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Biomedical subjects

D F Counts

Publications and source records attributed to D F Counts.

14 recordsLinked to original sources
PubMed1991

Effect of carbohydrate structure and concentration on the non-enzymatic glycosylation and subsequent cross-linking of collagen.

It has been previously demonstrated that non-enzymatic glycosylation and subsequent cross-linking of proteins can occur at high or greater than physiological concentrations of glucose. Soluble collagen was incubated in the presence of increasing glucose concentrations. The amount of cross-linked collagen was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Our findings reveal that cross-linking due to non-enzymatic glycosylation occurs at or near physiological concentrations of glucose (3.11-4.22 mM). In addition, this glucose induced cross-linking is a time dependent reaction. When collagen was incubated with a variety of different carbohydrates it was found that ketoses are more active cross-linking agents than aldoses. The addition of a reactive group (such as an amine) alpha to the aldehyde group on the carbohydrate increases the cross-linking activity of glucose 2.8 fold. Blockage of the reactive group alpha to the aldehyde (such as N-acetyl glucosamine or 2-deoxy-D-glucose) totally abolishes glycosylation activity. Both 5-C and 7-C carbohydrates are more active than 6-C carbohydrates. Thus, although glucose may be the most abundant carbohydrate capable of non-enzymatic glycosylation and subsequent cross-linking, it is not the most chemically reactive. However, the significance of these findings to the pathogenesis of diabetes needs to be defined.

Animals↗
PubMed1991

Acute hemolytic anemia after oral administration of L-tryptophan in ponies.

The hematologic and pathologic effects of orally administered L-tryptophan and indoleactic acid and of L-tryptophan administered IV were studied in ponies. Sixteen adult Shetland ponies were allotted into 4 experimental groups. Group 1 consisted of 5 ponies (1-5) given 0.6 g of tryptophan/kg of body weight in a water slurry via stomach tube. Group 2 included 4 ponies (6-9) given 0.35 g of tryptophan/kg orally. Group-3 ponies (10-13) were given 0.35 g of indoleacetic acid/kg orally. Group 4 consisted of 3 ponies (14-16) given a single 4-hour IV infusion of 0.1 g of tryptophan/kg. Restlessness, increased respiratory rate, hemolysis, and hemoglobinuria were detected in 4 of the 5 group-1 ponies. Only pony 7 in group 2 developed hemolysis, hemoglobinuria, and a significant increase in respiratory rate. Renal pathologic lesions, consistent with hemoglobinuric nephrosis, were seen in ponies 2, 4, 5, and 7. Bronchiolar degeneration was evident in 4 of 9 ponies given tryptophan orally. The importance of these respiratory lesions was unknown. Clinical or pathologic abnormalities were not noticed in the ponies of groups 3 and 4. Mean plasma tryptophan values increased significantly in groups 1 and 2 at 6 hours after dosing. A second peak of tryptophan was detected in both groups at 12 hours. Values returned to predose values by 48 hours. Plasma indole and 3-methylindole concentrations were detectable in only 2 ponies (4 and 7). In vitro incubations of cecal fluid from ponies 6, 8, and 9 yielded a percentage conversion of tryptophan to indole of 16.75%, 5.84%, and 7.96%, respectively. 3-Methylindole was not produced. These results suggested that indole was the major metabolite of orally administered tryptophan in these ponies.

Acute Disease↗
PubMed1991

Acute hemolytic anemia induced by oral administration of indole in ponies.

