Identification of an HLA-A24-restricted OY-TES-1 epitope recognized by cytotoxic T-cells

OY-TES-1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY-TES-1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY-TES-1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA-A24-binding OY-TES-1 peptide, TES(401-409) (KTPFVSPLL) recognized by CD8 T-cells. Purified CD8 T-cells from healthy donors stimulated in vitro with the peptide-pulsed autologous DC and PBMC produced IFNgamma in response to the peptide-pulsed PBMC and showed cytotoxicity against the peptide-pulsed autologous EBV-B specifically. Furthermore, cytotoxicity was also observed against an OY-TES-1 mRNA-expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV-B cells in recognition by CD8 CTL was the same as that of the fraction of the TES(401-409) peptide itself, suggesting that the TES(401-409) was a naturally processed peptide on LK79.     

2-Haloacrylate reductase, a novel enzyme of the medium chain dehydrogenase/reductase superfamily that catalyzes the reduction of a carbon-carbon double bond of unsaturated organohalogen compounds

A soil bacterium, Burkholderia sp. WS, grows on 2-chloroacrylate as the sole carbon source. To identify the enzymes metabolizing 2-chloroacrylate, we carried out comparative two-dimensional gel electrophoresis of the proteins from 2-chloroacrylate- and lactate-grown bacterial cells. As a result, we found that a protein named CAA43 was inducibly synthesized when the cells were grown on 2-chloroacrylate. The CAA43 gene was cloned and shown to encode a protein of 333 amino acid residues (M(r) 35,788) that shared a significant sequence similarity with NADPH-dependent quinone oxidoreductase from Escherichia coli (38.2% identity). CAA43 was overproduced in E. coli and purified to homogeneity. The purified protein catalyzed the NADPH-dependent reduction of the carbon-carbon double bond of 2-chloroacrylate to produce (S)-2-chloropropionate, which is probably further metabolized to (R)-lactate by (S)-2-haloacid dehalogenase in Burkholderia sp. WS. NADH did not serve as a reductant. Despite the sequence similarity to quinone oxidoreductases, CAA43 did not act on 1,4-benzoquinone and 1,4-naphthoquinone. 2-Chloroacrylate analogs, such as acrylate and methacrylate, were also inert as the substrates. In contrast, 2-bromoacrylate served as the substrate. Thus, we named this novel enzyme 2-haloacrylate reductase. This study revealed a new pathway for the degradation of unsaturated organohalogen compounds. It is also notable that the enzyme is useful for the production of (S)-2-chloropropionate, which is used for the industrial production of aryloxyphenoxypropionic acid herbicides.     

Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli

MacB is an ABC-type membrane protein that exports only macrolide compounds containing 14- and 15-membered lactones, cooperating with a membrane fusion protein, MacA, and a multifunctional outer membrane channel, TolC. We determined the membrane topology of MacB by means of site-specific competitive chemical modification of single cysteine mutants. As a result, it was revealed that MacB is composed of four transmembrane (TM) segments with a cytoplasmic N-terminal nucleotide binding domain of about 270 amino acid residues and a periplasmic large hydrophilic polypeptide between TM segments 1 and 2 of about 200 amino acid residues.     

