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W39F, F52Y, S98G, S98A, and S98C mutants of the neocarzinostatin apoprotein (apo-NCS) were newly prepared and investigated their physicochemical properties. The circular dichroism (CD) spectra of F78W, F52Y, S98A, S98G, S98C were superimposable with that of wild type 1R49 protein although the minor spectral change seemed to be in the ellipticity of W39F. The results suggest that position 52, 78, and 98 involving natural chromophore binding do not play a major role in the inducing overall structural changes of the protein. Conversely, the position 39 would be affected slightly. Ethidium bromide (EtdBr) binding to mutants was also evaluated by the monitoring of total fluorescence intensity and fluorescence polarization (FP). The observed dissociation constant in the FP study was 4.4 microM for wild type, 2.2 microM for S98A, 1.3 microM for S98G, 9.7 microM for S98C, respectively. When S98G and F52Y, the calculated maximum change of the total fluorescence intensity was increased, suggesting that the EtdBr binding to S98G or F52Y were slightly improved compared with the wild type. Then, a total of 14 amino acids randomly substituted phage displayed library of apo-NCS was successfully prepared, because substitution of the amino acid structured the chromophore-binding cavity were not change the overall structural features. The phages which bound glycyrrhetic acid conjugated bovine serum albumin were enriched from this library using phage display technique as the pilot experiments. Although more precision investigation still needs, it should be possible to select variants that have new functions not found in nature.

We developed a determination method for human urinary 12-hydroxyeicosatetraenoic acid (12-HETE) using LC–MS–MS. This method, which includes simple extraction and detection in the SRM mode, allows precise and accurate determination of 12-HETE. There was a significant sex difference in urinary 12-HETE levels. Chiral analysis of 12-HETE using LC–MS–MS with column-switching technique revealed that the major enantiomer was 12(S)-HETE. Furthermore, the urinary level in patients with diabetes mellitus (DM) was analyzed. The present in vivo findings indicate that there could be difference in production of 12(S)-HETE between genders and 12(S)-HETE may play a role in the pathogenesis of DM.

We have developed a simultaneous quantification method for prostaglandin (PG) E(2), PGD(2), PGF(2 alpha), 8-epi-PGF(2 alpha), 6-keto-PGF(1 alpha) and thromboxane (TX) B(2). Using [3,3,4,4-(2)H(4)]PGE(2), [3,3,4,4-(2)H(4)]PGD(2), [3,3,4,4-(2)H(4)]8-epi-PGF(2 alpha), [3,3,4,4-(2)H(4)]PGF(2 alpha), [3,3,4,4-(2)H(4)]6-keto-PGF(1 alpha) and [18,18,19,19-(2)H(4)]TXB(2) as internal standards (I.S.), the eicosanoids and their I.S. were simultaneously extracted by solid-phase extraction from cell-cultured medium, derivatized to methyl ester/methoxim/tert.-butyldimethylsilyl ether derivatives and analyzed using gas chromatography-mass spectrometry in the selected ion monitoring mode. The accuracy for the added eicosanoids ranged from 92 to 113%, and coefficients of variation ranged from 0.1 to 12.2%. Increased eicosanoids in RAW264.7 and U937 cells stimulated by lipopolysaccharide were suppressed by NS-398 and indometacin. This simultaneous quantification method can be applied routinely for assaying eicosanoids in vitro.

Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.

Objectives: The routine detection of polymorphisms affecting drug sensitivity in patients before treatment is important in the identification of drug responders or nonresponders, and patients at increased risk of drug toxicity. Here, we present an assay for the simultaneous and rapid genotyping of five polymorphisms influencing drug sensitivity. Design and methods: We used a hybridization probe assay on the LightCycler to detect five single nucleotide polymorphisms (SNPs): INPP1 (973C>A), ADRB2 (R16G and Q27E), HTR2A (102T>C), and mtDNA (1555A>G). Two fluorescent labeled hybridization probes were designed for the simultaneous detection of the five SNPs and detection of the variant alleles was performed by melting curve analysis. Results: All five SNPs were detected with a single thermocycle protocol within 40 min. The genotypes determined in this assay were identical to those obtained with conventional PCR and restriction fragment length polymorphism analysis. Conclusions: To our knowledge, we report here for the first time a method for simultaneous detection of five SNPs, on a single thermocycle protocol by the LightCycler. This method is rapid, highly sensitive, and high-throughput, and is thus suitable for routine clinical use and large-scale epidemiologic studies.

Queuosine is a modified nucleoside located at the first position of the tRNA anticodon, which is synthesized by tRNA-guanine transglycosylase (TGT). Although the levels of queuosine in cancer cells have been reported to be lower than those in normal cells, the expression levels of TGT remain to be determined. We determined the expression levels of a subunit of TGT (TGT60KD). Contrary of our expectations, the results revealed higher levels of expression of TGT60KD than that in normal cells, and the level of queuosine in the tRNA fraction corresponded with that of TGT60KD expression. These results suggest the possibilities that the expression levels of TGT60KD regulate TGT activity and the levels of queuosine, and that TGT60KD plays significant roles in carcinogenesis. To our knowledge, this is a first report of increased expression levels of TGT60KD in human cancer cells.

