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Featured researches published by June Nelson.


Metabolism-clinical and Experimental | 1994

Regulation of the glutamate dehydrogenase activity in rat islets of Langerhans and its consequence on insulin release

Jadwiga Bryla; Mariusz Michalik; June Nelson; Maria Erecińska

Kinetic properties of glutamate dehydrogenase (GDH) and the effects on its activity of several putative modulators were examined in mitochondrial extracts of rat pancreatic islets. In the presence of 40 mmol/L NH4Cl and 0.1 mmol/L NADH, stepwise elevation of the 2-oxoglutarate concentration from 0.005 to 0.05 mmol/L increased glutamate formation, whereas further increases led to a progressive decrease of the reaction velocity. Adenosine diphosphate (ADP) at 0.1 mmol/L partially and at 1 mmol/L completely reversed the inhibitory effect of 2-oxoglutarate. The sensitivity to activation by either ADP or leucine was dependent on 2-oxoglutarate concentrations. At higher concentrations of the latter, greater amounts of the activators were needed to attain maximal effect. In the absence of allosteric activators, sulfate or phosphate at 20 mmol/L partially released the inhibitory effect of 2-oxoglutarate levels and increased the maximal velocity (Vmax) for the reaction. In the presence of 0.1 mmol/L ADP, both anions prevented the inhibition by higher concentrations of 2-oxoglutarate, whereas with 1 mmol/L ADP their only effect was a slight increase in the Vmax. Mg2+ and naturally occurring polyamines decreased glutamate formation in a dose-dependent manner; with 0.1 mmol/L ADP, inhibition was seen at all 2-oxoglutarate concentrations studied, whereas with 1 mmol/L ADP, it was noticeable at substrate concentrations higher than 0.5 mmol/L. This inhibitory effect on GDH activity was partially attenuated by sulfate. Addition of either 2 mmol/L spermidine or extra magnesium (final 2.5 or 5 mmol/L) to the perifusion buffer markedly attenuated the insulin release elicited by alpha-ketoisocaproate. It is suggested that naturally occurring polyamines, magnesium, and phosphate act as physiological modulators of GDH activity in pancreatic beta cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolism-clinical and Experimental | 1989

Bioenergetic response of pancreatic islets to stimulation by fuel molecules

Martin D. Meglasson; June Nelson; David Nelson; Maria Erecińska

The relationship of fuel-stimulated insulin secretion and the beta-cell bioenergetic state was investigated in isolated rat islets. In islets perifused with 5 mmol/L glucose to maintain a high basal energy state, stimulation by 9 to 28 mmol/L glucose increased the [ATP]/[ADP] and [GTP]/[GDP]. The rise in the former occurred prior to, or coincident with, the onset of insulin secretion and was dependent on glucose concentration. The increase in the latter appeared to lag behind the alteration in the [ATP]/[ADP] and achieved statistical significance after 30 minutes of incubation. Addition of 20 mmol/L alpha-ketoisocaproic acid, a powerful secretagogue, also caused a rise in the [ATP]/[ADP]. By contrast, 20 mmol/L lactate, which affected insulin secretion only minimally, failed to alter nucleotide concentrations. These data support the hypothesis that an increase in the islet energy state is a metabolic signal linking fuel metabolism with initiation of insulin secretion.


Archives of Biochemistry and Biophysics | 1985

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages.

Mark W. Sutherland; June Nelson; Gerald Harrison; Henry Jay Forman

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.


