Effect of Dichloroacetate on Regional Energy Metabolites and Pyruvate Dehydrogenase Activity During Ischemia and Reperfusion in Gerbil Brain

The objective of this study was to determine whether administration of dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), improves recovery of energy metabolites following transient cerebral ischemia. Gerbils were pretreated with DCA, and cerebral ischemia was produced using bilateral carotid artery occlusion for 20 min, followed by reperfusion up to 4 h. DCA had no effect on the accumulation of lactic acid and the decrease in ATP and phosphocreatine (PCr) during the 20-min insult, nor on the recovery of these metabolites measured at 20 and 60 min reperfusion. However, at 4 h reperfusion, levels of ATP and PCr were significantly higher in DCA-treated animals than in controls, as PCr exhibited a secondary decrease in caudate nucleus of control animals. PDH was markedly inhibited at 20 min reperfusion in both groups, but was reactivated to a greater extent in DCA-treated animals at 60 min and 4 h reperfusion. These results demonstrate that DCA had no effect on the initial recovery of metabolites following transient ischemia. However, later in reperfusion, DCA enhanced the postischemic reactivation of PDH and prevented the secondary failure of energy metabolism in caudate nucleus. Thus, inhibition of PDH may limit the recovery of energy metabolism following cerebral ischemia.     

Isolation and characterization of the Escherichia coli mutL gene product

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 A is 139,000 daltons, indicating that MutL exists as a dimer in solution. In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP.     

Identification and regulation of whole-cell chloride currents in airway epithelium

We used the whole-cell patch-clamp technique to study membrane currents in human airway epithelial cells. The conductive properties, as described by the instantaneous current-voltage relationship, rectified in the outward direction when bathed in symmetrical CsCl solutions. In the presence of Cl concentration gradients, currents reversed near ECl and were not altered significantly by cations. Agents that inhibit the apical membrane Cl conductance inhibited Cl currents. These conductive properties are similar to the conductive properties of the apical membrane Cl channel studied with the single-channel patch-clamp technique. The results suggest that the outwardly rectifying Cl channel is the predominant Cl-conductive pathway in the cell membrane. The steady-state and non-steady-state kinetics indicate that current flows through ion channels that are open at hyperpolarizing voltages and close with depolarization. These Cl currents were regulated by the cAMP-dependent protein kinase: when the catalytic subunit of cAMP-dependent protein kinase was included in the pipette solution, Cl channel current more than doubled. We also found that reducing extracellular osmolarity by 30% increased Cl current, suggesting that cell-swelling stimulated Cl current. Studies of transepithelial Cl transport in cell monolayers suggest that a reduction in solution osmolarity activates the apical Cl channel: reducing extracellular osmolarity stimulated a short-circuit current that was inhibited by Cl-free solution, by mucosal addition of a Cl channel antagonist, and by submucosal addition of a loop diuretic. These results suggest that apical membrane Cl channels may be regulated by cell volume and by the cAMP-dependent protein kinase.     

The second messenger pathway for germ cell-mediated stimulation of Sertoli cells.

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.     

A role for tyrosine kinase activation in interleukin-1β induced nitric oxide production in the insulin producing cell line RINm-5F

The aim of this investigation was to study the putative role of protein phosphorylation in interleukin-1 (IL-1) induced signal transduction in insulin producing cells. For this purpose, insulin producing RINm-5F cells were exposed to IL-1 for 7 hours with or without different agonists and antagonists to protein kinases and phosphatases and the production of nitrite was subsequently determined. It has been shown earlier that IL-1 will stimulate the production of nitrite in such cells. It was found that EDTA, TPA and staurosporine did not affect IL-1 induced nitrite production. However, the tyrosine kinase antagonist tyrphostin inhibited, whereas sodium orthovanadate, okadaic acid and cyclosporin A, all inhibitors of protein phosphatases, potentiated IL-1 induced nitrite release to the medium. The tyrosine kinase antagonist genistein potentiated at a low concentration and inhibited at a high concentration the IL-1 effect. It is concluded that protein phosphorylation events, mediated either by protein kinases or phosphatases on both tyrosine and serine/threonine residues, may mediate or antagonize IL-1 induced signal transduction in insulin producing cells.     

Production of acetone - butanol from acid whey

Summary Clostridium acetobutylicum ATCC 824 was used to produce butanol and acetone by fermenting acid whey. Results showed that both autoclaving and agitation played roles in solvent production. Maximum production was obtained in 120 h using autoclaved, pH adjusted (6.0) acid whey at 37 °C in a fermentor that was not agitated.     

Polarized distribution of bradykinin receptors on airway epithelial cells and independent coupling to second messenger pathways.

Bradykinin stimulates Cl- secretion by airway epithelia, but different patterns of secretion result from addition to the mucosal and submucosal surfaces. Earlier work suggested that bradykinin activates two second messenger pathways: increasing inositol phosphates (InsP) via phosphatidylinositol bisphosphate hydrolysis and increasing cAMP via arachidonic acid metabolism. In this study, we measured arachidonic acid release and InsP production in cultured canine tracheal epithelial cells. Bradykinin increased the two second messengers via independent mechanisms: (a) dose-response curves with different incubation media demonstrated that each second messenger could be generated independently of the other; (b) phorbol ester inhibited InsP production but stimulated arachidonic acid release; (c) for polarized cultures, submucosal bradykinin stimulated production of both second messengers but mucosal bradykinin stimulated only arachidonic acid release. To determine if differences in second messenger formation at the two membranes resulted from differences in hormone-receptor interactions, we compared bradykinin binding to the apical and basolateral membranes. Both the binding capacities and affinity constants (KD) were different (basolateral KD, 257 +/- 53 pM; apical KD, 39 +/- 3 pM). These data demonstrate polarized coupling of bradykinin receptors to second messenger pathways in airway epithelial cells and suggest that this polarized coupling is due to different bradykinin receptors at the two membranes.