Stereoselective pharmacokinetics of RS-8359, a selective and reversible MAO-A inhibitor, by species-dependent drug-metabolizing enzymes

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]pyrimidine selectively and reversibly inhibits monoamine oxidase A (MAO-A). After oral administration of rac-RS-8359 to rats, mice, dogs, monkeys, and humans, plasma concentrations of the (R)-enantiomer were greatly higher than were those of the (S)-enantiomer in all species studied. The AUC((R)) to AUC((S)) ratios were 2.6 in rats, 3.8 in mice, 31 in dogs, and 238 in monkeys, and the (S)-enantiomer was almost negligible in human plasma. After intravenous administration of RS-8359 enantiomers to rats, the pharmacokinetic parameters showed that the (S)-enantiomer had a 2.7-fold greater total clearance (CL(t)) and a 70% shorter half-life (t(1/2)) than those for the (R)-enantiomer but had no difference in distribution volume (V(d)). No significant difference in the intestinal absorption rate was observed. The principal metabolites were the 2-keto form, possibly produced by aldehyde oxidase, the cis-diol form, and the 2-keto-cis-diol form produced by cytochrome P450 in rats, the cis-diol form in mice, RS-8359 glucuronide in dogs, and the 2-keto form in monkeys and humans. Thus, the rapid disappearance of the (S)-enantiomer from the plasma was thought to be due to the rapid metabolism of the (S)-enantiomer by different drug-metabolizing enzymes, depending on species.     

Effect of randomly interesterified triacylglycerols containing medium- and long-chain fatty acids on energy expenditure and hepatic fatty acid metabolism in rats

In our previous studies, medium- and long-chain triacylglycerols (MLCT), randomly interesterified triacylglycerols containing medium-chain and long-chain fatty acids in the same glycerol molecule, significantly reduced body fat accumulation in humans and rats. To clarify mechanism(s) for this effect of MLCT, we measured energy expenditure and hepatic fatty acid metabolism in rats by comparison with long-chain triacylglycerols (LCT) or medium-chain triacylglycerols (MCT). MLCT, compared with LCT, showed significantly lower body fat accumulation, higher 24-h energy expenditure and acyl-CoA dehydrogenase activity measured using octanoyl-CoA as a substrate, and similar lipogenic activity. MCT, compared with LCT, showed significantly higher energy expenditure, but fat accumulation was comparable. Additionally, MCT exhibited significantly higher lipogenic activity than the other oils. These data suggest that enhancement of energy expenditure and medium-chain fatty acids (MCFA) oxidation without activating de novo lipogenesis are responsible at least for the lower body fat accumulation in rats fed MLCT. The activation of hepatic lipogenesis by excessive intake of MCFA might counteract their preventive effects on body fat accumulation.     

Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.     

Colocalization of APC and DLG at the tips of cellular protrusions in cultured epithelial cells and its dependency on cytoskeletons

Adenomatous polyposis coli gene product (APC) is a tumor suppressor linked to familial adenomatous polyposis and is thought to be involved in cellular polarization and migration in moving epithelial cells. APC interacts with the mammalian homolog of Discs large (DLG). DLG is a member of the membrane-associated guanylate kinase superfamily and is thought to function as a scaffolding protein that coordinates the assembly of a lateral plasma membrane-localized protein complex in epithelial cells. We confirmed the suitability of several anti-APC antibodies for immunocytochemical analysis. Using these antibodies, we showed that APC clusters were colocalized with DLG protein at cellular protrusions of subconfluent MDCK cells. A portion of the clusters was found at the tips of microtubules extending into the cellular protrusions. In addition, actin stress fibers converged near the clusters. When microtubules were disrupted by nocodazole, the colocalization of APC and DLG was lost due to the disappearance of APC clusters. However, the coclusters remained after depolymerization of actin filaments with latrunculin A. This is the first report showing colocalization of APC and DLG in non-polarized epithelial cells. This colocalization suggests that DLG functions not only at the lateral cell–cell contact sites of polarized epithelial cells but also at the protrusions of non-polarized epithelial cells through the interaction with APC protein.     

