Effect of ramelteon on insomnia severity: evaluation of patient characteristics affecting treatment response

Sleep and Biological Rhythms - We conducted a multicenter, open-label, observational study to evaluate the effectiveness and safety of ramelteon in patients with insomnia, and patient...     

Electrochemical characteristics of a hyperthermophilic enzyme in microdroplets stirred and heated by surface acoustic waves

Micro total analysis system (μTAS) is expected to be applied in various fields. In particular, since electrochemical measurement is inexpensive and easy, electrochemical measurement can be integrated with a microchannel. However, electrochemical detection sensitivity in a microchannel is lowered because the diffusion of the detection target is limited. In an ordinary electrochemical detection system, using a stirrer in a beaker can overcome limited diffusion. We previously proposed a new detection system that combines a microliquid solution agitation technology using surface acoustic waves (SAWs) with the μTAS. The SAWs function as microstirrers, thus making electrochemical detection possible by overcoming limited diffusion of the sample. However, when the solution is stirred by the SAWs, the temperature of the solution increases to 70°C due to vibrational energy. This leads to enzyme inactivation and impaired electrochemical response. Therefore, in this study, we used a hyperthermophile-derived enzyme. Temperature and electrochemical characteristics of the detection system using SAWs and a multi-copper oxidase (MCO) derived from the hyperthermophilic archaea Pyrobaculum aerophilum were studied. Laccase, which is an MCO derived from the thermophilic fungus Trametes versicolor, was also studied. We also characterized the enzyme-electrochemical reaction using SAWs by comparing the magnitude of the reduction current obtained using the two enzymes with different heat resistances. We observed an increase in the electrochemical response with the SAWs, without impaired enzyme activity. Thus, the use of a thermostable enzyme is suitable for the development of a biosensor that uses SAWs for agitation.     

Integrated effects of thermal acclimation and challenge temperature on cellular immunity in the plusiine moth larvae Chrysodeixis eriosoma (Lepidoptera: Noctuidae)

Temperature is one of the most influential factors for animals. The acclimation (rearing) and challenge temperatures are often more important than the given temperature per se. These effects on physiological responses have been known, but not well understood on immune responses. Here, we investigated the integrated effects of rearing and challenge temperatures on haemocyte populations in larvae of a plusiine moth, Chrysodeixis eriosoma. We hypothesize that the haemocyte concentration is decreased (increased) at higher (lower) temperatures from rearing temperatures and that the proportions of haemocyte types exhibit directional changes at higher (lower) temperatures to compensate for immune reactions. We expect that increasing (decreasing) the challenge temperature from the rearing temperature enhances (reduces) phagocytic activity. We found that higher temperatures slightly decreased the haemocyte concentration. We detected small changes in the proportions of haemocyte types among rearing temperatures, but the changes were non‐directional and most of them were statistically insignificant. We also found the integrated effects only with increases in the challenge temperatures, which resulted in increased phagocytosis, whereas no apparent reactions were detected with decreases in the challenge temperatures. Our results show that the haemocyte concentration is significantly affected by the rearing temperature, which implies that haematopoiesis depends on the ambient temperature. We discuss some adaptive and non‐adaptive components for the positive integrated effects of increases in the challenge temperatures. We also discussed the obtained non‐responsiveness in the integrated effects with decreases in the challenge temperatures.     

Fluorescence detection of serum albumin with a turnover-based sensor utilizing Kemp elimination reaction

The Kemp elimination reaction is a well-known chemical reaction that is facilitated on a protein surface microenvironment, and in particular is highly accelerated in a unique binding pocket of serum albumin. We have designed and synthesized a fluorescently activatable coumarin derivative with a benzisoxazole scaffold to enable monitoring of the Kemp elimination reaction in terms of fluorescence change for the first time. We show that this fluorescent sensor can sensitively and selectively quantitate serum albumin in blood samples. It also works in a dry-chemistry format.     

