Direct demonstration of involvement of protein kinase Calpha in the Ca2+-induced platelet aggregation

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin alphaIIbbeta3. However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca2+-induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of alphaIIbbeta3, suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKCalpha and beta from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKCalpha. Furthermore, the addition of purified PKCalpha in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKCalpha is involved in the regulation of Ca2+-induced platelet aggregation.     

Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties

Sphere formation has been utilized as a way to isolate multipotent stem/progenitor cells from various tissues. However, very few studies on bone marrow-derived spheres have been published and assessed their multipotentiality. In this study, multipotent marrow cell populations were isolated using a three-step method. First, after elimination of hematopoietic cells, murine marrow-derived adherent cells were cultured in plastic dishes until small cells gradually appeared and multiplied. Cells were then cultured under non-adherent conditions and formed spheres that were immunopositive for a neural precursor marker, nestin. RT-PCR analysis also revealed that the spheres were positive for nestin in addition to PPARgamma, osf2, SOX9, and myoD, which are markers of precursors of adipocytic, osteoblastic, chondrocytic, and skeletal myeloblastic lineages, respectively. Finally, spheres were dissociated into single cells and expanded in adherent cultures. Under appropriate induction conditions, the sphere-derived cells acquired the phenotypic properties in vitro of neurons, skeletal myoblasts, and beating cardiomyocytes, as well as adipocytes, osteoblasts, and chondrocytes. Next, sphere-derived cells were transplanted into murine myocardial infarction models. One month later, they had become engrafted as cardiomyocytes, and cardiac catheterization showed significant functional improvements. Thus, sphere-derived cells represent a new approach to enhance the multi-differentiation potential of murine bone marrow.     

Polycystic ovary syndrome is associated with genetic polymorphism in the insulin signaling gene IRS-1 but not ENPP1 in a Japanese population

Recent studies indicate that insulin resistance resulting from altered post-receptor signaling is associated with polycystic ovary syndrome (PCOS). We hypothesized that insulin receptor substrate-1 (IRS-1) Gly972Arg polymorphism and/or ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) Lys121Gln polymorphism predisposes women to PCOS and that these polymorphisms also affect anthropometric variables, glucose metabolism and androgen synthesis. To test those ideas, we studied the genotypes, indexes of insulin resistance, and hormone profiles in 123 Japanese women with PCOS and 380 healthy Japanese controls. We found that there were significantly more IRS-1 972Arg carriers among the PCOS patients than among the healthy controls (10.6% vs. 4.8%, p=0.029), which is consistent with our finding that women carrying the IRS-1 972Arg allele had a significantly increased risk of developing PCOS (odds ratio: 3.31, 95% confidence interval: 1.49-7.35). By contrast, the ENPP1 Lys121Arg polymorphism was distributed equally among PCOS patients and controls. In addition, neither of these polymorphisms studied affected the anthropometric variables, metabolic parameters or androgen levels of women with PCOS. We conclude that the IRS-1 Gly972Arg polymorphism is associated with PCOS in the Japanese population.     

Similar fish species composition despite larger environmental heterogeneity during severe hypoxia in a coastal ecosystem

Environmental heterogeneity is one of the most influential factors that create compositional variation among local communities. Greater compositional variation is expected when an environmental gradient encompasses the most severe conditions where species sorting is more likely to operate. However, evidence for stronger species sorting at severer environment has typically been obtained for less mobile organisms and tests are scarce for those with higher dispersal ability that allows individuals to sensitively respond to environmental stress. Here, with the dynamics of fish communities in a Japanese bay revealed by environmental DNA metabarcoding analyses as a model case, we tested the hypothesis that larger environmental heterogeneity caused by severe seasonal hypoxia (lower concentration of oxygen in bottom waters in summer) leads to larger variation of species composition among communities. During summer, fish species richness was lower in the bottom layer, suggesting the severity of the hypoxic bottom water. In contrast to the prediction, we found that although the environmental parameters of bottom and surface water was clearly distinct in summer, fish species composition was more similar between the two layers. Our null model analysis suggested that the higher compositional similarity during hypoxia season was not a result of the sampling effect reflecting differences in the alpha or gamma diversity. Furthermore, a shift in the species occurrence from bottom to surface layers was observed during hypoxia season, which was consistent across species, suggesting that the severe condition in the bottom adversely affected fish species irrespective of their identity. These results suggest that larger environmental heterogeneity does not necessarily lead to higher compositional variation once the environmental gradient encompasses extremely severe conditions. This is most likely because individual organisms actively avoided the severity quasi‐neutrally, which induced mass effect‐like dispersal and lead to the mixing of species composition across habitats. By showing counter evidence against the prevailing view, we provide novel insights into how species sorting by environment acts in heterogeneous and severe conditions.     

