Molecular phylogeny and radiation time of Erysiphales inferred from the nuclear ribosomal DNA sequences

Phylogenetic relationships of Erysiphales within Ascomycota were inferred from the newly determined sequences of the 18S rDNA and partial sequences of the 28S rDNA including the D1 and D2 regions of 10 Erysiphales taxa. Phylogenetic analyses revealed that the Erysiphales form a distinct clade among ascomycetous fungi suggesting that the Erysiphales diverged from a single ancestral taxon. The Myxotrichaceae of the Onygenales was distantly related to the other onygenalean families and was the sister group to the Erysiphales calde, with which it combined to form a clade. The Erysiphales/Myxotrichaceae clade was also closely related to some discomycetous fungi (Leotiales, Cyttariales and Thelebolaceae) including taxa that form cleistothecial ascomata. The present molecular analyses as well as previously reported morphological observations suggest the possible existence of a novel evolutionary pathway from cleistothecial discomycetous fungi to Erysiphales and Myxotrichaceae. However, since most of these fungi, except for the Erysiphales, are saprophytic on dung and/or plant materials, the questions of how and why an obligate biotroph like the Erysiphales radiated from the saprophytic fungi remain to be addressed. We also estimated the radiation time of the Erysiphales using the 18S rDNA sequences and the two molecular clockes that have been previously reported. The calculation showed that the Erysiphales split from the Myxotrichaceae 190–127 myr ago. Since the radiation time of the Erysiphales does not exceed 230 myr ago, even when allowance is made for the uncertainty of the molecular clocks, it is possible to consider that the Erysiphales evolved after the radiation of angiosperms. The results of our calculation also showed that the first radiation within the Erysiphales (138–92 myr ago) coincided with the date of a major diversification of angiosperms (130–90 myr ago). These results may support our early assumption that the radiation of the Erysiphales coincided with the evolution of angiosperm plants. Contribution No. 152 from the Laboratory of Plant Pathology, Mie University     

Purification and characterization of S-modulin, a calcium-dependent regulator on cGMP phosphodiesterase in frog rod photoreceptors.

S-modulin is a 26 kDa protein that regulates light sensitivity of cGMP phosphodiesterase in a Ca(2+)-dependent manner in frog rod outer segments (ROSs). In the present study, we purified S-modulin by taking advantage of a hydrophobic interaction between Phenyl Sepharose and S-modulin at high Ca2+ concentrations. The yield was greater than 90%. 45Ca(2+)-binding experiment showed that S-modulin is a Ca(2+)-binding protein. At high Ca2+ concentrations, S-modulin binds to ROS membranes. The binding target of the Ca2+/S-modulin complex is possibly a ROS membrane lipid(s), but it was difficult to identify. The binding was observed mainly at greater than 1 microM Ca2+. The amino acid sequence deduced from proteolytic fragments of S-modulin was approximately 80% and 60% identical to those of recovering and visinin, respectively.     

Complementation of the protein transport defect of an Escherichia coli secY mutant (secY24) by Bacillus subtilis secY homologue

Bacillus subtilis SecY homologue shares 41.3% homology with that of E. coli and remarkably higher homologous regions (more than 80%) are present in the four cytoplasmic regions [(1990) J. Biochem. 107, 603-607]. Based on the formation of the mature form of OmpA in E. coli, we have shown that the protein transport defect of the E. coli secY mutant (secY24) is complemented by the gene product from the B. subtilis secY homologue, which is expressed under the lac promoter control. However, B. subtilis SecY could not restore growth of the E. coli mutant at nonpermissive temperature.     

Golgi/plastid‐type manganese superoxide dismutase involved in heat‐stress tolerance during grain filling of rice

Superoxide dismutase (SOD) is widely assumed to play a role in the detoxification of reactive oxygen species caused by environmental stresses. We found a characteristic expression of manganese SOD 1 (MSD1) in a heat‐stress‐tolerant cultivar of rice (Oryza sativa). The deduced amino acid sequence contains a signal sequence and an N‐glycosylation site. Confocal imaging analysis of rice and onion cells transiently expressing MSD1‐YFP showed MSD1‐YFP in the Golgi apparatus and plastids, indicating that MSD1 is a unique Golgi/plastid‐type SOD. To evaluate the involvement of MSD1 in heat‐stress tolerance, we generated transgenic rice plants with either constitutive high expression or suppression of MSD1. The grain quality of rice with constitutive high expression of MSD1 grown at 33/28 °C, 12/12 h, was significantly better than that of the wild type. In contrast, MSD1‐knock‐down rice was markedly susceptible to heat stress. Quantitative shotgun proteomic analysis indicated that the overexpression of MSD1 up‐regulated reactive oxygen scavenging, chaperone and quality control systems in rice grains under heat stress. We propose that the Golgi/plastid MSD1 plays an important role in adaptation to heat stress.     

