Pseudomonas aeruginosa is an opportunistic pathogen responsible for morbidity and mortality in humans, animals, and plants. This bacterium has been regarded to be widely present in terrestrial and freshwater environments, but not in open ocean environments. Our purpose was to clarify its presence in open ocean, and their genotypic and physiological characteristics were compared with those of isolates from clinical, animal, and freshwater sources. Water samples were collected from freshwater, bays, and offshore environments in Japan. Sixty-two isolates, including 26 from the open ocean, were identified as P. aeruginosa by phenotypic characteristics and the BD Phoenix System. Pulsed-field gel electrophoresis (PFGE) was performed on all strains, together with 21 clinical and 8 animal strains. The results showed that open ocean strains are composed of a few genotypes, which are separated from other strains. Although some clinical isolates made a cluster, other strains tended to mix together. Different antibiotypes were observed among marine isolates that had similar PFGE and serotyping patterns. Some were multidrug-resistant. Laboratory-based microcosm study were carried out to see the responses of P. aeruginosa toward increased NaCl concentrations in deionized water (DW). Marine strains showed better survival with the increase, whereas river and clinical strains were suppressed by the increase. These findings illustrate the potential significance of open ocean as a possible reservoir of P. aeruginosa, and there may be clones unique to this environment. To our knowledge, this is the first report on the presence and characterization of P. aeruginosa in the open ocean.

Syndecan-4 is a transmembrane heparan sulfate proteoglycan belonging to the syndecan family. Following intraperitoneal injection of lipopolysaccharide (LPS), syndecan-4-deficient mice exhibited high mortality compared with wild-type controls. Severe endotoxin shock was observed in the deficient mice: systolic blood pressure and left ventricular fractional shortening were lower in the deficient mice than in the wild-type controls 9 h after LPS injection. Although histological examinations revealed no apparent differences between two groups, the plasma level of interleukin (IL)-1␤ was higher in the deficient mice than in the wild-type controls 9 h after LPS injection. Consistent with the regulatory roles of syndecan-4, its expression in monocytes and endothelial cells of microvasculature increased in the wild-type mice after LPS administration. Although IL-1␤ was produced to the same extent by macrophages from syndecan-4-deficient and wild-type mice after LPS stimulation, inhibition of its production by transforming growth factor-␤1 was impaired in the syndecan-4-deficient macrophages. These results indicate that syndecan-4 could be involved in prevention of endotoxin shock, at least partly through the inhibitory action of transforming growth factor-␤1 on IL-1␤ production.

Depth-related changes in whole-community structure were evaluated in a coastal marine sediment using a molecular fingerprinting method, terminal restriction fragment length polymorphism (T-RFLP) analysis, and a chemotaxonomic technique (quinone profiling). Dendrograms derived from both T-RFLP analysis and quinone profiling indicated a significant variation in microbial community structure between the 0-2 cm layer and deeper layers. This corresponded to the dramatic change in the redox potential, acid-volatile sulphide-sulphur and bacterial numbers observed at 0-2 cm and 2-4 cm depths. A significant change in the number of terminal restriction fragments (T-RFs) was also detected at this transition depth. However, the change in major T-RFs with depth was not seen in electropherograms. The population changes were primarily variations in minor ribotypes. Most quinone homologues were detected at all depths, although the quinone composition changed with depth. Therefore, quinone profiling also suggested that the depth-related variation was primarily attributable to minor bacterial groups rather than change in the major population structure. 16S rDNA clone library analysis revealed that clones belonging to the genera Vibrio and Serratia predominated as major bacterial groups at all depths. Our data suggested that the sediment community might result from sedimentation effects of sinking particles. Overall, our results demonstrated that the combined methods of T-RFLP analysis and quinone profiling were effective for assessing depth-related microbial populations.

No abstract

The gene for Menkes disease, an X-linked disorder of copper transport, has recently been identified and shown to encode a copper-transporting P-type ATPase. The macular mutant mouse has been proposed as an animal model for Menkes disease. In the present study, we report the finding of a missense mutation in the mottled gene of the macular mouse. A single base change, T to C, at nucleotide position 4223, is predicted to result in an amino acid change from serine to proline at residue 1382 in the eighth transmembrane domain. This mutation differs from the 6-bp deletion we find in brindled cDNA. With validation of macular as an animal model of Menkes disease, we compared mottled gene expression in the intestine, kidney, and brain of macular and normal mice. In Northern analyses an 8.3-kb transcript was detected in the intestine, kidney, and brain of both normal and macular mice, with the level of transcript in macular approximately 80% that of normal. In situ hybridization studies revealed that the mottled gene was clearly expressed in intestinal epithelial cells, Paneth cells, and renal proximal tubular cells of both normal and macular mice. In normal brain, mottled gene expression was most intensely observed in the choroid plexus, in Ammon's born and the dentate gyrus in the hippocampus, in Purkinje cells, and the granular layer of the cerebellum. The intensity and localization of the signals in the brain of macular mice were similar to those of the controls. The distribution of expression of mottled is correlated with cells and tissues showing histopathology or abnormal copper sequestration in macular and other mutants.

Although aging accompanies specific pathological changes, including thrombosis and organ sclerosis, the underlying mechanisms of these processes remain to be elucidated. In the present study, we analyzed the gene expression of plasminogen activator inhibitor-1 (PAI-1), a key molecule in the development of thrombosis, in a murine model of aging, klotho mutant ( kl/kl) mice. Active PAI-1 antigen in plasma and PAI-1 mRNA in several tissues were strikingly elevated in kl/kl mice as compared with wild-type mice. This increased PAI-1 expression was age dependent and linked to the development of ectopic calcification and glomerular fibrin deposition in the kidneys. In situ hybridization analysis of kl/kl mice demonstrated that strong signals for PAI-1 mRNA were localized in renal tubular epithelial cells, cardiomyocytes, adrenal medullar cells, and smooth muscle and endothelial cells in Mönckeberg's arteriosclerotic vessels. Renal glomerular fibrin deposition, as evaluated immunohistochemically, was occasionally observed only in kl/kl mice, and the number of fibrin-positive glomeruli increased as the kl/kl mice aged. These observations suggest that in the process of aging the PAI-1 gene expression is increased, contributing to the development of thrombosis.

No abstract

Non-obese diabetic mice aged 30 to 60 days were treated orally with Cyclosporin at doses of 25, 15 and 2.5 mg/kg every 2 days until 160 days of age. Diabetes developed in 12 out of 18 oil-treated mice (67%), with partial to complete Langerhans' islet destruction associated with lymphocytic infiltration. The non-obese diabetic mice showed a plasma glucose concentration of 6.62 +/- 0.92 mmol/l (mean +/- SD) at 50 days of age. The plasma glucose level of oil-treated non-obese diabetic mice gradually increased after 130 days of age and reached 14.0 to 19.0 mmol/l at 160 days of age, while Cyclosporin-treated non-obese diabetic mice showed neither clear increase of plasma glucose levels nor development of insulitis. The cumulative incidence of diabetes in Cyclosporin-treated mice was significantly lower than that in oil-treated mice (p less than 0.01). Subsequently, Cyclosporin treatment was started after development of glucose intolerance. Twenty-five mg/kg of Cyclosporin was administered every 2 days for 35 days. Cyclosporin appeared to have little therapeutic effect on diabetes in non-obese diabetic mice.