Cynomolgus monkey cytochrome P450 2C43: cDNA cloning, heterologous expression, purification and characterization

The cDNA of cytochrome P450 (CYP) 2C43 was cloned from cynomolgus monkey liver by RT-PCR. The deduced amino acid sequence showed 93% and 91% identity to human CYP2C9 and CYP2C19, respectively. The cDNA was expressed in Escherichia coli and purified by a series of chromatography steps, yielding a specific content of 11.5 nmol P450/mg protein. The substrate specificity of the purified CYP2C43 was examined in a reconstitution system comprising NADPH-P450 reductase, lipid, cytochrome b(5) and CYP2C marker substrates. The purified CYP2C43 showed high activity for testosterone 17-oxidation and progesterone 21-hydroxylation, which were also observed for CYP2C19 but not CYP2C9. In addition, CYP2C43 showed activity for (S)-mephenytoin 4'-hydroxylation, a marker reaction for CYP2C19. With CYP2C9 marker substrates, CYP2C43 exhibited low activity for diclofenac 4'-hydroxylation and no activity for tolbutamide p-methylhydroxylation. Therefore, in terms of substrate specificity, our results indicate that CYP2C43 is similar to CYP2C19, rather than CYP2C9.     

Plasmodium falciparum: Development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer

Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488 nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.     

Efficacy of genotype notification to Japanese smokers on smoking cessation—An intervention study at workplace

Objectives: It is well-known that smoking causes many diseases including cancers. Informing smokers of their genotypes associated with the vulnerability to the harms of smoking may be effective measures for smoking cessation. The present study examined the effects of genotype notification of an oncogene (L-myc) genotype to smokers on their behavior to quit smoking. Methods: Subjects were 562 employees of a bank who answered to be a smoker for a questionnaire used at annual health checkup at workplace from July to December 2002. Those enrolled on August, October, and December were allocated into the genotype notification group (intervention group), and the rest into the controls. Among 286 smokers allocated into the intervention group, 257 participants (89.9%) agreed to genotype testing. One year after the enrollment, a follow-up questionnaire survey was conducted for all smokers including controls. Results: Those who stated to have quitted smoking were 22 (8.0%) among the 276 controls and 15 (5.8%) among the 257 genotype notified participants, providing that the odds ratio (OR) of cessation for the intervention was 0.64 (95% confidence interval, 0.32–1.28). No psychological problems associated with genotype notification were observed. Conclusion: The present study did not show positive effects of genotype notification on smoking cessation rate. To elevate the cessation rate, methods to explain and notify genotypes should be improved.     

Preparation, characterization, and biological evaluation of 6(I),6(IV)-di-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose and 6-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose

6(I),6(IV)-Di-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose (βCD) {6(I),6(IV)-di-O-[α-l-Fuc-(1→6)-β-d-GlcNAc]-βCD (5)} and 6-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-βCD {6-O-[α-l-Fuc-(1→6)-β-d-GlcNAc]-βCD (6)} were chemically synthesized using the corresponding authentic compounds, bis(2,3-di-O-acetyl)-pentakis(2,3,6-tri-O-acetyl)-βCD as the glycosyl acceptor and 2,3,4-tri-O-benzyl-α-l-fucopyranosyl-(1→6)-3,4-di-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-d-glucopyranosyl trichloroacetimidate as the fuco-glucosaminyl donor. NMR confirmed that α-l-Fuc-(1→6)-d-GlcNAc was bonded by β-linking to the βCD ring. To evaluate biological efficiency, the biological activities of the new branched βCDs were examined. The cell detachment activity of 5 was lower than that of 6 in real-time cell sensing (RT-CES) assay, indicating that 5 has lower toxicity. In SPR analysis, 5 had a higher special binding with AAL, a fucose-recognizing lectin. These results suggest that 5 could be an efficient drug carrier directed at cells expressing fucose-binding proteins.     

Association between Combined Lifestyle Factors and Non-Restorative Sleep in Japan: A Cross-Sectional Study Based on a Japanese Health Database

Background

Although lifestyle factors such as cigarette smoking, excessive drinking, obesity, low or no exercise, and unhealthy dietary habits have each been associated with inadequate sleep, little is known about their combined effect. The aim of this study was to quantify the overall impact of lifestyle-related factors on non-restorative sleep in the general Japanese population.

