Hemocyanin gene family evolution in spiders (Araneae), with implications for phylogenetic relationships and divergence times in the infraorder Mygalomorphae

Hemocyanins are multimeric copper-containing hemolymph proteins involved in oxygen binding and transport in all major arthropod lineages. Most arachnids have seven primary subunits (encoded by paralogous genes a–g), which combine to form a 24-mer (4 × 6) quaternary structure. Within some spider lineages, however, hemocyanin evolution has been a dynamic process with extensive paralog duplication and loss. We have obtained hemocyanin gene sequences from numerous representatives of the spider infraorders Mygalomorphae and Araneomorphae in order to infer the evolution of the hemocyanin gene family and estimate spider relationships using these conserved loci. Our hemocyanin gene tree is largely consistent with the previous hypotheses of paralog relationships based on immunological studies, but reveals some discrepancies in which paralog types have been lost or duplicated in specific spider lineages. Analyses of concatenated hemocyanin sequences resolved deep nodes in the spider phylogeny and recovered a number of clades that are supported by other molecular studies, particularly for mygalomorph taxa. The concatenated data set is also used to estimate dates of higher-level spider divergences and suggests that the diversification of extant mygalomorphs preceded that of extant araneomorphs. Spiders are diverse in behavior and respiratory morphology, and our results are beneficial for comparative analyses of spider respiration. Lastly, the conserved hemocyanin sequences allow for the inference of spider relationships and ancient divergence dates.     

The Ca channel TRPML3 specifically interacts with the mammalian ATG8 homologue GATE16 to regulate autophagy

TRPML3 is a Ca²⁺ permeable cation channel expressed in multiple intracellular compartments. Although TRPML3 is implicated in autophagy, how TRPML3 can regulate autophagy is not understood. To search interacting proteins with TRPML3 in autophagy, we performed split-ubiquitin membrane yeast two-hybrid (MY2H) screening with TRPML3-loop as a bait and identified GATE16, a mammalian ATG8 homologue. GST pull-down assay revealed that TRPML3 and TRPML3-loop specifically bind to GATE16, not to LC3B. Co-immunoprecipitation (co-IP) experiments showed that TRPML3 and TRPML3-loop pull down only the lipidated form of GATE16, indicating that the interaction occurs exclusively at the organellar membrane. The interaction of TRPML3 with GATE16 and GATE16-positive vesicle formation were increased in starvation induced autophagy, suggesting that the interaction facilitates the function of GATE16 in autophagosome formation. However, GATE16 was not required for TRPML3 trafficking to autophagosomes. Experiments using dominant-negative (DN) TRPML3(D458K) showed that GATE16 is localized not only in autophagosomes but also in extra-autophagosomal compartments, by contrast with LC3B. Since GATE16 acts at a later stage of the autophagosome biogenesis, our results suggest that TRPML3 plays a role in autophagosome maturation through the interaction with GATE16, by providing Ca²⁺ in the fusion process.     

Genistein promotes DNA demethylation of the steroidogenic factor 1 (SF-1) promoter in endometrial stromal cells

It has recently been demonstrated that genistein (GEN), a phytoestrogen in soy products, is an epigenetic modulator in various types of cells; but its effect on endometrium has not yet been determined. We investigated the effects of GEN on mouse uterine cells, in vivo and in vitro. Oral administration of GEN for 1 week induced mild proliferation of the endometrium in ovariectomized (OVX) mice, which was accompanied by the induction of steroidogenic factor 1 (SF-1) gene expression. GEN administration induced demethylation of multiple CpG sites in the SF-1 promoter; these sites are extensively methylated and thus silenced in normal endometrium. The GEN-mediated promoter demethylation occurred predominantly on the luminal side, as opposed to myometrium side, indicating that the epigenetic change was mainly shown in regenerated cells. Primary cultures of endometrial stromal cell colonies were screened for GEN-mediated alterations of DNA methylation by a high-resolution melting (HRM) method. One out of 20 colony-forming cell clones showed GEN-induced demethylation of SF-1. This clone exhibited a high proliferation capacity with continuous colony formation activity through multiple serial clonings. We propose that only a portion of endometrial cells are capable of receiving epigenetic modulation by GEN.     

Relationships in hydrogen metabolism between hydrogenase and nitrogenase in phototrophic bacteria.

Purple bacteria Rhodospirillum rubrum and Thiocapsa roseopersicina form two enzymes, hydrogenase and nitrogenase, which participate in hydrogen metabolism. H2 photoproduction in these bacteria is associated mainly or completely with the action of nitrogenase. The soluble and membrane-bound hydrogenases of T. roseopersicina have similar physicochemical properties (mol. weight, subunit composition, N-terminal amino acids, Fe2+ and S2- content, pl. Eo'). In comparison with other hydrogenases the enzyme from R. rubrum and T. roseopersicina evolve H2 with high rate from reduced cytochrome c3, but not from ferredoxins. H2 production and N2 fixation take place in the presence of NAD(P)H. NADP-reductase, ferredoxin and cytochrome c3 participate in this reaction. Possible relationships between hydrogenase-nitrogenase in the metabolism of molecular hydrogen are discussed.     