Eight ponies were allotted to 2 groups of 4. Group-1 ponies (1-4) were given 0.2 g of indole/kg of body weight orally and group-2 ponies (5 to 8) were given 0.1 g of indole/kg. Various physical, hematologic, and physiologic measurements were obtained after administration of indole. Intravascular hemolysis and hemoglobinuria were detected in both groups within 24 hours of dosing. Hemolysis was reflected by decreases in PCV, hemoglobin concentration, and RBC count, and an increase in indirect bilirubin. Erythrocyte fragility appeared to increase in both groups at 8 hours after dosing and peaked at 16 hours after dosing. At 72 hours after dosing, the RBC fragility value was less than predose measurements. Heinz body formation was noticed in group-2 ponies, but not in group 1. Plasma indole concentrations increased in both groups from the nondetectable predose concentrations. Group-1 values were 203% of group-2 values. In group 2, plasma indole was nondetectable by 12 hours, whereas low concentrations could still be measured in the group-1 ponies at 24 hours. Ponies in group 1 died or were euthanatized between 24 and 72 hours after dosing, whereas group-2 ponies were euthanatized between 48 and 120 hours. At necropsy, all body fat, mucous membranes, and elastic tissue were stained yellow. Hemoglobinuric nephrosis was the most prominent microscopic lesion. Results of this study indicated that indole, a metabolite of the amino acid tryptophan, causes acute intravascular hemolysis in ponies.

Acute Disease↗
PubMed1989

Experimental instability in the rabbit lumbar spine.

The authors performed mechanical, biochemical, and histologic analyses of changes in the rabbit lumbar spine occurring after instability had been induced by facet removal to find whether this intervention produced an experimental model for intervertebral disc degeneration. Sham operated animals and an unoperated control group were used for comparison. Half of the operated animals were housed under conditions to promote higher physical activity than the other animals housed individually in small cages. Acutely, the removal of facet joints increased the flexibility of intervertebral joints. Over the following year, this increase in flexibility was reduced to close to control levels in all groups of animals. Within the intervertebral discs, there was no significant change in proportions or solubility of collagen or proteoglycans after surgery, nor was there microscopic or macroscopic evidence of disc degeneration. The surgical procedure produced hypermobility of the spine, but there was a subsequent restabilization, and the intended disc degeneration was not produced. These findings indicate that some as yet unidentified soft tissue repair process, facilitated by activity, overcame the hypermobility created at surgery, so degenerative changes in the intervertebral discs did not result. We suggest that other animal models of disc degeneration may represent a failure of reparative response to acute injury.

Animals↗
PubMed1986

Proliferation of human peripheral blood lymphocytes induced by recombinant human interleukin 2: contribution of large granular lymphocytes and T lymphocytes.

Recombinant human interleukin 2 (rH IL-2) in the presence or absence of additional stimuli, was found to be able to induce and support the proliferation of human peripheral blood lymphocytes (PBLs). These proliferative effects were observed at low doses (less than or equal to 10 U/ml) of interleukin 2 (IL-2) only when additional signals (antigen, mitogen) were provided. However, higher doses (greater than or equal to 100 U/ml) of rH IL-2 significantly stimulated the proliferation of PBL even in the absence of exogenous lectin, antigen, or allogeneic serum. The subpopulation of lymphocytes most responsive to these higher doses of rH IL-2 was the large granular lymphocyte (LGL), the morphologic homologue of natural killer activity. After the separation of human PBLs on discontinuous Percoll gradients, cells from fraction 2 (greater than 90% LGLs) responded in a dose-dependent manner to rH IL-2 alone, whereas cells from fraction 6 (greater than 90% T cells) were only slightly responsive to rH IL-2 alone. A portion of the proliferation of cells from fraction 2 was dependent on the expression of the TAC receptor, because the prior removal of TAC-positive cells significantly reduced IL-2-induced lymphocyte proliferation. These results demonstrate that human LGL that have not been exogenously stimulated can proliferate in direct response to IL-2, and suggest that LGL are the major cellular phenotype in the proliferative response that has been observed clinically.

Cell Cycle↗
PubMed1986

Skin lysyl oxidase activity is not rate limiting for collagen crosslinking in the glucocorticoid-treated rat.

Lysyl oxidase activity in the skin of rats receiving triamcinolone diacetate (12 mg/kg) for three consecutive days was decreased by sixty-four percent as compared to control values. A decrease of lysyl oxidase activity was observed twelve hours after the initial glucocorticoid injection. The decreased lysyl oxidase activity was accompanied by a forty-nine percent decrease of acetic acid extractable collagen. There was also a forty-two percent decrease in the alpha/beta ratio of the acetic acid soluble skin collagen of glucocorticoid-treated animals. These data indicate that although skin lysyl oxidase activity is decreased by glucocorticoid treatment, the crosslinking of acid extracted collagen as measured by the alpha/beta ratio and collagen solubility is increased. Accordingly lysyl oxidase activity is not rate limiting for collagen crosslink formation in the skins of rats treated with glucocorticoids.