Non-homologous end-joining for repairing I-SceI-induced DNA double strand breaks in human cells

DNA double strand breaks (DSBs) are usually repaired through either non-homologous end-joining (NHEJ) or homologous recombination (HR). While HR is basically error-free repair, NHEJ is a mutagenic pathway that leads to deletion. NHEJ must be precisely regulated to maintain genomic integrity. To clarify the role of NHEJ, we investigated the genetic consequences of NHEJ repair of DSBs in human cells. Human lymphoblastoid cell lines TSCE5 and TSCE105 have, respectively, single and double I-SceI endonuclease sites in the endogenous thymidine kinase gene (TK) located on chromosome 17q. I-SceI expression generated DSBs at the TK gene. We used the novel transfection system (Amaxa Nucleofector) to introduce an I-SceI expression vector into the cells and randomly isolated clones. We found mutations involved in the DSBs in the TK gene in 3% of TSCE5 cells and 30% of TSCE105 cell clones. Most of the mutations in TSCE5 were small (1-30bp) deletions with a 0-4bp microhomology at the junction. The others consisted of large (>60) bp deletions, an insertion, and a rearrangement. Mutants resulting from interallelic HR also occurred, but infrequently. Most of the mutations in TSCE105, on the other hand, were deletions that encompassed the two I-SceI sites generated by NHEJ at DSBs. The sequence joint was similar to that found in TSCE5 mutants. Interestingly, some mutants formed a new I-SceI site by perfectly joining the two original I-SceI sites without deletion of the broken-ends. These results support the idea that NHEJ for repairing I-SceI-induced DSBs mainly results in small or no deletions. Thus, NHEJ must help maintain genomic integrity in mammalian cells by repairing DSBs as well as by preventing many deleterious alterations.     

Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase

Bacterial UMP kinases do not exhibit any sequence homology with other nucleoside monophosphate kinases described so far, and appear under oligomeric forms, submitted to complex regulation by nucleotides. We propose here a structural model of UMP kinase from Escherichia coli based on the conservation of the fold of carbamate kinase whose crystal structure was recently solved. Despite sequence identity of only 18% over 203 amino acids, alignment of UMP kinase from E. coli with carbamate kinase from Enterococcus faecalis by hydrophobic cluster analysis and threading suggested the conservation of the overall structure, except for a small subdomain (absent in UMP kinase). The modelled dimer suggested conservation of the dimer interface observed in carbamate kinase while interaction of UMP kinase with a monoclonal antibody (Mab 44-2) suggests a three in-plane dimer subunit arrangement. The model was analyzed in light of various modified forms of UMP kinase obtained by site-directed mutagenesis.     

Molecular genetic and chemotaxonomic characterization of the terrestrial cyanobacterium Nostoc commune and its neighboring species

The phylogeny of the terrestrial cyanobacterium Nostoc commune and its neighboring Nostoc species was studied using molecular genetic and chemotaxonomic approaches. At least eight genotypes of N. commune were characterized by the differences among 16S rRNA gene sequences and the petH gene encoding ferredoxin-NADP? oxidoreductase and by random amplified polymorphic DNA analysis. The genotypes of N. commune were distributed in Japan without regional specificity. The nrtP gene encoding NrtP-type nitrate/nitrite permease was widely distributed in the genus Nostoc, suggesting that the occurrence of the nrtP gene can be one of the characteristic features that separate cyanobacteria into two groups. The wspA gene encoding a 36-kDa water stress protein was only found in N. commune and Nostoc verrucosum, suggesting that these Nostoc species that form massive colonies with extracellular polysaccharides can be exclusively characterized by the occurrence of the wspA gene. Fifteen species of Nostoc and Anabaena were investigated by comparing their carotenoid composition. Three groups with distinct patterns of carotenoids were related to the phylogenic tree constructed on the basis of 16S rRNA sequences. Nostoc commune and Nostoc punctiforme were clustered in one monophyletic group and characterized by the occurrence of nostoxanthin, canthaxanthin, and myxol glycosides.