Although the role of an adhesion molecule such as P-selectin may be important in the pathogenesis of stroke, temporal, spatial, and cellular profiles of the expression of such a protein has not been fully studied in the case of the middle cerebral artery (MCA) occlusion and reperfusion in rat brain. Change in expression of immunoreactive P-selectin was examined in rat brain after transient MCA occlusion (MCAO) in comparison to that of permanent occlusion with an anti-P-selectin monoclonal antibody. Western blot analyses were performed to ensure the selective detection of immunoreactive P-selectin protein with the monoclonal antibody using brain homogenates before and after MCAO. Temporal, spacial, and cellular changes of P-selectin expressions were evaluated with rat brain sections at 2, 8h, 1 and 3 days of permanent MCAO, and at 2, 8h, 1, 3 and 7 days of reperfusion after 1 h of transient MCAO. Western blot showed a single band with a molecular weight of 140 kDa for both cases with permanent occlusion and reperfusion. P-selectin immunoreactivity was not normally present in rat brain sections. However, it was expressed mainly in the post-capillary venules of the cerebral cortex and caudate in the MCA territory with a peak at 2-8h after permanent occlusion and at 8h to 1 day after the reperfusion. The expression was diminished by 1 day of permanent occlusion and 3 days of reperfusion. The maximum staining in the case of permanent MCAO was stronger than the case with reperfusion. However, spacial distribution of the staining was similar in the cerebral cortex and caudate between the cases with permanent of transient MCAO. These results suggest a different temporal but similar spacial and cellular expression of P-selection immunoreactivity between permanent occlusion and reperfusion of MCA in rat brain.

We studied the steric course of the reaction catalyzed by the N-ethylmaleimide (NEM) reductase of Yarrowia (Candida) lipolytica (Y. lipolytica), using 4R-[4-2H1]NADPH and 4S-[4-2H1]NADPH as cofactors and N-ethylcitraconimide as substrate. Active substrates and inhibitors of NEM reductase and its subcellular distribution were also investigated to clarify the biochemical properties of this enzyme. NEM reductase catalyzes the reduction of N-ethylmaleimide to N-ethylsuccinimide with NAD(P)H as the cofactor. Several maleimide and cyclopentenone derivatives tested were also active substrates for NEM reductase of Y. lipolytica. Some pyrazolone derivatives, particularly 1-phenyl-5-pyrazolone, were found to be effective inhibitors of NEM reductase. Subcellular localization of NEM reductase was carried out using protoplast formation and differential centrifugation. Ninety-eight percent of the NEM reductase activity was recovered in the cytosolic fraction, indicating that NEM reductase in Y. lipolytica was the cytosolic enzyme. We also determined the stereochemical specificity of the reduction of N-ethylcitraconimide by NEM reductase in Y. lipolytica, showing that 4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by NEM reductase, and two hydrogen atoms from NADPH and H2O added to opposite faces of the double bond of N-ethylcitraconimide.

We established two monoclonal antibodies (MAbs, A6-1-E4, 1gM; κ and BC-D1, IgG1, λ), that specifically recognize different epitopes of rat Δ3, Δ2-enoyl-CoA isomerase (ECI). We then developed a specific and reliable sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using a capture monoclonal antibody and an indicator polyclonal antibody to evaluate ECI. The amounts of ECI were linearly determined in the ranges from 15 to 250 ng/well by this method. This assay can detect the amount of ECI in tissue homogenates without subfractionation. The ECI levels were 0.13 ng/μg homogenate in normal rat liver and 1.50 ng/μg homogenate in clofibrate-treated rat liver.

To evaluate the clinical usefulness of serum 1-methyladenosine, several modifications have been made in our previously established inhibition ELISA system. Horseradish peroxidase (HRP) labeled anti-mouse IgG and 3,3',5,5'-tetramethylbenzidine (TMBZ) were used as a secondary antibody and a substrate, respectively. The second blocking was done just before the addition of the secondary antibody. The standard curve of the modified ELISA system showed good linearity between 1 and 1,000 ng/ml, and the detection limit was 50 pg/well. Using the ultrafiltrated-serum samples, serum 1-methyladenosine levels in healthy individuals and cancer patients were determined. The mean level of 1-methyladenosine in 31 healthy individuals was 28.3 +/- 7.9 ng/ml, and cut off value was set at 44.1 ng/ml (Mean+ 2SD). In cancer patients, elevated levels of serum 1-methyladenosine above the cut off value were detected in 4 out of 25 cases tested, though 11 cases had elevated urinary 1-methyladenosine levels above the cut off value (3.23 nmol/mumol creatinine). Since 1-methyladenosine has no interaction with serum proteins and its molecular weight is quite low, it might be rapidly excreted into the urine.

Localization of delta 3, delta 2-enoyl-CoA isomerase (ECI) and NADPH-dependent-2,4-dienoyl-CoA reductase (DCR) in the rat kidney was investigated by immunocytochemical techniques. The kidneys were perfusion-fixed and embedded in Epon or LR White. For light microscopy, semi-thin sections of Epon-embedded materials were stained by the immunoenzyme technique after the epoxy resin was removed by treatment with sodium ethoxide. For electron microscopy, ultra-thin sections of LR White-embedded materials were stained by the protein A-gold technique. By light microscopy, the S1 segment of the proximal tubule was most heavily stained for ECI and DCR whilst S2 and S3 segments showed intermediate staining. A weak staining reaction was observed in the distal tubule and the medullary collecting tubule. In the cortical collecting tubule, heavily stained cells were present between weakly stained cells. By electron microscopy, gold particles showing the antigenic sites for ECI were confined mainly to the mitochondria, but few particles were observed in the peroxisomes. Gold labeling for DCR was localized both in the mitochondria and the peroxisomes. The labeling intensity of the peroxisomes was much higher than that of the mitochondria. The results suggest that metabolism of unsaturated fatty acids occurs mainly in the mitochondria and the peroxisomes of the proximal tubule in the kidney.