Construction of thermotolerant yeast expressing thermostable cellulase genes

Kluyveromyces marxianus NBRC1777 was identified as a thermotolerant yeast and was developed as a host for the expression of thermostable cellulase genes. The previously isolated genes for thermostable endo-beta-1,4-glucanase, cellobiohydrolase, and beta-glucosidase were introduced into the chromosome of K. marxianus and successfully expressed under the control of high-expression promoters. The recombinant K. marxianus expressing cellulase genes became able to grow in synthetic medium containing cellobiose or carboxymethyl-cellulose as the single carbon source. Moreover, the recombinant strain produced 43.4 g/L ethanol from 10% cellobiose. These results suggest that K. marxianus may afford a useful host system, which may be applicable to the simultaneous saccharification and fermentation and the foundation of cellulose consolidated bioprocessing.     

Evolution of condition-dependent dispersal: A genetic-algorithm search for the ESS reaction norm

Many insects produce two types (winged and wingless) of offspring that greatly differ in dispersal ability. The ratio of the two types often depends on the quality of the local habitat and the crowding experienced by the mother. Here we studied the condition-dependent dispersal that is evolutionarily stable. The model is also applicable to annual plants that produce two types of seeds differing in dispersal rates. The model assumptions are: the population is composed of a number of sites each occupied by a single adult. The total number of offspring produced by a mother depends on the environmental quality of the site that varies over the years and between sites. The ESS fraction of dispersing type as a function of the quality of the habitat (or ESS reaction norm) states that dispersers should not be produced if habitat qualitym is smaller than a critical valuek. Ifm is larger thank, the number of dispersers should increase withm and that of nondispersers should be kept constant. Second, we developed an alternative way of searching for the ESS: the reaction norm is represented as a three-layered neural network, and the parameters (weights and biases) are chosen by genetic algorithm (GA). This method can be extended easily to the cases of multiple environmental factors. There was an optimal (relatively wide) range of mutation rates for weights and biases, outside of which the convergence of the network to the valid ESS was likely to fail. Recombination, or crossing-over, was not effective in improving the success rate. The learned network often shows several characteristic ways of deviation from the ESS. We also examined the case in which the quality of different sites was correlated. In this case the ESS fraction of dispersers increases both with the quality of the site and with the average quality of the whole population in that year.     

Mechanisms of drought-induced dissipation of excitation energy in sun- and shade-adapted drought-tolerant mosses studied by fluorescence yield change and global and target analysis of fluorescence decay kinetics

Photosynthesis Research - Some mosses stay green and survive long even under desiccation. Dissipation mechanisms of excess excitation energy were studied in two drought-tolerant moss species...     

Apoptotic Effect of Ganciclovir on Primary Effusion Lymphoma Cells Infected with Kaposi's Sarcoma-Associated Herpesvirus

We evaluated the cytotoxic and apoptotic effects of two purine nucleoside analogues, acyclovir (ACV) and ganciclovir (GCV), on lymphoma cells stably harboring Kaposi's sarcoma-associated herpesvirus (KSHV). Colorimetric caspase assay, flow cytometry, and immunoblotting with antibodies against apoptosis-related molecules revealed that GCV has cytotoxic activity toward KSHV-infected primary effusion lymphoma cells, while ACV has weak or little activity. In addition to the GCV-induced cytotoxicity, apoptosis via caspase-7/8, cleavage of poly(ADP-ribose) polymerase, and accumulation of p53 and p21 were induced by GCV treatment. In contrast, neither ACV nor GCV have cytotoxicity- or apoptosis-inducing activities toward uninfected cells.