Functional characterization of 21 CYP2C19 allelic variants for clopidogrel 2-oxidation.

Genetic variations in cytochrome P450 2C19 (CYP2C19) contribute to interindividual variability in the metabolism of therapeutic agents such as clopidogrel. Polymorphisms in CYP2C19 are associated with large interindividual variations in the therapeutic efficacy of clopidogrel. This study evaluated the in vitro oxidation of clopidogrel by 21 CYP2C19 variants harboring amino acid substitutions. These CYP2C19 variants were heterologously expressed in COS-7 cells, and the kinetic parameters of clopidogrel 2-oxidation were estimated. Among the 21 CYP2C19 variants, 12 (that is, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.14, CYP2C19.16, CYP2C19.19, CYP2C19.22, CYP2C19.24 and CYP2C19.25) showed no or markedly low activity compared with the wild-type protein CYP2C19.1B. This comprehensive in vitro assessment provided insights into the specific metabolic activities of CYP2C19 proteins encoded by variant alleles, and this may to be valuable when interpreting the results of in vivo studies.

Safety evaluation of the use of calcineurin inhibitor to prenatal and postpartum women in Japan from a health administrative database.

BACKGROUND: To investigate the use of calcineurin inhibitors (CNIs) in pregnant Japanese women and to evaluate their safety in infants. METHODS: Data were extracted from the claims database of the Japan Medical Data Center. The prevalence of CNIs was evaluated 180 days before pregnancy onset, during pregnancy, and within180-days post partum. We investigated the characteristics of the infants, including the presence of major malformations and their diagnoses, for 1 year after birth. RESULTS: A total of 91,865 pregnancies in 80,049 women were included. Fifty-three women were prescribed CNIs between 180-day before pregnancy onset and 180-day postpartum; 35 of the 53 women were prescribed the drugs during pregnancy, and 10 of their infants were born preterm. Three were diagnosed with major congenital malformations, such as patent ductus arteriosus. Six preterm infants presented with infant respiratory distress syndrome. CONCLUSIONS: No congenital anomalies were clearly attributable to the use of CNIs during pregnancy.

Changes of simple and complex spike activity of cerebellar purkinje cells with sleep and waking.

Action potentials of cerebellar Purkinje cells were observed in intact monkeys during sleep and waking. Purkinje cells exhibit two sorts of action potentials, called simple and complex spikes, and these two sorts of spikes were differently affected by sleep. Simple-spike activity (generated by the parallel fiber inputs to the Purkinje cell) was highest during sleep with rapid eye movements as compared with both waking and sleep with electroencephalographic slow waves. In contrast, complex-spike activity (generated by the climbing fiber inputs to the Purkinje cell) was lowest during sleep with rapid eye movements. The complex action potential of the Purkinje cell consists of an initial large spike followed by one or more smaller secondary spikes, and the number of these secondary spikes was found to be independent of the background discharge frequency of the simple spike. This independence suggests a possible role of presynaptic factors rather than the excitability level of the Purkinje cell itself in determining the number of secondary discharges occurring in the complex spike.

OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract.

AIMS: In the present study, we focused on one of the Aeromonas veronii isolates that exhibited marked adhesion onto carp intestine and studied its membrane-associated proteins for their possible involvement in mucosal adhesion. METHODS AND RESULTS: We isolated a strain of Aer. veronii (CWP11) that exhibited a high degree of temperature-dependent adhesion activity onto carp intestinal tract and studied its adhesion factor. A proteomic analysis of the membrane-associated fraction showed the presence of multiple proteins that were specifically expressed in CWP11 cells cultured at 25 degrees C. Of these, a 30 kDa protein was identified to be OmpA by a mass fingerprint analysis. Cloning and nucleotide sequencing of the ompA region of CWP11 revealed the presence of two tandem ompA homologues (ompAI-ompAII). Escherichia coli that expressed either OmpAI or OmpAII exhibited marked adhesion onto carp intestinal surface. Disruption of ompAI by a homologous recombination technique resulted in marked reduction of the adhesion activity in CWP11. CONCLUSION: The OmpA homologue plays an important role in the adhesion of the Aer. veronii strain onto the surface of intestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: We successfully identified an OmpA homologue to be an adhesion factor of Aer. veronii, an optimistic pathogen that habituates in carp intestinal tract.

Binding characteristics of a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine, to bovine serum albumin as measured by fluorescence.

The interaction between a major thyroid hormone metabolite, 3,3',5'-triiodo-L-thyronine and bovine serum albumin was investigated by fluorescence measurements. The apparent binding constants were obtained at various pHs assuming the equivalence and independence of the interaction sites on the protein from the fluorescence titration curves. The maximum binding was attained at pH 8.0, and the apparent binding constant was (5.28 +/- 0.13).10(5) M-1 with one binding site per albumin molecule. Thermodynamic parameters were also determined from the van't Hoff plot of the apparent binding constants at pH 7.5. The free energy change, enthalpy change and entropy change were -7.70 +/- 0.09 kcal.mol-1, -4.59 kcal.mol-1 and 10.2 e.u., respectively.

