RhoGDIβ affects HeLa cell spindle orientation following UVC irradiation

The molecular signals that regulate mitotic spindle orientation to determine the proper division axis play a critical role in the development and maintenance of tissue homeostasis. However, deregulation of signaling events can result in spindle misorientation, which in turn can trigger developmental defects and cancer progression. Little is known about the cellular signaling pathway involved in the misorientation of proliferating cells that evade apoptosis after DNA damage. In this study, we found that perturbations to spindle orientation were induced in ultraviolet C (UVC)-irradiated surviving cells. N-terminal truncated Rho GDP-dissociation inhibitor β (RhoGDIβ), which is produced by UVC irradiation, distorted the spindle orientation of HeLa cells cultured on Matrigel. The short hairpin RNA-mediated knockdown of RhoGDIβ significantly attenuated UVC-induced misorientation. Subsequent expression of wild-type RhoGDIβ, but not a noncleavable mutant, RhoGDIβ (D19A), again led to a relative increase in spindle misorientation in response to UVC. Our findings revealed that RhoGDIβ impacts spindle orientation in response to DNA damage.     

Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

Inhibitory action of gemfibrozil on cholesterol absorption in rat intestine

This study was designed to determine whether gemfibrozil inhibits intestinal lipid absorption. Male Sprague-Dawley rats received an oral dose of 30 mg gemfibrozil/kg body weight for 14 days. Mesenteric lymph cannulation was performed, and a lipid infusion containing 40 micromol/h (35.4 mg/h) of radiolabeled triolein and 2.74 micromol/h (1.06 mg/h) of radiolabeled cholesterol with the addition of 1 mg/h of gemfibrozil was infused intraduodenally at a rate of 3 ml/h for 8 h. The lymph was collected, and the radioactivity levels of the lumen and gut mucosa were measured after the infusion. Lymph cholesterol transport was depressed in gemfibrozil-treated rats, in terms of mass measurements as well as radioactivity in a lesser degree. More radioactive cholesterol remained in the proximal portion of the intestinal lumen and mucosa in the treated rats than in the control rats. More radioactive triglycerides also remained in the proximal intestinal lumen of treated rats, although no difference in lymphatic triglyceride transport was observed between the groups. A significant portion of the radioactive cholesterol remained in the lumen in the gemfibrozil-treated rats. Gemfibrozil increased biliary cholesterol excretion. Thus, this study shows that gemfibrozil inhibits cholesterol absorption in rat intestine.     

Protective effect of NK1.1(+) T cells as well as NK cells against intraperitoneal tumors in mice

Peritoneal resident cells of mice normally contain small populations of NK cells and NK1.1(+) alphabetaT cells. These populations increased after either 3LL or EL4 tumor inoculations into the peritoneal cavity. In vivo depletion of NK cell alone by anti-asialo GM1 (ASGM1) Ab significantly decreased survival time of tumor-injected mice, while depletion of both NK cells and NK1.1(+) T cells by anti-NK 1.1 Ab greatly shortened mouse survival time. NK1. 1(+) T cells in peritoneal cavity consist of a larger proportion of double-negative T cells and smaller populations of CD4(+) T cells and Vbeta8(+) T cells compared with liver NK1.1(+) T cells and normally lack Vbeta2(+) T cells. Tumor inoculation induced rapid IL-12 and IFN-gamma mRNA in tumor-infiltrating mononuclear cells (TIM). Although anti-NK1 Ab pretreatment in vivo abrogated IFN-gamma mRNA expression and IFN-gamma production of TIM, NK cell depletion alone by anti-ASGM1 Ab pretreatment retained IFN-gamma mRNA expression and partly inhibited IFN-gamma production of TIM. Peritoneal NK cells as well as NK1.1(+) T cells but not NK1.1(-) T cells of 3LL cell- or EL4 cell-injected mice showed cytotoxicities against the same tumor cells. Further, either anti-IL-12 Ab or anti-IFN-gamma Ab ip injection significantly shortened EL4 cell-inoculated mouse survival time. Our findings suggest that peritoneal macrophages activated by tumors produce IL-12 which activates NK cells and NK1.1(+) T cells to produce IFN-gamma and both NK cells and NK1.1(+) T cells are important in suppressing the growth of the intraperitoneal tumors.     

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions of connectin and was compared to the incorporation of -actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with -actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.     

Thermo-labile stability of sigmaH (Spo0H) in temperature-sensitive spo0H mutants of Bacillus subtilis can be suppressed by mutations in RNA polymerase beta subunit

We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5, having E61K and G30E amino-acid substitutions within the sigmaH protein, respectively, and located in the highly conserved region, "2", among prokaryotic sigma factors that participates in binding to core enzyme of RNA polymerase. These mutants showed a sporulation-deficient phenotype at 43 degrees C. Moreover, we successfully isolated suppressor mutants that were spontaneously generated from the spo0H mutants. Our genetic analysis of these suppressor mutations revealed that the suppressor mutations are within the rpoB gene coding for the beta subunit of RNA polymerase. The mutations caused single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas the sigmaH-dependent expression of a spo0A-bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the suppressor mutants at 43 degrees C. Western blot analysis showed that the level of sigmaH protein in the wild type increased between T0 and T2 and decreased after T3, while the level of sigmaH protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant sigmaH proteins were rapidly degraded by some unknown proteolytic enzyme(s). The analysis of the half-life of sigmaH protein showed that the short life of sigmaH in spo0H mutants is prolonged in the suppressor mutants. These findings suggest that, at least to some extent, the process of E-sigmaH formation may be involved in stabilization of sigmaH at the onset of sporulation.     

Helicobacter pylori in Japanese river water and its prevalence in Japanese children

AIMS: The major transmission route of Helicobacter pylori remains unclear. In this study, we examined H. pylori in the environmental waters in Japan. METHODS AND RESULTS: A total of 24 water samples were collected from the upper, middle and downstream reaches of four Japanese rivers. Helicobacter pylori-specific DNA was examined using nested PCR. In addition, 224 children who lived near one river were studied by the stool antigen test for H. pylori prevalence. Helicobacter pylori DNA was detected in the water from the middle and downstream reaches of all four rivers, but not in the upper reaches. Helicobacter pylori was not found in cultured water samples with positive PCR results. Helicobacter pylori prevalence in the children examined was 9.8% for those living near the middle reaches and 23.8% nearby downstream, both of which were higher than the value in an area distant from the river (0%) (both, P < 0.01). CONCLUSIONS: Difference in H. pylori prevalence in the children may be related to the presence of H. pylori in the river. The results of this study showed that H. pylori DNA is frequently present in river water from the middle and downstream reaches in which the human biosphere is embedded. SIGNIFICANCE AND IMPACT OF THE STUDY: It is suggested that river water in the natural environment could be a risk factor for H. pylori transmission.     

Spontaneous transformation and its use for genetic mapping in Bacillus subtilis

Using a simple semi-synthetic competence and sporulation medium (CSM), we found evidence that Bacillus subtilis cells transformed in the competence phase can sporulate, indicating that genetic information acquired during the competence phase is inherited by the next generation after germination of the transformed spores. Moreover, the results from mixed cell culture experiments suggest that spontaneous genetic transformation can occur between competent cells and DNA released from lysed cells in the natural environment. We also found evidence that the spontaneous transformation system can be used for genetic mapping in B. subtilis.