Activation of insulin-secreting cells by pyruvate and halogenated derivatives.

Addition of pyruvate to rat islets perifused in the presence of 5 mM-glucose elicited an immediate pronounced biphasic stimulation of insulin secretion. At lower concentrations of glucose (2.5 mM), only the initial, transient, phase of secretion was observed. Pyruvate inhibited 45Ca2+ efflux from islets at 2.5 mM-glucose and stimulated efflux at 5 mM-glucose. Pyruvate also decreased the rate of efflux of 86Rb+ from perifused islets. A marked stimulation of insulin secretion and 45Ca2+ efflux rate was observed in response to 3-fluoropyruvate and 3-bromopyruvate, compounds which inhibited oxidative metabolism of [14C]glucose and [14C]pyruvate in islets. The stimulatory effects of 3-fluoro- and 3-bromo-pyruvate were associated with enhanced 86Rb+ efflux. Withdrawal of pyruvate or halogenated analogues from the perfusate resulted in a secondary stimulation of insulin release, 45Ca2+ efflux and, to some extent, 86Rb+ efflux rates. Pyruvate, 3-fluoropyruvate and 3-bromopyruvate were all effective in promoting intracellular acidification and a rise in cytosolic Ca2+ concentration, as judged from fluorescence measurements in HIT-T15 cells loaded with 2',7'-biscarboxyethyl-5'(6')-carboxyfluorescein and Quin 2 respectively. It is proposed that oxidative metabolism of pyruvate is not a prerequisite for its stimulatory actions on pancreatic beta-cells. An alternative mechanism of activation by pyruvate and its halogenated derivatives is proposed, based on the possible electrogenic flux of these anions across the cell membrane.     

Effect of Alternaria solani culture filtrate on adventitious shoot regeneration in potato

Summary The effect of Alternaria solani culture filtrate on adventitious shoot regeneration from tuber discs was evaluated using five potato cultivars, which were selected based on their field reaction to Alternaria solani and which represented a range of disease reactions. The culture filtrate stimulated regeneration, a response that could prove to be very useful in the wider utilization of transformation and in vitro selection technology.Research conducted at the Scottish Crop Research Institute during a transfer of work of the senior author     

Inward rectification in rat olfactory receptor neurons.

Inwardly rectifying currents in enzymically dissociated olfactory receptor neurons of rat were studied by using patch-clamp techniques. Upon hyperpolarization to membrane potentials more negative than -100 mV, small inward-current relaxations were observed. Activation was described by a single exponential with a time constant that decreased e-fold for a 21 mV hyperpolarization. The current was not reduced by the external application of 5 mM Ba2+, but was abolished by the addition of 5 mM Cs+ to the bath solution. Increasing the external K+ concentration ([K+]o) to 25 mM dramatically enhanced the current without affecting the voltage range or the kinetics of activation. In 25 mM [K+]o, tail currents reversed at -26 mV, significantly more positive than the K+ equilibrium potential of -44 mV. These characteristics are consistent with those of a mixed Na+/K+ inward rectification that has been reported in several types of neuronal, cardiac and smooth muscle cells. The current may contribute to controlling cell excitability during the response to some odorants.     

Brevibacterium liquefaciens adenylate cyclase and its in vivo stimulation by pyruvate.

Adenylate cyclase of Brevibacterium liquefaciens depends on pyruvate for activity. Growing in a simple medium containing glucose and DL-alanine, the microorganism excreted pyruvate, which reached 20 mM in the medium at stationary phase. Using [3H]adenosine to label the adenosine 5'-triphosphate pool, we showed that pyruvate in the medium stimulated adenylate cyclase of B. liquefaciens in vivo, in a manner similar to the stimulation observed in vitro. Adenylate cyclase in cells harvested at different phases of growth was equally responsive to exogenous pyruvate, indicating that the allosteric site for pyruvate was present in the enzyme throughout the various phases of cell growth. The specific activity of adenylate cyclase was highest in cells harvested at early log phase; thereafter it declined and was substantially lower at stationary phase. Although adenylate cyclase appears to be activated by pyruvate throughout the life span of the cell, the activity appears not to be critical to cell growth, which was comparable whether the medium contained high or low pyruvate.     

Melatonin excretion of man and rats: Effect of time of day,sleep, pinealectomy and food consumption

Pineal function was assessed in human subjects by measuring the excretion of melatonin. The hormone was extracted from the urine by adsorption on a nonionic polymeric resin and then elution with organic solvents; its concentration was determined with a bioassay based ontthe dermal melanophore response of larval anurans to melatonin in their bathing medium. Melatonin excretion among healthy, adult volunteers was 5- to 7-fold greater during the hours of sleep, darkness, and recumbancy (23:00–07:00 h) than during the active, waking hours (07:00–15:00 h or 15:00–23:00 h). When 2 subjects slept only during the daylight at odd, 8-hour intervals, their melatonin excretion was also greatest during their time of sleep. Three bedfast fracture patients (for whom sleep data were not available) failed to display melatonin rhythms. A new radioimmunoassay for melatonin was investigated in preliminary studies with experimental animals and was found to be expedient and sensitive; it was not as specific as the bioassay, however. Both analytical methods were used in studies on intact and pinealectomized rats, and the findings suggest that rhythmic melatonin excretion persists (although at a reduced amplitude) in the absence of the pineal. Fasting in the pinealectomized animal abolished the day/night variation in urinary melatonin, but did not have this effect in intact rats.     

Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin.

The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte.     

Molecular and genetic approaches to the analysis of the informational content of the mitochondrial genome in mammalian cells

Molecular and Cellular Biochemistry - Our laboratory has been involved in the last few years in investigations aiming at analyzing by molecular approaches the informational content of the...     

Complete retention of phospholipid acyl groups by mammalian cells in culture.

Radiolabeled phosphate, acetate, and glycerol are incorporated into strain L-fibroblast phospholipids. The acetate and glycerol specifically label the fatty acid and glycerol moieties, respectively, of the phospholipids. To study the metabolic fate of the various moieties of phospholipids, cells incubated with the above radiolabeled compounds were transferred to unlabeled medium, and the rate at which phospholipid radioactivity per 10(6) cells decreased was determined. The rate of decrease expected on the basis of cell division alone was estimated either by monitoring increases in cell number, or by measuring the rate at which radiolabeled DNA per 10(6) cells decreased. Both phospholipid phosphorus and glycerol are lost at a rate greater than can be accounted for by cell division alone. By contrast, nearly all phospholipid acyl chains were retained by the cell to the same extent as radiolabeled DNA. While presence of nonradioactive glycerol in the medium increased the rate at which glycerol was lost from phospholipid, the addition of exogenous fatty acid was without effect on the retention of phospholipid acyl groups. The acyl-glycerol bond of phosphatidylcholine is metabolically more labile than that of phosphatidylethanolamine. Together the data suggest that although L-fibroblast phospholipids undergo deacylation-reacylation reactions, the acyl chains do not equilibrate with either extracellular or intracellular pools of unesterified fatty acid.