Protein 4.1 G localizes in rodent microglia

Although it was reported that protein 4.1 G, a cytoskeletal protein characterized by its general expression in the body, interacts with some signal transduction molecules in the central nervous system (CNS), its distribution and significance in vivo remained to be elucidated. In the present study, we have identified 4.1 G-positive cells in the rodent CNS, and demonstrated its immunolocalization in the developing mouse CNS. In the rodent CNS, 4.1 G was colocalized with markers for microglia, such as CD45, OX-42 and ionized calcium-binding adapter molecule 1 (Iba1), but not with markers for neuronal or other glial cells. Additionally, colocalization of 4.1 G and A1 adenosine receptor was observed in the mouse cerebrum. In a mixed glial culture, most OX-42-positive microglia were positive for 4.1 G, and 4.1 G isoforms of the same molecular weight as in the rat brain were expressed in cultured microglia, where 4.1 G mRNA was detected by RT-PCR. In the developing mouse cerebral cortex, 4.1 G was detected in immature microglia, which were positive for Iba1. These results indicate that 4.1 G in the CNS is mainly distributed in microglia in vivo. Considering the interactions between 4.1 G and the signal transduction molecules, putative roles have been propsed for 4.1 G in microglial functions in the CNS.     

Optimization of a coagulation factor VIIa inhibitor found in factor Xa inhibitor library

An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a, was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in the TF-dependent extrinsic pathway.     

Biological activity of neurotrophins is dependent on recruitment of Rac1 to lipid rafts

The Rho family of small GTPases, key regulators of the actin cytoskeleton in eukaryotic cells, is implicated in the control of neuronal morphology. Here, we report that neurotrophin dependent cytoskeletal changes, characteristic of the phenotype of Rac1, in the hippocampal neurons or PC12 cells are inhibited by the disruption of lipid raft integrity. Activation of Rac1 induced by NGF is impaired in cholesterol-depleted PC12 cells. Pretreatment with gammaGTP shifted significant amount of Rac1, presumably in a GTP-bound form, from non-raft to raft fractions. Proper recruitment of activated Rac1 to lipid rafts, structures that represent specialized signaling organelles, is of fundamental importance in determining neurotrophins' bioactivity.     

Serotonin transporter polymorphisms affect human blood glucose control

We measured the effect of nutritional intervention on clinical data, including fasting blood glucose (FBG), and their association with polymorphisms of the serotonin transporter-linked polymorphic region (5-HTTLPR) which might affect adherence. Enrolled in the intervention program were 264 Japanese women not on medication for diabetes, hypercholesterolemia or hypertension. The 5-HTTLPR allele (S and L) frequencies among the subjects differed markedly from those of Caucasians: SS (n = 183), LS (n = 69), and LL (n = 12). The decrease in FBG (DeltaFBG) from the beginning to the end of the program (11 weeks; short-term study), and DeltaFBG from the beginning to a follow-up check performed between 2002 and 2004 (average of 23 years later; long-term study) was calculated. The SS homozygotes of 5-HTTLPR showed larger DeltaFBG (P = 0.01 and P < 0.0001 in the short- and long-term studies, respectively) than DeltaFBG with other genotypes.     

Differences in sensory projections between macro- and microchaetes in Drosophilid flies

From examination of the central axonal projections of sensory bristles on the notum of several species of Drosophilidae, we demonstrate different features that may indicate different functions for macro- and microchaetes. The large macrochaetes have conserved arborizations that correlate with their conserved position. Nevertheless, we find evidence for only two discrete projection patterns for bristles in the dorsocentral (DC) row, even when there may be four or five bristles present. We show that the small microchaetes of Drosophila melanogaster display regional specificity and subsets of contiguous bristles project to a common region in the thoracic ganglion. Interestingly, the axons of each of these subsets also form a specific fasciculation group on the scutum before joining the axon of a particular macrochaete. The positions of microchaetes on the scutum and the shape of the fasciculation groups vary between closely related species. There is no correlation between body size, bristle patterns, and fasciculation patterns. Furthermore, none of these traits correlate with the phylogenetic relationships between the species studied. We discuss the possibility that macro- and microchaetes may have different functions and that these have implications for evolutionary constraints on bristle patterns.