The type III secreted protein BopD in Bordetella bronchiseptica is complexed with BopB for pore formation on the host plasma membrane

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although BopB, BopN, BopD, and Bsp22 have been identified as type III secreted proteins, these proteins remain to be characterized. In this study, in order to clarify the function of BopD during Bordetella infection, a BopD mutant was generated. Although secretion of BopD into the culture supernatant was completely abolished by the bopD mutation, the secretion of other type III secreted proteins was not affected by this mutation. It has been reported that severe cytotoxicity, including cell detachment from the substrata, and release of lactate dehydrogenase (LDH) into the supernatant are induced in L2 cells by wild-type B. bronchiseptica infection, and these phenotypes are dependent on the type III secretion system. In contrast, neither cell detachment nor LDH release was induced in L2 cells infected with the BopD mutant. Furthermore, the hemolytic activity of the BopD mutant was greatly impaired compared with that of the wild-type strain. On the basis of the results of coimmunoprecipitation assays with anti-BopB antibodies, we conclude that BopD has the ability to associate with BopB. Finally, we show that the BopD-BopB complex is responsible for the pore formation in the host plasma membrane that functions as the conduit for the transition of effector proteins into host cells.     

Expression and characterization of Saccharomyces cerevisiae Cne1p, a calnexin homologue

The calnexin homologue (Cne1p) of Saccharomyces cerevisiae was expressed in Escherichia coli to evaluate its chaperone function. The chaperone function was examined as to the effects on the suppression of thermal denaturation and the enhancement of refolding, using citrate synthase (CS) as a nonspecific chaperone substrate. Cne1p effectively suppressed the thermal denaturation of CS and enhanced the refolding of thermally or chemically denatured CS in a concentration-dependent manner. In addition, the chaperone function of Cne1p was greatly affected in the presence of monoglucosylated oligosaccharides (G1M9) that specifically bind to the lectin site. These results indicated that Cne1p functions as a molecular chaperone in Saccharomyces cerevisiae.     

Enhancement of serum- and platelet-derived growth factor-induced cell proliferation by Necl-5/Tage4/poliovirus receptor/CD155 through the Ras-Raf-MEK-ERK signaling

Necl-5/Tage4/poliovirus receptor/CD155 has been shown to be the poliovirus receptor and to be up-regulated in rodent and human carcinoma. We have found previously that mouse Necl-5 regulates cell motility. We show here that mouse Necl-5 is furthermore involved in the regulation of cell proliferation. Studies using a specific antibody against Necl-5 and a dominant negative mutant of Necl-5 revealed that Necl-5 enhanced the serum-induced proliferation of NIH3T3, Swiss3T3, and mouse embryonic fibroblast cells. Necl-5 enhanced the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up-regulated cyclins D2 and E, and down-regulated p27(Kip1), eventually shortening the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 similarly enhanced the platelet-derived growth factor-induced activation of the Ras-Raf-MEK-ERK signaling and shortened the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 acted downstream of the platelet-derived growth factor receptor and upstream of Ras. Moreover, up-regulated Necl-5 was involved at least partly in the enhanced proliferation of transformed cells including NIH3T3 cells transformed by an oncogenic Ras or v-Src. These results indicate that Necl-5 plays roles not only in cell motility but also in cell proliferation.     

Association of the gene encoding wingless-type mammary tumor virus integration-site family member 5B (WNT5B) with type 2 diabetes

Recent reports have suggested that WNT signaling is an important regulator for adipogenesis or insulin secretion and might be involved in the pathogenesis of type 2 diabetes. To investigate possible roles of the WNT genes in conferring susceptibility to type 2 diabetes, we examined the association of the genes that encode members of the WNT family with type 2 diabetes in the Japanese population. First, 40 single-nucleotide polymorphism (SNP) loci within 11 WNT genes were analyzed in 188 subjects with type 2 diabetes (case-1) and 564 controls (control-1). Among them, six SNP loci exhibited a significant difference (P<.05) in the allele and/or genotype distributions between case and control subjects. These SNP loci were further analyzed in another set of case (case-2; n=733) and control (control-2; n=375) subjects to confirm their statistical significance. As a result, one SNP locus in the WNT5B gene was strongly associated with type 2 diabetes ( chi 2=15.6; P=.00008; odds ratio=1.74; 95% confidence interval 1.32-2.29). Expression of the WNT5B gene was detectable in several tissues, including adipose, pancreas, and liver. Subsequent in vitro experiments identified the fact that expression of the Wnt5b gene was increased at an early phase of adipocyte differentiation in mouse 3T3-L1 cells. Furthermore, overexpression of the Wnt5b gene in preadipocytes resulted in the promotion of adipogenesis and the enhancement of adipocytokine-gene expression. These results indicate that the WNT5B gene may contribute to conferring susceptibility to type 2 diabetes and may be involved in the pathogenesis of this disease through the regulation of adipocyte function.     

Simultaneous fluorometric measurement of histamine and tele-methylhistamine levels in rodent brain by high-performance liquid chromatography

An improved high-performance liquid chromatography (HPLC) method was developed for simultaneous analysis of histamine (HA) and tele-methylhistamine (tele-MHA) levels in mouse and rat brain. The method consists of a solid-phase extraction (SPE) and subsequent HPLC with postcolumn derivatization of the amines with o-phthalaldehyde. The recovery rates of HA and tele-MHA during the SPE procedure were 82.8+/-3.4 and 86.0+/-1.7%, respectively. The detection limits for HA and tele-MHA were 8 and 12pg, respectively, with sufficient linearity up to 30pg. Using this newly developed system, we observed that the brain tele-MHA levels in H3 receptor knockout mice were significantly higher than those of wild-type mice by 2.1-fold. Furthermore, we also observed that the brain HA and tele-MHA levels in Zucker rats were significantly lower than those of lean rats by 76.6+/-5.3 and 77.8+/-5.0%, respectively. These observations coincided well with those of previous studies using radioimmunoassay or HPLC with precolumn OPA derivatization, confirming the utilization of the assay system.     

Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.     

Isolation and Characterization of Novel Strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Serratia marcescens</Emphasis> Possessing High Efficiency to Degrade Gasoline,Kerosene, Diesel Oil,and Lubricating Oil

Bacteria possessing high capacity to degrade gasoline, kerosene, diesel oil, and lubricating oil were screened from several areas of Hokkaido, Japan. Among isolates, two strains, WatG and HokM, which were identified as new strains of Pseudomonas aeruginosa and Serratia marcescens species, respectively, showed relatively high capacity and wide spectrum to degrade the hydrocarbons in gasoline, kerosene, diesel, and lubricating oil. About 90-95% of excess amount of total diesel oil and kerosene added to mineral salts media as a sole carbon source could be degraded by WatG within 2 and 3 weeks, respectively. The same amount of lubricating oil was 60% degraded within 2 weeks. Strain HokM was more capable than WatG in degrading aromatic compounds in gasoline. This strain could also degrade kerosene, diesel, and lubricating oil with a capacity of 50-60%. Thus, these two isolates have potential to be useful for bioremediation of sites highly contaminated with petroleum hydrocarbons.