Comparison of species, virulence genes and clones of Aeromonas isolates from clinical specimens and well water in Okinawa Prefecture, Japan.

AIMS: To reveal the sources of Aeromonas infection in Okinawa Prefecture of Japan, the species, virulence genes and clones of strains isolated from clinical specimens and well water were compared. METHODS AND RESULTS: The properties of both isolates were investigated by sequencing of rpoD, detection of 10 virulence genes using PCR and genotyping with pulsed-field gel electrophoresis. In all, 68 clinical and 146 well water strains of Aeromonas were isolated and the main species were A. caviae, A. dhakensis, A. hydrophila and A. veronii biovar sobria. Aeromonas dhakensis possessed various virulence genes; however, A. caviae possessed only fla. The same or similar clones were distributed in certain areas of Okinawa and one clone had survived several months in the biliary system of two patients, respectively. CONCLUSION: Although the same Aeromonas clone was not isolated from clinical and well water samples, our study revealed the detected patterns of virulence genes in both isolates, the distribution of identical/similar clones in the Okinawan environment and long-time survival in patient's organs. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated the association between Aeromonas patients and well water exposure. This study provides the properties of species, virulence genes and clones of Aeromonas isolated from samples of these origins.

Approximate Bayesian computation analysis of EST-associated microsatellites indicates that the broadleaved evergreen tree Castanopsis sieboldii survived the Last Glacial Maximum in multiple refugia in Japan.

Climatic changes have played major roles in plants' evolutionary history. Glacial oscillations have been particularly important, but some of their effects on plants' populations are poorly understood, including the numbers and locations of refugia in Asian warm temperate zones. In the present study, we investigated the demographic history of the broadleaved evergreen tree species Castanopsis sieboldii (Fagaceae) during the last glacial period in Japan. We used approximate Bayesian computation (ABC) for model comparison and parameter estimation for the demographic modeling using 27 EST-associated microsatellites. We also performed the species distribution modeling (SDM). The results strongly support a demographic scenario that the Ryukyu Islands and the western parts in the main islands (Kyushu and western Shikoku) were derived from separate refugia and the eastern parts in the main islands and the Japan Sea groups were diverged from the western parts prior to the coldest stage of the Last Glacial Maximum (LGM). Our data indicate that multiple refugia survived at least one in the Ryukyu Islands, and the other three regions of the western and eastern parts and around the Japan Sea of the main islands of Japan during the LGM. The SDM analysis also suggests the potential habitats under LGM climate conditions were mainly located along the Pacific Ocean side of the coastal region. Our ABC-based study helps efforts resolve the demographic history of a dominant species in warm temperate broadleaved forests during and after the last glacial period, which provides a basic model for future phylogeographical studies using this approach.

Population structure and historical demography of Dipteronia dyeriana (Sapindaceae), an extremely narrow palaeoendemic plant from China: implications for conservation in a biodiversity hot spot.

Inferring past demography is a central question in evolutionary and conservation biology. It is, however, sometimes challenging to disentangle their roles of contemporary versus historical processes in shaping the current patterns of genetic variation in endangered species. In this study, we used both chloroplast microsatellite (cpSSR) loci and nuclear microsatellite (nSSR) loci to assess the levels of genetic differentiation, genetic effective population size, contemporary/historical levels of gene flow and demographic history for five populations sampled across the range of Dipteronia dyeriana, an endangered palaeoendemism from Southwestern China. We found that D. dyeriana had a mixed pattern of moderate genetic diversity and high inbreeding. Bayesian clustering divided D. dyeriana populations into two nSSR genetic clusters. Coalescent-based approximate Bayesian computation analyses suggest the western and eastern groups of D. dyeriana likely persisted in a long-term refuge in Southern China since the beginning of the last glacial period, whereas increasingly colder and arid climates at the onset of the last glacial maximum might have fostered the fragmentation of D. dyeriana within refugia. Following their divergence, the western group kept relatively stable effective population size, whereas the eastern group had experienced 500-fold population expansion during the Holocene. Although clear loss of genetic diversity by human activities was not suggested, recent habitat fragmentation has led to a reduction of population connectivity and increased genetic differentiation by ongoing genetic drift in isolated populations, possibly owing to decreased population size in recent dozen years. Finally, we discussed the implications of these results on conservation policies.

Impact of Subnormothermic Machine Perfusion Preservation in Severely Steatotic Rat Livers: A Detailed Assessment in an Isolated Setting.

The current drastic shortage of donor organs has led to acceptance of extended-criteria donors for transplantation, despite higher risk of primary nonfunction. Here, we report the impact of subnormothermic machine perfusion (SMP) preservation on the protection of >50% macrosteatotic livers. Dietary hepatic steatosis was induced in Wistar rats via 2-day fasting and subsequent 3-day re-feeding with a fat-free, carbohydrate-rich diet. This protocol induces 50-60% macrovesicular steatosis, which should be discarded when preserved via cold storage (CS). The fatty livers were retrieved and preserved for 4 h using either CS in histidine-tryptophan-ketoglutarate or SMP in polysol solution. Graft functional integrity was evaluated via oxygenated ex vivo reperfusion for 2 h at 37°C. SMP resulted in significant reductions in not only parenchymal alanine aminotransferase (p < 0.001), but also mitochondrial glutamate dehydrogenase (p < 0.001) enzyme release. Moreover, portal venous pressure (p = 0.047), tissue adenosine triphosphate (p = 0.001), bile production (p < 0.001), high-mobility group box protein-1 (p < 0.001), lipid peroxidation, and tissue glutathione were all significantly improved by SMP. Electron microscopy revealed that SMP alleviated deleterious alterations of sinusoidal microvasculature and hepatocellular mitochondria, both of which are characteristic disadvantages associated with steatosis. SMP could protect 50-60% macrosteatotic livers from preservation/reperfusion injury, and may thus represent a new means for expanding available donor pools.

