Ticks (Acari: Ixodidae) from medium-sized to large mammals in Ehime Prefecture, Japan

Ixodid ticks were collected from medium-sized to large mammals in Ehime Prefecture, Shikoku, Japan. Ten species of ticks (Amblyomma testudinarium, Dermacentor taiwanensis, Haemaphysalis flava, H. formosensis, H. hystricis, H. longicornis, H. megaspinosa, Ixodes nipponensis, I. ovatus, and I. tanuki) were collected from a total of 29 mammals comprising 11 species. Haemaphysalis hystricis, a possible vector of Japanese spotted fever in Ehime prefecture, was collected from Canis lupus familiaris (domestic dog), Martes melampus melampus, and Sus scrofa leucomystax. This is a first report of H. hystricis from the domesticated dog in the endemic area of Japanese spotted fever. This suggests that it is necessary to pay attention to dogs as a host of the vector ticks for Japanese spotted fever control. Nyctereutes procyonoides and Ma. melampus are new hosts for A. testudinarium. Nyctereutes procyonoides, Mustela itatsi, and Lepus brachyurus are new hosts for H. formosensis. Martes melampus is a new host for H. hystricis.     

Leaf carbon isotope ratio and water use efficiency of urban roadside trees in summer in Kyoto city

The effect of summer climate on leaf carbon isotope composition (δ¹³C) of the major roadside tree species Prunus × yedoensis (P. yedoensis) was investigated in Kyoto city, Japan, to explore the implications for alterations in urban environments. Temperature and the vapor pressure deficit were higher at sites of higher traffic volumes, possibly affected by a heat island effect. The leaf δ¹³C of P. yedoensis trees was affected strongly by leaf carbon isotope discrimination (Δ), with much less effect of δ¹³C on atmospheric CO₂. Leaf Δ values in the summer were smaller at sites of higher traffic volumes with high atmospheric temperatures, suggesting a higher long-term water use efficiency (WUE) at these sites. Gas exchange measurements of P. yedoensis leaves indeed suggested a higher intrinsic WUE at sites of higher traffic volumes with high atmospheric temperatures. These results suggest that leaf Δ is related to the response of WUE to summer climates, and that leaf δ¹³C in urban areas is a useful tracer for understanding the influences of urban environments on plant photosynthetic processes.     

High degree of genetic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus)

Populations of widespread marine organisms are typically characterized by a low degree of genetic differentiation in neutral genetic markers, but much less is known about differentiation in genes whose functional roles are associated with specific selection regimes. To uncover possible adaptive population divergence and heterogeneous genomic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus), we used a candidate gene‐based genome‐scan approach to analyse variability in 138 microsatellite loci located within/close to (<6 kb) functionally important genes in samples collected from ten geographic locations. The degree of genetic differentiation in markers classified as neutral or under balancing selection—as determined with several outlier detection methods—was low (F_ST = 0.033 or 0.011, respectively), whereas average F_ST for directionally selected markers was significantly higher (F_ST = 0.097). Clustering analyses provided support for genomic and geographic heterogeneity in selection: six genetic clusters were identified based on allele frequency differences in the directionally selected loci, whereas four were identified with the neutral loci. Allelic variation in several loci exhibited significant associations with environmental variables, supporting the conjecture that temperature and salinity, but not optic conditions, are important drivers of adaptive divergence among populations. In general, these results suggest that in spite of the high degree of physical connectivity and gene flow as inferred from neutral marker genes, marine stickleback populations are strongly genetically structured in loci associated with functionally relevant genes.     

Caveolar endocytosis is critical for BK virus infection of human renal proximal tubular epithelial cells

In recent years, BK virus (BKV) nephritis after renal transplantation has become a severe problem. The exact mechanisms of BKV cell entry and subsequent intracellular trafficking remain unknown. Since human renal proximal tubular epithelial cells (HRPTEC) represent a main natural target of BKV nephritis, analysis of BKV infection of HRPTEC is necessary to obtain additional insights into BKV biology and to develop novel strategies for the treatment of BKV nephritis. We coincubated HRPTEC with BKV and the cholesterol-depleting agents methyl beta cyclodextrin (MBCD) and nystatin (Nys), drugs inhibiting caveolar endocytosis. The percentage of infected cells (detected by immunofluorescence) and the cellular levels of BKV large T antigen expression (detected by Western blot analysis) were significantly decreased in both MBCD- and Nys-treated HPRTEC compared to the level in HRPTEC incubated with BKV alone. HRPTEC infection by BKV was also tested after small interfering RNA (siRNA)-dependent depletion of either the caveolar structural protein caveolin-1 (Cav-1) or clathrin, the major structural protein of clathrin-coated pits. BKV infection was inhibited in HRPTEC transfected with Cav-1 siRNA but not in HRPTEC transfected with clathrin siRNA. The colocalization of labeled BKV particles with either Cav-1 or clathrin was investigated by using fluorescent microscopy and image cross-correlation spectroscopy. The rate of colocalization of BKV with Cav-1 peaked at 4 h after incubation. Colocalization with clathrin was insignificant at all time points. These results suggest that BKV entered into HRPTEC via caveolae, not clathrin-coated pits, and that BKV is maximally associated with caveolae at 4 h after infection, prior to relocation to a different intracellular compartment.     

