Identification of important bases for the self-cleavage activity at two single-stranded regions of genomic HDV ribozyme.

In order to determine important bases at two single-stranded regions [SSrA (726-731 nt) and SSrB (762-766)] derived mainly from secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several point mutants at these two regions on the HDV88 molecule (683-770). Among the bases at SSrA and SSrB regions C763 was found to play an essential role during self-cleavage process since substitutions to any other bases viz. A or G or U completely abolished the activity.     

Effects of acorn abundance on density dependence in a Japanese wood mouse (Apodemus speciosus) population

We analysed the effects of Quercus crispula acorn abundance on the density dependence of the large Japanese wood mouse Apodemus speciosus using time series data (1992–2007). The data were obtained in a forest in northern Hokkaido, Japan, by live-trapping rodents and directly counting acorns on the ground. Acorn abundance in one year clearly influenced the abundance of wood mice in the following year in all models examined based on the Gompertz and Ricker model; in addition, the abundance of wood mice had effects on the population. Acorn abundance influenced the strength of density dependence (intraspecific competition) of the wood mouse population. When the abundance of acorns was high, density dependence was relaxed, and as a result the equilibrium density at which the population growth rate decreased to zero became higher. Those effects of acorn abundance were regarded as a nonlinear perturbation effect (sensu Royama 1992). The nonlinearity of density dependence was also detected; higher densities had stronger effects on population growth rates. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The giant mycoheterotrophic orchid Erythrorchis altissima is associated mainly with a divergent set of wood‐decaying fungi

The climbing orchid Erythrorchis altissima is the largest mycoheterotroph in the world. Although previous in vitro work suggests that E. altissima has a unique symbiosis with wood‐decaying fungi, little is known about how this giant orchid meets its carbon and nutrient demands exclusively via mycorrhizal fungi. In this study, the mycorrhizal fungi of E. altissima were molecularly identified using root samples from 26 individuals. Furthermore, in vitro symbiotic germination with five fungi and stable isotope compositions in five E. altissima at one site were examined. In total, 37 fungal operational taxonomic units (OTUs) belonging to nine orders in Basidiomycota were identified from the orchid roots. Most of the fungal OTUs were wood‐decaying fungi, but underground roots had ectomycorrhizal Russula. Two fungal isolates from mycorrhizal roots induced seed germination and subsequent seedling development in vitro. Measurement of carbon and nitrogen stable isotope abundances revealed that E. altissima is a full mycoheterotroph whose carbon originates mainly from wood‐decaying fungi. All of the results show that E. altissima is associated with a wide range of wood‐ and soil‐inhabiting fungi, the majority of which are wood‐decaying taxa. This generalist association enables E. altissima to access a large carbon pool in woody debris and has been key to the evolution of such a large mycoheterotroph.     

Sampling efficiency of the pale grass blue butterfly Zizeeria maha (Lepidoptera: Lycaenidae): A versatile indicator species for environmental risk assessment in Japan

The pale grass blue butterfly, Zizeeria maha (Lepidoptera, Lycaenidae), has successfully been employed as an indicator species to evaluate acute biological changes in polluted human-living environments after the Fukushima nuclear accident. Here, we quantitatively examined the field sampling efficiency of the butterfly and its associated sexual sampling bias under different conditions, which may provide important information for environmental risk assessment. Sampling data were obtained in 2014 and 2015 from 87 localities across Japan. Across the localities, the mean capture rate was 17 individuals per hour per person, and males represented approximately 80% of the collected butterflies. No significant difference was detected in the capture rate under different weather conditions. Among the four habitats, the riverside showed a significantly higher capture rate than the city park. A shorter sampling time tended to yield a higher capture rate. Comparisons among prefectures and districts of collection localities revealed that the Kanto district had significantly higher percentages of collected males than 4 other districts. Fukushima prefecture also had high percentage, although not significant. A generalized linear mixed model indicated that the capture rate was significantly negatively affected by the agricultural village, the city park, the rainy weather, and latitude and positively by longitude. Together, the present study showed high sampling efficiency and versatility of this butterfly as an indicator for environmental risk assessment but simultaneously suggested preferable conditions for a field sampling design. This study also suggested a possibility that the Fukushima nuclear accident or other factors might have affected behavioral or population dynamics of the butterfly.     