We established microdetermination methods of prostaglandin (PG) metabolites by GC-selected ion monitoring (GC-SIM) and applied them to the clinical investigations. At first the microdetermination of delta 17-6-keto-PGF1 alpha, a hydrolyzed metabolite of PGI2, is described. An authentic delta 17-6-keto-PGF1 alpha was prepared from eicosapentaenoic acid (EPA) incubated with a homogenate from the bovine aortic intima. [18O] delta 17-6-Keto-PGF1 alpha was synthesized to obtain an internal standard for GC-SIM of delta 17-6-keto-PGF1 alpha. A good linear response over the range of 10 pg-5 ng was demonstrated. Chromatographic conditions using a MP-65HT column presented nearly baseline separation of delta 17-6-keto-PGF1 alpha and 6-keto-PGF1 alpha. Furthermore, a monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-PGF1 alpha in the human blood sera. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in the human blood sera by GC/MS analysis. We were able to detect delta 17-6-keto-PGF1 alpha of the amount ranging from 6 to 26 pg/ml in the human blood plasma. The present method can be applied to the determination of delta 17-6-keto-PGF1 alpha in the human urine and plasma. Diabetes mellitus induces platelet alterations such as hyperaggregation. Variations in PG production seem to be related to this phenomenon but the changes in PG levels remain unclear. So we microanalyzed the 11-dehydrothromboxane B2 (TXB2) and 2,3-dinor-6-keto-PGF1 alpha, which were stable metabolites of TXA2 and PGI2, in the urine and investigated the relationship between the thromboxane/prostacyclin (TX/PGI) ratio and diabetes mellitus. The TX/PGI ratio in the urine of diabetics was higher than that of healthy volunteers. In murine, the TX/PGI ratio of STZ-induced mice was also higher than that of non-induced mice. The ratio of db/db mice also increased with the progress of diabetes mellitus. Furthermore, we investigated the relationship between the retinal vein occlusion (RVO), a thrombotic disease in which the retinal vein is blocked by blood aggregations, and the TX/PGI ratio. The TX/PGI level in patients with the RVO, who were not combine diabetes, was significantly higher than that in healthy volunteers. One of the causes of the RVO may be due to the variation of thromboxane production. This GC-SIM method can be used to determine the TX/PGI ratio in the urine.

Monoclonal antibodies (mAbs), 4H2 and 6H7, were prepared previously using a protein conjugate of a 1:1 epimeric mixture of the synthetic ABC-ring fragments of ciguatoxin (CTX), 3 and 4. Here, the interactions of these mAbs with the fragments of CTX and CTX3C, 3 and 5, were investigated by surface plasmon resonance (SPR) spectroscopy in an attempt to clarify an antigenic determinant. Compared with the previous synthesis, the fragment 3 possessing the 2S configuration was synthesized from tri-O-acetyl-D-glucal much more effectively. The mAb 4H2 was already known to show a dose-dependent binding to the bovine serum albumin (BSA) conjugate of 3, but not to that of 5. The present SPR study of 4H2 demonstrates that the A-ring side chain of 3 plays a decisive role as an epitope. Therefore, SPR can effectively replace the ELISA method for the analysis of mAbs.

Both aconite toxins (aconitine, mesaconitine, and hypaconitine) and a pufferfish toxin (tetrodotoxin, TTX) were detected in the blood of a legal autopsy case. In order to elucidate the in vivo influence of TTX on the toxic effects of aconitine, a mixture of aconitine and TTX was administered to male ICR mice orally or intraperitoneally. The animal experiments revealed that the time of death due to aconitine was significantly delayed in proportion to the dose of TTX compared with the case for aconitine alone, and that the mortality of aconitine was lowered by TTX when the dose ratio of the two toxins was in a particular range. Accordingly, it is thought that the toxic effects of aconitine are attenuated by TTX in vivo.

Neocarzinostatin (NCS), an antitumor protein antibiotic, is composed of apo-neocarzinostatin (apo-NCS) and neocarzinostatin-chromophore (NCS-chr), the principle of the biological activities of NCS. Apo-NCS having two tryptophan (Trp) residues at positions (39 and 83) was chemically modified by N-bromosuccinimide in a study on the correlation of the binding site(s) of NCS-chr. Selective oxidation of Trp residues was observed when NCS was titrated with N-bromosuccinimide. In contrast, non-selective oxidation of the two Trps on apo-NCS was observed and both Trp (39 and 83) of apo-NCS were titrated with N-bromosuccinimide. After selective oxidation, the remaining Trp residue of NCS was assigned as Trp (83). These results clearly indicate that the Trp (83) residue of apo-NCS changed from the "reactive type" to the "non-reactive type" after the binding of NCS-chr with apo-NCS. The fluorescence emission intensity of apo-NCS generated from the Trp (39) residue was quenched by NCS-chr. These data suggest that NCS-chr directly interacts with the Trp (39) residue and that a beta-sheeted loop containing the Trp (83) residue of apo-NCS changes the high-order structure upon binding with NCS-chr.

