Protein phosphorylation and calcium uptake into rat forebrain synaptosomes: modulation by the sigma ligand, 1,3-ditolylguanidine.

The sigma ligand 1,3-di-O-tolylguanidine (DTG) increased basal dynamin and decreased depolarization-stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose-dependently decreased the basal cytosolic free Ca2+ concentration ([Ca2+]i) and blocked the depolarization-dependent increases in [Ca2+]i. These effects were inhibited by the sigma antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8-(p-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate decreased basal [Ca2+]i and the KCI-evoked rise in [Ca2+]i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca2+]i was not mediated via downstream regulation of cyclic GMP levels. DTG increased 45Ca2+ uptake and efflux under basal conditions and inhibited the 45Ca2+ uptake induced by depolarization with KCI. The KCI-evoked rise in [Ca2+]i was inhibited by omega-conotoxin (omega-CgTx)-GVIA and -MVIIC but not nifedipine and omega-agatoxin-IVA. The effect of DTG on decreasing the KCI-evoked rise in [Ca2+]i was additive with omega-CgTx-MVIIC but not with omega-CgTx-GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca2+ levels via inhibition of N-type Ca2+ channels.

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells. Clonidine stimulates basal but inhibits nicotinic receptor evoked phosphorylation.

Clonidine inhibited the uptake of calcium and the overall phosphorylation of tyrosine hydroxylase induced by nicotinic receptor activation in bovine adrenal medullary chromaffin cells in culture. However, clonidine did not inhibit the increase in these parameters that accompanied K+ depolarisation of the cells. There was also no effect of clonidine on the overall phosphorylation of tyrosine hydroxylase when cells were stimulated by muscarine. Nicotinic receptor activation increased the phosphorylation of Ser-19, Ser-31, and Ser-40 on tyrosine hydroxylase, and this was inhibited by clonidine in a concentration-dependent manner. On the other hand, clonidine had no effect on calcium uptake, yet increased the phosphorylation of Ser-19 under basal conditions. Using calcium and calmodulin-stimulated protein kinase II obtained from rat brain clonidine increased the autophosphorylation of the alpha-subunit of the kinase by 37%, and also its activity against an exogenous peptide substrate by 29%. These data are consistent with the hypothesis that clonidine inhibits nicotinic receptor-induced tyrosine hydroxylase phosphorylation by decreasing calcium influx into chromaffin cells, perhaps by an action at the nicotinic receptor. Clonidine also increases the basal phosphorylation of tyrosine hydroxylase at Ser-19, perhaps by directly activating calcium and calmodulin-stimulated protein kinase II.

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of intracellular Ca2+ in the histamine H1 receptor-stimulated phosphorylation of Ser8, Ser19, Ser31, and Ser40.

Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H1 receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca2+. In contrast, the H1-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 microM) and significantly reduced by prior exposure to caffeine (10 mM for 10 min) to deplete intracellular Ca2+. Tryptic-phosphopeptide analysis by HPLC revealed that the H1 response in the presence or absence of extracellular Ca2+ resulted in a major increase in the phosphorylation of Ser19 with smaller increases in that of Ser40 and Ser31. In contrast, although a brief stimulation with nicotine (30 microM for 60 s) also resulted in a major increase in Ser19 phosphorylation, this response was abolished in the absence of extracellular Ca2+. These data indicate that the mobilization of intracellular Ca2+ plays a crucial role in supporting H1-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.

Amoxicillin treatment of bacterial vaginosis during pregnancy.

The purpose of this investigation was to evaluate the efficacy of amoxicillin for treatment of bacterial vaginosis during pregnancy. The diagnosis of bacterial vaginosis was established by clinical examination and microscopic examination of a Gram stain and saline preparation of vaginal secretions. In a double-blind, randomized manner, 108 patients at 15-25 weeks' gestation were assigned to treatment with oral amoxicillin, 500 mg three times daily for 14 days, or placebo. Patients were evaluated 2 weeks after treatment, at 34-36 weeks' gestation, and at delivery. There were no significant differences between the two groups with respect to any clinical or microbiologic measure of treatment outcome. There were also no significant differences in the frequency of obstetric complications. We conclude that amoxicillin is not effective therapy for bacterial vaginosis in pregnant women.

Effect of heat inactivation of HIV on specific serum proteins and tumour markers.

The effect on the concentrations of specific serum proteins and tumour markers, of heat-treating samples at 56 degrees C for 30 min to inactivate HIV, was investigated. Statistically significant decreases, that may affect clinical decisions, were observed for alpha-1-antitrypsin, beta-2-microglobulin, IgE, fibrinogen, C1-esterase inhibitor and CA125. Statistical, but not clinically significant, changes were observed for alpha-1-acid glycoprotein, C3, haptoglobin, IgA, IgG, IgM and transferrin. A significant decrease in the alpha-1 band was observed on protein electrophoresis.

Effectiveness of milk products in dietary management of lactose malabsorption.

Eleven lactose malabsorbers were studied to compare the effectiveness of commercially available products recommended for dietary treatment of lactose malabsorption. One product, a commercial lactase preparation, is added to milk for lactose hydrolysis before consumption. The other is a commercial milk product containing lactose-hydrolyzing, nonpathogenic bacteria, Lactobacillus acidophilus. Both of these products are presently recommended for management of lactose malabsorption, although such recommendations have not been validated by controlled studies. Lactose malabsorption was determined by breath H2 analyses after subjects drank four different test doses on 4 different days. The first test dose was 480 ml of low fat milk; the second was 480 ml of milk treated with a commercial lactase preparation; the third was 480 ml of a commercial L. acidophilus-containing milk; and the fourth was 480 ml of the L. acidophilus-containing milk after 1 wk of gastrointestinal exposure to this commercial bacteria-containing milk. The mean breath H2 response to the lactase-treated milk was significantly lower (p less than 0.001) than the mean response to regular milk. However, the mean breath H2 response to either of the test doses of the L. acidophilus-containing milk were not significantly different than responses to regular milk. It is concluded that the lactase-treated milk reduces breath H2 responses and symptomatic discomfort from malabsorption while the L. acidophilus-containing milk does not.