Molecular phylogeny reveals the presence of cryptic speciation within Erysiphe japonica (≡ Typhulochaeta japonica), a powdery mildew on Quercus spp.

Molecular phylogenetic analyses based on 28S, ITS and IGS rDNA sequences indicate that Erysiphe japonica (≡ Typhulochaeta japonica) consists of two different genetic groups, one group on Quercus aliena, Q. robur and Q. serrata, and another group on Q. crispula var. crispula and Q. crispula var. horikawae. As morphological difference between the two groups are not yet marked distinctly, we suppose that the process of speciation has not yet been finished and propose a new variety, E. japonica var. crispulae, for the latter group based on the difference in host range and the distinct genetic segregation. Quercus robur (pedunculate oak) is a new host of E. japonica.     

Long-term survival of transplanted induced pluripotent stem cell-derived neurospheres with nerve conduit into sciatic nerve defects in immunosuppressed mice

Since the advent of induced pluripotent stem cells (iPSCs), clinical trials using iPSC-based cell transplantation therapy have been performed in various fields of regenerative medicine. We previously demonstrated that the transplantation of mouse iPSC-derived neurospheres containing neural stem/progenitor cells with bioabsorbable nerve conduits promoted nerve regeneration in the long term in murine sciatic nerve defect models. However, it remains unclear how long the grafted iPSC-derived neurospheres survived and worked after implantation. In this study, the long-term survival of the transplanted mouse iPSC-derived neurospheres with nerve conduits was evaluated in high-immunosuppressed or non-immunosuppressed mice using in vivo imaging for the development of iPSC-based cell therapy for peripheral nerve injury. Complete 5-mm long defects were created in the sciatic nerves of immunosuppressed and non-immunosuppressed mice and reconstructed using nerve conduits coated with iPSC-derived neurospheres labeled with ffLuc. The survival of mouse iPSC-derived neurospheres on nerve conduits was monitored using in vivo imaging. The transplanted iPSC-derived neurospheres with nerve conduits survived for 365 days after transplantation in the immunosuppressed allograft models, but only survived for at least 14 days in non-immunosuppressed allograft models. This is the first study to find the longest survival rate of stem cells with nerve conduits transplanted into the peripheral nerve defects using in vivo imaging and demonstrates the differences in graft survival rate between the immunosuppressed allograft model and immune responsive allograft model. In the future, if iPSC-derived neurospheres are successfully transplanted into peripheral nerve defects with nerve conduits using iPSC stock cells without eliciting an immune response, axonal regeneration will be induced due to the longstanding supportive effect of grafted cells on direct remyelination and/or secretion of trophic factors.     

Evaluation of factors associated with the anxiety and depression of female infertility patients

Background

Because the primary aim of infertility treatment is to achieve pregnancy, mental health care during this treatment is often neglected. However, the inability to conceive children is stressful for couples throughout the world. Thus, the purpose of this study was to investigate factors related to the anxiety and depression of female infertility patients.

Methods

Participants included 83 Japanese women who initially visited the Reproduction Center of the Tokyo Dental College Ichikawa General Hospital to undergo testing and receive infertility treatment between February and April 2008. We administered two psychological tests, the Self-rating Depression Scale (SDS) test and the Hospital Anxiety and Depression Scale (HADS) test. We then examined the association of the test results with age, pregnancy and delivery history, employment status, duration of infertility, infertility treatment history, and male infertility.

Results

As patient age increased, total HADS and depression scores also increased. No correlation was observed between duration of infertility and SDS or HADS scores. Results were similar when the presence and absence of delivery history was compared. Patients who underwent infertility treatment were more likely to have high HADS depression scores compared to patients who had not undergone treatment. Additionally, patients whose husbands were infertile had significantly lower total HADS and anxiety scores than those whose husbands were not infertile.

Conclusions

Age and male infertility are factors that influence the presence of anxiety and depression in female infertility patients.     

Identification of Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a Regulator of N-Acetylglucosaminyltransferase GnT-IX (GnT-Vb)

Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.