Abnormal phosphorylation of Ser409/410 of TDP-43 in FTLD-U and ALS

A monoclonal antibody specific for phosphoserines 409 and 410 of TDP-43 (mAb pS409/410) has been produced. It strongly stained TDP-43-positive inclusions in brain of patients with frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but did not stain nuclei, in which normal TDP-43 is localized. It did not recognize TDP-43 rapidly extracted from brains of rats at various developmental stages, strongly suggesting that phosphorylation of Ser409/410 is an abnormal event. Analysis of postmortem changes of TDP-43 revealed that the amounts of Sarkosyl-insoluble, urea-soluble full-length TDP-43 and a 35 kDa N-terminal fragment increased time-dependently.     

The evolution of sex-independent transmission ratio distortion involving multiple allelic interactions at a single locus in rice

Transmission ratio distortion (TRD) is frequently observed in inter- and intraspecific hybrids of plants, leading to a violation of Mendelian inheritance. Sex-independent TRD (siTRD) was detected in a hybrid between Asian cultivated rice and its wild ancestor. Here we examined how siTRD caused by an allelic interaction at a specific locus arose in Asian rice species. The siTRD is controlled by the S(6) locus via a mechanism in which the S(6) allele acts as a gamete eliminator, and both the male and female gametes possessing the opposite allele (S(6)(a)) are aborted only in heterozygotes (S(6)/S(6)(a)). Fine mapping revealed that the S(6) locus is located near the centromere of chromosome 6. Testcross experiments using near-isogenic lines (NILs) carrying either the S(6) or S(6)(a) alleles revealed that Asian rice strains frequently harbor an additional allele (S(6)(n)) the presence of which, in heterozygotic states (S(6)/S(6)(n) and S(6)(a)/S(6)(n)), does not result in siTRD. A prominent reduction in the nucleotide diversity of S(6) or S(6)(a) carriers relative to that of S(6)(n) carriers was detected in the chromosomal region. These results suggest that the two incompatible alleles (S(6) and S(6)(a)) arose independently from S(6)(n) and established genetically discontinuous relationships between limited constituents of the Asian rice population.     

Development and application of a T-RFLP data analysis method using correlation coefficient matrices

Environmental microbiology studies commonly use terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, for example, to analyze changes in community structure in relation to changing physicochemical and biological conditions over space and time. Although T-RFLP is most useful for comparing samples from different environments, a large number of samples makes effective analysis difficult using the Web-based tools that are currently available. To resolve this dilemma, we used a new approach for calculating data from multiple T-RFLP samples by estimating terminal fragment combinations, then applying a correlation analysis using two different fluorescent dyes generated simultaneously from all samples. This calculation was based on the expectation that the proportions of two terminal fragments from one full-length polymerase chain reaction fragment would be nearly the same in each analysis. Using this program, the oral microflora in 73 human saliva samples were analyzed, and 24 bacterial groups, with peak areas of at least 0.5% and correlation coefficients of 0.55 or greater, were identified from the T-RFs within 40 s.     

Upregulation of GRAIL is associated with remission of ulcerative colitis

Abrogating tolerance against unidentified antigens is a critical step in the pathogenesis of ulcerative colitis (UC). T cell anergy, one of the main mechanisms of tolerance, has been shown to be induced by E3 ubiquitin ligases, such as gene related to anergy in lymphocytes (GRAIL), Itch, and c-Cbl in mice. However, it is not well known whether these E3 ligases play roles in human diseases. The pathophysiological role of the E3 ligases in patients with UC was investigated. At first, the expression of GRAIL, Itch, and c-Cbl in human anergic T cells was analyzed by quantitative RT-PCR and Western immunoblotting. Next, the mRNA expression of the E3 ligases was analyzed in peripheral CD4+ T cells of 20 patients with UC and 10 healthy volunteers (HV). mRNA expression was analyzed in patients with active UC before and after treatment with prednisolone and leukocytapheresis. Anergic human CD4+ T cells expressed significantly higher levels of GRAIL, Itch, and c-Cbl than nonanergic cells. GRAIL expression was significantly higher in patients with UC in remission than in patients with active disease and in HV (P < 0.01). The level of GRAIL expression was also significantly increased in patients with active disease whose clinical activity index scores improved after treatment (P < 0.05). There were no significant differences in Itch and c-Cbl expression among patients with active UC, patients with UC in remission, and HV. These data suggest that GRAIL plays an important role in maintaining remission in patients with UC.     

Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells

The recent finding that acrylamide (AA), a potent carcinogen, is formed in foods during cooking raises human health concerns. In the present study, we investigated the genotoxicity of AA and its metabolite glycidamide (GA) in human lymphoblastoid TK6 cells examining three endpoints: DNA damage (comet assay), clastogenesis (micronucleus test) and gene mutation (thymidine kinase (TK) assay). In a 4 h treatment without metabolic activation, AA was mildly genotoxic in the micronucleus and TK assays at high concentrations (> 10 mM), whereas GA was significantly and concentration-dependently genotoxic at all endpoints at > or = 0.5 mM. Molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity (LOH) mutation like spontaneous one while GA-induced primarily point mutations. These results indicate that the genotoxic characteristics of AA and GA were distinctly different: AA was clastogenic and GA was mutagenic. The cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), implying that the rat liver S9 did not activate AA. We discuss the in vitro and in vivo genotoxicity of AA and GA.     

Global gene expression analysis of yeast cells during sake brewing

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process.     

Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.     

Evaluation of performance of different surface-engineered yeast strains for direct ethanol production from raw starch

Two wood-dwelling ascomycetes, Xylaria hypoxylon and Xylaria polymorpha, were isolated from rotting beech wood. Lignin degradation was studied following the mineralization of a synthetic -labelled lignin in solid and liquid media. Approximately 9% of the synthetic lignin was mineralized by X. polymorpha during the growth on beech wood meal, and the major fraction (65.5%) was polymerized into water- and dioxan-insoluble material. Both fungi produced laccase (up to 1,200 U l⁻¹) in an agitated complex medium based on tomato juice; peroxidase activity (<80 U l⁻¹) was only detected for X. polymorpha in soybean meal suspension. The enzymatic attack of X. polymorpha on beech wood resulted in the formation of three fractions of water-soluble lignocellulose fragments with molecular masses of 200, 30 (major fraction) and 3 kDa, as demonstrated by high-performance size exclusion chromatography. This fragment pattern differs considerably from that of the white-rot fungus Bjerkandera adusta, which preferentially released smaller lignocellulose fragments (0.8 kDa). The finding that X. polymorpha produced large lignocellulose fragments, along with the fact that high levels of hydrolytic enzymes (esterase 630 U l⁻¹, xylanase 120 U l⁻¹) were detected, indicates the cleavage of bonds between the lignin and hemicellulose moieties.