High expression of KIBRA in low atypical protein kinase C-expressing gastric cancer correlates with lymphatic invasion and poor prognosis.

Overexpression of atypical protein kinase Cλ/ι (aPKCλ/ι), a regulator of cell polarity, is frequently associated with the poor prognoses of several cancers, including gastric cancer. Recent studies revealed a molecular link between aPKC and KIBRA, an upstream regulator of tumor suppressor Hippo pathway that regulates cell proliferation and apoptosis. Further, KIBRA directly inhibits the kinase activity of aPKC to regulate epithelial cell polarity. These observations suggest that the KIBRA-aPKC connection plays a role in cancer progression; however, clinical significance of the correlation between these factors remains unclear. Here we examined the correlation between KIBRA/aPKCλ/ι expression, as detected by immunohistochemistry, and clinicopathological outcomes in 164 gastric cancer patients using Fisher's exact test and Kaplan-Meier log-rank test. We found an intimate correlation between the expression level of KIBRA and aPKCλ/ι (P = 0.012). Furthermore, high expression of KIBRA is correlated with lymphatic (P = 0.046) and venous invasion (P = 0.039). The expression level of KIBRA by itself did not correlate with the prognosis; however, high expression of KIBRA in low aPKCλ/ι-expressing gastric cancer correlated with disease-specific (P = 0.037) and relapse-free survival (P = 0.041) by Kaplan-Meier with log-rank test and higher lymphatic invasion cases by Fisher's exact test (P = 0.042). Furthermore, overexpression of the aPKC-binding region of KIBRA disrupted tight junctions in epithelial cells. These results suggest that high expression of KIBRA in low aPKC-expressing cells causes massive loss of aPKC activity, leading to loss of polarity and invasiveness of gastric cancer cells.

The 8th and 9th tandem spectrin-like repeats of utrophin cooperatively form a functional unit to interact with polarity-regulating kinase PAR-1b.

Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor, dystroglycan (DG). The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal actin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polarity-regulating kinase, PAR-1b, interacts with the utrophin-DG complex, and positively regulates the interaction between utrophin and DG. In this study, we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b. Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin-DG complex. Interestingly, PAR-1b also binds and phosphorylates R8-9 of dystrophin, and colocalizes with dystrophin at the skeletal muscle membrane. These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain.

[Inactivation of Cryptosporidium parvum oocysts in copper tubing].

We studied whether the infectivity of Cryptosporidium parvum oocysts for suckling mice could be inactivated by copper tubing or by other types of tubing used to construct water distribution systems, including stainless steel, rigid polyvinyl chloride (PVC), PVC-lined steel, polyethylene (PE), cross-linked PE, and polybutene (PB), using glass tubing as the control. Oocysts were incubated in each tubings for 24 hours. The extent of inactivation of infectious oocysts by copper tubing was -1.303 log, which significantly inactivated of infectivity. In contrast, other types of tubing had no significant effect on some oocyst infectivity, although PB did show a maximum inactivation of -0.313 log. 25% of oocysts showed degeneration morphologically after passing through copper tubing, while 0.3% to 1.8% showed degeneration after passing through other tubing. Significant inactivation of infectious oocysts was not caused by water in which copper tubing had been let stand for 24 hours, although it had a cupric ion (Cu2+) concentration of 2.4 mg/L. The direct contact of oocysts with copper surface resulted in a decrease in the recovery percentage of oocysts and generation of hydrogen peroxide (0.5 mg/L) after 24 h of incubation. The percentage of degenerating oocysts was 29%. Such cryptosporidicidal effects of the copper surface on oocysts were completely inhibited by overlaying the surface with a Millipore filter before adding oocysts and incubating oocysts in the presence of catalase, an antioxidant enzyme. These findings suggest that copper tubing inactivates infectious C. parvum oocysts cytotoxically which may be due to oxygen radicals generated by the interaction between Cu2+ and hydrogen peroxide on the tubing surface.

