Effect of clutch size on male egg-fanning behavior and hatching success in the goby, Eviota prasina (Klunzinger)

In fish that exhibit paternal care, the females often choose their mates on the basis of male traits that are indicative of the parental ability of the males. In a marine goby, Eviota prasina, males tend their eggs within their nests until hatching, and females prefer males that have longer dorsal fins and exhibit courtship behavior with a higher frequency as their mates. In order to clarify the relationship between these sexually selected traits and the parental ability of males of E. prasina, the factors affecting the hatching success of eggs within male nests and the male parental care behavior were examined in an aquarium experiment. Females spawned their eggs in male nests and the clutch size of females showed a high individual variation (range = 88-833 eggs). The hatching success of eggs within male nests showed a positive correlation with the time spent by males in fanning eggs and the clutch size. In contrast to the prediction, however, the hatching success did not show a significant correlation with the sexually selected traits, i.e., the male dorsal fin length and the frequency of courtship displays. Moreover, multiple regression analysis indicated that the time spent by the males in fanning was the most important factor affecting the survival rate of the eggs. The time spent by males in fanning behavior was influenced by the clutch size within their nests; the fanning behavior of males occurred with a higher frequency when they tended larger clutches. Males are required to invest a greater effort in egg-tending behavior to achieve a higher hatching success when they receive larger clutches, probably due to the greater reward for their parental behavior. Based on their mate choice, females may obtain other benefits such as high quality offspring.     

Nitric oxide (NO) serves as a retrograde messenger to activate neuronal NO synthase in the spinal cord via NMDA receptors.

We have recently demonstrated that nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the spinal cord is involved in the maintenance of neuropathic pain. To clarify whether NO itself affected nNOS activity in the spinal cord as a retrograde messenger, we examined the involvement of the NO/cGMP signaling pathway in the regulation of nNOS activity by NADPH-diaphorase histochemistry. NO-generating agents NOR3 (t(1/2)=30min) and SNAP (t(1/2)=5h), but not NOR1 (t(1/2)=1.8min), significantly enhanced NADPH-diaphorase staining in the spinal cord. 8-Br-cGMP also enhanced it similar to that by NOR3, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NOR1 and NOR3 markedly increased the cGMP level in the spinal cord. The enhancement of NADPH-diaphorase staining by NOR3 was significantly inhibited by CPTIO, an NO scavenger, ODQ, a soluble guanylate cyclase inhibitor, and KT5823, an inhibitor of cGMP-dependent protein kinase. Additionally, the NOR3-enhanced nNOS activity was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the GluRepsilon2-selective antagonist CP-101,606, and was attenuated in GluRepsilon1(-/-) and GluRepsilon1(-/-)/epsilon4(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing GluRepsilon1 and GluRepsilon2 subunits.     

Identification of arylsulfonamides as Aquaporin 4 inhibitors

Carbonic anhydrase inhibitors AZA, EZA, and 4-acetamidobenzsulfonamide were found to inhibit human AQP4-M23 mediated water transport by 80%, 68%, and 23%, respectively, at 20 microM in an in vitro functional assay. AZA was found to have an IC50 against AQP4 of 0.9 microM. Phloretin was inactive under the same conditions.     

Interactions between peptides containing nucleobase amino acids and T7 phages displaying S. cerevisiae proteins

The importance of high-throughput analyses of protein abundances and functions is interestingly increasing in genomic/proteomic studies. In such postgenome sequencing era, a protein-detecting chip, in which a large number of molecules specifically capturing target proteins (capturing agents) such as antibodies, recombinant proteins, and small molecules are arrayed onto solid, wet, or semi-wet substrates, enables comprehensive analysis of proteomes by a single experiment. However, whole proteomes are generally complicated for comprehensive analyses so that alternative approaches to subproteome analysis categorized by protein functions and binding properties (focused proteome) would be effective. Approaching the goal of development of designed peptide chip for protein analysis, diversity increases in peptide structures and validation of target proteins are needed. We herein describe design and synthesis of nucleobase amino acid (NBA)-containing peptides, selection of nucleic acid-related proteins derived from S. cerevisiae, and detection of interactions between NBA-containing peptides and T7 phages displaying proteins by both enzyme-linked immunosorbent assays (ELISA) and label-free anomalous reflection of gold (AR) measurements. Twenty-eight phage clones were obtained by the phage-display method and sequenced. Ten of 28 clones were expected to be nucleic acid-related proteins including initiation factor, TYB protein, ribosomal proteins, elongation factor, ATP synthase subunit, GTP-binding protein, and ribonuclease. Other phage clones encoded several classes of enzymes such as reductase, oxidase, aldolase, metalloprotease, and hexokinase. Both ELISA and AR measurements suggested that the methodology of in vitro selection for recognition of the NBA-containing peptide presented in this study was successfully established. Such a combination of NBA and phage display technologies would be potential to efficiently confirm valuable target proteins binding specifically to capturing agents, to be arrayed onto solid surfaces to develop the designed peptide chip.     

