Inhibitory Effect on RT-PCR and Restriction Enzyme Activity by Ommochrome and Its Mechanism.

To explore the physiological role and/or pharmacological effects of ommochrome, which is a natural organic pigment widely distributed in Protostomia, we attempted to investigate the influence of ommochrome on RT-PCR and activities of restriction enzymes. It was found that ommin, an ommochrome purified from the diapause eggs of Bombyx mori, inhibited the RT-PCR and restriction enzyme activities. The mechanism of these inhibitory reactions is assumed to be the direct binding of ommochrome to DNA rather than acting against the enzymes because, similarly to actinomycin D, there is a phenoxazine ring in the structure of ommin that is known to be intercalated to DNA. To reveal the ommin/DNA interaction, it was investigated by computational approaches such as molecular docking, molecular dynamics simulation, and free energy calculation. From the computational analyses, it was expected that ommin would bind to DNA with almost the same strength as actinomycin D and intercalate into DNA. This is the first report on the pharmacological effect of ommochrome and its inhibitory mechanism obtained from biochemical and computational analyses.

Comparative Studies on Electrodes for Rumen Bacteria Microbial Fuel Cells.

Microbial fuel cells (MFCs) using rumen bacteria have been proposed as a power source for running devices inside cattle. In this study, we explored the key parameters of the conventional bamboo charcoal electrode in an attempt to improve the amount of electrical power generated by the microbial fuel cell. We evaluated the effects of the electrode's surface area, thickness, and rumen content on power generation and determined that only the electrode's surface area affects power generation levels. Furthermore, our observations and bacterial count on the electrode revealed that rumen bacteria concentrated on the surface of the bamboo charcoal electrode and did not penetrate the interior, explaining why only the electrode's surface area affected power generation levels. A Copper (Cu) plate and Cu paper electrodes were also used to evaluate the effect of different electrodes on measuring the rumen bacteria MFC's power potential, which had a temporarily higher maximum power point (MPP) compared to the bamboo charcoal electrode. However, the open circuit voltage and MPP decreased significantly over time due to the corrosion of the Cu electrodes. The MPP for the Cu plate electrode was 775 mW/m2 and the MPP for the Cu paper electrode was 1240 mW/m2, while the MPP for bamboo charcoal electrodes was only 18.7 mW/m2. In the future, rumen bacteria MFCs are expected to be used as the power supply of rumen sensors.

Demonstration of a spherical plasma mirror for the counter-propagating kilojoule-class petawatt LFEX laser system.

A counter-propagating laser-beam platform using a spherical plasma mirror was developed for the kilojoule-class petawatt LFEX laser. The temporal and spatial overlaps of the incoming and redirected beams were measured with an optical interferometer and an x-ray pinhole camera. The plasma mirror performance was evaluated by measuring fast electrons, ions, and neutrons generated in the counter-propagating laser interaction with a Cu-doped deuterated film on both sides. The reflectivity and peak intensity were estimated as ∼50% and ∼5 × 1018 W/cm2, respectively. The platform could enable studies of counter-streaming charged particles in high-energy-density plasmas for fundamental and inertial confinement fusion research.

Molecular Characterization and Tissue Distribution of Calcineurin Regulatory B Subunit during the Prevention of Diapause in the Silkworm, Bombyx mori.

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed calcineurin regulatory B subunit (CNB) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze CNB, which is known to associate with Ca2+. The gene expression level of CNB was increased by HCl treatment and the changes of the gene expression were almost the same as that in the non-diapause eggs. As for diapause eggs, almost no gene expression of CNB was confirmed except just after oviposition. In the assay for phosphorylation by protein kinase CK2, recombinant CNB (rCNB) was phosphorylated in vitro. Additionally, a Ca2+ binding assay indicated that rCNB shows affinity for Ca2+. The distribution of CNB was investigated with an immunohistochemical technique using antiserum against rCNB in diapause eggs and HCl-treated diapause eggs. CNB was localized in serosa cells and yolk cells in both eggs. These data may suggest that CNB is activated by intracellular Ca2+ or efflux Ca2+ resulting from HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

Petapascal Pressure Driven by Fast Isochoric Heating with a Multipicosecond Intense Laser Pulse.