Diabetes | 1993

GABA Production in Rat Islets of Langerhans

Mariusz Michalik; June Nelson; Maria Erecińska

Homogenates of pancreatic islets catalyzed breakdown of L-glutamate to GABA with a rate of 0.24 ± 0.04 nmol · min−1 · mg−1 protein at 37°C. The formation of GABA was stimulated by addition of pyridoxal phosphate in the range 0.05–1 microM (0.97 ± 0.02 nmol · min−1 · mg protein-1 at a saturating cofactor concentration), which indicates that the process was catalyzed by glutamic acid decarboxylase. The half-maximal effect was obtained with 0.1 microM PLP. Kinetic analyses of the results showed that the Vmax and Km for the reaction were 1.12 nmol · min−1 · mg protein−1 and 0.66 mM, respectively. The pH optimum was 7.0. Subcellular fractionation revealed that 51% of GAD activity was present in the cytosol, 17% in microsomes, 9% in secretory granules, 5% in mitochondria, and 11% in cell debris. Comparison of the kinetic properties of the cytosolic and microsomal forms of the enzyme showed that their Km for glutamate was the same, but that the cytosolic GAD had a lower Km for PLP. GABA synthesis in the nominal absence of PLP was enhanced by malate (twofold increase at 5 mM) and citrate (threefold increase at 5 mM), but was unaffected by ATP and chloride. However, if the islet homogenate was prepared and incubated in the presence of PLP, neither malate nor citrate influenced enzyme activity. Aspartate and AOA were powerful inhibitors of glutamate breakdown. Freshly isolated islets contained ∼4 mM GABA, whereas the concentration was < 0.1 mM in whole pancreas. Perifusion of islets in the absence of amino acids decreased GABA concentration by ∼40% in 60 min; this decline could be prevented by addition of either GABA itself or gabaculine, an inhibitor of GABA-transaminase. Inclusion of 5 mM GABA in the perifusion buffer raised the tissue ATP/A DP beyond the value observed in the absence of any external oxidizable substrate; the effect was eliminated by gabaculine. It is concluded that pancreatic islets contain active GAD, an enzymeresponsible for GABA formation; malate and citrate may act as physiological stimulators of GAD, whereas aspartate may function as its inhibitor; and GABA serves as a source of fuel for generation of intracellular ATP in islets perifused in the absence of glucose.


Metabolism-clinical and Experimental | 1992

Glutamate production in islets of Langerhans: Properties of phosphate-activated glutaminase

Mariusz Michalik; June Nelson; Maria Erecińska

Homogenates of rat pancreas, pancreatic islets, and HIT-T15 cells (a clonal line derived from B cells) catalyzed the breakdown of glutamine to glutamate. This activity was markedly stimulated by the addition of orthophosphate and was much greater in homogenates from islets and the B-cell-derived clonal cell line than in those from whole pancreas. Islet glutaminase was half-maximally stimulated with 40 mmol/L phosphate. Kinetic analyses of the rates of glutamine hydrolysis showed that the Vmax for the reaction increased with the increase in phosphate concentration, whereas the Km for glutamine (2.6 +/- 0.2 mmol/L) was unaltered. The pH optimum for enzyme activity was 8.0 to 8.5 at all phosphate concentrations studied. Glutamine breakdown was enhanced by adenosine triphosphate ([ATP] approximately 100% at 10 mmol/L) and citrate (approximately 30% at 10 mmol/L), but it was unaffected by malate, 2-oxoglutarate, lactate, and ammonia. Glutamate significantly inhibited glutamine hydrolysis. Freshly isolated islets had a low content of both glutamate and glutamine. After culturing for 1 hour in an amino acid-containing medium, the concentrations of glutamine and glutamate increased. Subsequent perifusion without amino acids caused a loss of glutamine and a concomitant increase in glutamate level. Perifusion with 1 mmol/L glutamine led to an increase in both internal glutamine and glutamate. The addition to the perifusion medium of either 10 mmol/L glutamine, 10 mmol/L orthophosphate, or both substantially enhanced insulin release evoked by 10 mmol/L leucine.(ABSTRACT TRUNCATED AT 250 WORDS)


Journal of Free Radicals in Biology & Medicine | 1985

Oxygen toxicity: Loss of lung macrophage function without metabolite depletion

Mark W. Sutherland; Mitchell Glass; June Nelson; Yuen Lyen; Henry Jay Forman

Hyperoxia inhibited concanavalin A stimulated O2- release (respiratory burst) of alveolar macrophages obtained by bronchoalveolar lavage from rats. After 36 h of normobaric 100% O2, a partial reversal (48%) of the inhibition was produced by addition of glucose. Since oxidant-induced, reversible NADPH depletion correlates with reversible inhibition of the respiratory burst, intracellular NADPH was assayed to determine whether irreversible inhibition of the respiratory burst was related to persistent changes in this metabolite. The cellular concentrations of ATP, glutathione, and ascorbate were also measured. After 36 h of hyperoxia, NADPH concentration in alveolar macrophages rose slightly while ATP and glutathione content remained at control levels. Ascorbate levels fell significantly but were not responsible for respiratory burst inhibition. Thus, irreversible loss of cellular function in hyperoxia is not due to persistent alterations in these metabolites. Significant amounts of both glutathione and ascorbate were found in extracellular fractions of lung washings, indicating high concentrations in the aqueous subphase in the lung fluid lining. There was no change in total content of these extracellular antioxidants following O2 exposure.