Structural basis of D-DOPA oxidation by D-amino acid oxidase: alternative pathway for dopamine biosynthesis

D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent endogenous ligand of N-methyl-D-aspartate type glutamate receptors. It also has been suggested that D-DOPA, the stereoisomer of L-DOPA, is oxidized by DAO and then converted to dopamine via an alternative biosynthetic pathway. Here, we provide direct crystallographic evidence that D-DOPA is readily fitted into the active site of human DAO, where it is oxidized by the enzyme. Moreover, our kinetic data show that the maximal velocity for oxidation of D-DOPA is much greater than for D-serine, which strongly supports the proposed alternative pathway for dopamine biosynthesis in the treatment of Parkinson's disease. In addition, determination of the structures of human DAO in various states revealed that the conformation of the hydrophobic VAAGL stretch (residues 47-51) to be uniquely stable in the human enzyme, which provides a structural basis for the unique kinetic features of human DAO.     

Capture and release of DNA using aminosilane-modified bacterial magnetic particles for automated detection system of single nucleotide polymorphisms

Bacterial magnetic particles (BMPs) were modified with 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to produce a dense amine surface. Modification of BMPs in a toluene solution resulted in an increased amine yield, and approximately 11.3 x 10(4) surface amines were detected on a single particle. The modified BMPs were capable of efficient electrostatic capture of DNA. The maximum amount of DNA captured on 10 microg of aminosilane-modified BMPs was 600 ng. A 10 mM phosphate buffer effectively released the captured DNA. This efficiency was dramatically enhanced by incubation at 80 degrees C and DNA recovery from aminosilane-modified BMPs approached 95%. DNA extraction from whole blood using these modified BMPs, followed by PCR, was successfully performed. Furthermore, automated single nucleotide polymorphism (SNP) detection of the aldehyde dehydrogenase 2 (ALDH2) was demonstrated.     

Molecular evolution of the nontandemly repeated genes of the histone 3 multigene family.

In some species, histone gene clusters consist of tandem arrays of each type of histone gene, whereas in other species the genes may be clustered but not arranged in tandem. In certain species, however, histone genes are found scattered across several different chromosomes. This study examines the evolution of histone 3 (H3) genes that are not arranged in large clusters of tandem repeats. Although H3 amino acid sequences are highly conserved both within and between species, we found that the nucleotide sequence divergence at synonymous sites is high, indicating that purifying selection is the major force for maintaining H3 amino acid sequence homogeneity over long-term evolution. In cases where synonymous-site divergence was low, recent gene duplication appeared to be a better explanation than gene conversion. These results, and other observations on gene inactivation, organization, and phylogeny, indicated that these H3 genes evolve according to a birth-and-death process under strong purifying selection. Thus, we found little evidence to support previous claims that all H3 proteins, regardless of their genome organization, undergo concerted evolution. Further analyses of the structure of H3 proteins revealed that the histones of higher eukaryotes might have evolved from a replication-independent-like H3 gene.     

Microtubule array observed in the posterior‐vegetal cortex during cytoplasmic and cortical reorganization of the ascidian egg

Body axis formation during embryogenesis results from asymmetric localization of maternal factors in the egg. Shortly before the first cleavage in ascidian eggs, cell polarity along the anteroposterior (A–P) axis is established and the cytoplasmic domain (myoplasm) relocates from the vegetal to the posterior region in a microtubule‐dependent manner. Through immunostaining, tubulin accumulation during this reorganization is observable on the myoplasm cortex. However, more detailed morphological features of microtubules remain relatively unknown. In this study, we invented a new reagent that improves the immunostaining of cortical microtubules and successfully visualized a parallel array of thick microtubules. During reorganization, they covered nearly the entire myoplasm cortical region, beneath the posterior‐vegetal cortex. We designated this microtubule array as CAMP (cortical array of microtubules in posterior vegetal region). During the late phase of reorganization, CAMP shrank and the myoplasm formed a crescent‐like cytoplasmic domain. When the CAMP formation was inhibited by sodium azide, myoplasmic reorganization and A–P axis formation were both abolished, suggesting that CAMP is important for these two processes.