Fast isochoric laser heating is a scheme to heat matter with a relativistic intensity (>10^{18} W/cm^{2}) laser pulse for producing an ultrahigh-energy-density (UHED) state. We have demonstrated an efficient fast isochoric heating of a compressed dense plasma core with a multipicosecond kilojoule-class petawatt laser and an assistance of externally applied kilotesla magnetic fields for guiding fast electrons to the dense plasma. A UHED state of 2.2 PPa is achieved experimentally with 4.6 kJ of total laser energy that is one order of magnitude lower than the energy used in the conventional implosion scheme. A two-dimensional particle-in-cell simulation confirmed that diffusive heating from a laser-plasma interaction zone to the dense plasma plays an essential role to the efficient creation of the UHED state.

Molecular Characterization and Tissue Distribution of Mitochondrial Ca2+ -Dependent Solute Carrier Protein during Prevention of Diapause by HCl Treatment in the Silkworm, Bombyx mori.

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed mitochondrial Ca2+ -dependent solute carrier protein (MCSC) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze MCSC, which is known to associate with Ca2+ . The isolated cDNA of B. mori MCSC (BmMCSC) had an open reading flame (ORF) of 667 amino acid residues, and the ORF contained two EF-hand calcium-binding domains and three characteristic features of the mitochondrial solute carrier superfamily. The gene expression level of BmMCSC increased by HCl treatment. A Ca2+ binding assay indicated that recombinant BmMCSC (rBmMCSC) shows an affinity with Ca2 + . The distribution of BmMCSC was investigated with an immunohistochemical technique using antisera against BmMCSC in diapause eggs and HCl-treated diapause eggs. BmMCSC was localized in serosa cells in both eggs. These data may suggest that BmMCSC is activated by intracellular Ca2+ or efflux Ca2+ by HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

Li-ion diffusion in Li intercalated graphite C6Li and C12Li probed by µ+SR.

In order to study a diffusive behavior of Li+ in Li intercalated graphites, we have measured muon spin relaxation (µ+SR) spectra for C6Li and C12Li synthesized with an electrochemical reaction between Li and graphite in a Li-ion battery. For both compounds, it was found that Li+ ions start to diffuse above 230 K and the diffusive behavior obeys a thermal activation process. The activation energy (Ea) for C6Li is obtained as 270(5) meV, while Ea = 170(20) meV for C12Li. Assuming a jump diffusion of Li+ in the Li layer of C6Li and C12Li, a self-diffusion coefficient DLi at 310 K was estimated as 7.6(3) × 10-11 (cm2 s-1) in C6Li and 14.6(4) × 10-11 (cm2 s-1) in C12Li.

Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori.

HCl Treatment for Preventing Diapause Causes Ca2+ Efflux in Bombyx mori Eggs.

To elucidate the mechanism for preventing entry into embryonic diapause or breakdown of diapause in Bombyx mori by HCl and dimethyl sulfoxide (DMSO) treatment or a combination of cold and HCl treatment, we performed quantitative analysis of Ca2+ and Mg2+ in the chorion and egg content using inductively coupled plasma atomic emission spectrometry (ICP-AES). When diapause eggs that had been incubated at 25°C for 2 days from oviposition and at 4°C for an additional six days were treated with HCl solution, the amount of Ca2+ in the chorion and egg content after HCl treatment was reduced to one-seventh, as compared with the amount before treatment. In contrast, there was no change in the amount of Mg2+ with HCl treatment. The amount of Ca2+ in the HCl solution after the diapause eggs were treated increased 7.5-fold, as compared with that of eggs treated with water. Even when 17-day-old diapausing eggs were treated with HCl, which did not break diapause, the amount of Ca2+ in the chorion and egg content was reduced to one-fifth, as compared with the control. Meanwhile, changes in Ca2+ and Mg2+ contents were not observed in 12-hr-old diapause-destined eggs before or after treatment with DMSO, which effectively prevents diapause. These data may suggest that Ca2+ efflux from diapause eggs by HCl is not directly associated with preventing entry into diapause or breaking of diapause. In addition, we discovered that the amount of Ca2+ in diapause-destined eggs was more than 2.4-fold larger than in non-diapause-destined eggs.

A single consumption of curry improved postprandial endothelial function in healthy male subjects: a randomized, controlled crossover trial.