Context dependent discharge characteristics of saccade-related Purkinje cells in the cerebellar hemispheres of the monkey.

In the previous paper (Mano et al, 1991), we reported the discharge patterns of saccade-related Purkinje cells during visually guided saccade task, which were recorded from posterior cerebellar hemisphere, the Crus IIa. In the present study, we analysed these P-cell's simple spike activity during the spontaneous saccade in inter-trial intervals (ITI) of visually guided saccade task, comparing with the activity during the visually guided saccade. We found that the modulation of simple spike discharges during spontaneous saccade was weaker than the modulation during the visually triggered saccade. We recorded single unit discharges of Purkinje cells from cerebellar posterior hemisphere (Crus IIa) in awake Japanese monkeys (Macaca fuscata), trained to perform simple reaction time saccade task gazing at a small light rear-projected on to a tangent screen 54 cm in front of the monkey. Horizontal and vertical eye positions were measured by a corneal search coil method. Comparison of simple spike activity associated with spontaneous saccade during ITI to the activity during visually triggered saccade clarified that the discharge patterns of simple spikes are basically the same during both types of the saccades, but the amount of the phasic modulation (increase of decrease of discharge rate) were larger for all directions (up, down, left and right) during visually guided saccade than that during spontaneous saccade in all saccade-related Purkinje cells so far examined in two monkeys. The modulation, however, cannot be assumed to have been induced by the visual stimulus per se. Because, the maximum increase of simple spike discharge rate aligned at saccade onset is larger than that aligned at target jump. And, the half width of the change was wider when aligned at target light jump than when aligned at the onset of saccades, in all the four directions, indicating the changes of the firing rate were more time-locked to the onset of saccadic eye movements than to the triggering visual stimulus. The present findings suggest that the cerebellar hemisphere plays a more important role in the control of externally triggered voluntary eye movements than in the control of self-initiated, self-paced eye movements. We discussed these findings combining with previous findings on limb movement-related P-cells (Mano et al, 1980, 1986, 1989), from view point of the general role of the cerebellar hemisphere in the control of voluntary movements.

Electrodes modified with nitrofluorenone derivatives as a basis for new biosensors.

We report about the electrocatalytic properties of electrodes modified by adsorption of nitro-fluorenone derivatives. The stable, adherent monolayer of these catalyst precursors can be transformed electrochemically into the corresponding hydroxylamine compounds (R-NO(2)+4e+4H(+)-->R-NHOH+H(2)O). The completely reversible two electron oxidation of the hydroxylamine leads to the nitroso compounds (R-NHOH-->R-NO+2e+2H(+)) that exhibit high catalytic activity in the electrooxidation of NADH at low overpotentials (-30 mV vs. Ag/AgCl) and therefore constitute a new family of efficient redox mediators for biosensor applications. A significant increase in catalytic activity (up to 500%) is observed after addition of calcium ions to the electrolyte. This is explained by a specific and bridging complexation between the coenzyme's phosphate groups and a carboxyl group present in the catalyst molecule. The interaction favours the contact between NADH and the surface confined catalyst, leading to a higher electron transfer efficiency. This interaction can be used in an approach of molecular level design for controlled monolayer deposition of catalyst, Ca(2+), NAD+ and enzyme. A very simple and inexpensive modification scheme, essentially based on electrostatic attraction, leads to electrodes that can be employed as reagentless biosensors for the electrochemical detection of common and commercially interesting analytes like glucose.

Vitamin B12 affects non-photic entrainment of circadian locomotor activity rhythms in mice.

Administration of vitamin B12 (VB12) has been reported to normalize human sleep-wake rhythm disorders such as non-24-h sleep-wake syndrome (HNS), delayed sleep phase syndrome (DSPS) or insomnia. However, the mechanisms of the action of VB12 on the rhythm disorders are unknown. In the present study, therefore, effects of VB12 on circadian rhythms of locomotor activity were examined in mice. In the first experiment, CBA/J mice were maintained under continuous light condition (LL) or blinded, and after free-running rhythms became stable, the mice were intraperitoneally injected with either VB12 or saline at a fixed time every day. In all the mice with tau > 24 h, saline injections resulted in entrainment of circadian rhythms, whereas not all the mice with tau < 24 h entrained to the injection. In contrast to saline injections, VB12 injections did not always induce entrainment and about half of the mice with tau > 24 h free-ran during the injection. In the second experiment, the amount of phase advances of circadian rhythms induced by a single injection of saline at circadian time (CT) 11 under LL was compared between the mice with and without VB12 silastic tubes. The results showed that the amplitude of phase advances was smaller in the mice with VB12 than those without VB12. In the third experiment, daily injections of saline were given to the mice with VB12 silastic tubes maintained under LL. In this chronic treatment of VB12 as well, attenuating effects of VB12 on saline-induced entrainment were observed. These results suggest that VB12 affects the mechanisms implicated in non-photic entrainment of circadian rhythms in mice.

Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid CoA thioester.

The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-18O3]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and 18O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed delta 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The delta 24-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.

Plasma direct injection high-performance liquid chromatographic method for simultaneously determining E3810 enantiomers and their metabolites by using flavoprotein-conjugated column.

A high-performance liquid chromatographic method using the column-switching technique was developed for the simultaneous determination of E3810 and its metabolites (M1-M4). An avidin column was used for in-line pretreatment to exclude plasma proteins, allowing direct injection of a large volume of plasma. A flavoprotein-conjugated chiral stationary phase in a gradient elution mode then gave baseline separation of E3810 and the four metabolites. The enantiomers of E3810 and M3 were also separated. The method is simple, rapid, accurate, and precise. Since no extraction procedure is employed, which might involve different recoveries of different metabolites, no internal standard is necessary. The method was applied to analyze E3810 and its metabolites in plasma after intravenous injection of the drug in beagle dogs.

Affinity assembled multilayers for new dehydrogenase biosensors.

We propose a novel approach, which allows the control of the spatial arrangement of redox mediator, coenzyme and enzyme on the electrode at a molecular level, using essentially electrostatic interactions. The first step consists of adsorbing a monolayer of molecules out of a new family of redox mediators, substituted nitrofluorenones. In a second step, a monolayer of calcium cations is immobilized at the interface. It serves as a bridge between the redox mediator and the subsequently adsorbed coenzyme. The weak interaction between a carboxyl group of the redox mediator and the coenzyme's phosphate groups, revealed by QCM measurements, allows the coenzyme to keep its natural activity in the adsorbed state. In the last step, we use the intrinsic affinity of this monolayer of NAD(+) for dehydrogenases to build up a supramolecular sandwich composed of mediator/Ca(2+)/NAD(+)/dehydrogenase. This simple modification procedure, which might constitute a versatile approach for the low cost assembly of well-defined biosensors surfaces, has been successfully applied to the enzymatic detection of glucose, glutamate and alcohol.

Mediator-modified electrodes for catalytic NADH oxidation: high rate constants at interesting overpotentials.

Carbon paste electrodes were modified with a nitrofluorenone derivative, 2,4,7-trinitro-9-fluorenone, adsorbed on zirconium phosphate (ZP). After electrochemical reduction of the fluorenone derivative, it turns into a very efficient mediator for electrocatalytic NADH oxidation, with a formal potential of about +250 mV vs. Ag/AgCl. The electrochemistry and the electrocatalytic properties of the mediator were investigated with cyclic voltammetry and rotating disk electrode methodology. The second order rate constant with NADH was evaluated and found to be higher than 10(6) M(-1) s(-1), thus approaching true diffusion controlled currents for NADH oxidation.

Characterization of Japanese eel immunoglobulin M and its level in serum.

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790,000; it contained an equimolar heavy chain and light chain with molecular weights of 72,000 and 25,000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by D-mannose and could be abolished by alpha-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml(-1); it amounted to 10.3% of the total serum protein.

Resolution of 4-(4-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepin-4yl)-1(2H)-phthalazi none enantiomers in plasma with frit-FAB LC-MS using a conalbumin column.

A new column-switching method for analysis of drug enantiomers in plasma has been developed with liquid chromatography-frit fast atom bombardment mass spectrometry in combination with a chiral resolution column, which consists of conalbumin (egg-white glycoprotein) immobilized on silica gel and can be used in the reversed-phase separation mode. This method makes it possible to inject a large volume of deproteinized plasma and obtain resolution of drug enantiomers with high sensitivity. The optimum mobile phase, including a non-volatile buffer such as phosphate buffer, for separation of drugs from a large amount of endogenous compounds can be used, because of inclusion of a trapping column with a desalting function. This method is very simple and rapid, and should be very powerful in studies requiring high-sensitivity analysis with chiral separation of drugs from biological samples such as plasma.

Infection with Edwardsiella tarda causes hypertrophy of liver cells in the Japanese flounder Paralichthys olivaceus.