Methods and Findings

A cross-sectional study of 243,767 participants (men, 39.8%) was performed using the Specific Health Check and Guidance System in Japan. A healthy lifestyle score was calculated by adding up the number of low-risk lifestyle factors for each participant. Low risk was defined as (1) not smoking, (2) body mass index<25 kg/m², (3) moderate or less alcohol consumption, (4) regular exercise, and (5) better eating patterns. Logistic regression analysis was used to examine the relationship between the score and the prevalence of non-restorative sleep, which was determined from questionnaire responses. Among 97,062 men (mean age, 63.9 years) and 146,705 women (mean age, 63.7 years), 18,678 (19.2%) and 38,539 (26.3%) reported non-restorative sleep, respectively. The prevalence of non-restorative sleep decreased with age for both sexes. Compared to participants with a healthy lifestyle score of 5 (most healthy), those with a score of 0 (least healthy) had a higher prevalence of non-restorative sleep (odds ratio, 1.59 [95% confidence interval, 1.29–1.97] for men and 2.88 [1.74–4.76] for women), independently of hypertension, hypercholesterolemia, diabetes, and chronic kidney disease. The main limitation of the study was the cross-sectional design, which limited causal inferences for the identified associations.

Conclusions

A combination of several unhealthy lifestyle factors was associated with non-restorative sleep among the general Japanese population. Further studies are needed to establish whether general lifestyle modification improves restorative sleep.     

A new class of type I protein geranylgeranyltransferase (GGTase I) inhibitor

Replacement of the thiol groups in 1, a potent and highly selective Candida albicans GGTase I inhibitor discovered through screening, with an imidazole ring was achieved by using solid phase synthesis. A non-thiol compound, 7, was found as a representative of a new class of potent C. albicans GGTase I inhibitor with high selectivity against human GGTase I.     

Dissociable anterograde amnesic effects of retrosplenial cortex and hippocampal lesions on spontaneous object recognition memory in rats

The amnesic effects of excitotoxic lesions of the rat retrosplenial cortex (RS) and hippocampus (HPC) in the spontaneous object recognition (SOR) performance were investigated. The SOR test consisted of the sample-exposure session(s) and a test session. First, to test retrograde amnesia, rats received four sample-exposure sessions within a day at 4 weeks and 1 day before the surgery, respectively. In the test sessions conducted 1 week after the surgery, both lesion groups showed a temporally ungraded retrograde amnesia. Second, to test anterograde amnesia, 1- and 4-week retention intervals were inserted between the four sample-exposure sessions and the test session. The RS-lesioned rats showed a retention interval-dependent impairment in the test sessions, while the HPC-lesioned rats showed an impairment regardless of the retention interval. Finally, to test short-term recognition memory, 5- or 30-min delay was interposed between the single sample-exposure session and the test session. Both lesion groups performed normally irrespective of the delay length. These results suggest that both the RS and HPC are important for long-term object recognition memory, but these areas have different roles in it. ? 2012 Wiley Periodicals, Inc.     

Acrolein produces nitric oxide through the elevation of intracellular calcium levels to induce apoptosis in human umbilical vein endothelial cells: implications for smoke angiopathy.

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde, the levels of which are increased in the blood of smokers. To determine if acrolein is involved in the pathology of smoke angiopathy, the effect of acrolein on human umbilical vein endothelial cells (HUVEC) was examined. Intracellular nitric oxide (NO) levels, determined using diaminofluorescein-2 diacetate (DAF-2 DA), an NO sensitive fluorescent dye, were found to be increased after treatment in HUVEC with 10 microM acrolein. The measurement of nitrite with 2,3-diaminonaphthalene and a Western blot analysis revealed that nitrite and S-nitroso-cysteine levels were increased in a dose-dependent manner, confirming that NO production is increased by acrolein. The increase was not reduced by treatment with 10mM N-acetyl-l-cysteine (NAC), an anti-oxidant, but was reduced with 10 microM of the intracellular calcium chelator, 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester. Acrolein-stimulated NO production was significantly reduced by pretreatment with 1mM N(G)-nitro-l-arginine-methyl ester (L-NAME), an NO synthase inhibitor. The cytotoxicity of acrolein was reduced by pretreatment with 10 microM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), an intracellular NO scavenger, or 1mM L-NAME, whereas it was not reduced by 10mM NAC, 20 microM Curcumin, another peroxide scavenger, or 100 microM Mn(III)TMPyP, a superoxide dismutase mimic. Nuclear staining and a Western blot analysis using an anti-cleaved caspase 3 antibody revealed that the reduced viability of HUVEC by acrolein was due to apoptosis, which was reversed after pretreatment with 0.1mM carboxy-PTIO or 1mM L-NAME. Thus, acrolein increases intracellular calcium production to induce intracellular NO production by a calcium-dependent NO synthase, possibly eNOS, and the excess and rapid increase in NO might lead to the apoptosis of HUVEC. These data suggest that acrolein might be involved in the pathology of smoke angiopathy through the NO-induced apoptosis of endothelial cells.     