Biochemical Pharmacology | 1991

Relationships between energy level and insulin secretion in isolated rat islets of langerhans: A study at various pH values

Ohta Mitsuaki; David Nelson; June Nelson; Martin D. Meglasson; Maria Erecińska

To define better the role of [ATP]/[ADP] in insulin release from pancreatic islets, changes in the adenine nucleotide ratios elicited by alterations in external pH were correlated with the secretion profiles produced by administration of two metabolic secretagogues, 16 mM glucose and 10 mM alpha-ketoisocaproic acid. Experiments were carried out in buffers with and without bicarbonate, in the pH range 6.5-7.7. Insulin release was dependent on pHe irrespective of the secretagogue used. Secretion profiles for alpha-ketoisocaproic acid were the same both with and without bicarbonate; the release was decreased below pH 7.1 but maintained at 7.4-7.7. The same pattern was seen with glucose in media buffered with Hepes. With bicarbonate present, secretion caused by high glucose showed a bell-shaped dependence on [H+], with reductions at the acid and alkaline sides of pH 7.1-7.4. [ATP] and [ADP] were higher when Hepes was the buffer, at all pH values studied. The [ATP]/[ADP] declined with increasing pH under both basal and stimulated conditions; the values were always larger after stimulation although at pH 7.7 with bicarbonate present and glucose as the stimulant the difference was very small. It is concluded that: (i) the [ATP]/[ADP] in pancreatic islets is markedly dependent on pHe; (ii) there is no straight-forward correlation between either [ATP] or the absolute value for [ATP]/[ADP] and insulin secretion; and (iii) a rise in [ATP]/[ADP] is necessary for glucose-stimulated insulin release although it is not always the rate-determining event.


Biochimica et Biophysica Acta | 1996

SECRETAGOGUE-INDUCED PROTEOLYSIS OF CAMP-DEPENDENT PROTEIN KINASE IN INTACT RAT ALVEOLAR EPITHELIAL TYPE II CELLS

Un-Jin P. Zimmerman; Mengshu Wang; June Nelson; Felix S. Ekwunife; Lin Liu

Stimulation of secretion from rat alveolar epithelial type II cells by the beta-adrenergic agonist terbutaline activates cAMP-dependent protein kinase (PKA). The same secretagogue also activates endogenous protease calpain in type II cells. In this study, we investigated the effect of calpain activation on PKA and its phosphorylation activity in stimulated type II cells. Type II cells were either pretreated with cell-permeable calpain specific inhibitor (N-acetyl-leucyl-leucyl-methioninal) or untreated, and subsequently stimulated with terbutaline. Stimulus-induced phosphorylation activity was assayed using the PKA-specific substrate Kemptide. Maximum PKA activity was observed within 1-3 min of stimulation. Peak activity of the untreated cells was 20-25% higher and longer than that of the inhibitor-treated cells. The stimulus-induced phosphorylation activity of both cell groups was suppressable by PKA-specific inhibitor. Concomitant photoaffinity labeling with radioactive 8-azido-cAMP revealed that a 39 kDa proteolytic fragment was generated in response to stimulation by terbutaline. Stimulus-induced activation of PKA resulted in the phosphorylation of two endogenous proteins, p112 and p47. Phosphorylation of p112 and p47 was modulated in cells pretreated with calpain inhibitor or in the presence of PKA inhibitor. Aggregate results indicate that stimulus-induced proteolysis of pKA occurs in type II cells, suggesting that limited proteolysis of PKA by endogenous calpain may convert an initial transient signal to sustained and augumented phosphorylation activity for secretion.


Free Radical Biology and Medicine | 1996

Reactions of nitric oxide and peroxynitrite with organic molecules and ferrihorseradish peroxidase: Interference with the determination of hydrogen peroxide

Harry Ischiropoulos; June Nelson; Daniel Duran; Abu B. Al-Mehdi


Journal of Applied Physiology | 1982

Hyperoxia inhibits stimulated superoxide release by rat alveolar macrophages

Henry Jay Forman; Jay J. Williams; June Nelson; R. P. Daniele; Aron B. Fisher

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Henry Jay Forman

University of Southern California

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Maria Erecińska

University of Pennsylvania

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Carl D. Bennett

University of Pennsylvania

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Mariusz Michalik

University of Pennsylvania

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David Nelson

University of Pennsylvania

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Abu B. Al-Mehdi

University of Pennsylvania

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Aron B. Fisher

University of Pennsylvania

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