Induction of increase in intracellular calcium concentration of embryonic cells and acceleration of morphogenetic cell movements during amphibian gastrulation by a 50-Hz magnetic field

The influence of an alternating electromagnetic field (EMF) on early development of amphibian embryos was examined. When the embryos developed under the influence of a low-frequency EMF (50 Hz, 5-30 mT), the rate of early development was accelerated. The effect of EMF was exerted preferentially at the gastrula stage, and the period of gastrulation was shortened. Histological observations showed that EMF promoted morphogenetic cell movements during the gastrulation. The concentration of intracellular free Ca2+ ([Ca2+]i) in the embryonic cells under the influence of EMF was analyzed using Fura-2, an indicator of the intracellular concentration of calcium ions. The influence of EMF on [Ca2+]i was analyzed in embryonic cells isolated from blastula, gastrula, and neurula, EMF increased a [Ca2+]i particularly in the cells isolated from gastrula. Our results suggest that EMF specifically increased the [Ca2+]i of gastrula cells, thereby, accelerating the rate of morphogenetic cell movements during gastrulation.     

Early development in utero of bovine nuclear transfer embryos using early G1 and G0 phase cells

Bovine somatic cell nuclear transfer (NT) embryos can develop to normal calves, but the success rates are still quite low. Recently, enhanced development of bovine NT embryos to full term has been achieved using fibroblasts at the early G1 phase instead of cells at the quiescent (G0) phase. In the present study, we examined the morphological development in utero of NT embryos using early G1 phase cells (eG1-NT embryos) and G0 phase cells (G0-NT embryos). We produced eG1- and G0-NT blastocysts, and then they were transferred to recipient heifers for transient development in utero up to day 14 of gestation. In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. The rate of formation of embryonic disks of the recovered embryos was the same among the groups of eG1-NT, IVF, and AI embryos (p>0.05). The formation rate in eG1-NT embryos was significantly higher than that in G0-NT embryos (p<0.05). The lengths of eG1-NT embryos were the same as those of IVF, parthenogenetic, and AI embryos (p>0.05), but significantly shorter than those of G0-NT embryos (p<0.01). We conclude that the morphological development of day 14 embryos derived from eG1-NT embryos was mostly similar to that of AI embryos, but that the morphological development of G0-NT embryos was abnormally large and different from that of AI and eG1-NT embryos.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.     

HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci,induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum ‘N509’

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram-negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease-resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E-deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum ‘N509’. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C-terminal truncated HopAZ1 abolished HopAZ1-dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.     

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales,Rhodophyta) from East Asia

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.     

Effect of circadian rhythm type on serum lipid levels in shift workers: A 5-year cohort study

We investigated how differences in circadian rhythm type affect the health of workers engaged in shift work. Employees, who were newly hired in a steel company between 2007 and 2011, received the Morningness–Eveningness Questionnaire (MEQ) survey. The target participants were 153 male shift workers who were not being treated with any antihyperlipidemic drugs and underwent periodic physical examinations including blood tests at least twice. According to the score of the MEQ at the time of joining the company, we classified the subjects into five types. Longitudinal changes in serum lipid level were estimated among the circadian rhythm types adjusted for age, BMI, and other covariates using a linear mixed model. The regression coefficient of total cholesterol level in the “definitely and moderately morning” group was ?17.83 (95% confidence interval (CI): ?33.42 to ?2.23), and in the “intermediate ‘group’ was ?16.84 [95% CI: ?30.40 to ?3.28], compared to the moderate evening type.” The total cholesterol level was higher in the moderately evening type than in any of the other groups. Between the Morningness–Eveningness (ME) type and Low-density lipoprotein (LDL) cholesterol levels, compared with the “moderately evening type” group, the regression coefficient in the “intermediate type” group was ?16.08 (95% CI: ?28.79 to ?3.37), and in the “definitely and moderately morning type” group was ?17.50 [95% CI: ?32.11 to ?2.88]. The “moderately evening type” group had a higher LDL cholesterol level than any of the other groups. Evening-type circadian rhythm type shift workers are more prone to elevated serum lipid levels.     

Activation of human T lymphocytes by ganglioside-containing liposomes

We investigated the in vitro stimulatory effect of ganglioside (G_M3, G_D1a, G_D1b, G_T1b, or G_Q1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca²⁺]₁) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The G_D1a- and G_T1b-containing liposomes significantly increased [Ca²⁺]₁ of human T lymphocytes compared with the G_M3-, G_D1b- and G_Q1b-containing ones. The response of CD8⁺ and CD4⁺ cells was significantly higher than that of CD20⁺ cells. Our results show that the increase in [Ca²⁺]_i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8⁺ and CD4⁺ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.     

Thermodynamic analyses of calcium binding to troponin C, calmodulin and parvalbumins by using microcalorimetry

Results of microcalorimetric titrations of calcium-binding proteins with calcium or magnesium have been reviewed and evaluated. Results were analyzed mostly in terms of heat capacity changes, which is most closely related to the structural changes of the molecule on metal binding. Two high-affinity sites of rabbit skeletal troponin C are distinguishable in terms of their affinity to calcium and associated enthalpy changes. Heat capacity changes on calcium binding to one of the two high-affinity sites is negative and is in the range ascribed to the ligand binding. In contrast, that to the other of the high-affinity sites is large and positive, indicating that a substantial area of hydrophobic groups become exposed to the solvent. In frog skeletal troponin C, the anomalous positive heat capacity changes occur in one of the low-affinity calcium-specific sites, so that this may be involved in the regulation of contraction. Unlike skeletal troponin C, both of the two high-affinity sites of cardiac troponin C show negative heat capacity changes. In calmodulin, heat capacity changes are positive but small, indicating that calcium binding may induce clustering of the hydrophobic residues on the surface of the molecule. In parvalbumins, heat capacity changes are negative, characteristic of most ligand binding.