Growth, nitrogen fixation and mineral acquisition of Alnus sieboldiana after inoculation of Frankia together with Gigaspora margarita and Pseudomonas putida

The role of tetrapartite associations among Frankia, Gigaspora margarita (an arbuscular mycorrhizal fungus), Pseudomonas putida (rhizobacterium), and Alnus sieboldiana in growth, nitrogen fixation, and mineral acquisition of A. sieboldiana was investigated. Seedlings of A. sieboldiana were inoculated with Frankia isolated from root nodules of alder, followed by inoculation of G. margarita and P. putida, and were grown for 5 months in a greenhouse. The seedlings inoculated with Frankia and G. margarita together produced the highest biomass of shoots and root nodules. Nitrogen-fixation activity, measured by acetylene reduction assay, was observed when Frankia was inoculated. The activity, on a per-nodule gram basis, decreased after G. margarita inoculation, but on a per-plant basis there was no significant difference in the activity among inoculation treatments. The mineral content in the seedlings changed after inoculation with Frankia, but not after inoculation with P. putida and/or G. margarita. The results showed a synergistic interaction among Frankia, the mycorrhizal fungus, and the rhizobacterium on the growth of A. sieboldiana.     

Prevention of immune responses to human erythropoietin in cynomolgus monkeys (Macaca fascicularis)

Genes and proteins of human origin are often administered to monkeys for research purposes, however, it can be difficult to obtain sufficient levels of the products in vivo due to immunological clearance. In this study, we showed that human erythropoietin (hEPO) induces generation of anti-hEPO antibody in cynomolgus macaques (n=2), although 92% of amino acid residues are common between the human and macaque EPO. The administered hEPO was thus eliminated from the animals. On the other hand, when an immunosuppressant, cyclosporin A (CyA), was administered (6 mg/kg) intramuscularly every other day in combination with hEPO (n=2), no anti-hEPO antibody was generated and high serum levels of hEPO were obtained during administration of hEPO, resulting in an increase in serum hemoglobin levels. No adverse effects associated with CyA were observed. Thus, CyA treatment is useful for prevention of immune responses associated with the administration of human proteins in monkeys.     

Graded regulation of the Kv2.1 potassium channel by variable phosphorylation

Dynamic modulation of ion channels by phosphorylation underlies neuronal plasticity. The Kv2.1 potassium channel is highly phosphorylated in resting mammalian neurons. Activity-dependent Kv2.1 dephosphorylation by calcineurin induces graded hyperpolarizing shifts in voltage-dependent activation, causing suppression of neuronal excitability. Mass spectrometry-SILAC (stable isotope labeling with amino acids in cell culture) identified 16 Kv2.1 phosphorylation sites, of which 7 were dephosphorylated by calcineurin. Mutation of individual calcineurin-regulated sites to alanine produced incremental shifts mimicking dephosphorylation, whereas mutation to aspartate yielded equivalent resistance to calcineurin. Mutations at multiple sites were additive, showing that variable phosphorylation of Kv2.1 at a large number of sites allows graded activity-dependent regulation of channel gating and neuronal firing properties.     

Pigment pattern formation by contact-dependent depolarization

Although recent experimental studies have suggested that the interactions among the pigment cells play a key role in the skin pattern formation, details of the mechanism remain largely unknown. By using an in vitro cell culture system, we have detected interactions between the two pigment cell types, melanophores and xanthophores, in the zebrafish skin. During primary culture, the melanophore membrane transiently depolarizes when contacted with the dendrites of a xanthophore. This depolarization triggers melanophore migration to avoid further contact with the xanthophores. Cell depolarization and repulsive movement were not observed in pigment cells with the jaguar mutant, which shows defective segregation of melanophores and xanthophores. The depolarization-repulsion of wild-type pigment cells may explain the pigment cell behaviors generating the stripe pattern of zebrafish.     

Agronomic traits and gene containment capability of cleistogamous rice lines with the superwoman1-cleistogamy mutation

Pollen-mediated transgene flow is a major concern for the production of genetically modified (GM) rice. Cleistogamy is a useful tool for preventing this form of gene flow. We previously identified the cleistogamous rice mutant superwoman1-cleistogamy (spw1-cls) and determined its molecular genetic mechanism. In the present study, we cultivated spw1-cls over five years to examine effects of cleistogamy on agronomic traits. Simultaneously, we cultivated cleistogamous backcross lines created by continuous backcrossing with “Yumeaoba” (a japonica cultivar) as the recurrent parent and by application of a DNA marker. In these experimental cultivations, spw1-cls and its backcross lines showed almost equal or slightly lower, but acceptable, agronomic traits compared with each control line. We also conducted natural crossing tests in paddy fields to assess the gene containment capability of spw1-cls. In a series of field experiments, there was no natural crossing between spw1-cls (pollen donor) and pollen recipient lines, but the wild-type donor and recipient lines were crossed. Thus, the cleistogamy of the spw1-cls mutation is able to inhibit natural crossing effectively, without significant loss of commercial benefits, such as yield. We conclude that spw1-cls cleistogamy is a practical tool for gene containment in GM rice cultivation.     