BACKGROUND: Curry, one of the most popular foods in Japan, contains spices that are rich in potentially antioxidative compounds, such as curcumin and eugenol. Oxidative stress is thought to impair endothelial function associated with atherosclerosis, a leading cause of cardiovascular events. The aim of this study was to determine whether a single consumption of curry meal would improve endothelial function in healthy men. METHODS: Fourteen healthy male subjects (BMI 23.7 ± 2.7 kg/m2; age 45 ± 9 years) were given a single serving of curry meal or spice-free control meal (180 g of curry or control and 200 g of cooked rice; approximately 500 kcal in total) in a randomized, controlled crossover design. Before and 1 hr after the consumption, fasting and postprandial flow-mediated vasodilation (FMD) responses and other parameters were measured. RESULTS: The consumption of the control meal decreased FMD from 5.8 ± 2.4% to 5.1 ± 2.3% (P = 0.039). On the other hand, the consumption of the curry meal increased FMD from 5.2 ± 2.5% to 6.6 ± 2.0% (P = 0.001), and the postprandial FMD after the curry meal was higher than that after the control meal (P = 0.002). Presence of spices in the curry did not alter significantly the systemic and forearm hemodynamics, or any biochemical parameters including oxidative stress markers measured. CONCLUSIONS: These findings suggest that the consumption of curry ameliorates postprandial endothelial function in healthy male subjects and may be beneficial for improving cardiovascular health. TRIAL REGISTRATION: UMIN Clinical Trials Registry 000012012.

Chronic intake of onion extract containing quercetin improved postprandial endothelial dysfunction in healthy men.

BACKGROUND: Epidemiologic studies have shown that dietary flavonoids reduce the risk of cardiovascular events. Onion is rich in quercetin, a strong antioxidant flavonoid. In some in vitro studies, quercetin improved endothelial function associated with atherosclerosis, a leading cause of cardiovascular events. OBJECTIVE: The aim of this study was to determine whether chronic onion extract intake would improve postprandial endothelial dysfunction induced by an oral maltose load in healthy men. METHODS: Healthy men (44±10 years, n=23) received 4.3 g of onion extract (containing 51 mg of quercetin) once a day for 30 days. Before and after the chronic onion extract intake, fasting and postprandial flow-mediated vasodilation (FMD) responses were measured. RESULTS: Maltose loading significantly decreased FMD both before and after chronic onion extract intake (p=0.000037 and p=0.0035, respectively). The chronic onion extract intake did not significantly affect fasting FMD (p=0.069) but improved the postprandial FMD significantly from 5.1%±2.2% to 6.7%±2.6% (p=0.00015). The chronic onion extract intake did not alter systemic and forearm hemodynamics. CONCLUSION: These findings suggest that chronic onion extract intake ameliorates postprandial endothelial dysfunction in healthy men and may be beneficial for improving cardiovascular health.

Differences in the roles of a glutamine amidotransferase subunit of pyridoxal 5'-phosphate synthase between Bacillus circulans and Bacillus subtilis.

BtrC2 of the butirosin producer Bacillus circulans is a non-catalytic subunit of 2-deoxy-scyllo-inosose (DOI) synthase that is involved in butirosin biosynthesis, and also a homolog of glutamine amidotransferase subunit (PdxT) of pyridoxal 5'-phosphate (PLP) synthase of Bacillus subtilis. BtrC2 has been found to have functions in B. circulans both in primary and secondary metabolism. In this study, we investigated the properties of PdxT of B. subtilis in order to determine whether the property of enzyme stabilization is universal among PdxT homologs. Complementation with PdxT in the btrC2 disruptant of B. circulans restored the growth and short-term production of antibiotics, but long-term production of antibiotics cannot be restored. Additionally, PdxT did not bind physically with or stabilize BtrC. Our results indicate that the function of BtrC2 in secondary metabolism is specific properties, not universal among PdxT homologs.

Simulation of collaborative studies for real-time PCR-based quantitation methods for genetically modified crops.

To study impacts of various random effects and parameters of collaborative studies on the precision of quantitation methods of genetically modified (GM) crops, we developed a set of random effects models for cycle time values of a standard curve-based relative real-time PCR that makes use of an endogenous gene sequence as the internal standard. The models and data from a published collaborative study for six GM lines at four concentration levels were used to simulate collaborative studies under various conditions. Results suggested that by reducing the numbers of well replications from three to two, and standard levels of endogenous sequence from five to three, the number of unknown samples analyzable on a 96-well PCR plate in routine analyses could be almost doubled, and still the acceptable repeatability RSD (RSDr < or = 25%) and the reproducibility RSD (RSDR < 35%) of the collaborative study could be met. Further, RSDr and RSD(R) were found most sensitive to random effects attributable to inhomogeneity among blind replicates, but they were little influenced by those attributable to DNA extractions. The proposed models are expected to be useful for optimizing standard curve-based relative quantitation methods for GM crops by real-time PCR and their collaborative studies.