Cytochrome P450 4B1 (CYP4B1) is involved in the metabolism of several xenobiotics, such as 2-aminofluorene, 2-naphthylamine and benzidine. CYP4B1 allelic variants CYP4B1*1‐ *7 were recently identified. We thus hypothesized that CYP4B1 genotypes may modify bladder cancer risk. We examined the CYP4B1 genotypes in 169 bladder cancer cases and 190 hospital controls using a hybridization probe assay. Among the CYP4B1 genotypes observed, the most frequent genotypes in both the groups were CYP4B1*1/*1, *1/*2, *1/*3 and *2/*2. Logistic regression analysis revealed that the subjects carrying the CYP4B1*1/*2 or *2/*2 genotypes had a 1.75-fold increased risk of bladder cancer (95% CI¼1.03‐2.95, P ¼ 0.038) compared with the subjects carrying the CYP4B1*1/*1 genotype. We demonstrated the first genetic study regarding the association of CYP4B1 with bladder cancer. Our results suggest that CYP4B1 genotypes might have an effect on the risk of bladder cancer.

Satratoxin H, a mycotoxin, is thought to induce apoptosis of PC12 cells through the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in a glutathione (GSH)-sensitive manner. The present study was undertaken to further elucidate the mechanism by which satratoxin H induces cell death in PC12 cells. Satratoxin H caused apoptosis of PC12 cells within 24-h, as determined by DNA fragmentation and flow cytometric analysis. Satratoxin H increased reactive oxygen species (ROS) production and lipid peroxidation, as determined by malondialdehyde formation. These effects were attenuated by incubation of cells with GSH, suggesting that satratoxin H-induced increase in apoptosis of serum-deprived PC12 cells may be partially mediated through the generation of ROS.

The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3,trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxyacyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240,000. Two types of subunits, with molecular weights of 73,000 and 42,000, were identified. The complex was composed of two copies each of the 73,000- and 42,000-Da subunits. The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.

Aldehyde oxidase catalysed 2-oxidation activity of the (S)-enantiomer of RS-8359, a selective and reversible monoamine oxidase A (MAO-A) inhibitor, was investigated in liver cytosolic fractions from ten rat strains. Remarkably large strain differences were observed with approximately a 230 variation between the highest activity in the Wistar-Imamichi strain and the lowest activity in the Slc:Wistar strain. The activities of Crj:SD and Slc:SD strain rats were considerably low, and that of the F344/DuCrj strain was very low. Among six Wistar strains, Crj:Wistar, Slc:Wistar, WKY/Izm, WKAH/Hkm, Jcl:Wistar and Wistar-Imamichi, the Slc:Wistar strain rats showed exceptionally low 2-oxidation activity that was comparable to that of the F344/DuCrj strain. The rat strain differences in the catalytic activity of aldehyde oxidase could correlate in part with the expressed levels of protein based on the mRNA of aldehyde oxidase. However, no small discrepancy existed in the almost negligible catalytic activity and the fairly high expression levels of protein and mRNA in the F344/DuCrj and Slc:Wistar strain rats. Some genetic factors might possibly be one of reasons for the discrepancy.

Using the gene mapping membrane technique, we identified a gene (nemA) that encodes N-ethylmaleimide reductace in Escherichia coli. The open reading frame encodes a polypeptide of 365 amino acids with a molecular mass of 39, 514 Da. The deduced amino acid sequence showed a high degree of homology (87% identical) with the pentaerythritol tetranitrate reductase of Enterobacter cloacae and the morphinone reductase of Pseudomonas putida (52% identical).

Of a number of DNA marker typing techniques for personal identification in the field of forensic medicine, polymorphic short tandem repeat (STR) typing is currently the most frequently used technique. However, the multiplex STR method is time consuming. In contrast, single nucleotide polymorphism (SNP) detection methods are relatively rapid and amenable to high throughput. The discrimination power of each SNP is inferior to that of an STR, but a combination of many SNPs could realize a high discriminating power. In this regard, 16 highly informative SNP markers were selected in the introns of genes whose alleles had a proportion of 0.4-0.6 in the Japanese SNP database. The 16 SNPs were sequentially detected within 40 min using the hybridization probe assay on the LightCycler system. The allele and genotype frequencies of these SNPs were determined in a group comprising 64 unrelated Japanese subjects. Based on the frequency data of this group, the combined matching probability, defined as the estimated probability that two unrelated individuals selected at random would possess identical multilocus genotypes, was calculated with the 16 SNPs in the Japanese population and was found to be 2.025 × 10−7. This system is an effective tool in the forensic medicine to obtain information on personal identification.

There is wide individual variation in drug responses and adverse effects. As the main causes of the variation in drug responses, attention has focused on the genetic polymorphisms that encode metabolic enzymes regulating pharmacodynamics and receptors modulating the affinity with the responsive sites. Tailor-made drug therapy analyzes genetic polymorphisms involved in drug responses before drug administration and selects drugs and doses suitable for the individual genetic background. Establishment of tailor-made drug therapy is expected to contribute to medical economy by avoiding wasteful drug administration. To promote such medical practice, it is necessary to use simple genetic testing that is clinically convenient. Currently, genetic testing using real-time PCR has been frequently employed at laboratories with its clinical application anticipated. As to the many genes involved in drug responses, to date, the application of patient genetic information to tailor-made drug therapy has been achieved at the practical level. Information on pharmacogenetics will be a critical factor in medical practice in the near future.

