NADPH-cytochrome P450 reductase.

The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s^?1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min^?1 (mg of protein)^?1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min^?1 (mg of protein)^?1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase.     

In vitro construction of different oligomeric forms of lambdadv DNA and studies of their transforming activities.

Plasmid lambdadv1, which is in a dimeric form, was converted to a linear monomer duplex by the action of EcoRI restriction endonuclease that incises at a unique site in this plasmid genome. The resulting products were then joined by Escherichia coli DNA ligase to produce molecules with various oligomeric forms, and from these monomeric, dimeric, or trimeric circular molecules were purified. By transformation of cells with these DNAs, clones were obtained that carried lambdadv1 in a monomeric or dimeric form. The former type of clones have not been generated in vivo, except for one in a different host strain, and carriers of timeric or tetrameric lambdadv1's have not been obtained so far. It was observed that a considerable fraction of these oligomeric circular DNAs were converted to lower oligomers (e.g., from trimer to dimer) during transformation. The characteristics of the monomeric lambdadv1 carriers obtained were compared with those of dimeric lambdadv1 carriers. The stabilities of the plasmids of the two forms were the same. However, the monomeric plasmid carriers were less tolerant to lambdavir phage infection and perpetuated about 30% less plasmid genomes in monomer units. Furthermore, dimeric plasmid carriers appeared spontaneously and accumulated in cultures of the monomeric lambdadv1 carriers.     

Identification and Characterization of a Novel aac(6′)-Iag Associated with the bla IMP-1–Integron in a Multidrug-Resistant Pseudomonas aeruginosa

In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-β-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6′)-Iag, bla _IMP-1, a truncated form of bla _IMP-1, and a truncated form of aac(6′)-Iag. The aac(6′)-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6′)-Iz. Recombinant AAC(6′)-Iag protein showed aminoglycoside 6′-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6′)-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for bla _IMP-1 and aac(6′)-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.     

Role of PPARα and HNF4α in Stress-Mediated Alterations in Lipid Homeostasis

Stress is a risk factor for several cardiovascular pathologies. PPARα holds a fundamental role in control of lipid homeostasis by directly regulating genes involved in fatty acid transport and oxidation. Importantly, PPARα agonists are effective in raising HDL-cholesterol and lowering triglycerides, properties that reduce the risk for cardiovascular diseases. This study investigated the role of stress and adrenergic receptor (AR)-related pathways in PPARα and HNF4α regulation and signaling in mice following repeated restraint stress or treatment with AR-antagonists administered prior to stress to block AR-linked pathways. Repeated restraint stress up-regulated Pparα and its target genes in the liver, including Acox, Acot1, Acot4, Cyp4a10, Cyp4a14 and Lipin2, an effect that was highly correlated with Hnf4α. In vitro studies using primary hepatocyte cultures treated with epinephrine or AR-agonists confirmed that hepatic AR/cAMP/PKA/CREB- and JNK-linked pathways are involved in PPARα and HNF4α regulation. Notably, restraint stress, independent of PPARα, suppressed plasma triglyceride levels. This stress-induced effect could be attributed in part to hormone sensitive lipase activation in the white adipose tissue, which was not prevented by the increased levels of perilipin. Overall, this study identifies a mechanistic basis for the modification of lipid homeostasis following stress and potentially indicates novel roles for PPARα and HNF4α in stress-induced lipid metabolism.     

Predictors of Beta-Blocker Intolerance and Mortality in Patients After Acute Coronary Syndrome

Purpose

To investigate the predictors of intolerance to beta-blockers treatment and the 6-month mortality in hospitalized patients with acute coronary syndrome (ACS).

Methods

This was a single-center, prospective, and longitudinal study including 370 consecutive ACS patients in Killip class I or II. BBs were prescribed according to international guidelines and withdrawn if intolerance occurred. The study was approved by the institutional ethics committee of our university. Statistics: the clinical parameters evaluated at admission, and the related intolerance to BBs and death at 6 months were analyzed using logistic regression (p<0.05)in PATIENTS.

Results

BB intolerance was observed in 84 patients and was associated with no prior use of statins (OR: 2.16, 95%CI: 1.26–3.69, p= 0.005) and Killip class II (OR: 2.5, 95%CI: 1.30-4.75, p=0.004) in the model adjusted for age, sex, blood pressure, and renal function. There was no association with ST-segment alteration or left anterior descending coronary artery plaque. Intolerance to BB was associated with the greatest risk of death (OR: 4.5, 95%CI: 2.15–9.40, p<0.001).

Conclusions

After ACS, intolerance to BBs in the first 48 h of admission was associated to non previous use of statin and Killip class II and had a high risk of death within 6 months.     

Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.     

Basal and Ischemia-Induced Transcardiac Troponin Release into the Coronary Circulation in Patients with Suspected Coronary Artery Disease

Background

Cardiac troponin is a specific biomarker for cardiomyocyte necrosis in acute coronary syndromes. Troponin release from the coronary circulation remains to be determined because of the lower sensitivity of the conventional assay. We sought to determine basal and angina-induced troponin release using a highly sensitive troponin assay.

Methods and Results

The cardiac troponin T levels in serum sampled from the peripheral vein (PV), the aortic root (AO), and the coronary sinus (CS) were measured in 105 consecutive stable patients with coronary risk factor(s) and suspected coronary artery disease (CAD) and in 33 patients without CAD who underwent an acetylcholine provocation test. At baseline, there was a significant increase in the troponin levels from AO [9.0 (6.4, 13.1) pg/mL for median (25^th, 75^th percentiles)] to CS [10.3 (7.3, 15.5) pg/mL, p<0.001] in 96 (91.4%) patients and the difference was 1.1 (0.4, 2.1) pg/mL, which reflected basal transcardiac troponin release (TTR). TTR was positively correlated with PV levels (r = 0.22, p = 0.03). Male sex, left ventricular hypertrophy determined by echocardiography, T-wave inversion, and CAD correlated with elevated TTR defined as above: median, 1.1 pg/mL. A significant increase in TTR was noted in 17 patients with coronary spasms [0.6 (0.2, 1.2) pg/mL, p<0.01] but not in 16 patients without spasms [0.0 (−0.5, 0.9) pg/mL, p = 0.73] after the acetylcholine provocation.

Conclusion

Basal TTR in the coronary circulation was observed in most of the patients with suspected CAD and risk factor(s). This sensitive assay detected myocardial ischemia-induced increases in TTR caused by coronary spasms.