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.

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.

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.

Phylogenetic analysis of intestinal bacteria and their adhesive capability in relation to the intestinal mucus of carp.

AIMS: The aims of the present study are to characterize the intestinal microbial community displaying a high-adhesive capability in fish, and to evaluate the relationship between mucosal adhesion of intestinal bacteria and fish health and disease. METHODS AND RESULTS: A total of 707 aerobic bacteria isolated from carp intestine that were maintained under either feeding (feeding group) or no-feeding (no-feeding group) conditions and were performed adhesive assay. Isolates were divided into three categories on the basis of adhesive capability: high-, medium-, and low- adhesive capabilities. The average proportions of isolates with high-adhesive capability in the feeding and no-feeding groups were 30% and 32%, respectively. A phylogenetic analysis using a partial 16S rRNA gene demonstrated that most isolates with high-adhesive capability in both groups were classified as belonging to an Aeromonas group, and populations of isolates within high- and low-adhesive categories were markedly different. CONCLUSIONS: Intestinal bacteria with a high-adhesive capability in relation to intestinal mucous always colonize on the surface of intestinal mucosa and grow in the intestinal tract of feeding carp. The adhesive capability of intestinal bacteria is essential for colonization and growth in the intestinal tract of fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that members of the Aeromonas group with adhesive capability always colonize on the surface of intestinal mucosa.

Molecular lego for the assembly of biosensing layers.

We propose a procedure to assemble monolayers of redox mediator, coenzyme, enzyme and stabilizing polyelectrolyte on an electrode surface using essentially electrostatic and complexing interactions. In a first step a monolayer of redox mediator, substituted nitrofluorenones, is adsorbed. In a second step, a layer of calcium cations is immobilized at the interface. It establishes a bridge between the redox mediator and the subsequently adsorbed coenzyme NAD(+). In the next step we use the intrinsic affinity of the NAD(+) monolayer for dehydrogenases to build up a multilayer composed of mediator/Ca(2+)/NAD(+)/dehydrogenase. The so obtained modified electrode can be used as a biosensor. Quartz crystal microbalance measurements allowed us to better understand the different parameters responsible for the adsorption. A more detailed investigation of the system made it possible to finally stabilize the assembly sufficiently by the adsorption of a polyelectrolyte layer in order to perform rotating disk electrode measurements with the whole supramolecular architecture on the electrode surface.

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.

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.

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.

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.

The enantioselective immunoaffinity extraction of an optically active ibuprofen-modified peptide fragment.

Acyl glucuronides are known to produce the covalently bound protein adducts which may be the cause of hypersensitivity and toxic responses to acidic drugs. The structural analysis of the drug-protein adducts is therefore needed. From this point of view, we developed an enantioselective immunoaffinity extraction method, which employs an immobilized antibody to specifically isolate peptide fragments that have been modified with optically active ibuprofen. Rabbits were immunized with (S)-ibuprofen coupled to bovine serum albumin through a beta-alanine group. The elicited antibody strongly recognizes the asymmetric center and the isobutylphenyl moiety of (S)-ibuprofen and its conjugates but has a low affinity for their anti podes. A 0.5-mL aliquot of the immunosorbent (11.5 mg of IgG/mL gel) prepared by immobilization of the antibody was capable of retaining up to 1 microg of (S)-ibuprofen. When a mixture of substance P with (R)- and (S)-ibuprofen-modified substance P was loaded on the immunosorbent, the (S)-ibuprofen-modified substance P was selectively retained. The modified peptide was quantitatively recovered by elution with 10 mM ammonium acetate buffer (pH 5.0)/methanol (5:95, v/v). The proposed method would be useful for the structural characterization of optically active ibuprofen-modified human serum albumin.

Sequelae after limb-sparing surgery with major vascular resection for tumor of the lower extremity.