The repeated administration of methamphetamine (MAP) causes behavioral sensitization in animals. We previously reported that the high accumulation of MAP was observed in the MAP-sensitized animal brain, which suggested that this phenomenon is an important factor in the development or expression of behavioral sensitization. The purpose of the present study is to elucidate the MAP distribution in the MAP-sensitized rat using gas chromatography/mass spectrometry (GC/MS). As a result, the MAP distribution in the heart at 10 min when showing a high accumulation of MAP in the MAP-sensitized rat brain was significantly higher than that of the control rat, whereas no significant differences in the liver, kidney, abdominal muscle, femoral muscle and blood were observed. In the brain and heart, there was no different distribution at 1 min, reflecting only the influx process from blood to brain and heart. On the contrary, there was the significant difference at 10 min, reflecting both the influx and efflux process, suggesting that the efflux process of MAP from brain or heart to blood may be slow due to MAP sensitization. In conclusion, it was clear that the brain and heart specific alteration of the MAP distribution occurred in the MAP sensitization. It was considered that the high accumulation of MAP in the MAP-sensitized rat brain may be related to the expression of behavioral sensitization and that the delayed efflux of MAP in the MAP-sensitized rat heart may be connected with the cardiac toxicity.

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.

Objective. The goal of this study was to determine the frequencies of allelic variants of CYP2B6 and CYP3A5 in the Japanese population. Methods. Genotyping of CYP2B6(*2, *3, *4, *5, *6, and *7) and CYP3A5 (*2, *3, *4, *5, and *6) was carried out in 265 unrelated Japanese subjects by polymerase chain reaction (PCR), restriction fragment length polymorphism and allele-specific, real-time PCR assays. Results. Allele frequencies for CYP2B6*2, *3, *4, *5, *6, and *7 in 256 Japanese subjects were 0.047, 0, 0.093, 0.011, 0.164, and 0, respectively. Ethnic variation in allele frequencies relative to that in Caucasian subjects was observed for CYP2B6*4 (0.093 vs 0.040), *5 (0.011 vs 0.109), *6 (0.164 vs 0.256), and *7 (0 vs 0.030). Allele frequencies for CYP3A5*2, *3, *4, *5, and *6 in 265 Japanese subjects were 0, 0.740, 0, 0.004, and 0, respectively. The frequency of the CYP3A5*1 allele is 2.8 times higher in Japanese than in Caucasians. Conclusions. Our results contribute to a better understanding of the molecular basis of ethnic differences in drug response, which may help to improve individualization of drug therapy and offer a preliminary basis for more rational use of drugs that are substrates for CYP2B6 and CYP3A5 in the Japanese population.

To evaluate the usefulness of urinary modified nucleosides as markers for malignancy, we prepared monoclonal antibodies (MoAbs) specific for 1-methyladenosine, termed AMA-2, and pseudouridine, termed APU-6. In patients with leukemia and other forms of cancer, significantly elevated levels of these nucleosides were detected, and these nucleosides clearly reflected the disease status of the patients with leukemia and lymphoma. The immunohistochemical analysis of esophagus cancer tissues with AMA-2 and APU-6 showed that cancer cells with pathological evidence were specifically stained with the MoAbs. Chemical identification of the cellular components reactive with the MoAbs revealed that AMA-2-associated antigens were mainly tRNA, and APU-6-associated antigens were mainly rRNA. The constant expression of APU-6-associated antigens among several cell lines suggest that the increase of intensity in malignant cells may be partly attributed to a quantitative increase rather than a hypermodification of tumor cell RNA. These results suggest that AMA-2 and APU-6 would be useful tools for clinical and biological studies of cancer.

We have developed an allele-specific fluorogenic 5' nuclease chain reaction assay for detecting polymorphisms in the following human drug-metabolizing enzyme genes : CYP2C9 (CYP2C9*2 and *3), CYP2C19(CYP2C19*2 and *3), CYP2D6 (CYP2D6*4, *10, *14, *18, and *21(C8)), N-acetyltransferase 2 (NAT2*5B, *6A, and *7B), thiopurine methyltransferase (TPMT*3C), and aldehyde dehydrogenase2 (ALDH2*2). This method is a marriage of two emerging technologies, the use of allele-specific amplification primers for target DNA and hybridization of the TaqMan probe. The TaqMan probe is labeled with both a fluorescent reporter dye and a quencher dye. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. All assays are performed using a single thermocycling protocol. This genotyping method is rapid and highly sensitive and yields a high throughput. It could be applied toward automated large-scale genotyping.

Thiopurine methyltransferase (TPMT) catalyzes the metabolism of important drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. The identification and frequency distributions of several variant TPMT alleles (TPMT*2--*8) have been described recently in many ethnic groups. We have recently demonstrated that TPMT*3C is the most common allele in Japanese subjects; however, it remains to be elucidated whether TPMT*4--*8 variants also exist in Japanese subjects. To detect polymorphisms in the TPMT gene (TPMT*4--*8), we have developed a mismatch polymerase chain reaction and restriction fragment length polymorphism method and conducted a population study of Japanese subjects. Genotyping of these variant forms was carried out in 192 Japanese healthy volunteers. The TPMT*4, TPMT*5, TPMT*6, TPMT*7, and TPMT*8 variants were not detected in any of the samples analyzed. This study provides the first analysis of the TPMT*4--*8 variants in a sample of the Japanese population and indicates that TPMT*4--*8 variants do not occur or are rare alleles in this population.