To study the direct cause of liver enlargement in the Japanese flounder Paralichthys olivaceus infected with Edwardsiella tarda, the fish were challenged with E. tarda and reared without feeding. The liver of fish exposed to the bacteria was markedly enlarged compared to that of the controls while no severe histopathological change appeared in the organ during the experiments. No notable difference was observed in the crude fat, glycogen, and water content of the liver between challenged and control fish. The size of liver cells and nuclei of the challenged fish was apparently larger than that of the controls. Analysis of crude DNA in the liver suggested that the number of liver cells of starved control fish significantly decreased during the experiment while that of the challenged fish was maintained at a level of the initial control. RNA/DNA ratio of the liver of challenged fish clearly increased while it decreased in the control fish during the experiment. These observations suggest that liver enlargement of flounder infected with E. tarda, at least in the early stage of infection, is not a result of any readily observable histopathological changes and that E. tarda infection causes hypertrophy of the cells, as well as preventing decrease in liver cell number.

Direct injection method for quantitation of endogenous leukotriene E4 in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry with a column-switching technique.

A new analytical method employing liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a column-switching system was developed for quantitative determination of leukotriene E4 (LTE4) in human urine. A column-switching system using a trapping column, which concentrates the analyte and removes salts and other water-soluble contaminants, allowed direct injection of human urine. Because simultaneously eluted endogenous contaminants suppressed the ionization efficiency of LTE4, good liquid chromatographic separation was very important for establishing this method, notwithstanding the high selectivity of MS/MS. The calibration curve was linear over the range from 10 to 3000 pg/mL, and the method showed good accuracy and precision. This method should therefore be very useful for determination of LTE4 amounts in human urine in studies on leukotriene metabolism and the efficacy of antileukotriene drugs.

Simultaneous detection of cholyl adenylate and coenzyme A thioester utilizing liquid chromatography/electrospray ionization mass spectrometry.

It has been proposed that acyl adenylate is first formed during activation of the carboxy group into the acyl CoA thioester, an intermediate in the formation of amino acid conjugates. Acyl CoA synthetases may be responsible for this acyl adenylate formation. Recently, we hypothesized the preferential formation of cholic acid adenylate, a major bile acid, preceding production of the corresponding CoA thioester in incubations with rat liver microsomal fractions. To verify this biosynthetic mechanism, monitoring of the incubation mixture of acyl adenylate together with both substrate and acyl CoA thioester is needed. We have developed a detection method for the simultaneous detection of these cholic acid derivatives utilizing liquid chromatography/electrospray ionization mass spectrometry. The CoA thioester of cholic acid forms a chelation complex with the divalent cations remaining on the silica gel packed into the analytical column. Both the addition of a chelating agent, such as EDTA, to the mobile phase and an adjustment of the mobile phase pH to a weak alkaline effectively removed such chelate formation, producing a sharp CoA thioester peak. For a simultaneous mass spectrometric analysis of cholic acid, the corresponding adenylate and CoA thioester, the combined use of a 300 A particle diameter ODS column and 20 mM ammonium acetate buffer (pH 9.0)/2-propanol/acetonitrile as the mobile phase have been proved to be preferable. To avoid any degradation of the chemically unstable adenylate produced in the incubation, we employed a direct injection of the sample onto a preconcentration column. The obtained results indicated a high sensitivity of this method.

[Pharmacokinetic and clinical study on cefodizime in obstetrics and gynecological field].

Clinical investigation of cefodizime (CDZM, THR-221), a newly developed cephem antibiotic, was carried out with regard to its distributions to genital organs, and the drug was evaluated clinically against infections in obstetric and gynecological fields. 1. Distributions to genital organs One gram of CDZM was administered to each patient who received simple total hysterectomy by 1 hour intravenous drip infusion and concentrations of CDZM in genital organs such as antecubital vein, uterine artery, ovary, oviduct, endometrium, myometrium, cervix uteri and portio vaginalis were examined. Serum concentrations were elevated to an average of 90.63 micrograms/ml in 15 minutes after administrations of CDZM and decreased gradually at fixed times. CDZM was distributed in concentration ranges of 45.32-10.96 micrograms/g in ovary, 26.58-10.20 micrograms/g in oviduct, 42.20-9.80 micrograms/g in endometrium, 31.28-11.72 micrograms/g in myometrium, 42.20-12.52 micrograms/g in cervix uteri and 45.32-9.40 micrograms/g in portio vaginalis, and these high concentrations lasted more than 3 hours after administrations. 2. Clinical evaluations CDZM was given to 10 patients, including 7 cases with pelvioperitonitis and 1 case each with pyometra, Bartholin's abscess and puerperal fever, at a dose level of 1 or 2 gram 2 times daily by 30-60 minutes intravenous drip infusion. Overall clinical efficacies were excellent in 3 cases and good in 7 and the efficacy rate was very high, at 100%. Bacteriological efficacies were eradicated in 6 cases, and unknown in 2, and the eradication rate was 100%.(ABSTRACT TRUNCATED AT 250 WORDS)