Structural characterization of GnRH loci in the medaka genome

To help clarify the origin of a third gonadotropin-releasing hormone (GnRH) paralog found only in the teleost lineage, we have characterized GnRH loci in a teleost species, the medaka Oryzias latipes, and compared corresponding regions of the medaka and human genomes. Three GnRHs for medaka-type GnRH (mdGnRH), chicken-II-type GnRH (cGnRH-II), and salmon-type GnRH (sGnRH) exist as single-copy genes and reside on separate chromosomes in the medaka genome. Both medaka mdGnRH and human mGnRH are closely linked to FLJ20038 encoding a hypothetical protein, and both cGnRH-IIs in the medaka and humans are adjacent to PTP(alpha) for protein tyrosine phosphatase alpha. These conserved syntenies demonstrate that mdGnRH and cGnRH-II in teleosts are orthologous to mGnRH and cGnRH-II in tetrapods, respectively. On the other hand, the third paralogous GnRH in the medaka, sGnRH, is adjacent to PTP(epsilon), a paralog of PTP(alpha). Although humans possess PTP(epsilon) on 10q26, no sGnRH-like sequence was found in the human genome databases. Therefore a gene duplication that gave rise to the third paralogous GnRH likely occurred before the divergence of teleosts and tetrapods, and it has been lost only in the tetrapod lineage. Additionally, together with the prior observations that like GnRH, PTP(alpha)/PTP(epsilon) are strongly expressed in neural and tumor cells and that GnRH can increase PTP activity, the current data suggests that the physically linked cGnRH-II/sGnRH and PTP(alpha)/PTP(epsilon) are also functionally linked.     

Processing of bisphenol A by plant tissues: glucosylation by cultured BY-2 cells and glucosylation/translocation by plants of Nicotiana tabacum

Bisphenol A (BPA, 4,4'-isopropylidenediphenol), an endocrine disrupter with estrogenic properties, was supplied to tobacco BY-2 cells in suspension culture and the chemical nature of its metabolites was investigated. The concentration of BPA in the culture medium decreased rapidly and became undetectable at 2.5 h after the application. Four metabolites of BPA were observed in a methanol extract of the cells when the culture was supplemented with [(14)C]BPA. The most abundant metabolite was determined to be 4,4'-isopropylidenediphenol-O-beta-D-glucopyranoside (BPAG) by mass spectrometry, nuclear magnetic resonance spectroscopy and by hydrolysis with beta-glucosidase. This identification was confirmed by synthesis. When [(14)C]BPA was administrated to tobacco seedlings from their roots, radioactivity was incorporated in BPAG and three unidentified metabolites. These metabolites were accumulated in the leaves after 4 h exposure, indicating that tobacco seedlings absorbed BPA through their root systems, metabolized to its beta-glucoside and translocated the metabolites to their leaves.     

Isolation of an ozone-sensitive and jasmonate-semi-insensitive Arabidopsis mutant (oji1)

A novel ozone-sensitive mutant was isolated from Arabidopsis T-DNA tagging lines. This mutant revealed severe foliar injury and higher ethylene emission than the wild type under ozone exposure. The ozone-induced injury and ethylene emission were suppressed by pretreatment with aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis, both in this mutant and wild-type plants. Pretreatment with methyl-jasmonate (MeJA) at 10 micro M, however, suppressed the ozone-induced ethylene emission and foliar injury only in the wild-type plants. This mutant was less sensitive to jasmonate than the wild type, estimated by the MeJA-induced inhibition of root elongation and ozone-induced expression of AtVSP1, a jasmonate-inducible gene. Thus, this mutant was named oji1 (ozone-sensitive and jasmonate-insensitive 1). These results suggest that the ozone sensitivity of oji1 is caused by the increase in ozone-induced emission of ethylene as a result of low sensitivity to jasmonate, which plays defensive roles under stress conditions.     

Profile analysis of 24-hours measurements of blood pressure

A method is proposed for classifying subjects according to their convex, flat, or concave change patterns of 24-hours blood pressure measurements. To obtain such a classification is useful for detecting subjects who show abnormal change patterns and giving them appropriate medical treatments. Therefore, an appropriate statistic is proposed for detecting a systematic change along the time axis, as well as a statistic with its inverse characteristic appropriate for evaluating the noise variation. The method is based on the ratio of those two types of statistics; it is verified to work well on real data, giving a classification of subjects into four types of subgroups: extreme dipper, dipper, nondipper, and inverted dipper. It also suggests that there might be an ultra-extreme dipper subgroup.     

Common anti-apoptotic roles of parkin and alpha-synuclein in human dopaminergic cells

Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson's disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of alpha-synuclein (alpha-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and alpha-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.