Structural evidence that alanine racemase from a D-cycloserine-producing microorganism exhibits resistance to its own product

Alanine racemase (ALR), an enzyme that catalyzes the interconversion of Ala enantiomers, is essential for the synthesis of the bacterial cell wall. We have shown that it is harder to inhibit the catalytic activity of ALR from D-cycloserine (DCS)-producing Streptomyces lavendulae than that from Escherichia coli by DCS. To obtain structural evidence for the fact that Streptomyces ALR displays resistance to DCS, we determined the precise nature of the x-ray crystal structures of the cycloserine-free and cycloserine enantiomer-bound forms of Streptomyces ALR at high resolutions. Streptomyces ALR takes a dimer structure, which is formed by interactions between the N-terminal domain of one monomer with the C-terminal domain of its partner. Each of the two active sites of ALR, which is generated as a result of the formation of the dimer structure, is composed of pyridoxal 5'-phosphate (PLP), the PLP-binding residue Lys(38), and the amino acids in the immediate environment of the pyridoxal cofactor. The current model suggests that each active site of Streptomyces ALR maintains a larger space and takes a more rigid conformation than that of Bacillus stearothermophilus ALR determined previously. Furthermore, we show that Streptomyces ALR results in a slow conversion to a final form of a pyridoxal derivative arising from either isomer of cycloserine, which inhibits the catalytic activity noncompetitively. In fact, the slow conversion is confirmed by the fact that each enzyme bound cycloserine derivative, which is bound to PLP, takes an asymmetric structure.     

Radiosensitization by hyperthermia in the chicken B-lymphocyte cell line DT40 and its derivatives lacking nonhomologous end joining and/or homologous recombination pathways of DNA double-strand break repair

Hyperthermia has a radiosensitizing effect, which is one of the most important biological bases for its use in cancer therapy with radiation. Although the mechanism of this effect has not been clarified in molecular terms, possible involvement of either one or both of two major DNA double-strand break (DSB) repair pathways, i.e. nonhomologous end joining (NHEJ) and homologous recombination (HR), has been speculated. To test this possibility, we examined cells of the chicken B-lymphocyte cell line DT40 and its derivatives lacking NHEJ and/or HR: KU70(-/-), DNA-PKcs(-/-/-), RAD54(-/-) and KU70(-/-)/RAD54(-/-). Radiosensitization by hyperthermia could be seen in all of the mutants, including KU70(-/-)/RAD54(-/-), which lacked both NHEJ and HR. Therefore, radiosensitization by hyperthermia cannot be explained simply by its inhibitory effects, if any, on NHEJ and/or HR alone. However, in NHEJ-defective KU70(-/-) and DNA-PKcs(-/-/-), consisting of two subpopulations with distinct radiosensitivity, the radiosensitive subpopulation, which is considered to be cells in G(1) and early S, was not sensitized. Substantial sensitization was seen only in the radioresistant subpopulation, which is considered to be cells in late S and G(2), capable of repairing DSBs through HR. This observation did not exclude possible involvement of NHEJ in G(1) and early S phase and also suggested inhibitory effects of hyperthermia on HR. Thus partial contribution of NHEJ and HR in radiosensitization by hyperthermia, especially that depending on the cell cycle stage, remains to be considered.     

Studies on virulence of HIV and development of non-virulent live AIDS vaccine using monkeys

A great effort for developing AIDS vaccine has been carried out in the world, designed by various new ideas based on basic research information obtained in recent virology and immunology. Withall it, to obtain effective AIDS vaccine is considered skeptical. One of the reasons of its difficulty is a lack of experimental animals susceptible to HIV-1. In our laboratory, we have succeeded in developing chimeric SIV having 3' half of HIV-1 genome including env (SHIV), which is infectious to macaque monkeys. One of SHIVs has been proved nonpathogenic in monkeys from various aspects and it afforded protective immunity to monkeys against pathogenic SHIV challenge infection. Now, we are trying to develop anti-HIV live attenuated vaccines using the nonpathogenic SHIV as a starting material. In the history of virus vaccine, live attenuated vaccines have been proved most effective in measles and polio-myelitis. However, it is not clear whether nonpathogenic HIV exists or not. Futhermore, even if nonpathogenic HIV could be obtained, there is possibility that it will easily mutate to pathogenic one. Therefore, to develop live attenuated AIDS vaccine is considered dangerous. In this article, We will introduce our research on SHIV pathogenicity using monkeys and hypothesize possibility to obtain nonpathogenic HIV which is speculated from the origin and evolution of HIV/SIV. To clarify virulence and nonvirulence of HIV and to obtain nonpathogenic virus are not only applied research but also basic science to dissolve the fundemental question why HIV can induce the disease.     

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8'-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8'-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8'-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.     

Identification of new prostate stem cell antigen-derived peptides immunogenic in HLA-A2<Superscript>+</Superscript> patients with hormone-refractory prostate cancer

Purpose Prostate cancer refractory to hormonal manipulation requires new treatment modalities. In the present study we attempted to identify prostate stem cell antigen (PSCA)-derived peptides immunogenic in HLA-A2⁺ prostate cancer patients in order to develop peptide-based immunotherapy against hormone-refractory prostate cancer (HRPC).Methods Eleven different PSCA-derived peptides, which were prepared based on the HLA-A2 binding motif, were examined to determine whether they would be recognized by cellular and humoral immune responses in 12 HLA-A2⁺ patients (11 with HRPC and 1 with non-HRPC).Results Among the PSCA-derived peptides, PSCA 7–15 and PSCA 21–30 peptides effectively induced HLA-A2-restricted peptide-specific and tumor-reactive cytotoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells (PBMCs) of HLA-A2⁺ patients. The PSCA 21–30 peptide was capable of inducing peptide-specific CTLs in both cancer patients and healthy donors, whereas the PSCA 7–15 peptide was immunogenic in only cancer patients. Immunoglobulin G (IgG) reactive to the PSCA 21–30 peptide was detected in plasma of most patients and healthy donors, whereas IgG reactive to PSCA 7–15 was undetectable in all cases. These results indicate that the former peptide elicits both cellular and humoral immune responses in both patients and healthy donors, whereas the latter elicits only cellular responses in patients.Conclusion These two PSCA peptides should be considered for use in clinical trials of immunotherapy for HLA-A2⁺ HRPC patients.     

Emergence and serovar transition of Vibrio parahaemolyticus pandemic strains isolated during a diarrhea outbreak in Vietnam between 1997 and 1999

We characterized 523 Vibrio parahaemolyticus strains isolated during a survey of diarrhea patients in Khanh Hoa province, Vietnam between 1997 and 1999. Forty-nine percent of the strains were judged to belong to the pandemic strains that emerged around 1996 and spread to many countries. These strains were positive in the GS-PCR assay and carried the tdh gene. The ORF8 of the f237 phage genome, a possible marker of the pandemic clone, was absent in 10% of these strains. Eleven O: K serovars were detected among the pandemic strains and the strains representing all 11 serovars of pandemic strains were shown to be closely related regardless of the ORF8 genotype using arbitrarily primed PCR and pulsed field gel electrophoresis analyses. It was clear that a transition of major serovars occurred among the pandemic strains represented by the emergence of O3: K6 in 1997, O4: K68 in 1998, and O1: K25 in 1998 and 1999.