QUANTITATIVE STUDIES ON ISOLATED PANCREATIC ISLETS OF MAMMALS: ADENOSINE TRIPHOSPHATASE ACTIVITY IN NORMAL AND OBESE-HYPERGLYCEMIC MICETÄLJEDAL, INGE-BERT; HELLMAN, BO; HELLERSTRÖM, CLAES
doi: 10.1177/12.7.491pmid: 14209984
Microchemical and histochemical methods were used for the characterization, localization and assay of adenosine triphosphate (ATP) splitting enzymes in homogenates and sections of the endocrine pancreas from obese-hyperglycemic mice and their lean litter mates. The following observations were made:1. Dephosphorylation of ATP was maximal at pH 9.1. It was strongly stimulated by magnesium ions at an optimal concentration of 1 mM. ATP cleavage was inhibited by adenosine diphosphate, sodium azide and p-chloromercuribenzoic acid. The addition of l-cysteine, sodium cyanide or sodium fluoride to the substrate medium had no effect on the enzyme activity. Substitution of ATP by equimolar amounts of other phosphate esters in the medium considerably reduced the substrate cleavage. These results are taken as evidence for the presence of sulfhydryl-dependent adenosine triphosphatase (ATPase) in the islet tissue.2. Histochemical staining revealed a strong ATP splitting enzyme activity in the capillaries and walls of larger blood vessels throughout the pancreas; a rather weak and diffuse cytoplasmic reaction being found in the islet cells.3. Microchemical assays revealed a lower cleavage of ATP in the islets as compared with the exocrine pancreas and the liver. The cleavage of ATP was more intense in the islets of the obese-hyperglycemic mice than in those of the lean litter mates.4. Starvation for 7 days induced no significant changes in the enzyme activity of the endocrine pancreas.
EFFECT OF FREEZING AND THAWING ON CERTAIN NUCLEAR AND MITOCHONDRIAL ENZYMES OF MOUSE LIVERWARAVDEKAR, VAMAN S.; GOLDBLATT, PETER J.; TRUMP, BENJAMIN F.; GRIFFIN, CHARLES C.; STOWELL, ROBERT E.
doi: 10.1177/12.7.498pmid: 14209985
1. The effects produced by a rapid freezing and thawing on the activities of some enzymes of nuclear (nicotinamide adenine dinucleotide (NAD)-pyrophosphorylase) and mitochondrial (succinoxidase, succinic dehydrogenase, and glutamic dehydrogenase) origin are presented. These effects are compared with those produced by sonic treatment of fresh tissue.2. The activity of NAD-pyrophosphorylase was unaffected by the freezing procedures employed, namely dry ice at –79°C, isopentane at –155°C, and propane at –175°C, or by sonic treatment of homogenates of fresh tissue.3. Succinoxidase activity was decreased to some extent by all the procedures employed. Sonic treatment produced a 47% loss in the enzyme activity, while freezing on dry ice, in isopentane, or in propane produced losses of 26, 29, and 37%, respectively.4. The activity of succinic dehydrogenase was least affected by sonic treatment or by freezing on dry ice. Quenching in isopentane or in propane, however, produced 11% and 23% loss respectively in the dehydrogenase activity.5. Free glutamic dehydrogenase activity, which is low in fresh-tissue preparations, was increased to the same extent by all methods employed. The unsedimentable activity depended upon the procedure used, however. Sonic treatment and freezing with propane, isopentane, or dry ice gave 78, 48, 33, and 20%, respectively, of the total enzyme activity.6. The fact that the increase in unsedimentable glutamic dehydrogenase activity roughly parallels a decrease in succinoxidase activity suggests that a rapid freezing followed by thawing produces alterations in the mitochondrial membranes.
HISTOCHEICAL ALKYLATION: A STUDY OF METHYL IODIDE AND ITS EFFECT ON TISSUESTERNER, JACOB Y.
doi: 10.1177/12.7.504pmid: 14209986
The use of methyl iodide as a histochemical alkylating agent is described. Methyl iodide selectively esterifies carbohydrate and other carboxyl groups. Sulfate half esters are not affected. Phosphate esters are esterified by methyl iodide, although the latter reaction is more variable; in several tissues nuclear basophilia could not be abolished. Saponification with barium hydroxide reverses the methyl iodide blockade of basophilia. Sulfhydryl and phenol reactions are prevented by methylation with methyl iodide; amines are alkylated and the usual blockade of acidophilia by acylation or deamination is prevented. Saponification does not reverse the blockade of these latter groups. The periodic acid-Schiff and Sakaguchi reactions are not affected. Marked increases in acidophilia were noted after methylation with methyl iodide. The hypothesis that a large number of tissue acids and amines are ordinarily prevented from binding basic and acid dyes respectively by intra- or intermolecular salt linkages is set forth. Similarly, the inability of protein end groups to demonstrate chemically determined pK values (to 2.5 for carboxyl groups and to 12.0 for arginine) in their interaction with dyes is attributed to interfering salt linkages.
