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Rat hepatic flavin-containing monooxygenase (FMO) 1A1 expressed in yeast catalysed the S-oxidation of flosequinan sulfide (7-fluoro-1-methyl-3-methylthio-4-quinolone) to R (+)-flosequinan (sulfoxide form, R(+)-7-fluoro-1-methyl-3-methylsulphinyl-4-quinolone) but not to S (−)-flosequinan, and did not catalyse the oxidation of R (+)- and S (−)- flosequinan to flosequinan sulfone. The K m and V max for the stereoselective S-oxidation were 33 μM and 6.2 nmol per min per mg of microsomal protein, respectively. The S-oxidation was inhibited by 1-(1-naphthyl)-2-thiourea and thiobenzamide. n -Octylamine activated the S-oxidation with little change in stereoselectivity. The ability of the recombinant yeast to produce R (+)-flosequinan from flosequinan sulflde could be maintained for at least 2 days and exemplifies the value of a recombinant yeast expressing FMO as a stereoselective bioreactor.

1. In order to study the pharmacokinetics of flosequinan enantiomers ((+-)-7-fluoro-1-methyl-3-methylsulphinyl-4-quinolone) containing chiral sulphur, plasma levels of (+)-(R)- and (-)-(S)-flosequinan (R-FSO and S-FSO) and two metabolites (flosequinan sulphide (FS) and flosequinan sulphone (FSO2)) were measured after oral and i.v. administration of racemic flosequinan (rac-FSO), R-FSO and S-FSO in male rat. 2. The pharmacokinetic parameters of the enantiomers were different after oral and i.v. administration of R-FSO and S-FSO. The plasma clearance of R-FSO was higher than S-FSO. 3. The major metabolites of boh R-FSO and S-FSO was FSO2. A minor metabolite, FS, was also detected in plasma. 4. Interconversions occurred after the oral and i.v. administration of R-FSO and S-FSO. The amount of interconversion from S-FSO and R-FSO was greater than that from R-FSO to S-FSO. The rate of interconversion after oral administration was higher than that after i.v. administration. 5. After i.v. administration of FS, R-FSO and S-FSO were detected in plasma, suggesting that the interconversion occurred via formation of FS. 6. The pharmacokinetic parameters of R-FSO after administration of rac-FSO differed from that after administration of R-FSO, indicating the interaction between each enantiomer.

1. 7-Ethyl-10-[4-(piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), a potent anticancer agent currently under development for clinical use, is metabolized in vivo to 7-ethyl-10-hydroxycamptothecin (SN-38), which is subsequently conjugated to 7-ethyl-10-hydroxycamptothecin glucuronide (SN-38-glucuronide). The SN-38-glucuronide was hydrolysed by beta-glucuronidase from E. coli to aglycones and glucuronic acid. 2. Four purified natural glucuronides including baicalin, wogonoside, luteolin-3'-glucuronide, and glycyrrhizin, inhibited beta-glucuronidase using SN-38-glucuronide as substrate. The inhibition potencies of these natural glucuronides toward beta-glucuronidase were similar to that of saccharic acid 1,4-lactone. 3. These results indicate that plant materials of Kampo (Japanese herbal) medicines containing these glucuronides could be used in vivo to decrease the enterohepatic circulation of SN-38 and possibly that of other drugs.

The Long Evans Cinnamon (LEC) rat, which has been established as a strain showing hereditary hepatitis and hepatic carcinoma, was found to possess autoimmune antibodies to liver microsomal proteins, particularly to a protein with the molecular weight of 56kD. The antibodies also recognized a protein(s) in liver microsomes from Long Evans Agouti and Sprague-Dawley rats. About 42 and 15 percent of respective female and male LEC rats died within a week after acute hepatitis; sera from all of the animals contained the antibodies. About 43 and 0 percent of the surviving female and male LEC rats possessed the antibodies, respectively. These results suggest that the autoantibodies occur in association with acute lethal hepatitis in the LEC rats.

Seven DNA probes which recognize single-locus hypervariable region (HVR) were applied to a paternity test in which the putative father and his wife were deceased. Three legitimate children, an illegitimate child and her mother were available for analysis. The cumulative paternity index of the illegitimate child derived from 15 conventional blood group markers was 18.71 and from 7 DNA probes 92,572.08, that is, 4,948 times higher than the former. Thus the DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the child. The application of highly discriminating polymorphisms of DNA which recognize single HVR loci is considered to be extremely informative in cases of disputed parentage.

Multiple forms cytochrome P450 (P450 or CYP) enzymes play important roles in the oxidation of structurally diverse xenobiotics and endobiotics. P450s are not self-sufficient enzymes and require a reductase as an electron carrier to function as monooxygenases. Recently recombinant P450 enzymes from different sources, e.g., microsomes of human lymphoblastoid cells, of yeast, and insect cells infected with baculovirus systems, and Escherichia coli membranes containing P450 and reductase coexpressed, have been widely used for drug metabolism research. However, the marker activities or kinetic parameters of human P450 enzymes reported from different research laboratories and commercial enzymes manufacturers are not always similar. These differences may be a critical factor for understanding roles of human P450 enzymes involved in drug metabolism. Troglitazone, a new oral antidiabetic drug, is reported not to be oxidized by P450 enzymes. However, cDNA-expressed CYP2C8 and CYP3A4 were active in catalyzing formation of a quinonetype metabolite. Intensity of inhibitory effects of specific P450 inhibitors and anti-P450 antibodies on the quinone-type metabolite formation depended on human liver samples and their P450 status. Azelastine, an antiallergy drug, was N-demethylated by CYP2D6, CYP3A4 and CYP1A2 in human liver microsomes biphasically. Human intrinsic clearance was predictable from P450 contents or kinetic parameters using cDNA-expressed P450 enzymes. The results suggest that different P450 enzymes in human liver have major roles in quinone-type metabolite formation from troglitazone and azelastine N-demethylation and that the hepatic contents of these P450 forms determine which P450 enzymes play the major roles in drug metabolism in individual humans.

