A RecA-mediated exon profiling method

We have developed a RecA-mediated simple, rapid and scalable method for identifying novel alternatively spliced full-length cDNA candidates. This method is based on the principle that RecA proteins allow to carry radioisotope-labeled probe DNAs to their homologous sequences, resulting in forming triplexes. The resulting complex is easily detected by mobility difference on electrophoresis. We applied this exon profiling method to four selected mouse genes as a feasibility study. To design probes for detection, the information on known exonic regions was extracted from public database, RefSeq. Concerning the potentially transcribed novel exonic regions, RNA mapping experiment using Affymetrix tiling array was performed. As a result, we were able to identify alternative splice variants of Thioredoxin domain containing 5, Interleukin1β, Interleukin 1 family 6 and glutamine-rich hypothetical protein. In addition, full-length sequencing demonstrated that our method could profile exon structures with >90% accuracy. This reliable method can allow us to screen novel splice variants from a huge number of cDNA clone set effectively.     

Association of the -112A>C polymorphism of the uncoupling protein 1 gene with insulin resistance in Japanese individuals with type 2 diabetes

The -112A>C polymorphism (rs10011540) of the gene for uncoupling protein 1 (UCP1) has been associated with type 2 diabetes mellitus in Japanese individuals. The aim of the present study was to investigate the effects of this polymorphism, as well as the well-known -3826A>G polymorphism (rs1800592), on clinical characteristics of type 2 diabetes. We determined the genotypes of the two polymorphisms in 93 Japanese patients with type 2 diabetes. Intramyocellular lipid content and hepatic lipid content (HLC) were measured by magnetic resonance spectroscopy. No significant differences in age, sex, BMI, or HbA1c level were detected between type 2 diabetic patients with the -112C allele and those without it. However, homeostasis model assessment for insulin resistance (p=0.0089) and HLC (p=0.012) was significantly greater in patients with the -112C allele. We did not detect an association of the -3826A>G polymorphism (rs1800592) of UCP1 gene with any measured parameters. These results suggest that insulin resistance caused by the -112C allele influences the susceptibility to type 2 diabetes.     

Reduced responsiveness is an essential feature of chronic fatigue syndrome: A fMRI study

Background  

Although the neural mechanism of chronic fatigue syndrome has been investigated by a number of researchers, it remains poorly understood.     

Roles of Ataxin-2 in Pathological Cascades Mediated by TAR DNA-binding Protein 43 (TDP-43) and Fused in Sarcoma (FUS)

The RNA-binding proteins TDP-43 and Fused in Sarcoma (FUS) play central roles in neurodegeneration associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Both proteins are components of messenger ribonucleoprotein (mRNP) granules and show cytoplasmic mislocalization in affected tissues. Recently, ataxin-2 was identified as a potent modifier of TDP-43 toxicity in an RNA-dependent manner. This study investigated to clarify how ataxin-2 modifies the TDP-43 and FUS pathological pathway. The expression of cytoplasmic TDP-43, the 35-kDa C-terminal fragment (TDP-p35f), and mutant FUS recruited ataxin-2 to mRNP granules, whereas increased ataxin-2 inhibited the mRNP granule formation of the 35-kDa C-terminal fragment and mutant FUS. A subcellular compartment analysis showed that the overexpressed ataxin-2 increased the cytoplasmic concentrations of both proteins, whereas it decreased their nuclear distributions. These data indicate that increased ataxin-2 impairs the assembly of TDP-43 and FUS into mRNP granules, leading to an aberrant distribution of RNA-binding proteins. Consequently, these sequences may exacerbate the impairment of the RNA-quality control system mediated by amyotrophic lateral sclerosis/frontotemporal lobar degeneration-associated RNA-binding proteins, which forms the core of the degenerative cascade.     

