A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Purification and characterization of malate:quinone oxidoreductase from thermophilic Bacillus sp. PS3

Several bacteria possess membrane-bound dehydrogenases other than cytosolic dehydrogenases in their respiratory chains. In many cases, the membrane-bound malate:quinone oxidoreductases (MQOs) are essential for growth. However, these MQOs are absent in mammalian mitochondria, and therefore may be a potential drug target for pathogenic bacteria. To characterize the kinetic properties of MQOs, we purified MQO from Bacillus sp. PS3, which is a gram-positive and thermophilic bacterium, and cloned the gene encoding MQO based on the obtained partial N-terminus sequence. Purified MQOs showed a molecular mass of ~90 kDa, which was estimated using gel filtration, and it consists of two subunits with a molecular mass of ~50 kDa. Phylogenetic analysis showed a high similarity to the MQO of the Geobacillus group rather than the Bacillus group. Additionally, the purified enzyme was thermostable and it retained menaquinol reduction activity at high temperatures. Although it is difficult to conduct experiments using menaquinol because of its instability, we were able to measure the oxidase activity of cytochrome bd-type quinol oxidase by using menaquinol-1 by coupling this molecule with the menaquinol reduction reaction using purified MQOs.     

Expression of a single-chain antibody against indole-3-acetic acid in Escherichia coli

A hybridoma cell line that produces a monoclonal antibody specific for indole-3-acetic acid (IAA) was prepared. The DNA fragments coding the variable regions of the light and the heavy chains of the antibody were prepared by PCR using the cDNA of the antibody as a template. A chimera DNA for a single chain variable fragment (scFv) was constructed, and expressed in Escherichia coli. The scFv antibody expressed in E. coli as well as the original monoclonal antibody showed a specific binding to IAA.     

Metabolism of medroxyprogesterone acetate (MPA) via CYP enzymes in vitro and effect of MPA on bleeding time in female rats in dependence on CYP activity in vivo

Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced breast cancer, although it is known to cause thrombosis as a serious side effect. Recently, we found that cytochrome P450 3A4 (CYP3A4) mainly catalyzed the metabolism of MPA via CYP in human liver microsomes. However, the metabolic products of MPA in humans and rats have not been elucidated. In addition, it is not clear whether thrombosis could be induced by MPA itself or by its metabolites. In this study, we determined the overall metabolism of MPA as the disappearance of the parent drug from an incubation mixture, and identified the enzymes catalyzing the metabolism of MPA via CYP in rats. Moreover, the effects of CYP-modulators on MPA-induced hypercoagulation in vivo were examined. Intrinsic clearance of MPA in rat liver microsomes was increased by treatment with CYP3A-inducers. The intrinsic clearance of MPA in liver microsomes of rats treated with various CYP-inducers showed a significant correlation with CYP3A activity, but not CYP1A activity, CYP2B activity or CYP2C contents. Among the eight recombinant rat CYPs studied, CYP3A1, CYP3A2 and CYP2A2 catalyzed the metabolism of MPA. However, since CYP3A2 and CYP2A2 are male-specific isoforms, CYP3A1 appears to be mainly involved in the metabolism of MPA in liver microsomes of female rats. In an in vivo study, pretreatment of female rats with SKF525A, an inhibitor of CYPs including CYP3A1, significantly (p < 0.05) enhanced MPA-induced hypercoagulation, whereas pretreatment with phenobarbital, an inducer of CYPs including CYP3A1, reduced it. These findings suggest that CYP-catalyzed metabolism of MPA is mainly catalyzed by CYP3A1 and that MPA-induced hypercoagulation is predominantly caused by MPA itself in female rats.     

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.     

Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/ Ribavirin Therapy in Chronic Hepatitis C

Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3− NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed.　Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.     

Efficient cross-presentation of soluble exogenous antigens introduced into dendritic cells using a weak-based amphiphilic peptide

To develop a novel dendritic cell (DC)-based vaccine for inducing antigen-specific CD8⁺ T cell responses by cross-presentation, we tested a novel antigen delivery system that introduces soluble antigens into the cytosol of cells by an endocytosis-mediated mechanism which avoids damaging the plasma membrane (“Endo-Porter”™). Proteins released from endosomes into the cytoplasm are degraded by the proteasome, and fragmented antigenic peptides are presented to the classical cytosolic MHC class I pathway. DCs pulsed with OVA protein in the presence of Endo-Porter efficiently stimulate OVA peptide-specific CD8⁺ T (OT-I) cells. Although this agent diverts some of the endocytosed antigens away from the classical MHC class II-restricted presentation pathway to the class I pathway, the activation of CD4⁺ T cells was found not to be hampered by Endo-Porter-mediated antigen delivery. On the contrary, it was rather augmented, probably due to the increased uptake of antigen. Because specific CD4⁺ T cell help is required to license DCs for cross-priming, Endo-Porter-mediated antigen delivery is a promising approach for developing more efficient cancer vaccines targeting both CD4⁺ and CD8⁺ T cells.     

Biochemical Characterization and Phylogenetic Analysis Based on 16S rRNA Sequences for V-Factor Dependent Members of <Emphasis Type="Italic">Pasteurellaceae</Emphasis> Derived from Laboratory Rats

Phylogenetic analysis based on 16S rRNA sequences with sequence data of some bacterial species of Pasteurellaceae related to rodents deposited in GenBank was performed along with biochemical characterization for the 20 strains of V-factor dependent members of Pasteurellaceae derived from laboratory rats to obtain basic information and to investigate the taxonomic positions. The results of biochemical tests for all strains were identical except for three tests, the ornithine decarboxylase test, and fermentation tests of D(+) mannose and D(+) xylose. The biochemical properties of 8 of 20 strains that showed negative results for the fermentation test of D(+) xylose agreed with those of Haemophilus parainfluenzae complex. By phylogenetic analysis, the strains were divided into two clusters that agreed with the results of the fermentation test of xylose (group I: negative reaction for xylose, group II: positive reaction for xylose). The clusters were independent of other bacterial species of Pasteurellaceae tested. The sequences of the strains in group I showed 99.7–99.8% similarity and the strains in group II showed 99.3–99.7% similarity. None of the strains in group I had a close relation with Haemophilus parainfluenzae by phylogenetic analysis, although they showed the same biochemical properties. In conclusion, the strains had characteristic biochemical properties and formed two independent groups within the “rodent cluster” of Pasteurellaceae that differed in the results of the fermentation test of xylose. Therefore, they seemed to be hitherto undescribed taxa in Pasteurellaceae.