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M J KARNOVSKY

Publications and source records attributed to M J KARNOVSKY.

18 recordsLinked to original sources
PubMed1964

THE LOCALIZATION OF CHOLINESTERASE ACTIVITY IN RAT CARDIAC MUSCLE BY ELECTRON MICROSCOPY.

A method has been developed for localizing sites of cholinesterase activity in rat cardiac muscle by electron microscopy. The method utilizes thiocholine esters as substrates, and is believed to be dependent on the reduction of ferricyanide to ferrocyanide by thiocholine released by enzymatic activity. The ferrocyanide thus formed is captured by copper to form fine, electron-opaque deposits of copper ferrocyanide, which sharply delineate sites of enzymatic activity at the ultrastructural level. Cholinesterase activity in formalin-fixed heart muscle was localized: (a) in longitudinal elements of the sarcoplasmic reticulum, but not in the T, or transverse, elements; and (b) in the A band, with virtually no activity noted in the M band, or in the H zone. The I band was also negative. No activity was detected in the sarcolemma, or in invaginations of the sarcolemma at the level of the Z band. The perinuclear element of the sarcoplasmic (endoplasmic) reticulum was frequently strongly positive. Activity at all sites was completely abolished by omitting the substrates, or by inhibition with eserine 10(-4)M and diisopropylfluorophosphate 10(-5)M. Eserine 10(-5)M completely inhibited reaction in the sarcoplasmic reticulum, and virtually abolished that in the A band. These observations, together with the use of the relatively specific substrates and suitable controls to eliminate non-enzymatic staining, indicate that cholinesterase activity was being demonstrated. The activity in rat heart against different substrates was that of non-specific cholinesterases, in accordance with biochemical data. The activity in the A band was considered to be probably due to myosincholinesterase. It is proposed that the localization of cholinesterases in myocardium at the ultrastructural level should be taken into account in considering the possible functions of these myocardial enzymes, and it is hoped that knowledge of their localization will open up new avenues of approach in considering their physiological role in myocardium, which at present is not definitely known.

Animals↗
PubMed1961

Metabolic effects of lathyrogenic agents on cartilage in vivo and in vitro.

The effects of lathyrogenic agents in vivo and in vitro are described, in respect to some biochemical indices of cartilage metabolism. Lathyrogenic agents in vivo inhibited the incorporation of radiosulfate into rat epiphyseal cartilage and the isolated chondroitin sulfate. No significant changes in hydroxyproline or hexosamine content of epiphyseal cartilage were found, but there was a marked increase in water content. The content of chondroitin sulfate, measured as uronic acid, was decreased. The importance of taking growth rate differences between control and experimental rats into account in assessing the effects of lathyrogenic agents in vivo is emphasized. In an in vitro system, utilizing fresh calf costal cartilage slices, the presence of low concentrations of lathyrogenic agents markedly affected various metabolic events. The incorporation into cartilage slices of sulfate-S(35), glucose-U-C(14), and glycine-1-C(14) was significantly depressed, as was the production of organic acids, including lactic acid. In general, these effects were more severe under anaerobic conditions. Glutamine restored the activities of the slices treated with lathyrogenic agents to control values obtained in the absence of either lathyrogen or glutamine.

Animals↗
PubMed1961

Electron microscopy of osteoclasts in healing fracturees of rat bone.

Osmium-fixed, undecalcified, callus tissue from healing fractures of rat tibias was sectioned with a diamond knife for study with the electron microscope. Large multinucleated cells were found adjacent to bone. A characteristic labyrinthine infolded border was consistently seen in parts of the cells close to the bone surface. The innermost parts of this "ruffled border" gave rise to vacuoles. The bone surface was always disrupted under the "ruffled border" of the cells. Needle-like crystals were seen at the osseous fringe, within folds in the ruffled border as well as within vacuoles deeper in the cells. Collagen fibers denuded of crystals were never observed. Mitochondria, containing clusters of fine granules, were abundant. The part of the cell away from bone contained rough endoplasmic reticulum and the cell membrane was thrown into irregular microvilli. These observations are discussed in relation to current concepts of osteoclastic resorption of bone.

Animals↗
PubMed1961

The histochemical demonstration of glyceraldehyde-3-phosphate dehydrogenase activity.