CARBOHYDRATE HISTOCHEMISTRY OF THE SALIVARY GLANDS AND PANCREAS IN CYSTIC FIBROSISSHACKLEFORD, JOHN M.; BENTLEY, HERSCHEL P.
doi: 10.1177/12.7.512pmid: 14209987
Various histochemical methods for demonstrating complex tissue carbohydrates were applied to salivary gland and pancreatic tissue from four known cases of cystic fibrosis. Aside from the usual methods, e.g., periodic acid Schiff and alcian blue, the p-hydrazinobenzenesulfonic acid reaction was found to be particularly valuable as a survey method for estimating concentrations of vicinal hydroxyl groups. In the pancreas, submandibular and parotid glands striking changes in carbohydrate histochemistry occur in the small intralobular (intercalated) ducts which normally secrete relatively little or no mucopolysaccharide. The lumina of salivary gland striated ducts and large excretory ducts frequently contain inspissated mucus. In the sublingual glands the inspissated mucus is mixed with considerable amounts of deoxyribonucleic acid. Vibrio cholera neuraminidase significantly reduces acid group staining in most of the epithelial mucus although some resistance to digestion by this enzyme is evident. Some acid group substances appear in the interstices of sublingual glands which exhibit the histochemical characteristics of epithelial mucin. This reaction is consistent with a "leakage" of some of the sialomucins from the acini into the interstitial areas.
EXTRACTION RESISTANCE OF SUDAN STAINED MAST CELLS AFTER PREVIOUS ACID TREATMENTFEAGLER, JOHN R.; MCMANUS, J. F. A.
doi: 10.1177/12.7.530pmid: 14209989
1. Sudan black B alone stains the mast cells very faintly. The granules do not appear very distinct or numerous.2. The periodic acid Schiff (PAS) technique stains the mast cells; however, the results are variable. When stained, the mast cell granules appear slightly darker than the cytoplasm.3. When PAS and Sudan black are used together, the mast cell granules gain an avidity for the dye and are stained black.4. Brief acid treatment prior to immersion in Sudan black results in granules gaining an avidity for the Sudan dye that is extraction resistant. The reaction appears dependent upon modifying the mast cell granules by acid treatment. It appears as though the hydrogen ion changes some physical or chemical property of the granules so that they gain an avidity for the dye.5. Acetylated Sudan black shows an affinity for the mast cell granules greater than Sudan black alone. This is not increased by previous acid treatment. Acetylated Sudan black also stains the mast cell nucleus.
DEMONSTRATION OF PEROXIDASE ACTIVITY IN TISSUE SECTIONSWACHSTEIN, MAX; MEISEL, ELIZABETH
doi: 10.1177/12.7.538pmid: 14217926
By using an improved benzidine technique, peroxidase activity can be demonstrated in various locations in mammalian tissues. A relatively formalin resistant enzyme is found in hemoglobin and is also associated with mitochondria of striated muscle and heart. A somewhat less formalin resistant peroxidase occurs in the granules of myeloid and mast cells. A relatively formalin sensitive peroxidase is present in a number of additional locations, e.g. the acinar cells in thyroid and salivary gland, the medulla of the kidney, in hair follicles of the guinea pig skin and Kupffer cells of the liver.
NEW SUBSTRATES OF TRYPSIN AND SOME TRYPSIN-LIKE ENZYMESGLENNER, G. G.; HOPSU, V. K.; COHEN, L. A.
doi: 10.1177/12.7.545pmid: 14209991
The results of the investigation of two new naphthyl substrates of trypsin and the trypsinlike enzyme(s) in human mast cells, carbobenzyloxy-l-arginine and benzoyl-l-arginine β-naphthylamide, have led directly to the synthesis and study of a further substrate, carbobenzyloxy-l-arginine methyl ester. The activity of trypsin against the carbobenzyloxy ester is greater than that previously described biochemically for other synthetic substrates. The inhibition of the amidase and esterase activity of trypsin and the tryptic enzyme in human mast cells by ε-aminocaproic acid derivatives is characterized and evidence is presented to show that this enzymic activity in human mast cells is more closely related to trypsin than to plasmin (fibrinolysin).