[Antimicrobial ceramic for killing Legionella pneumophila in hot spring waters].

Killing of Legionella pneumophila by an antimicrobial ceramic was evaluated during culture in nine kinds of hot spring water at 40 degrees C. After 24 hours, the efficacy against L. pneumophila varied, depended on water quality. The strongest antibacterial effect was seen in chloride hot spring water from Wakayama and in deionized water. In four hot spring water samples (sulfur and hydrogen carbonate springs from Fukushima, simple thermals from Mie, and radioactive spring from Tottori), the decrease was < -2 log cfu after 48 hours. These results suggest that the antimicrobial ceramic is able to eradicate Legionella from hot spring waters.

[Physicochemical factors influencing distribution of Legionella species in Japanese hot springs].

We examined the relationship between the distribution of Legionella bacteria and various physicochemical characteristics of hot springs in Japan. Legionella bacteria were isolated from 52 (49.5%) out of 105 water samples, particularly from outdoor hot springs (67.3%). The bacterial count in the water samples positive for Legionella (86.5%) ranged from 10(1) to < 10(3) cfu/100 mL. L. pneumophila serogroup (SG) 4 (27.8%) was predominant in the water samples, followed by SG 5 (12.2%). The pulsefield gel electrophoresis (PFGE) patterns of chromosomal DNA for L. pneumophila SG 4 isolated from different parts of a hot spring resort were identical. Isolation of Legionella species from hot spring waters did not occur at pH 1.8-3.3, SO4(2-): > 780 mg/L, and H2SiO3: > 146 mg/L. The hot water-recirculating systems were applied to 18 out of 20 (90%) hot spring facilities which were found positive for Legionella. These results indicate that Legionella species are widespread in hot springs throughout Japan, except for waters with a low pH and non-recirculating waters, and that a single strain of L. pneumophila SG 4 is predominant in a particular hot spring resort.

[Disinfection of water of remove Legionella species: evaluation of an antimicrobial ceramic].

To evaluate the efficacy of an antimicrobial ceramic for killing Legionella strains in vitro, bacteria were exposed to the ceramic soaked in PBS at 25 degrees C or 42 degrees C. The number of L. pneumophila began to decrease significantly after 4 h of exposure at 25 degrees C and reached < 10 log cfu/ml after 12 h. A similar significant decrease was also observed after exposure at 42 degrees C. Furthermore, it was found that the antimicrobial ceramic showed bactericidal activity against six strains of Legionella isolated from various water sources, including L. pneumophila (serotype 1-4), L. micdadei, and L. dumoffii, after 24 h of exposure. The antimicrobial activity against L. pneumophila of the supernatant obtained by soaking the ceramic in PBS for 24 h was also assessed. Bactericidal activity of this supernatant was also noted. Analysis of the supernatant by ICP-MS resulted in the detection of eight metals (Mg, Al, Ca, Mn, Zn, Sr, Ag, and Ba) at a maximum concentration of 2.5 mg/l. When reconstituted PBS was made with all eight metals at the same concentrations as in the supernatant, the reconstituted PBS containing Ag alone and all metals showed significantly bactericidal activity against L. pneumophila, but PBS with only one metal component except Ag or a combination of Ag with Zn and/or Ca did not. These findings suggest that the antimicrobial ceramic possesses strong bactericidal activity against Legionella species and that eight metals released from the ceramic have a synergistic bactericidal effect against Legionella. When the antimicrobial ceramic was placed in hot spring water or cooling tower water instead of PBS, the number of L. pneumophila in the water decreased to < 10 log cfu/ml after 24 h of exposure and the bactericidal activity persisted for 5 weeks. These results indicate that the antimicrobial ceramic can be used to eradicate Legionella species contaminating various water sources.

Pretreatment of Pseudomonas aeruginosa with a sub-MIC of imipenem enhances bactericidal activity of neutrophils.