Comparative expression analysis of the H3K27 demethylases, JMJD3 and UTX, with the H3K27 methylase, EZH2, in Xenopus

The regulated removal of the gene-silencing epigenetic mark, trimethylation of lysine 27 of histone H3 (H3K27me3), has been shown to be critical for tissue-specific activation of developmental genes; however, the extent of embryonic expression of its demethylases, JMJD3 and UTX, has remained unclear. In this study, we investigated the expression of jmjd3 and utx genes in Xenopus embryos in parallel with that of the H3K27 methylase gene, ezh2. At the blastula stage, jmjd3, utx and ezh2 showed similar expression patterns in the animal cap and marginal zone that give rise to the ectoderm and mesoderm, respectively. The three genes maintained similar expression patterns in the neural plate, preplacodal ectoderm and axial mesoderm during the gastrula and neurula stages. Later, expression was maintained in the developing brain and cranial sensory tissues, such as the eye and ear, of tailbud embryos. These findings suggest that the H3K27 demethylases and methylase may function continuously for progressive switching of genetic programs during neural development, a model involving the simultaneous action of both of the demethylases for the de-repression of silent genes and the methylase for the silencing of active genes.     

Self-interaction, nucleic acid binding, and nucleic acid chaperone activities are unexpectedly retained in the unique ORF1p of zebrafish LINE

Long interspersed elements (LINEs) are mobile elements that comprise a large proportion of many eukaryotic genomes. Although some LINE-encoded open reading frame 1 proteins (ORF1ps) were suggested to be required for LINE mobilization through binding to their RNA, their general role is not known. The ZfL2-1 ORF1p, which belongs to the esterase-type ORF1p, is especially interesting because it has no known RNA-binding domain. Here we demonstrate that ZfL2-1 ORF1p has all the canonical activities associated with known ORF1ps, including self-interaction, nucleic acid binding, and nucleic acid chaperone activities. In particular, we showed that its chaperone activity is reversible, suggesting that the chaperone activities of many other ORF1ps are also reversible. From this discovery, we propose that LINE ORF1ps play a general role in LINE integration by forming a complex with LINE RNA and rearranging its conformation.     

Discovery of novel 5-alkynyl-4-anilinopyrimidines as potent, orally active dual inhibitors of EGFR and Her-2 tyrosine kinases

5-Alkenyl or 5-alkynyl-4-anilinopyrimidines were prepared and evaluated for in vitro inhibition of EGFR/Her-2 kinase activity and the growth of tumor cell lines (BT474 and N87). Several of these compounds inhibited the growth of BT474 and N87 at concentrations below 200nM. Structure-activity relationship studies revealed a critical role for the 5-alkynyl moieties. The representative compound 19 exhibited significant antitumor potency in a mouse xenograft model.     

Single nucleotide polymorphisms in Cryptomeria japonica: their discovery and validation for genome mapping and diversity studies

In order to develop a large set of single-nucleotide polymorphisms (SNPs) in Cryptomeria japonica, for a wide range of applications, we adopted a systematic EST (expressed sequence tags) re-sequencing approach. We examined a group of four genotypes comprising parents of a mapping population as well as representatives of two main lines from natural populations. We sequenced 5,170 gene fragments, representing analysis of over 1.3?Mb of DNA sequences in C. japonica. This analysis leads to the discovery of 13,413 SNPs in 3,744 amplicons, with an average of one SNP for every 101.0?bp (one SNP for every 78.3?bp in introns and for every 106.7?bp in exon regions). Nucleotide diversity in C. japonica (???=?0.0045) was found to be similar to values recorded in highly polymorphic forest tree species such as pine. We also validated the use of the SNPs as molecular markers for genetic diversity studies using the high throughput SNP genotyping platform GoldenGate. From 1,536 candidate SNP sites tested, 1,164 (75.8?%) were confirmed to be polymorphic. We anticipate that the genome-wide SNP markers reported here will be useful for evaluating the species?? range-wide genetic structure and in marker-assisted selection used as part of the C. japonica tree improvement program.