Changes in genotypic composition of Myzus persicae (Hemiptera: Aphididae) on tobacco during the past two decades in Japan

Ninety-nine and 476 clones of Myzus persicae (Sulzer) were sampled on tobacco at Kyoto (Shimogamo) (1996-2000) and at 23 other localities (1998-1999) in Japan, respectively. The clones were classified into colour-esterase forms, distinguished by combinations of body colour and electrophoretically detectable esterases, to verify the changes in genotypic composition during the past two decades. Fifteen and 31 colour-esterase forms were found at Kyoto (Shimogamo) and at 23 other localities, respectively. Fourteen (representing c. 95% of the total clones sampled) and 24 (c. 44%) colour-esterase forms, respectively, were different from those found on tobacco during 1978-1985. The frequency of the green form and the very highly insecticide-resistant form increased. More than half of the colour-esterase forms found in the present survey were newly detected. The factors associated with these changes are discussed.     

Highly sensitive quantification of vancomycin in plasma samples using liquid chromatography-tandem mass spectrometry and oral bioavailability in rats

We developed a highly sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS-MS) for a glycopeptide antibacterial drug, vancomycin (VCM), in rat plasma. After precipitating 100 micro l of plasma with 300 micro l of 10% trifluoroacetic acid-methanol (2:1, v/v), the supernatant was diluted with 300 micro l of distilled water and was passed through a filter. LC-MS-MS equipped with electrospray ionization in the positive ion mode used a pair of ions at 725/144 m/z for VCM in the multiple reaction-monitoring mode with a sample injection volume of 20 micro l. The calibration curve had a linear range from 0.01 to 20 micro g/ml when linear least square regression was applied to the concentration versus peak area plot. The drug in the sample was detected within 5 min. Precision, accuracy and limit of quantitation indicated that this method was suitable for the quantitative determination of VCM in rat plasma. Using this method, we defined for the first time that the oral bioavailability of VCM in rats was 0.069%. This method can be applied to basic pharmacokinetic and pharmaceutical studies in rats.     

Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP

Using biochemical assays, it has been demonstrated that expression of Ebola virus VP40 alone in mammalian cells induced production of particles with a density similar to that of virions. To determine the morphological properties of these particles, cells expressing VP40 and the particles released from the cells were examined by electron microscopy. VP40 induced budding from the plasma membrane of filamentous particles, which differed in length but had uniform diameters of approximately 65 nm. When the Ebola virus glycoprotein (GP) responsible for receptor binding and membrane fusion was expressed in cells, we found pleomorphic particles budding from the plasma membrane. By contrast, when GP was coexpressed with VP40, GP was found on the filamentous particles induced by VP40. These results demonstrated the central role of VP40 in formation of the filamentous structure of Ebola virions and may suggest an interaction between VP40 and GP in morphogenesis.     

Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (DeltaPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas DeltaPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or DeltaPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3alpha/beta, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither DeltaPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3alpha/beta pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or DeltaPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.     

Identification of a link between the tumour suppressor APC and the kinesin superfamily

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC is involved in the proteasome-mediated degradation of beta-catenin, through its interaction with beta-catenin, GSK-3 beta and Axin. APC also interacts with the microtubule cytoskeleton and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends. However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. We show here that APC interacts with the kinesin superfamily (KIF) 3A-KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and beta-catenin are transported along microtubules by KAP3-KIF3A-KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration.     

Sex-dependent pharmacokinetics of S(-)-hydroxyhexamide,a pharmacologically active metabolite of acetohexamide,in rats

The pharmacokinetic profile of S(-)-hydroxyhexamide (S-HH), a pharmacologically active metabolite of acetohexamide, was examined in male and female rats. S-HH was eliminated more rapidly from plasma in the males than in the females. A significant sex difference was observed in the pharmacokinetic parameters of S-HH in rats. Testectomy caused significant alteration in these parameters of S-HH in male rats, whereas ovariectomy did not in the females. The co-administration of sulfamethazine significantly decreased the plasma clearance (CL(p)) of S-HH in male rats, but had no effect in the females. The plasma concentrations of acetohexamide generated from S-HH showed no sex-related difference. Furthermore, there was no difference in the accumulation of S-HH by renal cortical slices from male and female rats. We propose the possibility that the sex-dependent pharmacokinetics of S-HH in rats is mediated through the male-specific hydroxylation of the cyclohexyl ring catalyzed by a major cytochrome p450 (CYP) isoform (CYP2C11), although the detailed mechanism remains to be elucidated.