Estimation of outcrossing rates at hierarchical levels of fruits, individuals, populations and species in Magnolia stellata.

In plant species with mixed mating systems, differences in diverse factors-including their pollination system, flowering phenology, life form and susceptibility to inbreeding depression-cause variation in outcrossing rates among fruits within individuals, among individuals within populations and among populations within species. To quantify this hierarchical variation, we examined outcrossing rates at the seed stage in five populations of Magnolia stellata, a self-compatible, insect-pollinated and protogynous tree species. For this purpose, we sampled 1498 seeds within 204 fruits obtained from 56 individuals of the five populations, determined genotypes of the sampled seeds and maternal individuals at six polymorphic microsatellite loci, then estimated outcrossing rates and their variance components at four hierarchical levels (fruits, individuals, populations and species) using a nested analysis of variance-type linear model with a Bayesian approach. The species-level outcrossing rate was 0.730 (95% credible interval, 0.595-0.842), indicating that this species has a mixed mating system. Outcrossing rates were not significantly different among populations, but were significantly different among individuals within populations. Variance components at the levels of individual and fruit were statistically supported and were highest for the former. Thus, factors influencing outcrossing rates at the individual level, such as differences in flowering phenology and early-stage inbreeding depression, appear to have important effects within these M. stellata populations, but not among them. The method of hierarchically estimating outcrossing rates using a Bayesian approach, as applied in this study, is compared with conventional methods for estimating outcrossing rates, and the statistical properties of the Bayesian approach are discussed.

Genetic variation and differentiation in populations of a threatened tree, Magnolia stellata: factors influencing the level of within-population genetic variation.

Genetic variation and differentiation in Magnolia stellata were studied in 20 populations distributed across most of the species' range using 10 microsatellite markers, and the factors influencing their levels of within-population genetic variation were examined. Generally, populations distributed intermittently from southern Gifu to central Aichi Prefectures showed substantially higher levels of genetic variation (exceptions included populations located at unusually high altitude sites or western and southern edges of the range) than more isolated populations on the Atsumi Peninsula of southern Aichi Prefecture and in northern Mie Prefecture. Significant isolation-by-distance patterns were detected in genetic differentiation among the studied populations, and a neighbor-joining tree based on D(A) distances among the populations reflected well the geographical positions of the populations. The level of within-population genetic variation was significantly influenced not only by the size of the populations (represented by the number of reproductive individuals) but also by their degree of isolation (represented by the number of populations within a radius of 0.5 km around them). Therefore, areas within radii of 0.5 km may encompass M. stellata metapopulations, in which gene flow may usually occur. We suggest that this area may be a suitable standard for constructing conservation units for the species.

[Activities of antimicrobial agents against 5,180 clinical isolates obtained from 26 medical institutions during 1998 in Japan. Levofloxacin--Surveillance Group].

The surveillance study was conducted to determine the antimicrobial activity of fluoroquinolones (ofloxacin, levofloxacin, ciprofloxacin, tosufloxacin) and other 20 antimicrobial agents against 5,180 clinical isolates obtained from 26 medical institutions during 1998 in Japan. The resistance to fluoroquinolones was remarkable in Enterococci, methicillin-resistant staphylococci and Pseudomonas aeruginosa from UTI. However, many of the common pathogens such as Streptococcus pneumoniae including penicillin-resistant isolates, methicillin-susceptible Stahylococcus aureus, Moraxella catarrhalis, the family of Enterobacteriaceae, Haemophilus influenzae including ampicillin-resistant isolates have been kept to be susceptible to fluoroquinolones. About 90% of P. aeruginosa isolates from RTI were susceptible to fluoroquinolones. In conclusion, the results from this surveillance study suggest that fluoroquinolones are useful in the treatment of various bacterial infections including respiratory infections.

Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.

Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling.

Involvement of protein-tyrosine phosphorylation and dephosphorylation in sperm-induced Xenopus egg activation.

We have analyzed tyrosine-phosphorylated proteins in Xenopus laevis eggs before and after fertilization by immunoblotting with anti-phosphotyrosine antibody. A number of egg proteins with different subcellular distribution became tyrosine-phosphorylated or dephosphorylated within 30 min after insemination. Tyrosine kinase-specific inhibitors genistein and herbimycin A were found to inhibit sperm-induced egg activation judged by the egg cortical contraction. Surprisingly, sodium orthovanadate, a tyrosine phosphatase inhibitor, also inhibited the egg activation. Moreover, we found that fertilization-dependent tyrosine dephosphorylation of 42-kDa mitogen-activated protein kinase was inhibited in genistein-treated eggs. These results suggest that both protein-tyrosine phosphorylation and dephosphorylation pathways play an important role in the sperm-induced Xenopus egg activation.