Inhibition of human low density lipoprotein oxidation by flavonols and their glycosides

Antioxidative effects of the flavonols and their glycosides, i.e., quercetin (Q), quercetin galactopyranoside (QG), quercetin rhamnolpyranoside (QR), rutin (R), morin (MO), myrecetin (MY), kaempferol (K) and kaempferol glucoside (KG), against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either by a water-soluble initiator 2,2'-azobis(2-amidino propane hydrochloride) (AAPH), or by cupric ion (Cu2+). The reaction kinetics were monitored either by the uptake of oxygen and the depletion of alpha-tocopherol (TOH) presented in the native LDL, or by the formation of thiobarbituric acid reactive substances (TBARS). Kinetic analysis of the antioxidation process demonstrates that these flavonols and their glycosides are effective antioxidants against AAPH- and Cu(2+)-initiated LDL peroxidation, the flavonols bearing ortho-dihydroxyl groups possess significantly higher antioxidant activity than those bearing no such functionalities, and the glycosides are less active than their parent aglycones.     

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.     

Assessment of genetic differentiation in the large yellow croaker, Pseudosciaena crocea Richardson,and its first hybrid filial generations with AFLP markers

The genetic diversity (the digital characteristics) of four populations (120 individuals) of breeding large yellow croakers, Pseudosciaena crocea Richardson, was analyzed with amplified fragment length polymorphism (AFLP) markers. Ten primer combinations amplified 248 bands, of which 39.52% were polymorphic. Shannon's information index of the Daiqv (DQ) population was higher than that of the Minyu (MY) population, at 0.2167 and 0.2074, respectively. Additionally, the Shannon's information index value for the minus-hybrid (DQ population♂ × MY population♀) first hybrid generation was higher than the value for the plus-hybrid (MY population♂ × DQ population♀) first hybrid generation at 0.1687 and 0.1613, respectively. The F_ST of the parental generation was lower than that of the filial generation at 0.0329 and 0.0891, respectively. Gene flow was very high according to F_st values in both parental and filial generations. The UPGMA clustering analysis based on genetic similarity organized the four populations into three groups. The minus-hybrid stock was the most distinct as compared to the other populations.     

Phylogenetic Relationships Among JC Virus Strains in Japanese/Koreans and Native Americans Speaking Amerind or Na-Dene

Abstract Many genetic studies using human mtDNA or the Y chromosome have been conducted to elucidate the relationships among the three Native American groups speaking Amerind, Na-Dene, and Eskimo-Aleut. Human polyomavirus JC (JCV) may also help to gain insights into this issue. JCV isolates are classified into more than 10 geographically distinct genotypes (designated subtypes here), which were generated by splits in the three superclusters, Types A, B, and C. A particular subtype of JCV (named MY) belonging to Type B is spread in both Japanese/Koreans and Native Americans speaking Amerind or Na-Dene. In this study, we evaluated the phylogenetic relationships among MY isolates worldwide, using the whole-genome approach, with which a highly reliable phylogeny of JCV isolates can be reconstructed. Thirty-six complete sequences belonging to MY (10 from Japanese/Koreans, 24 from Native Americans, and 2 from others), together with 54 belonging to other subtypes around the world, were aligned and subjected to phylogenetic analysis using the neighbor-joining and maximum-likelihood methods. In the resultant phylogenetic trees, the MY sequences diverged into two Japanese/Korean and five Native American clades with high bootstrap probabilities. Two of the Native American clades contained isolates mainly from Na-Denes and the others contained isolates mainly from Amerinds. The Na-Dene clades were not clustered together, nor were the Amerind clades. In contrast, the two Japanese/Korean clades were clustered at a high bootstrap probability. We concluded that there is no distinction between Amerinds and Na-Denes in terms of indigenous JCVs, although they are linguistically distinguished from each other.     

Kitasatospora sp. MY5-36产ε-聚赖氨酸分批补料发酵动力学

以北里孢菌(Kitasatospora sp.)MY 5-36为供试菌株,对ε-聚赖氨酸分批补料发酵动力学模型进行研究。建立了该菌株发酵合成ε-聚赖氨酸的菌体生长、产物合成和总糖消耗的动力学模型,并通过Origin 8.1软件对模型参数进行非线性拟合。结果表明:菌体量和聚赖氨酸的产量分别为16.25和13.15 g/L,产物合成与菌体生长的关系为部分耦联型。经验证,预测值与实验值有良好的拟合性,拟合度分别为0.999、0.995和0.992,说明所构建模型能够较好地反映ε-聚赖氨酸分批补料发酵过程。