A histochemical method for demonstration of glyceraldehyde-3-phosphate dehydrogenation by tissues is described. The method utilizes Nitro BT as an indicator, glyceraldehyde-3-phosphate obtained from hydrolysis of commercially obtainable glyceraldehyde-3-phosphate diethylacetal (monobarium salt) as substrate, and (ethylenediamine)tetraacetic acid acid disodium as an activating agent in a medium buffered to pH 7.2 by 0.2 M sodium phosphate. The heat lability, substrate and coenzyme specificity, and sulfhydryl and phosphate dependence of the tissue component catalyzing this reaction indicate that glyceraldehyde-3-phosphate dehydrogenase activity is being demonstrated. The disparity between the known pH optimum of this enzyme and that determined histochemically, and the anomalous histochemical localization to mitochondria of this enzyme which has been found in the soluble fraction by differential centrifugation, are thought to result from the diaphorase dependence of the tetrazolium methods and to emphasize the need for caution in the interpretation of histochemically determined intracellular localization of dehydrogenating enzymes. The evidence gathered by previous workers concerning the feasibility of demonstrating specific dehydrogenases with Nitro BT, and the correspondence of the distribution of glyceraldehyde-3-phosphate dehydrogenase determined histochemically with available quantitative data, suggest that at the cellular level the histochemical results accurately reflect the distribution of this enzyme.

Glyceraldehyde 3-Phosphate↗
PubMed1961

Oxidative and hydrolytic enzymes in the nephron of Necturus maculosus. Histochemical. biochemical. and electron microscopical studies.

The distribution of oxidative and hydrolytic enzyme activities along the nephron of Necturus maculosus Rafinesque was studied histochemically. The proximal tubule possessed all the demonstrable enzyme activities associated with the hexose-monophosphate shunt and glycolysis, but lacked detectable succinic dehydrogenase and cytochrome oxidase activities. Krebs cycle enzymes other than succinic dehydrogenase were easily detectable. The distal tubule, on the other hand, possessed no detectable hexose-monophosphate shunt enzyme activities, but all demonstrable glycolytic and Krebs cycle enzymes and cytochrome oxidase were present in high activity. These data indicate that the proximal tubule of Necturus probably cannot depend, as can the distal tubule, on the Krebs cycle and cytochrome system to provide energy for its transport processes, an inference supported, in general, by available physiological evidence. The question of the importance of the hexose shunt to proximal tubular function arises. Evidence is presented that the proximal tubular blood supply is primarily venous in nature, a hypothesis which would correlate well with its anaerobic metabolic pattern. In addition, the absence of cytochrome oxidase and succinic dehydrogenase from the proximal tubular cells implies either that they possess very few mitochondria, or that their mitochondria have a very unusual enzymatic pattern. Electron microscopical observations and data obtained from the measurement of the enzyme activities of homogenates of Necturus kidney are presented which indicate that the second hypothesis is more probably correct.

Animals↗
PubMed1960

A histochemical method for distinguishing between side-chain and terminal (alpha-acylamido) carboxyl groups of proteins.

The specificity of the Barrnett-Seligman method for the histochemical demonstration of alpha-acylamido carboxyl groups (C terminal) of proteins is dependent on the conversion of such groups to ketones by the action of acetic anhydride and absolute pyridine. Studies on model compounds show that the side-chain carboxyl groups also react in the method and that most of the final color developed can be attributed to these carboxyls, rather than to the C terminal carboxyl groups. It is postulated that the side-chain carboxyls react by formation of mixed anhydrides in the presence of acetic anhydride and pyridine. This mixed anhydride then could link with a hydrazide to form a dihydrazide, which is capable of coupling with a diazo dye. Acetic anhydride treatment alone, without pyridine, also yields mixed anhydride. The mixed anhydride derived from the side-chain carboxyls can be destroyed by base, whereas the methyl ketone derived from the C terminal carboxyl is unaffected, and this treatment makes the method specific for C terminal carboxyl groups. Tissues treated in such a fashion demonstrate that all the color reaction obtained in the method is due to side-chain carboxyls, and that C terminal groups yield little or no staining as would be expected for "average" molecular weight proteins.

Acetic Anhydrides↗