Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.     

Pleuromutilin derivatives having a purine ring. Part 1: new compounds with promising antibacterial activity against resistant Gram-positive pathogens

In the course of our research aimed at the discovery of metabolic stable pleuromutilin derivatives with more potent antibacterial activity against Gram-positive pathogens than previous analogues, a series of compounds bearing a purine ring were prepared and evaluated. From SAR studies, we identified two promising compounds 85 and 87, which have excellent in vitro activity against a number of Gram-positive pathogens, including existing drug-resistant strains, and potent in vivo efficacy.     

Osmolyte effect on the stability and folding of a hyperthermophilic protein

Proteins are known to be stabilized by naturally occurring osmolytes such as amino acids, sugars, and methylamines. Here, we examine the effect of trimethylamine-N-oxide (TMAO) on the conformational stability of ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII), which inherently possesses high conformational stability. Heat- and guanidine hydrochloride-induced unfolding experiments demonstrated that the conformational stability of Tk-RNase HII in the presence of 0.5M TMAO was higher than that in the absence of TMAO at all examined temperatures. TMAO affected the unfolding and refolding kinetics of Tk-RNase HII to a similar extent. These results indicate that proteins are universally stabilized by osmolytes, regardless of their robustness, and suggest a stabilization mechanism by osmolytes, caused by the unfavorable interaction of osmolytes with protein backbones in the denatured state. Our results also imply that the basic protein folding principle is not dependent on protein stability and evolution.     

Latitudinal compensation in female reproductive rate of a geographically widespread reef fish

Latitudinal variation in fitness-related traits has often been attributed to local adaptation to climates. In poikilotherms including fishes, lower temperatures and shorter reproductive seasons at high latitudes would be expected to cause a reduction in annual reproductive output of an individual. Theories of latitudinal compensation predict that organisms at high latitudes should evolve compensatory responses for these climatic effects. Therefore, latitudinal compensation in female reproductive rate (egg production rate), that individuals from high latitudes produce eggs at higher rates than those from lower latitudes, is likely to occur. I tested this hypothesis with a latitudinally widespread reef fish Pomacentrus coelestis that is a multiple batch spawner, from three different localities, from temperate to subtropical waters, within Japan. I used common-environment experiments at three different temperatures to compare reproductive capacity among local populations. In the experiments, average inter-spawning intervals were the shortest and average size-specific clutch weight was the heaviest in fish from the most northern locality across all temperatures, showing clear latitudinal clines. Thus, the northern fish can achieve higher reproductive output per unit time both by shortening inter-spawning intervals and increasing size-specific clutch weight. Additionally, faster egg production rate of the northern fish did not result from increased food consumption. This finding suggests that gross egg production efficiency was higher in the northern fish and that northern fish had a superior capacity for reproduction within a season. These results support the prediction that latitudinal compensation occurs in the female reproductive rate of P. coelestis. As the reproductive season of this species decreases drastically with increasing latitude, the observed cline in the reproductive rate must be an adaptive response to the local selective regime, i.e., length of the reproductive season. Such latitudinal compensation in female reproductive rates may be a common pattern in latitudinally widespread fishes.     

Phylogeography of Japanese water crowfoot based on chloroplast DNA haplotypes

Water crowfoot, Ranunculus subgenus Batrachium, are submerged macrophytes in the Ranunculaceae. We aimed to infer the Quaternary history of these aquatic macrophytes in the Japanese archipelago. We studied 212 individuals of three perennial and one annual species from 46 populations covering the entire geographic range and found eight haplotypes based on approximately 1800 bp of four spacers in chloroplast DNA. The relationships among haplotypes were resolved using maximum parsimony and parsimony network analyses. To identify the zones of clear genetic boundaries, Monmonier's algorithm was used. The eight haplotypes were distinguished from adjacent haplotypes by one substitution or indel. Each of the 46 populations was fixed for a single haplotype. Inconsistency between cpDNA haplotypes and Batrachium taxa was found, except in one annual species. The distribution of the haplotypes in perennial species was highly geographically structured. An abrupt genetic change was detected between the Tohoku region and more southerly regions. Since the perennial Batrachium are cold-adapted, this genetic differentiation may be due to historical changes in their distributions caused by Quaternary climatic oscillations: interglacial retreats in colder refugia and glacial range expansions. The single haplotype composition of each population may have been shaped by founder effects during colonization and/or by genetic drift. The interglacial refugial populations must have been small enough to deplete haplotype diversity. Inconsistency between cpDNA haplotypes and Batrachium taxa may be due to incomplete lineage sorting of ancestral polymorphic haplotypes.     

TRAF6 is a critical signal transducer in IL-33 signaling pathway

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.     

Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect

Introduction  

Current cell therapy for cartilage regeneration requires invasive procedures, periosteal coverage and scaffold use. We have developed a novel transplantation method with synovial mesenchymal stem cells (MSCs) to adhere to the cartilage defect.     

IRS1-independent defects define major nodes of insulin resistance

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.     

Differences in endemic insect assemblages among vegetation types on a small island of the oceanic Ogasawara Islands

Natural vegetation is often replaced by invasive alien plants on isolated oceanic islands. To determine how invasive alien plants affect insect diversity, we compared flying insects captured using Malaise traps among different vegetation types on a small island (Nishijima; 0.49 km²) in the oceanic Ogasawara (Bonin) Islands in the north‐western Pacific. The numbers of individuals and species, and the species composition of pollinators (bees), predators (wasps) and wood borers (cerambycid, mordellid and elaterid beetles) were compared among three vegetation types: Casuarina equisetifolia (an invasive alien tree) forest, natural forest and natural grassland (forest edge), during two seasons (June and October–November 2005). In traps, 80.0, 66.7, 87.5, 85.7 and 100.0% of bee, wasp, cerambycid, mordellid and elaterid beetle species, respectively, were endemic to the Ogasawara Islands. Grassland had the highest wasp and bee species richness, whereas natural forest had the highest species richness of wood‐boring beetles. The C. equisetifolia forest had the poorest species richness for most insect groups (except mordellid beetles). More individuals of most insect groups (except bees) were captured in June than in October–November. More individual bees and wasps were captured in grassland than in forests, whereas more individual mordellid and elaterid beetles were captured in forests than in grassland. The number of cerambycid individuals did not differ among vegetation types. Redundancy analysis suggested that most insect species preferred natural forest or grassland to alien forest. Therefore, further invasion of natural grassland and forest by the alien tree C. equisetifolia may negatively affect the endemic insect fauna of Nishijima.