Developmental changes in the localization of protein kinase CK2 in non-diapause and diapause eggs of the silkworm, Bombyx mori.

To analyze the role of protein kinase CK2 (CK2) during early embryogenesis in non-diapause and diapause of the silkworm, the distribution and localization of Bombyx mori CK2 (BmCK2) were investigated by an immunohistochemical technique using antibodies against the α- and ß-subunits of BmCK2. Both were localized in blastoderm cells of non-diapause and diapause eggs until 24 h after oviposition. More than 24 h after oviposition, however, the distribution of BmCK2 was different in non-diapause and diapause eggs. In non-diapause eggs, BmCK2 was mainly localized in yolk cells. In contrast, in diapause eggs, the localization was mainly observed in germ-band cells. Furthermore, we confirmed that the RNA helicase-like protein that was localized together with BmCK2 in non-diapause eggs was phosphorylated by BmCK2 in vitro. These data suggest that the role of BmCK2 is different in non-diapause and diapause eggs.

Roles of a 20 kDa protein associated with a carbocycle-forming enzyme involved in aminoglycoside biosynthesis in primary and secondary metabolism.

2-Deoxy-scyllo-inosose (DOI) synthase participates in the biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The enzyme is expected to be of industrial use, because it converts a sustainable resource (glucose 6-phosphate) into carbocycle (DOI), which easily aromatizes to yield catechol. In the present study, we clarified the physiological role of a non-catalytic 20 kDa protein, BtrC2, associated with DOI synthase from the butirosin-producer Bacillus circulans. Based on the results of complementation analysis using btrC2 disruptant and western analysis against catalytic 40 kDa protein (BtrC) and BtrC2, it is suggested that BtrC2 has two functions. It is involved in the vitamin B(6) biosynthesis in primary metabolism, like homologous protein (Pdx2) in Bacillus subtilis. Additionally, it takes part in butirosin biosynthesis as stabilizer of DOI synthase by forming a heterodimer with BtrC, and qualifies B. circulans for stable and constant production of butirosin for long periods.

Chronic pancreatitis in farmed pigs fed excessive zinc oxide.

Most of the pigs on a farm in Aichi Prefecture, Japan had chronic diarrhea and severe wasting. The pigs had consumed 8,000 ppm zinc oxide (ZnO) as a feed additive. The pancreas of each of 4 autopsied pigs was less than half the normal size. Acinar cells were considerably decreased. Epithelial duct-like cells were increased and tested positive for cytokeratin AE1/AE3, Ki67, PGP9.5, and Sox9. Pancreatic islet cells were decreased and shrunken. The α and δ cells were relatively decreased, and their distribution was abnormal. Islet cells were positive for PGP9.5. The livers and kidneys had high accumulations of zinc (Zn; 788 µg/g and 613 µg/g, respectively). Copper was deficient in the liver, likely as a result of Zn poisoning. Our immunohistologic examination suggested that the high dose of ZnO could influence the function of islet cells in addition to that of acinar cells. Given that colistin sulfate has been banned as a feed additive in order to reduce antimicrobial use in Japan, the use of ZnO in the livestock industry is expected to increase. Zn supplementation of pig feed must be monitored to prevent Zn poisoning and contamination of soil and water.

Quantum transport evidence of Weyl fermions in an epitaxial ferromagnetic oxide.

Magnetic Weyl semimetals have novel transport phenomena related to pairs of Weyl nodes in the band structure. Although the existence of Weyl fermions is expected in various oxides, the evidence of Weyl fermions in oxide materials remains elusive. Here we show direct quantum transport evidence of Weyl fermions in an epitaxial 4d ferromagnetic oxide SrRuO3. We employ machine-learning-assisted molecular beam epitaxy to synthesize SrRuO3 films whose quality is sufficiently high to probe their intrinsic transport properties. Experimental observation of the five transport signatures of Weyl fermions-the linear positive magnetoresistance, chiral-anomaly-induced negative magnetoresistance, π phase shift in a quantum oscillation, light cyclotron mass, and high quantum mobility of about 10,000 cm2V-1s-1-combined with first-principles electronic structure calculations establishes SrRuO3 as a magnetic Weyl semimetal. We also clarify the disorder dependence of the transport of the Weyl fermions, which gives a clear guideline for accessing the topologically nontrivial transport phenomena.

