the peak GH after glucagon; in seven subjects the peak GH was suppressed but in three it was not. CONCLUSIONS: (1) The GH response after glucagon is usually due to a fall in serum glucose after the initial rise in serum glucose induced by glucagon. (2) Nevertheless, since glucose does not consistently inhibit the GH response after glucagon, a second mechanism probably exists by which glucagon stimulates GH secretion. (3) Glucose completely suppresses the propranolol-induced increase in the GH response to glucagon; an adrenergic mechanism may be involved in the control of GH secretion by glucose.

Adolescent↗
PubMed1975

Heparan sulfate of skin fibroblasts grown in culture.

Primary cultures of normal human skin fibroblasts were examined for glycosaminoglycan content. Heparan sulfate was found in the growth medium of these cells, in fractions obtained by sequential collagenase and trypsin treatments, and in the remaining intact cells. Heparan sulfate was found to be the major sulfated glycosaminoglycan of the trypsin fraction but appeared as a smaller proportion of the collagenase fraction. The heparan sulfate of the growth medium, the collagenase fraction, and the trypsin fraction appeared to be proteoglycan while intracellular material appeared to be mainly free polysaccharide. The collagenase fraction is thought to be representative of "matrix" material produced by the cells, while the trypsin fraction may represent external cell surface material. The trypsin fraction heparan sulfate polysaccharide was relatively homogeneous in size with an average molecular weight of approximately 40,000 relative to a chondroitin sulfate standard. It was also relatively homogeneous in sulfate content, containing an average of 0.8 sulfate groups per disaccharide repeating unit. Approximately 50% of this was N-sulfate.

Cells, Cultured↗
PubMed1975

Double-antibody radioimmunoassay of serum insulin: effect of use of hormone-depleted human serum.

Human serum can be depleted of insulin and growth hormone by treatment with dextran-coated charcoal or by dialysis, or both. Inclusion of such hormone-depleted serum in the standard curve of a double-antibody radioimmunoassay for immunoreactive insulin more nearly mimics the conditions under which an unknown human serum sample is assayed. Compared to the usual standard curve in which serum albumin is the only protein, the addition of hormone-depleted serum can cause an increase (by an average 64% under the conditions we used) in the absolute value for apparent insulin in serum. The effect of hormone-depleted serum should be tested in the standard curves of double-antibody radioimmunoassays and included routinely in these standard curves if it changes the results. When used in conjunction with an eventual reference standard for human insulin, this modification of the insulin assay may make the measured values of human serum insulin from different laboratories more comparable.

Blood↗