Quantitative analysis of the kinetics of phospholipase A2 using fast atom bombardment mass spectrometry

Fast atom bombardment mass spectrometry that can directly analyze lysophospholipids was used to quantitatively determine the kinetics of phospholipase A2. This method is 1250 times more sensitive than the colorimetric assay.     

New lead compounds for brassinosteroid biosynthesis inhibitors

The first brassinosteroid biosynthesis inhibitor is reported. Among newly synthesized triazole derivatives, 4-(4-chlorophenyl)-2-phenyl-3-(1,2,4-triazoyl)butan-2-ol (6) was found to inhibit the growth of cress seedlings, and this inhibition was recovered by the treatment of brassinolide, suggesting that compound 6 primarily inhibits brassinosteroid biosynthesis.     

Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization

With the development of a direct visualization of sex chromosome in a single sperm by fluorescence in situ hybridization (FISH) technique, the frequency of aberration (aneuploidy) in spermatozoa in several mammals has been investigated. However, there is no report in the incidence of X-Y aneuploidy in the sperm population of dogs. Therefore, in this study, the aneuploidy in dog spermatozoa was examined by multicolor FISH using specific molecular probes for canine sex chromosomes and autosome. Semen from eight male Labrador retrievers was used as specimen. For decondensation of sperm nuclei, the specimen was treated with 1 M NaOH for 4 minutes at room temperature. Probes for chromosomes X, Y, and 1, labeled with SpectrumGreen, Cy3 and Cy5, respectively, were hybridized with decondensed spermatozoa. Fluorescence in situ hybridization signals in sperm heads were clearly detected in each specimen, regardless of the sperm donor. The FISH signal of at least one of the three probes was detected in all sperm heads examined. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X and Y chromosomes in spermatozoa of all the eight dogs. Mean percentage of sex chromosome aneuploidy was 0.127% (ranged between 0% and 0.316%). This percentage of canine sex chromosome aneuploidy was lower than the one reported in cattle, horses, river buffalo, and goats sperm, but higher than that observed in mice and sheep.     

Anaphase DNA bridges induced by lack of RecQ5 in Drosophila syncytial embryos

Drosophila melanogaster RecQ5, a member of the RecQ family, is expressed in early embryos. The loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos. We demonstrate that the mitotic defects are derived from anaphase DNA bridges. Pairs of daughter nuclei that had been linked by the bridges concurrently exited from the cycle and were eliminated by Chk2-dependent centrosome inactivation. These results suggest that the lack of RecQ5 leads to spontaneous double-stranded DNA breaks (DSBs). RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during interphase in syncytial Drosophila embryos.     

Conservation of a gliding motility and cell invasion machinery in Apicomplexan parasites

Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.     

Design,synthesis, and evaluation of polyamine-memantine hybrids as NMDA channel blockers

N-Methyl-d-aspartate (NMDA) receptors have been implicated in learning and memory, and may also play a central role in various conditions leading to neuronal degradation. NMDA receptor antagonists could therefore be of therapeutic benefit for a number of neurological disorders. We have designed hybrid compounds of polyamines and memantine, both of which function as NMDA channel blockers. The triamine derivative with a guanidine moiety showed more potent antagonistic activity than memantine.     

Job stress, social support at work, and insomnia in Japanese shift workers

A cross-sectional study was conducted to clarify the contribution of psychological job stress to insomnia in shift workers. A self-administered questionnaire concerning job stress, sleep, depressive symptoms and lifestyle factors was submitted to a sample of 530 rotating shift workers of age 18-59 years (mean age 27) in an electric equipment manufacturing company. Perceived job stress, i.e., job demands, job control and social support at work, was assessed using the Japanese version of the Job Content Questionnaire. Insomnia was regarded as prevalent if the workers had at least one of the following symptoms in the last year; less than 30 minutes to fall asleep, difficulty in maintaining sleep, or early morning awakening almost everyday. Overall prevalence was 37.8%. Logistic regression analyses while adjusting relevant factors showed that lower social support at work was significantly associated with a greater risk of insomnia than the higher social support (adjusted OR 2.5). Higher job strain with lower social support at work increased the risk, compared to lower strain with higher support at work (crude OR 1.8; adjusted OR 1.5). Our findings suggest the low social support at work independently associated with insomnia in shift workers.     

Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type

We examined whether auxin/indole-3-acetic acid (Aux/IAA) proteins, which are key players in auxin-signal transduction, are involved in brassinosteroid (BR) responses. iaa7/axr2-1 and iaa17/axr3-3 mutants showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ-dependent manner. Two auxin inhibitors were tested in terms of BR responses. Yokonolide B inhibited BR responses, whereas p-chlorophenoxyisobutyric acid did not inhibit BR responses. DNA microarray analysis revealed that 108 genes were up-regulated, while only eight genes were down-regulated in iaa7. Among the genes that were up- or down-regulated in axr2, 22% were brassinolide-inducible genes, 20% were auxin-inducible genes, and the majority were sensitive neither to BR nor to auxin. An inhibitor of BR biosynthesis, brassinazole, inhibited auxin induction of the DR5-GUS gene, which consists of a synthetic auxin-response element, a minimum promoter, and a beta-glucuronidase. These results suggest that Aux/IAA proteins function in auxin- and BR-signaling pathways, and that IAA proteins function as the signaling components modulating BR sensitivity in a manner dependent on organ type.     

Enhanced expression of human cDNA by phosphoglycerate kinase promoter-puromycin cassette in the mouse transthyretin locus

To produce a humanized mouse, it is critical to obtain a correct expression of a human gene/cDNA after insertion into a mouse locus. We previously generated a targeted allele in which the PGK-neo cassette, flanked by lox71 and loxP, was inserted into the first exon of the mouse endogenous transthyretin (Ttr) gene in ES cells. Using these ES cells, we showed that a human transthyretin (TTR) cDNA with the PGK-puro cassette can be efficiently inserted into this locus by Cre-mediated recombination, and that the human TTR cDNA was expressed in a tissue-specific manner under the control of the mouse endogenous Ttr promoter. To examine whether the PGK-puro cassette or IRES could affect the expression of human TTR cDNA, we generated four mouse lines using Cre and Flp-mediated recombination. The mouse line containing the PGK-puro cassette, but not IRES, exhibited quantitatively and temporally similar expression of human TTR cDNA. Removal of the PGK-puro cassette significantly downregulated the expression of the cDNA. The insertion of IRES sequence upstream of the human TTR cDNA resulted in decreased expression, even in the presence of the PGK-puro cassette. The mouse line containing IRES, but not PGK-puro, showed the lowest level of expression. These results suggest that the PGK-puro cassette is necessary to obtain the enhanced expression of a co-existing human cDNA in the mouse Ttr locus, even though the expression of co-existing cDNA was under the control of the mouse endogenous promoter.