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.     

Circadian rhythm of atrioventricular conduction predicts long-term survival in patients with chronic atrial fibrillation

The R-R interval of the electrocardiogram during atrial fibrillation (AF) appears absolutely irregular. However, the Poincaré plot of the R-R interval reveals a sector shape of distribution that is unique to AF. Furthermore, the height of lower envelope (LE1.0) of the distribution and the degree of scatter above the envelope (scattering index) may reflect the refractoriness and concealment of atrioventricular (AV) conduction, respectively. We previously observed that both the LE1.0 and scattering index show clear circadian rhythms in patients with chronic AF and that the rhythms are blunted in those with congestive heart failure and chronic AF. In the present study, we examined if the blunted circadian rhythm of the AV conduction has prognostic value in patients with chronic AF. We studied a retrospective cohort of 120 patients who underwent 24h Holter monitoring at baseline. During an observation period of 33 +/- 16 mon, there were 25 deaths (21%) including 13 cardiac and 8 stroke deaths. All patients showed significant circadian rhythms in both LE1.0 and scattering index with acrophases occurring at night; however, patients dying subsequently from cardiac causes, but not those from fatal stroke were blunted in the circadian rhythms (the amplitudes were < 55% of those in surviving patients). Furthermore, the reduced circadian amplitude of scattering index was an increased risk for cardiac death even after adjustment of coexisting cardiovascular risks [adjusted relative risk (95% confidence interval) per 1-SD decrement, 4.24 (1.54-11.6)]. When patients were divided by the circadian amplitude of the scattering index of 36.5 msec (mean minus 1-SD), the 5yr cardiac mortality below and above the cutoff was 57 and 6%, respectively (log-rank test, p < 0.001). We conclude that the blunted circadian rhythm of AV conduction is an independent risk for cardiac death in patients with chronic AF.     

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.     

The role of ANG II and endothelin-1 in exercise-induced diastolic dysfunction in heart failure

The diastolic dysfunction present at rest in congestive heart failure (CHF) is exacerbated during exercise (Ex). Increases in circulating ANG II and endothelin-1 (ET-1) during Ex may contribute to this response. We assessed the effect of Ex on circulating plasma levels of ANG II and ET-1 and left ventricular (LV) dynamics before and after pacing-induced CHF at rest and during Ex in nine conscious, instrumented dogs. Before CHF, there were modest increases in circulating levels of ANG II (but not ET-1) during Ex. LV diastolic performance was enhanced during Ex with decreases in the time constant of LV relaxation (tau), LV end-systolic volume (V(ES)), and LV minimum pressure with a downward shift of the LV early diastolic portion of the pressure-volume (P-V) loop. This produced an increase in peak LV filling rate without an increase in mean left atrial (LA) pressure. After CHF, the resting values of ANG II and ET-1 were elevated and increased to very high levels during Ex. After CHF, mean LA pressure, tau, and LV minimum pressure were elevated at rest and increased further during Ex. Treatment with L-754,142, a potent ET-1 antagonist, or losartan, an ANG II AT(1)-receptor blocker, decreased these abnormal Ex responses in CHF more effectively than an equally vasodilatory dose of sodium nitroprusside. Combined treatment with both ANG II- and ET-1-receptor blockers was more effective than either agent alone. We conclude that in CHF, circulating ANG II and ET-1 increase to very high levels during Ex and exacerbate the diastolic dysfunction present at rest.     

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.