Inhibition of sporulation, glycopeptide antibiotic production and resistance in Streptomyces toyocaensis NRRL 15009 by protein kinase inhibitors.

Production of the glycopeptide antibiotic A47934 by Streptomyces toyocaensis NRRL 15009 begins in the late exponential phase in liquid culture and peaks in the early stationary phase. The pattern of cellular phosphoprotein production changes upon onset of A48934 production with the appearance of several novel phosphoproteins only when an antibiotic is being produced. Phosphoamino acid analysis revealed that S. toyocaensis NRRL 15009 produces proteins phosphorylated on His, Ser, Thr and Tyr, with most being membrane-associated. Addition of the isoflavones genistein or quercetin abolishes A47934 production in liquid culture and sporulation on solid medium. Furthermore, genistein slows the onset of inducible glycopeptide antibiotic resistance in S. toyocaensis NRRL 15009. These results support the participation of protein kinase pathways in A47934 biosynthesis and resistance and cell differentiation in S. toyocaensis NRRL 15009.

A marked diurnal rhythm of melatonin ML1A receptor mRNA expression in the suprachiasmatic nucleus.

Melatonin (ML1A) receptor binding in the suprachiasmatic nucleus (SCN) exhibits a diurnal rhythm, with significant increases during the daytime. In order to determine whether the diurnal variation in binding is reflected by a cycling of melatonin ML1A receptor mRNA levels, we utilized a semiquantitative reverse transcriptase-polymerase chain reaction procedure to examine receptor expression throughout the light-dark cycle. Here we report the first evidence of a significant diurnal variation in ML1A receptor mRNA levels within the SCN. Furthermore, this mRNA expression occurs approximately 3 h prior to, but is tightly correlated to the diurnal rhythm in high-affinity melatonin binding in the SCN.

PK 11195 blockade of benzodiazepine-induced inhibition of forskolin-stimulated adenylate cyclase activity in the striatum.

1. The effects of benzodiazepine receptor antagonists on the inhibition of forskolin-stimulated adenylate cyclase (AC) activity by various benzodiazepine (BZ) and indoleamine agonists in the rat striatum were investigated. 2. A biphasic inhibition of forskolin-stimulated AC activity by the peripheral-type agonist, Ro5-4864, and a multiphasic inhibition by the non-selective BZ, diazepam, was observed. One phase of AC inhibition is consistent with a Gi-coupled receptor-mediated action, whereas the other phases appear to involve a direct effect on the enzyme itself. 3. While the central-type antagonist, flumazenil, had no effect on the ability of Ro5-4864 to inhibit AC activity, the peripheral-type receptor ligand, PK 11195, abolished the first phase of inhibition. 4. PK 11195 and pertussis toxin were found to block the inhibitory effect of various BZs and the indoleamines, melatonin and 2-iodomelatonin, on induced AC activity. 5. Saturation binding studies, conducted at 30 degrees C with [3H]-diazepam revealed a single binding site in the rat striatum (KD = 19.3 +/- 0.80 nM) which significantly decreased in affinity in the presence of GTP (KD = 30.5 +/- 2.6 nM; P < 0.05). No significant change in Bmax was observed. 6. These findings indicate the presence of Gi-coupled BZ receptors in the rat striatum. Thus, suppression of cyclic AMP production may contribute to the diverse neuropharmacological effects of BZs, melatonin and related drugs.

Induction of CYP3A and associated terfenadine N-dealkylation in rat hepatocytes cocultured with 3T3 cells.

Long-term culture of hepatocytes has been challenged by the loss of differentiated functions. In particular, there is a rapid decline in cytochrome P450 (CYP). In this study, we cocultured rat hepatocytes with 3T3 fibroblasts for 10 days, and examined hepatocyte viability, morphology, and expression of CYP3A. Terfenadine was incubated with the cultures, and its biotransformation was quantitatively analyzed by HPLC. Terfenadine is metabolized by two major pathways: C-hydroxylation to an alcohol metabolite which is further oxidized to a carboxylic acid, and N-dealkylation to azacyclonol. In rat liver, only the N-dealkylation pathway appears to be mediated by CYP3A since anti-rat CYP3A antibody inhibited azacyclonol but not alcohol metabolite formation in incubations of terfenadine with liver microsomes. Freshly isolated rat hepatocytes were seeded on top of confluent 3T3 cells. Cultures were maintained in Williams' E medium supplemented with 10% fetal bovine serum and either 0.1 mumol/L or 5 mumol/L dexamethasone. In pure hepatocyte cultures, viability, as determined by lactate dehydrogenase (LDH) activity, decreased steadily to less than 30% of initial levels by day 10. In cocultures, LDH activity remained high and was 70% of initial levels on day 10. The half-life of terfenadine disappearance was optimally maintained in cocultures treated with 5 mumol/L dexamethasone, and was associated with the increased formation of azacyclonol. On day 5, nearly 50% of added 5 mumol/L terfenadine was converted to azacyclonol within 6 h, whereas the conversion was only 4% on day 1. Western and RNA-slot blot analyses confirmed that treatment with 5 mumol/L dexamethasone induced CYP3A mRNA expression and CYP3A protein expression. This coculture system could offer a useful approach in the study of drugs and xenobiotics metabolized by CYP3A.