Characteristics of a pyrimidine-specific 5'-nucleotidase in human erythrocytes.

A 5'-nucleotidase with unique specificity has been identified in the soluble fraction of normal human erythrocytes. It mediates the hydrolytic dephosphorylation of pyrimidine 5'-ribosemonophosphates but is catalytically ineffective with purine nucleotides or with the 2'-, 3'-, or cyclic isomers of pyrimidine nucleotides. Activities at 37 degrees in dialyzed hemolysates of nromal human erythrocytes averaged 7.3 and 6.2 mumol of Pi liberated per hour per g of hemoglobin for the substrates UMP and CMP, respectively. Activity with TMP as substrate was approximately one-half as much as with UMP or CMP. Apparent Michaelis constants were 0.33 mM UMP, 0.15 mM CMP, and 1.0 mM TMP. Magnesium was required for optimal activity, and this cation could not be replaced by Mn2+. Maximum activity was obtained between pH 7.0 and 7.5 with rapid decreases in more alkaline media and moderate decreases with acidification. The enzyme was quite sensitive to heat and was strongly inhibited by AMP, by some purine bases, and by both purine and pyrimidine nucleosides. Divalent cations of heavy metals were also strongly inhibitory, as were agents active against sulfhydryl groups. The presence of substrates and/or 2-mercaptoethanol provided considerable protection against some of these deleterious agents and conditions. Pyrimidine 5'-nucleotidase activity in hemolysates was clearly distinguishable from erythrocyte acid phosphatase and from leukocyte and serum alkaline phosphatases and nucleotidases.     

Streptozotocin treatment of streptozotocin-induced islet cell adenomas in rats.

Hypoglycemic rats bearing insulin-secreting islet-cell adenomas produced by the combined action of streptozotocin and nicotinamide were treated with streptozotocin. Antitumor response was demonstrated by elevation of blood glucose, reduction in plasma and tumor IRI, and histopathologic changes in the beta-cell neoplasm. The rodent tumor model may serve as a predictive system for selection and investigation of mechanisms of action of future antitumor agents to be used in the treatment of malignant insulinoma in man.     

Effect of glucose feeding on net transport of plasma free fatty acids

The effect of a single glucose feeding upon the net inflow and outflow transport of plasma free fatty acids (FFA) has been studied in 75 unanesthetized rats. The animals were fasted for 22 +/- 2 hr; then 50 rats were refed 2 ml of 50% glucose by gastric intubation. At 0, 10-15, and 30-35 min after glucose refeeding, the rats were injected with palmitate-1-(14)C complexed to rat serum. The tracer dose included (131)I-labeled albumin. Plasma FFA concentration, (131)I concentration, and FFA-(14)C were measured at five time intervals after injection of the tracer dose. From these data the irreversible disposal rate, or net outflow transport, and the net inflow transport of plasma FFA were calculated. Estimations were based upon a special case of a general solution for measuring net inflow and outflow transport of a circulating metabolite. The general solution is independent of the number of compartments, how they are interconnected, the number of nonradioactive inflows, and where the inflows enter the system. Net inflow = net outflow transport = 7.6 micro eq/min in the fasted state and 3.5 micro eq/min in the new steady state that is reached 30-40 min after glucose refeeding. A very slight imbalance between the rates of net inflow and outflow transport could account for the rapid fall in plasma FFA concentration that results from a single glucose feeding. Theoretical and practical problems associated with studying inflow and outflow transport by means of the technique using a single injection of racer are discussed.     

A Histological Analysis of Regeneration in Watermelon

The optimization of regeneration protocol for different genotypes increases the yield in transformation studies. Cotyledon explants of watermelon [Citrullus lanatus (Thunb) Matsum & Nakai] cv Crimson Sweet were cultured on MS medium containing combinations of benzyl adenine (BA) (0, 5, 10, 20 µM) and indole-3-acetic acid (IAA) (0, 0.5, 5 µM). Maximum shoot growth and subsequent rooting from explants on regeneration medium were obtained from the media containing 10 µM BA + 0.5 µM IAA and 20 µM BA (75 and 78%) by direct organogenesis, respectively. Histological analysis showed that cell division was observed in the epidermal and subepidermal layers. Protuberant structures were observed in tissues between 7 and 12 days in culture. Meristematic structures were observed after 12days in culture which later developed into buds.     

Effect of auto-oxidized phospholipids on oxidative enzyme assays based on tetrazolium salt reduction

The influence of auto-oxidized phospholipids on the reduction of the tetrazolium salt MTT coupled to the NAD+-dependent lactate dehydrogenase reaction was studied. The following results were obtained: (1) peroxidized phosphatidylcholine interfered in the time-course of the lactate dehydrogenase-mediated MTT reduction; (2) there was a time-dependent decrease in the hydroperoxide content of phosphatidylcholine vesicles during the incubation; (3) the diminution of phosphatidylcholine hydroperoxides required the presence of all the components of the system except MTT; (4) hydroperoxide diminution and MTT reduction were mediated by the superoxide radical O2-, since both processes were inhibited by superoxide dismutase; (5) EDTA inhibited the hydroperoxide decrease and abolished the interference of peroxidized phosphatidylcholine with MTT reduction. It was concluded that hydroperoxides compete with MTT for the electrons coming from substrate oxidation. The superoxide radical O2- and traces of some contaminating metal ion are involved in the process. This is a potential complication in the study of the effect of lipids on enzymatic activities assayed by the tetrazolium salt method.