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.     

Delayed sleep/wake rhythm and excessive daytime sleepiness correlate with decreased daytime brain activity during cognitive task in university students

Insufficient sleep and irregular sleep/wake rhythm are common problems among university students. We investigated the effect of sleep/wake rhythm and excessive daytime sleepiness (EDS) on the cortical oxygenation as measured by near-infrared spectroscopy (NIRS) and cognitive performance in university students. Peak- and integral values by a word fluency task were measured with NIRS. EDS was evaluated by the Epworth sleepiness scale (ESS), and performance function was evaluated using N-back task. Peak cerebral oxygenation was significantly correlated with ESS, bedtime, wake-up time, and median time of sleep. Accuracy on 2-back task was significantly correlated with integral value. Peak- and integral values were significantly lower, and bedtime and median time of sleep were significantly delayed in the EDS group than in the non-EDS group. EDS accompanied by delayed sleep/wake rhythm and short sleep duration may play an important role in decreasing daytime brain activity and cognitive performance.     

Reclassification of Pichia scaptomyzae and Pichia galeiformis

Based on the base composition of nuclear DNA and DNA/DNA hybridization, Pichia galeiformis IFO 10718T was reclassified as a synonym of Pichia manshurica, and Pichia scaptomyzae IFO 1073 1T was confirmed to be a synonym of Pichia membranifaciens. Comparison of 18S rRNA gene sequences indicated that IFO 10731T (P. scaptomyzae) is identical to P. membranifaciens IFO 10215T and IFO 10725, and IFO 10718T (P. galeiformis) is identical to P. manshurica IFO 10726T. These data were consistent with the view that P scaptomyzae and P membranifaciens should be conspecific, as should P. galeiformis and P manshurica. Variation among 26S rRNA gene domain D1/D2 sequences from three P membranifaciens strains indicated that this species encompasses a genetically heterogeneous population.     

Effects of protein transport inhibitors on the distribution and secretion of the fusion protein RntA-EGFP in Aspergillus oryzae

The distribution of the secreted protein ribonuclease T1 (RntA) fused with the enhanced green fluorescent protein (EGFP), RntA-EGFP, was visualized in hyphae of Aspergillus oryzae in the presence of a protein transport inhibitor, brefeldin A, cytochalasin A, or nocodazole. During treatment with the protein transport inhibitors, the distribution of RntA-EGFP changed and distinct patterns of fluorescence accumulation were observed. The addition of brefeldin A caused RntA-EGFP fluorescence to appear in reticular networks, and the disruption of the polymerization of actin filaments by cytochalasin A caused an increase in RntA-EGFP fluorescence intensity in the hyphae without accumulation in a specific cellular component. In contrast, RntA-EGFP fluorescence was distributed in different parts of a hypha during treatment with nocodazole, a compound that depolymerizes microtubules. In addition, quantitative analysis was performed using the RntA-EGFP visualization system to analyze the relative amount of RntA-EGFP secreted into the culture medium during treatment with the protein transport inhibitors.     

Oocyte mitochondria: Strategies to improve embryogenesis

Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.     

Synthesis of anilino-monoindolylmaleimides as potent and selective PKCbeta inhibitors

We report herein synthesis of PKCbeta-selective inhibitors possessing the novel pharmacophore of anilino-monoindolylmaleimide. Several compounds of this series exhibited IC50's as low as 50 nM against human PKCbeta2. One of the most potent compounds, 6l, inhibited PKCbeta1 and PKCbeta2 with IC50 of 21 and 5 nM, respectively, and exhibited selectivity of more than 60-fold in favor of PKCbeta2 relative to other PKC isozymes (PKCalpha, PKCgamma, and PKCepsilon).     

Reactive oxygen species impair Slo1 BK channel function by altering cysteine-mediated calcium sensing

Vascular dysfunction is a hallmark of many diseases, including coronary heart disease, stroke and diabetes. The underlying mechanisms of these disorders, which are intimately associated with inflammation and oxidative stress caused by excess reactive oxygen species (ROS), have remained elusive. Here we report that ROS are powerful inhibitors of vascular smooth muscle calcium-dependent Slo1 BK or Maxi-K potassium channels, an important physiological determinant of vascular tone. By targeting a cysteine residue near the Ca(2+) bowl of the BK alpha subunit, H(2)O(2) virtually eliminates physiological activation of the channel, with an inhibitory potency comparable to a knockout of the auxiliary subunit BK beta 1. These results reveal a molecular structural basis for the vascular dysfunction involving oxidative stress and provide a solid rationale for a potential use of BK openers in the prevention and treatment of cardiovascular disorders.