Devising Assisted Reproductive Technologies for Wild-Derived Strains of Mice: 37 Strains from Five Subspecies of Mus musculus

Wild-derived mice have long offered invaluable experimental models for mouse genetics because of their high evolutionary divergence from laboratory mice. A number of wild-derived strains are available from the RIKEN BioResource Center (BRC), but they have been maintained as living stocks because of the unavailability of assisted reproductive technology (ART). In this study, we sought to devise ART for 37 wild-derived strains from five subspecies of Mus musculus maintained at the BRC. Superovulation of females was effective (more than 15 oocytes per female) for 34 out of 37 strains by treatment with either equine chorionic gonadotropin or anti-inhibin serum, depending on their genetic background (subspecies). The collected oocytes could be fertilized in vitro at mean rates of 79.0% and 54.6% by the optimized protocol using fresh or frozen-thawed spermatozoa, respectively. They were cryopreserved at the 2-cell stage by vitrification with an ethylene glycol-based solution. In total, 94.6% of cryopreserved embryos survived the vitrification procedure and restored their normal morphology after warming. A conventional embryo transfer protocol could be applied to 25 out of the 35 strains tested. In the remaining 10 strains, live offspring could be obtained by a modified embryo transfer protocol using cyclosporin A treatment and co-transfer of ICR (laboratory mouse strain) embryos. Thus, ART for 37 wild-derived strains was devised successfully and is now routinely used for their preservation and transportation. The information provided here might facilitate broader use and wider distribution of wild-derived mice for biomedical research.     

Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91(phox)) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca(2+). However, the significance of Ca(2+) binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca(2+) ionophore that induces Ca(2+) influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca(2+) binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.     

Novel anticancer agents, kayeassamins A and B from the flower of Kayea assamica of Myanmar

The CHCl(3)-soluble fraction of 70% EtOH extract of the flower of Kayea assamica completely killed human pancreatic PANC-1 cancer cells preferentially under nutrient-deprived conditions at 1 microg/mL. Bioassay-guided fractionation and isolation afforded two novel compounds, kayeassamins A (1) and B (2). Their structures were elucidated using extensive spectroscopic methods and the modified Mosher method. Each compound showed 100% preferential cytotoxicity (PC(100)) against PANC-1 cells under nutrient-deprived conditions at 1 microM. Furthermore, both compounds inhibited the migration of PANC-1 cells in the wound closure assay.     

Differences in parasitism of Meloidogyne incognita and two genotypes of M. arenaria on Solanum torvum in Japan

Two genotypes of root‐knot nematode, Meloidogyne arenaria (A2‐O and A2‐J), are found in Japan. They were distinguished from each other based on mitochondrial DNA sequences. The primer set (C2F3/1108) amplified a 1.7‐kb fragment from A2‐J, whereas a 1.1‐kb fragment was amplified from A2‐O. M. arenaria (A2‐O) was detected in local regions of southern Japan, whereas M. arenaria (A2‐J) was widespread from the Kyushu region to the Tohoku region. The distribution of M. arenaria (A2‐J) overlaps with the cultivation area of eggplant. Solanum torvum is used worldwide as a rootstock for eggplant cultivation, and it is resistant to Meloidogyne spp. In particular, it is reported that S. torvum is resistant to M. arenaria outside Japan. In this study, we inoculated S. torvum rootstock cultivars with M. arenaria (A2‐J), M. arenaria (A2‐O) and Meloidogyne incognita populations. Although M. incognita and M. arenaria (A2‐O) produced only a few egg masses on S. torvum, thereby confirming its resistance, the four geographical populations of M. arenaria (A2‐J) produced large numbers of egg masses on S. torvum. This study confirmed that S. torvum is resistant to M. incognita and M. arenaria (A2‐O) populations, but susceptible to populations of M. arenaria (A2‐J) in the eggplant production area of Japan.     

Cytoskeletal pattern changes during branch formation in a centrifugedAdiantum protonema

A protonemal branch was induced on a side wall of a fern filamentous protonema by cell centrifugation and subsequent polarized-red light irradiation as described in a previous paper (Wada 1995, J. Plant Res. 108: 501–509). Changes in microtubule (MT) and microfilament (MF) patters during the branch development were observed under fluorescence microscopy. A ring-like band of cortical MTs (MT-ring) and MFs similar to a preprophase band or a subapical ring structure (Murataet al. 1987) appeared transiently at the future branching site before cell swelling, the first visible step of branch formation. At this stage, the nucleus was located far from the branching site and the MT-ring appeared to be connected to the nucleus by endoplasmic MFs as well as with endoplasmic MTs. The MT-ring disappeared when cell wall swelling occurred. When the cell wall swelling began, a fan-like pattern of cortical MTs emanating from the new growing tip was established and the MTs reached the opposite flank of the protonema. When a new branch started to elongate and the nucleus moved into the branch, a faint subapical ring of MTs appeared at the subapical part of the new branch. Strands of MTs and MFs emanating from the nuclear front end reached a part of the subapical ring.     

Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor

We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.     

A role of melatonin in the initial stage of photoperiodism in the Japanese quail

To estimate whether melatonin is involved in gonadal activity in the male quail, the dynamics of plasma melatonin at an early stage of the photoperiodic response were investigated. Nocturnal levels of melatonin were manipulated by treatment with anti-melatonin (anti-M). By means of 4 additional hours of photic stimulation of the brain (provided by a red light-emitting diode inserted through the back of the head) after the environmental lights (8L:16D, lights-on, 1000 h) were turned off, the elevation of levels of melatonin after lights-off was significantly suppressed on Days 1 and 2 (p less than 0.01); after 5 days of brain-lighting, gonadal growth first became noticeable. However, 4 h of brain-lighting before lights-on elicited no change in levels of melatonin or in gonadal growth. The injections of anti-M just before lights-off (at 1800 h) for the first 3 days caused significant gonadal growth (p less than 0.01), whereas injections at 2200, 0200, or 0600 h were without effect. In addition, 4 h of brain-lighting before lights-on became gonadostimulatory (p less than 0.01) when it was accompanied by the injection of anti-M at 1800 h, but remained without effect when anti-M was injected at 0600 h. These results suggest that melatonin is involved in the initial stage of photoperiodism in birds, and the timing of suppression of the elevation of melatonin levels is critical in gonadal development.     

Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase

Both vanadate and hydrogen peroxide (H2O2) are known to have insulin-mimetic effects. We previously reported that the mixture of vanadate plus H2O2 results in the generation of a peroxide(s) of vanadate, which strongly enhances IGF-II binding to rat adipocytes (Kadota et al., 1987b). We now report that pervanadate mimics insulin in isolated rat adipocytes to (1) stimulate lipogenesis, (2) inhibit epinephrine-stimulated lipolysis, and (3) stimulate protein synthesis. The efficacy of pervanadate is comparable to that of insulin. However, it is 10(2)-10(3) times more potent than vanadate alone. Exposure of intact rat adipocytes to pervanadate was found to activate the WGA-purified insulin receptor tyrosine kinase assayed with the exogenous substrate poly(Glu80/Tyr20) in a dose-dependent manner to a maximum of 1464% of control at 10(-3) M compared with a maximum insulin effect of 1046% at 10(-6) M. In contrast, in vitro assayed autophosphorylation of the WGA-purified extract was increased 3-fold after exposure of intact cells to insulin but not significantly increased after pervanadate. Furthermore, high concentrations of pervanadate (10(-5) M) inhibited subsequent in vitro added insulin-stimulated autophosphorylation. In vitro addition of pervanadate to WGA-purified receptors could not stimulate autophosphorylation or exogenous tyrosine kinase activity and did not inhibit insulin-stimulated autophosphorylation. Labeling of intact adipocytes with [32P]orthophosphate followed by exposure to 10(-4) M pervanadate increased insulin receptor beta-subunit phosphorylation (7.9 +/- 3.0)-fold, while 10(-7) M insulin and 10(-4) vanadate increased labeling (5.3 +/- 1.8)- and (1.1 +/- 0.2)-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blue- and red-light action in photoorientation of chloroplasts in Adiantum protonemata

An action spectrum for the low-fluencerate response of chloroplast movement in protonemata of the fern Adiantum capillus-veneris L. was determined using polarized light vibrating perpendicularly to the protonema axis. The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm, the blue peaks being higher than the red one. The red-light action was suppressed by nonpolarized far-red light given simultaneously or alternately, whereas the bluelight action was not. Chloroplast movement was also induced by a local irradiation with a narrow beam of monochromatic light. A beam of blue light at low energy fluence rates (7.3·10^-3-1.0 W m^-2) caused movement of the chloroplasts to the beam area (positive response), while one at high fluence rates (10 W m^-2 and higher) caused movement to outside of the beam area (negative response). A red beam caused a positive response at fluence rates up to 100 W m^-2, but a negative response at very high fluence rates (230 and 470 W m^-2). When a far-red beam was combined with total background irradiation with red light at fluence rates causing a low-fluence-rate response in whole cells, chloroplasts moved out of the beam area. When blue light was used as background irradiation, however, a narrow far-red beam had no effect on chloroplast distribution. These results indicate that the light-oriented movement of Adiantum chloroplasts is caused by red and blue light, mediated by phytochrome and another, unidentified photoreceptor(s), respectively. This movement depends on a local gradient of the far-red-absorbing form of phytochrome or of a photoexcited blue-light photoreceptor, and it includes positive and negative responses for both red and blue light.Abbreviations BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - UV ultraviolet