Evidence for multiple caeruloplasmin components in human serum.
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Biomedical subjects
Publications and source records attributed to E P BENDITT.
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A method is described by which mast cells harvested from the rat peritoneal cavity can be deposited on the surface of a Millipore filter without gross injury, permitting observation of morphological and chemical changes induced by a variety of agents. These isolated peritoneal mast cells respond to 48/80 and to polymyxin B but not to dextran or ovomucoid with degranulation and histamine release. Thus four agents which in vivo appear to have similar activities have been found by means of in vitro analysis to operate by at least two different mechanisms.
Estradiol in large amounts reduces the incorporation of radioactive sulfate into cartilage and aortas of rats. This reduction becomes apparent within 3 days for cartilage and 3 weeks for aorta. The effect is not mediated through suppression of testosterone secretion and the hypophysis is not necessary for the effect to be demonstrated. The thoracic segment of aortas from normal rats incorporates more sulfate than does the abdominal segment and this difference is reduced following the administration of estradiol.
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Mast cells contain an enzyme which hydrolyzes 3-chloroacetoxy-2-naphthoic acid anilide. By using highly purified mast cells isolated by differential centrifugation in high density sucrose solutions we have been able to study this enzymatic activity in more detail. The enzyme has properties similar to those of chymotrypsin: Chymotrypsin will hydrolyze the histochemical substrate, and the chymotrypsin and mast cell activities with this substrate are similarly inhibited by diisopropylfluorophosphate. The mast cell enzyme is capable of hydrolyzing the N-acetyl esters of tryptophan, tyrosine, and phenylalanine, the relative rates of hydrolysis being similar to those seen with chymotrypsin. A characteristic trypsin substrate, p-toluenesulfonyl arginine methyl ester, is not acted upon by the mast cell enzyme or chymotrypsin. The pH activity curve of the new cell enzyme is similar to that of chymotrypsin as determined with N-acetyl-L-tryptophan ethyl ester as substrate.
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The enterochromaffin cells of the guinea pig duodenum and 5-hydroxytryptamine incorporated into gelatin models gave, after formalin fixation, nearly identical reactions with methenamine silver, ferric-ferricyanide, and diazotized o-aminoazotoluene. Following formaldehyde fixation the indole reaction with p-dimethylaminobenzaldehyde was negative in tissue enterochromaffin cells; similarly the indole reaction was negative after formaldehyde fixation of gelatin models. In the duodenum of the guinea pig 5-hydroxytryptamine appears to be largely, if not entirely, contained in the enterochromaffin cells. Its intracellular concentration estimated from relative cell volume and assay of concentration in the whole tissue, was not less than 5 mg./gm. of cells, and as judged by the intensity of staining reaction may have been as high as 10 to 20 mg./gm. of cells. Reserpine administered parenterally caused release of the chromogenic material of duodenal enterochromaffin cells and a parallel reduction in the amount of extractable 5-hydroxytryptamine.
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Each of the four agents, ovomucoid, dextran, 48/80, and testis extract, when injected beneath the skin of the dorsa of the paws of rats produces a local vascular injury characterized by a protein-rich edema. Each agent also produces damage to mast cells. Either 5-hydroxytryptamine or histamine produces a response similar in the gross to that elicited by the agents which damage mast cells; however, neither of these two agents produces mast cell damage. On a weight basis 5-hydroxytryptamine is a much more potent edema-producing agent than histamine. The edema-producing action of 5-hydroxytryptamine can be differentiated from the similar action of histamine by the use of specific antagonists; dibenamine is a 5-hydroxytryptamine antagonist and pyrilamine a histamine antagonist. The edema produced by the mast cell-damaging agents is partially inhibited by dibenamine but is not diminished by pyrilamine. It is completely inhibited by treatment of rats with both drugs. The drugs which inhibit edema do not prevent mast cell damage by ovomucoid, dextran, 48/80, or testis extract. The observations are consistent with the hypothesis that agents which damage mast cells, "release" both 5-hydroxytryptamine and histamine and that in the rat the edema associated with mast cell damage is mediated largely by 5-hydroxytryptamine.
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