Acetates↗
PubMed1985

Collagen accumulation in the neonatal rat skin: absence of fibrillar collagen degradation during normal growth.

Collagen-bound collagenase activity was assayed in the entire skins of growing neonatal rats. The levels of fibrillar collagen degradation were found to be extremely low throughout the first six days of life. Less than one percent of the collagen accumulated during one day's growth could be degraded by the collagenase bound to the extracellular fibrils of the skin. Control experiments, which included the addition of purified rat uterine collagenase to the skins both before and after homogenization, showed that collagenase activity is easily detectable in the tissue when it is present. It therefore appears that the vast majority of skin collagen, once deposited as fibrils in the skin, fails to turn over during growth. Support for this concept was provided by experiments in which neonatal animals were injected with the potent synthetic glucocorticoid, triamcinolone. Very low levels of collagen-bound collagenase, comparable to those observed in control animals, were found in the steroid-treated animals. Furthermore, the inhibition of collagen synthesis in these animals resulted in a constant chemical content of collagen as well as a constant amount of proteinaceous [14C]-hydroxyproline after injection of [14C]-proline over a 72-hour period. Our results strongly suggest that the bulk of fibrillar collagen does not participate in a dynamic equilibrium between synthesis and degradation during normal neonatal growth. In addition, the results in steroid-treated animals suggest that the rate of collagen accumulation during this period appears to be essentially a function only of collagen synthesis.

Animals↗
PubMed1978

Prolyl hydroxylase half reaction: peptidyl prolyl-independent decarboxylation of alpha-ketoglutarate.

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) is a mixed-function oxygenase that hydroxylates peptidyl proline with the simultaneous and stoichiometric decarboxylation of alpha-ketoglutarate to succinate and CO2. It has been found that highly purified preparations of the enzyme can decarboxylate alpha-ketoglutarate in the absence of a peptidyl proline substrate. The uncoupled decarboxylation proceeds at only a fraction of the rate of the whole reaction and for study requires substrate quantities of the pure enzyme, as well as oxygen, ferrous ion, and ascorbate. No hydroxyproline is formed under these conditions. Immobilized antiserum to prolyl hydroxylase was found to remove both activities from enzyme preparations. However, addition of free antiserum during incubation inhibits only the complete reaction. Poly(L-proline), a specific inhibitor of prolyl hydroxylation, enhances the uncoupled decarboxylation of alpha-ketoglutarate without itself being hydroxylated. All of these findings prove that alpha-ketoglutarate can serve as substrate in the absence of peptidyl proline and is most likely the initial site of attack by oxygen. In the coupled reaction an oxidized form of the keto acid, perhaps a peroxy acid, then attacks prolyl residues in the unhydroxylated substrate.

Animals↗
PubMed1977

Collagen and non-collagen protein synthesis in the lungs of rats exposed to a trypsin aerosol.

In rat lungs, 24 h after a 10 min inhalation of a nebulized 1% (w/v) trypsin solution, there was a 25% increase in lung weight. The incorporation of 3H-tryptophane and 2,3-[3H]-proline into trichloroacetic acid insoluble material was decreased although there was no alteration in prolyl hydroxylase activity. Although hydroxyproline formation was decreased, this decrease was probably due to the general decrease in protein synthesis. Ninety-six hours after inhalation of the trypsin solution there was an increase in non-collagen protein biosynthesis. Proline incorporation and hydroxyproline formation were both increased more than the tryptophane incorporation increase at this same time point. These increases were accompanied by an increase in prolyl hydroxylase activity. These experiments indicate that major changes in protein biosynthesis occur in lung tissues after inhalation of proteolytic enzymes and demonstrate the temporal biochemical changes which occur in lung injury.

Aerosols↗