The presence of anti-sheep red blood cell heterophile antibodies and their characteristics in murine schistosomiasis japonica

Sensitive methods of an enzyme-linked immunosorbent assay (ELISA) using red blood cells (RBC) have been developed and were applied to the detection of anti-sheep red blood cell (SRBC) heterophile antibodies (Ab) present in sera of Schistosoma japonicum (SJ)-infected mice. The indirect hemagglutination test (IHA) was used for the purpose as well. By these methods a significant increase in the heterophile Ab levels was demonstrated in the mice particularly after 6-10 weeks of infection. The heterophile Ab in SJ-infected mice were predominantly immunoglobulins resistant to 2-mercaptoethanol treatment and had temperature-independent reactivity. In an attempt to investigate the immunological specificity of the heterophile Ab, various absorption tests were performed; Davidsohn's differential absorption test revealed that the heterophile Ab were distinct from Forssman antibody, Paul-Bunnell antibody and heterophile agglutinins known to appear in serum sickness. The heterophile Ab were absorbed only with SRBC and goat red blood cells, not with other species of RBC such as human O Rh-, O Rh+, A Rh+, B Rh+, mouse, ox, chicken, horse, rabbit, guinea pig, and rat RBC, or syngeneic tissue homogenates including brain, lung, liver, spleen, kidney, muscle, and thymus. This heterophile antibody response is not a consequence of a specific immune response directed to the antigens of SJ parasites, since absorption of the heterophile Ab with SJ adult worms or an egg preparation did not reduce the heterophile Ab level.     

Triiodothyronine concomitantly inhibits calcium overload and postischemic myocardial stunning in diabetic rats.

Acute effects of triiodothyronine (T3) on postischemic myocardial stunning and intracellular Ca2+ contents were studied in the isolated working hearts of streptozotocin-induced diabetic rats and age-matched controls. After two weeks of diabetes, serum T3 and T4 levels were decreased to 62.5% and 33.9% of control values. Basal preischemic cardiac performance did not differ between diabetic and control rats. In contrast, during reperfusion after 20-min ischemia, diabetic rats exhibited an impaired recovery of heart rate (at 30-min reperfusion 57.5% of baseline vs. control 88.5%), left ventricular (LV) systolic pressure (44.1% vs. 89.5%), and cardiac work (23.1% vs. 66.0%). When 1 and 100 nM T3 was added before ischemia, heart rate was recovered to 77.2% and 81.8% of baseline, LV systolic pressure to 68.3% and 81.9%, and cardiac work to 50.8% and 59.0%, respectively. Diabetic rat hearts showed a higher Ca2+ content in the basal state and a further increase after reperfusion (4.96+/-1.17 vs. control 3.78+/-0.48 micromol/g, p<0.01). In diabetic hearts, H+ release was decreased after reperfusion (5.24+/-2.21 vs. 8.70+/-1.41 mmol/min/g, p<0.05). T3 administration caused a decrease in the postischemic Ca2+ accumulation (lnM T3 4.66+/-0.41 and 100 nM T3 3.58+/-0.36) and recovered the H+ release (lnM T3 16.2+/-3.9 and 100 nM T3 11.6+/-0.9). T3 did not alter myocardial O2 consumption. Results suggest that diabetic rat hearts are vulnerable to postischemic stunning, and T3 protects the myocardial stunning possibly via inhibiting Ca2+ overload.