The influence of the pretreatment of Pseudomonas aeruginosa strain O1 (PAO-1) with a sub-minimum inhibitory concentration (MIC) of imipenem on bactericidal activity, phagocytosis, the production of oxygen radical intermediates, and the induction of apoptosis in murine peritoneal neutrophils, as well as the catalase activity in the bacteria in comparison with that of ceftazidime-treated bacteria were studied. Bacteria treated with imipenem at (1/4) MIC were killed at significantly higher rates by neutrophils than ceftazidime-treated and nontreated bacteria. However, antibiotic-treated bacteria showed similar numbers of bacteria-phagocytized neutrophils to those in untreated bacteria. Imipenem pretreatment of bacteria led to an increase in the production of oxygen radical intermediates by neutrophils and the inhibition of neutrophilic apoptosis following incubation, whereas these features did not occur in neutrophils incubated with nontreated and ceftazidime-treated bacteria. The catalase activity of bacteria was not suppressed by pretreatment with either antibiotic at (1/4) MIC. These findings suggest that the exposure of P. aeruginosa to a sub-MIC of imipenem enhances the susceptibility of the bacteria to neutrophilic killing and effectively modifies the physiological activities of neutrophils, but does not decrease bacterial catalase activity. These actions may account for the postantibiotic leukocyte enhancement (PALE) effect of a sub-MIC of imipenem in the host.

Apoptosis of intestinal crypt epithelium after Cryptosporidium parvum infection.

Using a neonatal mouse model of Cryptosporidium parvum infection, we investigated whether apoptosis of epithelial cells was induced in the small intestine. At the time when the number of C. parvum oocysts in the ileum was maximal, columnar goblet cells and absorptive cells showed a decrease in the ileal epithelium that was accompanied by a significant reduction in the height of the villi. A few apoptotic epithelial cells were also observed in the vicinity of the basal crypts where C. parvum was proliferating. Morphological changes of the villous structure and apoptotic epithelial cells associated with proliferation of the parasite were scarcely detected in the duodenum, cecum, and colon of the infected mice. These findings suggest that the loss of absorptive cells and goblet cells, and the apoptosis of intestinal epithelial cells, are common events in the ileum after C. parvum infection, and that epithelial apoptosis may have a significant role in the pathogenesis of cryptosporidiosis.

[Effect of the mixed-oxidant solution on infectivity of Cryptosporidium parvum oocysts in a neonatal mouse model].

Cryptosporidium parvum oocysts were exposed to the mixed-oxidant solution, which was electrochemically generated by Miox Water Disinfection Unit, and sodium hypochlorite in phosphate buffered saline (PBS, pH 7.2) or biologically treated wastewater at 25 degrees by using concentrations of residual chlorine of up to 5 mg/l and contact times of up to 8 h. The effect of two disinfectants on infectivity of the oocysts in a neonatal murine model was comparatively evaluated by determining the total number of oocysts recovered from the intestine. Exposure to the mixed-oxidant solution at 2 and 5 mg/l (residual chlorine) yielded a significant inactivation of infectivity in the dose- and exposure time-dependent manner, while exposure to 5 mg/l (residual chlorine) of sodium hypochlorite for contact times of up to 4 h produced no measurable inactivation of infectivity. Morphological examination also revealed a picture of degenerating oocysts after exposure to 5 mg/l (residual chlorine) of the mixed-oxidant solution, but not with sodium hypochlorite. When the oocysts were exposed to either biologically treated wastewater--or PBS-diluted the mixed-oxidant solution at 5 mg/l (residual chlorine) for 4 h, the disinfectants produced a significant inactivation of infectious oocysts. The decrease number of the oocysts was 0.8 log 10 in the former and 2.1 log 10 in the latter. These results demonstrate that the mixed-oxidant solution may be a useful disinfectant against Cryptosporidium oocysts, but appropriate applications need to be validated.