Gene expression profiling of functional murine embryonic stem cell-derived cardiomyocytes and comparison with adult heart: profiling of murine ESC-derived cardiomyocytes.

Although embryonic stem cell (ESC)-derived cardiomyocytes may be a powerful tool in drug discovery, their potential has not yet been fully explored. Nor has a detailed comparison with adult heart tissue been performed. We have developed a method for efficient production of cardiomyocyte-rich embryoid bodies (EBs) from murine ESCs. Analysis of global gene expression profiles showed that EBs on day 7 and/or 21 of differentiation (d7CMs and d21CMs, respectively) were similar to adult heart tissue for genes categorized as regulators of muscle contraction or voltage-gated ion channel activity, although d21CMs were more mature than d7CMs for contractile components related to morphological structures. Calcium and sodium channel blockers altered Ca2+ transients, and isoproterenol, a beta-adrenergic compound, increased the rate of beating in d7CMs and d21CMs. Our gene analytic system therefore enabled us to identify genes that are expressed in the physiological pathways associated with ion channels and structural components in d7CMs and d21CMs. We conclude that EBs might be of use for the basic screening of drugs that might affect contractile function through ion channels.

Similar compounds searching system by using the gene expression microarray database.

Numbers of microarrays have been examined and several public and commercial databases have been developed. However, it is not easy to compare in-house microarray data with those in a database because of insufficient reproducibility due to differences in the experimental conditions. As one of the approach to use these databases, we developed the similar compounds searching system (SCSS) on a toxicogenomics database. The datasets of 55 compounds administered to rats in the Toxicogenomics Project (TGP) database in Japan were used in this study. Using the fold-change ranking method developed by Lamb et al. [Lamb, J., Crawford, E.D., Peck, D., Modell, J.W., Blat, I.C., Wrobel, M.J., Lerner, J., Brunet, J.P., Subramanian, A., Ross, K.N., Reich, M., Hieronymus, H., Wei, G., Armstrong, S.A., Haggarty, S.J., Clemons, P.A., Wei, R., Carr, S.A., Lander, E.S., Golub, T.R., 2006. The connectivity map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929-1935] and criteria called hit ratio, the system let us compare in-house microarray data and those in the database. In-house generated data for clofibrate, phenobarbital, and a proprietary compound were tested to evaluate the performance of the SCSS method. Phenobarbital and clofibrate, which were included in the TGP database, scored highest by the SCSS method. Other high scoring compounds had effects similar to either phenobarbital (a cytochrome P450s inducer) or clofibrate (a peroxisome proliferator). Some of high scoring compounds identified using the proprietary compound-administered rats have been known to cause similar toxicological changes in different species. Our results suggest that the SCSS method could be used in drug discovery and development. Moreover, this method may be a powerful tool to understand the mechanisms by which biological systems respond to various chemical compounds and may also predict adverse effects of new compounds.

Isolation of dihydrocurcuminoids from cell clumps and their distribution in various parts of turmeric (Curcuma longa).

In addition to well-known curcuminoids, three colored metabolites were isolated from cultured cell clumps that had been induced from buds on turmeric rhizomes. The isolated compounds were identified as dihydro derivatives of curcuminoids, dihydrocurcumin (dihydroCurc), dihydrodesmethoxycurcumin-a (dihydroDMC-a), and dihydrobisdesmethoxycurcumin (dihydroBDMC). The cell clumps did not contain dihydroDMC-b, an isomer of dihydroDMC-a. A comparison of the distribution profiles of curcuminoids and dihydrocurcuminoids in the cell clumps with those in the rhizomes, leaves, and roots revealed the following differences: Unlike rhizomes, the cell clumps, leaves, and roots contained dihydrocurcuminoids as the major colored constituents. Whereas dimethoxy compounds, curcumin and dihydrocurcumin, respectively, were most abundant in the rhizomes and leaves, one of the monomethoxy derivatives, dihydroDMC-a, was found most abundantly in the cell clumps and roots. While both dihydroDMC-a and b were detected in the rhizomes, dihydroDMC-b was not detectable in the cell clumps, leaves, or roots. The occurrence of only one of the two possible isomers of dihydroDMC suggests biosynthetic formation of dihydrocurcuminoids in turmeric.