Fusion peptide P-9 was isolated from bovine serum albumin by controlled pepsin degradation in the presence of caprylic acid, followed by Sephadex G-75 gel filtration and ion-exchange chromatography of CM-cellulose. By this procedure, P-9 could be strictly separated from peptic fragment P-Phe, which has a molecular weight close to that of P-9. P-Phe has no fusogenic activity. The addition of P-9 to phosphatidylcholine (PC) liposomes containing cholesterol (Chol) gave rise to an increase of absorption intensity at around pH 4.0. The increase of turbidity by P-9 addition did not decrease with increasing pH, indicating P-9-mediated fusion of PC liposomes. The extent of the fusion of PC liposomes was strongly dependent on the PC chain length and temperature. The membrane fluidity close to the polar head groups of the fatty acyl chains of PC affected markedly the extent of P-9-mediated liposome fusion. However, there was no correlation between membrane fluidity near the hydrophobic end of the fatty acyl chains and the extent of liposome fusion. The rate of liposome fusion was dependent on both lipid composition and PC chain length. These results suggest that a contact or an interaction of P-9 with liposomal membrane occurs in the rigid regions. The character of the membrane-water interface region in the liposome controls a triggering effect for P-9-mediated fusion.

We demonstrate that glycyrrhizin (GL) enhanced Fas-mediated apoptotic body formation and DNA fragmentation in T cell lines although GL alone did not induce apoptosis. The enhancement effect of Fas-mediated apoptosis by GL was dose-dependent above 0.3 microM. Time course study revealed that simultaneous co-treatment of GL and anti-Fas antibody was crucial for the enhancement of apoptosis and pretreatment with GL was not effective. Anti-Fas antibody elicited caspase-3-like activity. However caspase-3-like activity with co-treatment of GL and anti-Fas antibody was the same level as the antibody alone. Glycyrrhetic acid, the aglycon of GL, did not enhance Fas-mediated apoptosis. The amphipathic property of GL might enable it to interact with the plasma membrane and lead to the enhancement of apoptosis.

The elevation of urinary modified nucleosides levels in urine is found in patients with cancers. In the precent study, we have tested 616 urine samples randomly collected from non-malignant cases. Thirty-two percent (194/616) and 11% (68/616) had elevated levels of 1-methyladenosine and pseudouridine, respectively (They are designated as false-positive cases). To elucidate the cause on non-specific elevation of the nucleosides, the correlation between creatinine excretion level and urinary nucleosides levels were determined. The result revealed that false-positive cases were frequently detected in patients with lower creatinine excretion levels. The mean creatinine levels of false-positive cases were significantly lower than those of negative cases. From these results, the false-positive of urinary 1-methyladenosine and pseudouridine might be due to the low creatinine excretion mainly caused by the renal dysfunction. Creatinine excretion in each individual should be taken into consideration in case of determining urinary modified nucleosides.

The selectin family of adhesion molecules is involved in adhesion of leukocyte to microcirculatory system and the transmigration into brain parenchyma. Although the role of P-selectin may be important in the pathogenesis of brain ischemia, a possible protective effect on ischemic brain injury by blocking P-selectin has not been reported. We have examined the effects of a novel anti-P-selectin antibody on ischemic brain injury after 24 h of permanent middle cerebral artery occlusion (MCAO) in rat. Male Wistar rats were subjected to MCAO by an insertion of a silicone rubber cylinder for 24 h. Anti-rat P-selectin monoclonal antibody, ARP 2-4, was injected intravenously at a dose of 1 mg kg-1 at 5 min before the induction of MCAO. Control animals received the same volume of vehicle solution. Regional cerebral blood flow (rCBF) was measured immediately after and at 8 h of MCAO. At decapitation of rats at 24 h of permanent MCAO, infarct size was compared between the antibody and vehicle treated group. In addition, immunohistochemistry for leukocyte infiltration and HSP72, and histochemistry for TUNEL were also, compared. Pretreatment with ARP 2-4 improved rCBF at 8 h of MCAO (55.4% +/- 11.7% of control, n = 5) as compared to vehicle group (24.2% +/- 11.8%, n = 5, p < 0.02). Although leukocyte infiltration was not normally detected by monoclonal antibodies for CD11a and CD18, it became remarkably evident at 1 day of MCAO. Although HSP72 and TUNEL were not also detected in sham control brains, they were induced in neurons of the MCA area at 1 day of MCAO. Treatment with ARP 2-4 significantly reduced the numbers of leukocyte and neurons with positive HSP72 and TUNEL stainings. These results demonstrated that an administration of a monoclonal antibody against P-selectin improved rCBF, and attenuated infarct size that was associated with reduction of leukocyte infiltration. Furthermore, treatment with the antibody reduced both HSP72 and TUNEL stainings. These data suggest an important role of P-selectin in ischemic brain damage, and a future therapeutic potential to human stroke patients.

Molecularly imprinted polymer (MIP) was prepared against phenobarbital using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross linking monomer. We analyzed the recognition properties of the phenobarbital MIP. In some organic solvents, imprinted polymer showed selective binding to phenobarbital. Two dissociation constants of binding were calculated by Scatchard plot analyses; Kd values were 1.8, 121.7 microM, and the number of binding sites was 8.3, 92.3 mumol/g MIP in toluene-heptane-acetic acid (25 : 75 : 1, v/v), respectively. The relationship between the binding affinity to phenobarbital MIP and the polarity of the solvent system, as well as the structure of the template molecule is also discussed.