PURPOSE: Limb-sparing procedures have recently replaced amputations as the treatment for tumors invading major vessels of the lower extremity. Major arteries must be reconstructed for limb salvage. The veins are not usually reconstructed. This study was undertaken to investigate the sequelae such as chronic venous disease after venous resection for tumors. METHODS: Ten patients who underwent limb-sparing surgery for a tumor of the lower extremity or retroperitoneum that required major vascular resection were studied. The median follow-up period was 48 months. After combined resection of a major artery and vein, arterial reconstruction was performed. The veins were not reconstructed. The resected veins included the inferior vena cava (n = 2), the external iliac and common femoral veins (n = 3), the superficial femoral vein (n = 3), and the popliteal vein (n = 2). The main outcome measures were clinical classification of chronic venous disease in 10 patients and air plethysmography in seven patients. RESULTS: Clinical classification was C(0A) in 6 patients, C(3A) in 1 patient, C(3S) in 2 patients, and C(4S) in 1 patient. Venous claudication with uncontrollable edema was observed in two patients with C(3S) disease. Pain and itching with inflammatory skin changes were observed in one patient with C(4S) disease. These three patients had undergone resection of the femoral vein, including the deep femoral vein along with proximal adductor muscles. Air plethysmography revealed that the ejection fraction was significantly lower and the residual volume fraction was significantly higher in the three patients with symptoms than in symptom-free patients. CONCLUSIONS: Significant chronic venous disease was observed in the patients who underwent combined resection of the femoral vein, the deep femoral vein, and the adductor muscles for a tumor.

Bile acid acyl adenylate: a possible intermediate to produce a protein-bound bile acid.

The non-enzymatic production of a protein-bound adduct by the action of the acyl adenylate of bile acids is described. On incubation of deoxycholyl adenylate with substance P in phosphate buffer, peptides covalently bound with one or two molecules of the bile acid were detected. The modified peptides were structurally characterized by time-of-flight mass spectrometry with matrix-assisted laser desorption/ionization (MALDI-TOFMS) in the post-source decay mode, and by liquid chromatography/electrospray ionization MS/MS. The deoxycholic acid was bound on substance P through the amino group at Arg-1 and/or Lys-3. The adenylate of cholic acid also produced the protein-bound bile acid on incubation with lysozyme, and the binding sites of the cholic acid appeared to be the lysine residues at 1, 33, 97 and 116. The results clearly suggest that bile acid adenylates in vivo may act as active intermediates to produce covalently bound bile acid adducts with peptides and proteins by nucleophilic displacement of the 5'-adenylic acid through the free amino groups.

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.

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.

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.

Structural analysis of N-linked carbohydrate chains of funnel web spider (Agelenopsis aperta) venom peptide isomerase.

The structure of the N-linked carbohydrate chains of peptide isomerase from the venom of the funnel web spider (Agelenopsis aperta) has been analyzed. Carbohydrates were released from peptide isomerase by hydrazinolysis and reductively aminated with 2-aminopyridine. The fluorescent derivatives were purified by phenol/chloroform extraction, followed by size-exclusion HPLC. The structure of the purified pyridylamino (PA-) carbohydrate chains were analyzed by a combination of two-dimensional HPLC mapping, sugar composition analysis, sequential exoglycosidase digestions, and mass spectrometry. The peptide isomerase contains six kinds of N-linked carbohydrate chains of truncated high-mannose type, with a fucose alpha 1-6 linked to the reducing N-acetylglucosamine in approximately 80% of them.

Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry.

Sphingolipid metabolites ceramide, sphingomyelin, sphingosine, psycosine, sphingosylphosphorylcholine, and dimethylsphingosine were separated and simulataneously quantitated by liquid chromatography/ionspray ionization tandem mass spectrometry (LC/MS/ MS). The use of glassware throughout minimized losses due to adsorption and the pretreatment of this method consisted of simple liquid-liquid extraction procedure with a mixture of chloroform and methanol. After separation on a short C18 silica column eluted in a gradient mode, the metabolites were detected by MS/ MS. This assay allows simultaneously quantification of these metabolites over a range of at least 0.1 to 100 ng/ 10(6) cells. The LC/MS/MS analyses took 10 to 15 min per sample and we could examine up to 50 samples per day. We also detected endogenous sphingosine 1-phosphate in HL-60 cells. The utility of the method was demonstrated by examining changes in metabolites levels in HL-60 cells after treatment with sphingomyelinase. It was found that sphingomyelinase from Bacillus cereus may have selectivity for acyl chain length.