Lectin binding pattern of gastric mucosa of pacific white-sided dolphin, Lagenorhynchus obliquidens

The stomach of the Pacific white-sided dolphin is divided into three parts: forestomach, proper gastric gland portion, and pyloric chamber. The histological features of the dolphin stomach are similar to those of terrestrial mammal stomachs, although the distribution of glycoconjugates in mucosal cells of the dolphin stomach is unknown. To learn about glycoconjugates in cetacean gastric mucosa, the glycoconjugate distribution in the mucous epithelium of the Pacific white-sided dolphin was studied using 21 lectins. Among the lectins tested, GSL-I and DBA specifically labelled the superficial layer of the forestomach epithelium. GSL-I, SBA, RCA-I, VVA, GSL-II, DSL, LEL, STL, s-WGA, WGA, PNA, and Jacalin labelled the luminal surface of the chief cells in the proper gastric gland. GSL-I, SBA, RCA-I, DSL, LEL, STL, s-WGA, PNA, and LCA labelled tubular structures in the cytoplasm of parietal cells. The surface portion of the pits in the pyloric chamber strongly reacted with RCA-I, GSL-II, WGA, PNA, LCA, PHA-L, and UEA-I, whereas the neck portion reacted weakly. Although lining one tubular portion, individual secretory cells in the pyloric gland displayed a heterogeneous reaction. This is the first report on the lectin histochemistry of a cetacean stomach and reveals GSL-I and DBA as specific marker lectins for the cornified stratified squamous epithelium cells of the Pacific white-sided dolphin. The stomachs of cetaceans and terrestrial mammals have similar histological features and mucous glycoconjugate content.     

Reference values of hematological and biochemical parameters for the world smallest microminipigs

In this study, we demonstrated growth curves and reference values for hematological and serum biochemical parameters of Microminipigs, the world smallest experimental minipigs. In both male and female animals, the body weights (BWs) at 3 and 6 months of age were <5 kg and <10 kg, respectively, and growth curve revealed almost plateau (approximately 20 kg BW) after 18 months of age. Major hematological and serum biochemical parameters showed no gender differences and the values were very similar to those in G?ttingen and Yukatan minipigs. The values obtained in this study can serve as fundamental reference, and thereby facilitate the use of Microminipig in life science research.     

Estimation of breeding values from large‐sized routine carcass data in Japanese Black cattle using Bayesian analysis

Volumes of official data sets have been increasing rapidly in the genetic evaluation using the Japanese Black routine carcass field data. Therefore, an alternative approach with smaller memory requirement to the current one using the restricted maximum likelihood (REML) and the empirical best linear unbiased prediction (EBLUP) is desired. This study applied a Bayesian analysis using Gibbs sampling (GS) to a large data set of the routine carcass field data and practically verified its validity in the estimation of breeding values. A Bayesian analysis like REML‐EBLUP was implemented, and the posterior means were calculated using every 10th sample from 90 000 of samples after 10 000 samples discarded. Moment and rank correlations between breeding values estimated by GS and REML‐EBLUP were very close to one, and the linear regression coefficients and the intercepts of the GS on the REML‐EBLUP estimates were substantially one and zero, respectively, showing a very good agreement between breeding value estimation by the current GS and the REML‐EBLUP. The current GS required only one‐sixth of the memory space with REML‐EBLUP. It is confirmed that the current GS approach with relatively small memory requirement is valid as a genetic evaluation procedure using large routine carcass data.     

Consecutive monitoring for conidiogenesis by Oidium neolycopersici on tomato leaves with a high-fidelity digital microscope

Conidiogenesis by Oidium neolycopersici KTP-01 on tomato leaves was vitally monitored with a high-fidelity digital microscope. Conidiophores were initially formed 3 days after inoculation and then elongated to a maximum length within at least 12h. The apical part was split into two cells after two successive septations, accompanied by apical expansion. These cells subsequently developed into primary and secondary conidia. An additional septation at the stem portion of the conidiophores produced a generative and a foot cell. Subsequent conidiation occurred during repeated cycles of splitting of the generative cell, maturation of the apical cell into a conidium, and abstriction of the conidium. To our knowledge, this report is the first on the developmental process of conidiogenesis by powdery mildew on host leaves as revealed with the digital microscope.     

Development of an efficient method for recovery of Puumala and Puumala-related viruses by inoculation of Mongolian gerbils

Puumala (PUU) virus and PUU-related viruses are difficult to isolate in cell culture. To determine whether animal inoculation would be a better alternative for virus recovery, the Sotkamo strain of PUU virus was inoculated into several animal species. Newborn Mongolian gerbils (MGs), mice, and rats were infected with the Sotkamo strain by intracerebral (ic), intraperitoneal (ip), and subcutaneous (sc) inoculation. Antibodies to PUU appeared in MGs at 30 days post-infection (dpi), and in mice and rats at 15 dpi. Interestingly, virus appeared at 7 dpi in lung and brain of MGs inoculated via ic and ip routes. Virus was detected in all tested tissues of MGs at 15 dpi, with a peak level of 1.36 x 10 (5) focus forming units (FFU)/g in brain tissue. The virus titer declined with the onset of the antibody response and became undetectable by 75 dpi, when the antibody titer reached the maximum level. The appearance of the virus in mice and rats was delayed as compared to MGs, and the virus titer was apparently lower, at approximately 4 to 8 x 10(3) FFU/g, at 15 dpi. In addition, lung homogenates of antibody-positive Clethrionomys (C.) rufocanus (captured in Tobetsu, Hokkaido, Japan) were inoculated into MGs by the ic route. PUU-related viral RNA was detected at 16 dpi in the brains of MG inoculated with the lung homogenate, and antibodies were detected at 45 dpi. These findings indicate that newborn MG inoculation is an efficient method to recover PUU and PUU-related viruses.