A monoclonal antibody specific for a modified nucleoside, 5-methyl-2'-deoxycytidine (m5dCyd), was prepared using 5-methylcytidine (m5Cyd)-keyhole limpet haemocyanin (KLH) conjugate, and was characterized. Termed FMC9, the antibody reacts with m5dCyd and slightly with m5Cyd and 5-methylcytosine (m5Cyt) but not with other nucleosides tested in this investigation. FMC-9 was used in an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of m5dCyd levels. Sensitivity was in the picomole range. Methylation levels in peripheral blood cells of healthy donors were determined by inhibition ELISA. The percentage of m5dCyd in peripheral blood cells of 10 healthy donors was 5.08 +/- 0.50%. These results suggest that the inhibition ELISA using FMC9 is useful to monitor m5dCyd levels in the peripheral blood cells.

The induction of interferon (IFN) and interleukin-1 (IL-1) production in murine macrophages by a phosphopolysaccharide, produced by a dairy lactic acid bacteria, Lactococcus lactis ssp. cremoris, was investigated. When the phosphopolysaccharide was added into macrophage cultures at concentrations from 1 to 200 micrograms/ml, substantial IFN titers (6.2-79.2 IU/ml) were detected. Using the reverse transcription-polymerase chain reaction (RT-PCR), the expression of mRNA encoding IFN-gamma was verified in spleen macrophage cultures. Macrophages stimulated with the phosphopolysaccharide also produced IL-1 alpha at a concentration of 50 micrograms/ml. This study showed for the first time that phosphopolysaccharide derived from a dairy lactic acid bacterium can induce IFN-gamma and IL-1 alpha production in macrophages. These findings strongly suggest that the phosphopolysaccharide is a type of 'biological response modifier' and the fermented dairy foods containing Lactococcus lactis ssp. cremoris can be designated as a physiologically functional food.

The subcellular distribution of 2,4-dienoyl-CoA reductase (EC. 1.3.1.34, DCR) in rat liver was studied biochemically and immunochemically after the induction of clofibrate. DCR activity was mainly detected in the mitochondrial fraction by sucrose density gradient centrifugation in the livers of both normal and clofibrate-treated rats. It was also shown that the polyclonal antibody against purified DCR detected the enzyme in the mitochondrial fraction. However, the antibodies, which were affinity purified using the purified mitochondrial DCR or were epitope-selected using the fusion-polypeptide expressed from the mitochondrial cDNA clone (lambda gt11-RDR181), were cross-reacted with the peroxisomal DCR. These results suggest that peroxisomal DCR is immunochemically indistinguishable from mitochondrial DCR.

A monoclonal antibody against 5-methylcytidine was prepared and characterized. This antibody, termed AMC, was reactive with compounds that had 5-methylcytosine structure (i.e. 5-methyl-2'-deoxycytidine, 5-methylcytidine and 5-methylcytosine). AMC had the highest reactivity to 5-methyl-2'-deoxycytidine among reactive compounds and had no or very slight cross-reactivity to cytidine-related compounds and any other compounds. Analysis of immunoreactive materials in urine revealed that 5-methyl-2'-deoxycytidine rather than 5-methylcytidine was, contrary to our expectation, the major component. Then the inhibition ELISA system using AMC was established and urinary levels of 5-methyl-2'-deoxycytidine in healthy individuals and cancer patients were determined. The mean excretion levels of healthy individuals was 0.90 +/- 0.43 nmol/mumol creatinine and the cut-off value was set at the mean + 2 S.D. of healthy individuals (1.76 nmol/mumol creatinine). Among various types of cancer tested, elevated levels of 5-methyl-2'-deoxycytidine were detected in leukemia patients. From these results, urinary 5-methyl-2'-deoxycytidine might be applicable as a biologic marker for leukemia.

Hinokitiol (I) and tropolone (II) showed characteristic cytotoxic effects in vitro on five kinds of human and murine cell lines and blastic lymphocytes from mouse splenocytes. The cytotoxic effect of I on the growth of murine and human tumor cell lines, including RL male-1, MH134, HL60, K562 and KATO-III was definite when examined by thymidine incorporation into DNA and its 50% inhibitory concentration (IC50) on all cells was 0.3-0.6 microgram/ml. Compound II also showed comparable cytotoxic effects on these cell lines, indicating a little lower activity when compared to I. Furthermore, I and II also completely suppressed the [3H]thymidine ([3H]TdR) incorporation of mitogen-induced blastic lymphocytes. The suppressive activity on mouse lymphocyte proliferative response to concanavaline-A was also found with both compounds at a low concentration of 0.32 microgram/ml. As compound I is known to be of fairly low toxicity (LD50: 453 + 24 mg/kg in mice), the antitumor and immuno-suppressive effect of hinokitiol (I) should be further investigated.

A simple and sensitive semi-micro high-performance liquid chromatography (HPLC) was established for determining the serum levels of glycyrrhizin (GL) in humans. Butyl p-hydroxybenzoate was used as the internal standard and serum was deproteinized by methanol. The samples were separated on a Capcell Pak C18 UG120 column (150 x 1.5 mm i.d.; particle size, 5 microm). The detection limit of GL in serum was 100 ng/ml, which enables determination of serum levels of GL after administration of a therapeutic dose. The time-course study suggested that the elimination rate of GL differed between subjects for the same administered dose, although the sample was too small to allow a meaningful comment. In clinical practice, GL is used for its antiviral and anti-inflammatory effects. Excessive administration of GL can induce pseudoaldosteronism; however the optimal GL concentration in serum remains to be determined. The determination method reported here is expected to aid in the safe and efficient use of the drug in clinical practice.

The subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the peroxisomal isomerase, revealing a single band with an apparent molecular weight of 30,000. However, the isomerase was induced by clofibrate administration mainly in the mitochondrial fraction. These results suggest that delta 3,delta 2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.

N-Oxidation of pyrimidines having unsymmetrically substituted 4-and 6-positions with hydrogen peroxide in acetic acid gave mixture of their 1-oxides and 3-oxides. Ring transformation of these 1-oxides and 3-oxides into isoxazoles occurred on treatment with mineral acid, and provided information on the position of the N-oxide group in the pyrimidine N-oxides. Nuclear magnetic resonance spectroscopy with a lanthanide shift reagent was also found to be useful in identifying the pyrimidine 1-and 3-oxides.

Lipase [triacylglycerol lipase, EC 3.1.1.3] has been purified to homogeneity from Rhizopus japonicus NR 400 by chromatography on hydroxylapatite, octyl-Sepharose and Sephacryl S-200. It showed a molecular weight of about 30,000 by SDS-PAGE and a specific activity of 68,900 units/mg protein. The enzyme catalyzed the hydrolysis of tricapryn and tricaprylin rapidly in comparison with other triglycerides. This lipase had an optimum pH of around 5, and albumin enhanced its activity between pH 3 and 8. The composition of fatty acids liberated from linseed oil by the lipase was similar to that in the case of pancreatic lipase. The lipase activity was not affected by the addition of 1 mM metal ions or bile salts. Stimulation of the lipase activity was observed upon addition of albumin to the reaction mixture. Immunotitration experiments were also performed with antibodies raised against the purified lipase.

N-Acetylcysteamine and coenzyme A esters of cis-2-alkenoic acids have been found to undergo isomerization to the corresponding trans-isomers during their preparation by the mixed anhydride method and also during their alkaline hydrolysis. The isomerization might proceed by interaction of the free thiol group and the cis-double bond of 2-alkenoic thiol esters. The use of pyridine as a base and three or more equivalents of the mixed anhydride to the thiol compound prevented the formation of the trans-isomer. Addition of hydrogen peroxide during alkaline hydrolysis also prevented the isomerization completely.

2,4-Dienoyl-CoA reductase has been separated from Escherichia coli grown in the presence of linoleic acid and purified to homogeneity. The enzyme has a molecular weight close to 50,000 as determined by gel filtration on Sephacryl S-200 Super-fine. The reductase was rather stable in a buffer containing citric acid and kept its full activity on heating at 55 degrees C for 10 min in the pH range of 5.5 to 6.5, but was completely inactivated on heating at 58 degrees C for 10 min. Phosphocellulose column chromatography revealed that the reductase was not involved in the multi-enzyme complex (molecular weight of 260,000) of fatty acid oxidation.

Nuclear magnetic resonance spectroscopy with a lanthanide chemical shift reagent was applied for the structural determination of cis-and trans-isomers of 3-alkenoic acids, prepared as substrates for some enzyme reactions related to fatty acid metabolism. The configuration of N-acetylcysteamine derivatives of cis-2-alkenoic acids was also determined by the same technique.

Characteristics of the secondary structure of neocarzinostatin apoprotein (apo-NCS) were examined by various means. Gaussian analysis of the Fourier-transform infrared (FT-IR) curve and curve-fitting of the circular dichroism (CD) spectrum for apo-NCS revealed that this peptide was abundant in beta-structures. In the presence of sodium dodecyl sulfate (SDS), the CD bands of NCS originating from phenylalanyl, tyrosyl, and cystinyl residues decreased, indicating a conformational change around the chromophore (CNS-chr). On the other hand, apo-NCS, in the SDS system, showed no change of these bands. We showed that the major parts of the protein moiety consist of beta-structures by measurements of the FT-IR and CD spectra of apo-NCS and a prediction of the secondary structure based on the amino acid sequence of the peptide. It seems that properties of the protein may be important to the hydrophobic interaction between NCS-chr and apo-NCS.

The potassium permanganate oxidation of 3-methylthio-5, 6-diphenyl-as-triazine in the presence of a phase-transfer catalyst afforded 3-methylsulfonyl-5, 6-diphenyl-as-triazine. The 3-sulfonyl-as-triazine readily reacted not only with active methylene compounds but also with methyl or methylene ketones under basic conditions, and 5, 6-diphenyl-as-triazines containing a functionalized carbon substituent at the 3-position were obtained. Similarly, 2-substituted 4, 6-dimethylpyrimidines were synthesized by the nucleophilic substitution of 4, 6-dimethyl-2-phenylsulfonylpyrimidine with various ketones.

NADPH-dependent trans-2-enoyl-CoA reductase was purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was estimated to be 80,000 by gel filtration and that of the subunit was estimated to be 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was most active toward trans-2-hexenoyl-CoA and trans-2-octenoyl-CoA but was less active toward longer chain substrates, whereas the Km values decreased progressively with increasing carbon chain length of substrates. The reductase appears to have a functional thiol group. The enzyme activity was rapidly decreased by p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid), and slowly by N-ethylmaleimide. The enzyme was protected from inhibition by these SH-reagents by the addition of NADPH. The enzyme was also inhibited by saturated acyl-CoA esters.