Identification and characterization of XPC-binding domain of hHR23B.

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.     

Expression of heat shock protein in host macrophages correlates with a protective potential against infection with Leishmania major in mice

We studied the relationship between the expression of 65-kDa heat shock protein (HSP65) and resistance of mice to infection with Leishmania major (L. major), an obligate intracellular protozoan. C57BL/6 (B6) mice, a strain genetically resistant to L. major infection, expressed high level of HSP65 in their peritoneal and draining lymph node macrophages after infection, whereas susceptible BALB/c mice expressed only slightly at the early stage of infection. This protein was not expressed in the parasite itself and macrophages of non-infected mice. We examined the role of T cells in the expression of HSP65 by using SCID mice grafted with the fetal thymus from B6 or BALB/c mice (B6-TG or BALB-TG mice, respectively). Either BALB-TG or B6-TG mice were reconstituted with T cells derived from each grafted fetal thymus. B6-TG mice were markedly resistant against infection with L. major, compared with BALB-TG mice. Furthermore, the HSP65 expression in macrophages of thymus-grafted mice was similar to that of the thymus-donor type. That is, B6-TG mice expressed a high level of this protein, whereas BALB-TG mice did in lower level than B6-TG mice. These results show that T cells are necessary to express HSP65 and the expression correlates with a protective potential of T cells against infection with L. major.     

Interpopulation variation in reproductive traits of female masu salmon, Oncorhynchus masou

Several life history models predict that larger eggs and lower fecundity should be favored in a low-growth environment. We applied the model of Sibly et al. to seven Japanese populations of masu salmon ( Oncorhynchus masou ) in order to test the predictions that populations in which individual growth rate is low are characterized by larger eggs and lower fecundity. Two populations, Shumarinai and Shikaribetsu, had the lowest growth rates of individuals, largest egg sizes and lowest fecundities of all populations examined. In contrast, the Shiribetsu, Chitose and Uono populations had highest growth rates of individuals, smallest egg sizes and highest fecundities. The Shibetsu and Toya populations were intermediate between these two groups. The gonadsomatic index (GSI) of the Shumarinai and Shikaribetsu populations was smaller than that of all other populations. Correlation analysis indicated that populations with lower individual growth rates had larger eggs and lower fecundities. The results were consistent with the predictions of the Sibly et al. model: increased egg size which results in decreased fecundity is probably an adaptation to low-growth environments. Therefore, in masu salmon, growth differences among populations may explain interpopulation variation in egg size and fecundity.     

Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids.

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.     

Variation and association of fibronectin‐binding protein genes fnbA and fnbB in Staphylococcus aureus Japanese isolates

Fibronectin‐binding proteins A and B (FnBPA and FnBPB) mediate adhesion of Staphylococcus aureus to fibrinogen, elastin and fibronectin. FnBPA and FnBPB are encoded by two closely linked genes, fnbA and fnbB, respectively. With the exception of the N‐terminal regions, the amino acid sequences of FnBPA and FnBPB are highly conserved. To investigate the genetics and evolution of fnbA and fnbB, the most variable regions, which code for the 67th amino acids of the A through B regions (A67–B) of fnbA and fnbB, were focused upon. Eighty isolates of S. aureus in Japan were sequenced and 19 and 18 types in fnbA and fnbB, respectively, identified. Although the phylogeny of fnbA and fnbB were found to be quite different, each fnbA type connected with a specific fnbB type, indicating that fnbA and fnbB mutate independently, whereas the combination of both genes after recombination is stable. Hence those fnbA–fnbB combinations were defined as FnBP sequence types (FnSTs). Representative isolates of each FnST were assigned distinct STs by multilocus sequence typing, suggesting correspondence of FnST with genome lineage. Linkage disequilibrium (LD) analysis of the A67–B region revealed that subdomains N2, N3 and FnBR1 form a LD block in fnbA, whereas N2 and N3 form two independent LD blocks in fnbB. N2–N3 three‐dimensional structural models indicated that not only the variable amino acid residues, but also well‐conserved amino acid residues between FnBPA and FnBPB, are located on the surface of the protein. These results highlight a molecular process of the FnBP that has evolved by mingled mutation and recombination with retention of functions.