Molecular survey of Babesia microti, Ehrlichia species and Candidatus neoehrlichia mikurensis in wild rodents from Shimane Prefecture, Japan

A significant number of patients are diagnosed with "fevers of unknown origin" (FUO) in Shimane Prefecture in Japan where tick-borne diseases are endemic. We conducted molecular surveys for Babesia microti, Ehrlichia species, and Candidatus Neoehrlichia mikurensis in 62 FUO cases and 62 wild rodents from Shimane Prefecture, Japan. PCR using primers specific for the Babesia 18S small-subunit rRNA (rDNA) gene and Anaplasmataceae groESL amplified products from 45% (28/62) and 25.8% (16/62) of captured mice, respectively. Of the 28 18S rDNA PCR positives, 23 and five samples were positive for Hobetsu- and Kobe-type B. microti, respectively. In contrast, of the 16 groESL PCR positives, eight, one and seven samples were positive for Ehrlichia muris, Ehrlichia sp. HF565 and Candidatus N. mikurensis, respectively. Inoculation of selected blood samples into Golden Syrian hamsters indicated the presence of Hobetsu- and Kobe-type B. microti in four and one sample, respectively. Isolation of the latter strain was considered important as previous studies suggested that the distribution of this type was so far confined to Awaji Island in Hyogo Prefecture, where the first case of transfusion-associated human babesiosis originated. DNA samples from 62 FUO human cases tested negative for B. microti 18S rDNA gene, Anaplasmataceae groESL gene, Rickettsia japonica 17K genus-common antigen gene and Orientia tsutsugamushi 56K antigen gene by PCRs. We also conducted seroepidemiological surveys on 62 human sera collected in Shimane Prefecture from the FUO patients who were suspected of carrying tick-borne diseases. However, indirect immunofluorescent antibody tests using B. microti- and E. muris-infected cells detected IgG against E. muris in only a single positive sample. This study demonstrates the presence of several potentially important tick-borne pathogens in Shimane Prefecture and suggests the need for further study on the causative agents of FUOs.     

Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina,an orchid mainly utilizing saprotrophic rhizoctonia

The majority of chlorophyllous orchids form mycorrhizal associations with so‐called rhizoctonia fungi, a phylogenetically heterogeneous assemblage of predominantly saprotrophic fungi in Ceratobasidiaceae, Tulasnellaceae, and Serendipitaceae. It is still a matter of debate whether adult orchids mainly associated with rhizoctonia species are partially mycoheterotrophic. Here, we investigated the nutritional modes of green and albino variants of Goodyera velutina, an orchid species considered to be mainly associated with Ceratobasidium spp., by measuring their ¹³C and ¹⁵N abundances, and by molecular barcoding of their mycorrhizal fungi. Molecular analysis revealed that both green and albino variants of G. velutina harbored a similar range of mycobionts, mainly saprotrophic Ceratobasidium spp., Tulasnella spp., and ectomycorrhizal Russula spp. In addition, stable isotope analysis revealed that albino variants were significantly enriched in ¹³C but not so greatly in ¹⁵N, suggesting that saprotrophic Ceratobasidium spp. and Tulasnella spp. are their main carbon source. However, in green variants, ¹³C levels were depleted and those of ¹⁵N were indistinguishable from the co‐occurring autotrophic plants. Therefore, we concluded that the albino G. velutina variants are fully mycoheterotrophic plants whose C derives mainly from saprotrophic rhizoctonia, while the green G. velutina variants are mainly autotrophic plants, at least at our study site, in spite of their additional associations with ectomycorrhizal fungi. This is the first report demonstrating that adult nonphotosynthetic albino variants can obtain their nutrition mainly from nonectomycorrhizal rhizoctonia.     

Class I Myosins Have Overlapping and Specialized Functions in Left-Right Asymmetric Development in Drosophila

The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left–right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.     

5'-,3'-inverted thymidine-modified antisense oligodeoxynucleotide targeting midkine. Its design and application for cancer therapy

Oligodeoxynucleotides modified at both 5'- and 3'-ends with inverted thymidine (5'-,3'-inverted T) were introduced as new reagents for antisense strategies. These modifications were performed to make the oligodeoxynucleotides resistant to nucleases. The effectiveness of these oligodeoxynucleotides was evaluated in terms of inhibition of synthesis of midkine (MK), a heparin-binding growth factor, and consequent inhibition of growth of CMT-93 mouse rectal carcinoma cells. 5'-,3'-Inverted T antisense MK suppressed synthesis of MK by CMT-93 cells and their growth in culture. Furthermore, 5'-,3'-inverted T oligodeoxynucleotides exhibited less cytotoxicity and better stability than phosphorothioate oligodeoxynucleotides. When 5'-,3'-inverted T antisense MK was mixed with atelocollagen, and injected into CMT-93 tumors pregrown in nude mice, tumor growth was markedly suppressed as compared with tumors injected with sense controls. The suppressive effect of 5'-,3'-inverted T antisense MK on tumor growth was stronger than that of phosphorothioate antisense MK. These findings indicated the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides.     

Comparison of the substrate specificity of the two bacterial desulfurization systems

Using cell-free extracts of a desulfurizing mesophile, Rhodococcus erythropolis KA2-5-1 (the Dsz system) and Escherichia coli JM109, which possesses the desulfurizing genes of a thermophile Paenibacillus sp. A11-2 (the Tds system), the reactivity of desulfurizing enzymes toward 4,6-dialkyl dibenzothiophenes (4,6-dialkyl DBTs) and 7-alkyl benzothiophenes (7-alkyl BTs) was investigated. Both systems desulfurized all the 4,6-dialkyl DBTs, except 4,6-dibutyl DBT. Although some alkylated BTs were degraded by the Dsz system, no desulfurized compounds were detected. The reactivity of the Tds system toward alkylated BTs was higher than that of DBT. In contrast to the Dsz system, the Tds system yielded desulfurized compounds from all of the alkylated BTs examined.