The effect of menadione epoxide on the experimental immune arthritis in the rabbit.

It was shown previously that the experimentally induced arthritis in the rabbit can be largely nullified by subsequent treatment with menadione (by gavage). It is now shown that menadione epoxide, as is produced in the vitamin K cycle, also exerts a beneficial effect histologically and biochemically. Such treatment decreased both the glucose 6-phosphate dehydrogenase and the 6-phosphogluconolactonase activities in the synovial lining cells of the challenged joints towards values found in the unchallenged joints; it had only equivocal effects on the 6-phosphogluconate dehydrogenase activity. The results indicated that the epoxide might be interfering primarily with the lactonase activity.

A quantitative cytochemical method for ornithine decarboxylase activity.

Although decarboxylases, particularly ornithine decarboxylase, are of considerable importance in cell metabolism, it has been impossible to demonstrate their activity histochemically, as this depends on trapping carbon dioxide at neutral pH values. A new reagent, lead hydroxyisobutyrate, has been shown capable of such trapping. It has been applied to the demonstration of ornithine decarboxylase activity in mouse kidney. Optimal concentrations of substrate, co-factor and trapping agent, as well as the pH optimum, have been determined for cryostat sections stabilized with a collagen polypeptide. The activity was inhibited by the specific ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine.

The use of cationic polyelectrolytes in the preparation of cell monolayers for automated cell scanning and diagnostic cytopathology.

The introduction of cationic polyelectrolytes as cellular adherents has significantly advanced the preparation of cervical scrape specimens for automated cell scanning and has also provided an efficient technique for the preparation of cell monolayers of other cytologic specimens, e.g., breast cyst fluids, urines and serous effusions, for diagnostic cytopathology. Variable thickness of cell preparation and cell overlap have both been resolved by laying cells onto glass slides coated with the cationic polyelectrolyte poly-L-lysine. We have determined the optimal conditions for pH, molecular weight, concentration and temperature for the application of poly-L-lysine as a cell-to-slide adhesive.

Aerobic glycolysis of bone and cartilage: the possible involvement of fatty acid oxidation.

The apparent paradox of aerobic glycolysis has been investigated in bone and in cartilage. A new cytochemical procedure for hydroxyacyl dehydrogenase (HOAD) activity showed that the maximal activity of this enzyme in both tissues was equivalent to the maximal activity of glyceraldehyde 3-phosphate dehydrogenase (GAPD). The sum of these activities gave a measure of the maximum amount of acetyl-coenzyme A that could be produced. In these tissues, but not in liver which does not exhibit aerobic glycolysis, this summed value exceeded the maximal activity of succinate dehydrogenase (SDH). Consequently, it suggested that where fatty acid oxidation is sufficient to supply all the acetyl-coenzyme A required for the Krebs' cycle, that derived from fatty acid oxidation may inhibit pyruvate dehydrogenase causing accumulation of pyruvate which must be converted to lactate if pentose-shunt activity is to be maintained.

A quantitative cytochemical method for the measurement of beta-hydroxyacyl CoA dehydrogenase activity in rat heart muscle.

Although cytochemical methods exist for measuring dehydrogenases that act on substrates involved in the production of chemical energy from sugars, virtually no methods exist for measuring the dehydrogenases that act on fatty acids. Yet the oxidation of fatty acids accounts for over 60% of the oxidative activity of cardiac muscle. Consequently a new quantitative cytochemical method, based on a new substrate (DL-S-beta-hydroxybutyryl-N-acetyl cysteamine), has been developed for measuring the activity of hydroxy-acyl coenzyme A dehydrogenase, which is the penultimate step of the beta-oxidation of fatty acids to acetyl-coenzyme A that is used in the Krebs' cycle. Menadione or phenazine methosulphate is used as the intermediate hydrogen-acceptor, with neotetrazolium chloride as the final acceptor. The medium contains nitroprusside, ostensibly to react with any cysteamine liberated by hydrolysis of the substrate. As a control, cysteamine is substituted for the substrate. The concentrations of reactants have been optimized for cardiac muscle; the reaction is linear with thickness of the sections and with time of reaction from 15 to 60 min.

The use of a hidden metal-capture reagent for the measurement of Na+--K+-ATPase activity: a new concept in cytochemistry.

The original lead-trapping method for demonstrating Na+--K+-ATPase activity was discredited because of the effect that lead ions can have on the substrate and on the enzyme. Current methods, that measure this activity by the related K+-dependent phosphatase activity, do not appear to measure activity that is known, from microchemistry, to occur in proximal convoluted tubules. The disadvantages of using lead appear to have been overcome by the use of a new reagent in which the lead is complexed with ammonium citrate ions; phosphate, liberated enzymatically, successfully competes with these ions. The activities of total ATPase and of the ouabain sensitive Na+--K+-ATPase have been measured in three regions of the nephron in the guinea-pig and in the rat. The relative activities found, by this method, in the different regions of the latter, appear to be comparable with results found by others, using microchemical methods applied to isolated regions of the nephron.