CYP2A6 is known as an enzyme responsible for the metabolism of coumarin. As its toxicological significance, aflatoxin B1 and NNK are metabolically activated by this CYP to mutagens. In our recent studies, we found that a newly developed drug, SM-12502, was a specific substrate of CYP2A6, and 3 out of 28 Japanese were poor metabolizers (PM) of this drug. Genomic analysis of CYP2A6 gene in the PM indicated that an existence of a new CYP2A6 gene variant which lacks the entire region of open reading frame for the enzyme. The frequency of the individual who possess the mutation homozygously was estimated as 3-4% in Japanese. During the course of investigation of the frequency, we found an another CYP2A6 variant whose 60 by region in the 3'-untranslated region was substituted by the corresponding sequences of CYP2A7 gene. In this study, we investigated the frequency of these polymorphic alleles as well as reported v1 (CYP2A6*2) variant among different race.

A paternity case involving a putative father, a child, and the mother was referred to our laboratory for testing. Parentage was not excluded with 23 kinds of standard blood group markers and HLA, but the putative father requested more-affirmative evidence of paternity. Seven kinds of DNA probes that recognize hypervariable loci were applied. On the basis of the allelic frequencies and their confidence intervals previously reported among unrelated Japanese individuals, as well as confirmed codominant segregation of the polymorphism, the exclusion probability and paternity index were calculated for this case. The cumulative paternity index from the seven DNA probes was 1.4 × 10(6), which was 316 times higher than that from the 23 standard blood group markers and HLA. Accordingly, DNA polymorphism is considered to be informative enough for paternity testing.

Seven kinds of hypervariable DNA probes, which recognize hypervariable loci, were applied for individual identification of human bloodstains. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and codominant segregation of the polymorphism was confirmed in family studies. One-month-old bloodstains from ten individuals were investigated in the present study. The probability of matching was calculated using the database among Japanese population. Cumulative probability of matching from 7 kinds of hypervariable DNA probes was 1.1 x 10(-11), which was 4.5 x 10(9) times higher than that from 6 kinds of common blood group markers such as ABO, MN, Rh-Hr, P1, Hp and PGM1. Accordingly, DNA polymorphism is considered to be informative enough for individual identification of bloodstains.

The stability of DNA in human bloodstains and various post mortem tissues has been investigated. High molecular weight (HMW) DNA was usually recovered from dried bloodstains, even those up to a few years old, but very rapid degradation was found to occur post mortem in the liver, pancreas, spleen and kidney. Other tissues such as the heart, thyroid and skeletal muscle were found to give a reasonable yield of HMW DNA during the first few days after death. The feasibility of using DNA extracted from forensic bloodstain specimens for the detection of DNA polymorphisms was explored using a human haptoglobin (Hp) α chain specific probe. Using HindIII and XbaI digests the Hp genotypes Hp2, Hp1F and Hp1S were distinguished by Southern blot analysis in DNA prepared from 1 cm2 bloodstains up to 15–18 months old.

Midazolam (MDZ) is metabolized by CYP3A. Glucocorticoids are potent inducers of CYP3A in humans. The possible interaction between intravenous MDZ and chronically administered glucocorticoids was investigated during surgery in patients. MDZ (0.2 mg/kg) was administered intravenously to 8 patients taking glucocorticoid chronically and 10 patients not taking glucocorticoid. In patients taking glucocorticoid, the AUC0-infinity and CL of MDZ was decreased to 63.9% (16.3 +/- 10.5 vs 25.5 +/- 20.7 microg x min/mL) and increased to 127.5% (16.7 +/- 10.7 vs 13.1 +/- 8.3 mL/min/kg) of that in the control group, respectively. The terminal t1/2 values of MDZ were similar in two groups. In patients taking glucocorticoid, the AUC0-infinity of 1'-hydroxymidazolam (1'-OH MDZ) was 66.7% of that in the control group (7.6 +/- 2.6 vs 11.4 +/- 9.7 microg x min/mL), and the terminal t1/2 of 1'-OH MDZ was significantly (p < 0.01) decreased (1.8 +/- 0.5 vs 3.0 +/- 0.8 hr). Accumulative urinary excretion of 1'-OH MDZ glucuronide was increased to 157.6%. These observations might be results from induction of CYP3A4 and/or UDP-glucuronosyltransferase by glucocorticoids.

A recombinant DNA probe (pHY10) hybridizing specifically to human DNA family DYZ1, 3,000 copies of which are present on the long arm of the Y chromosome, was used for probing human genome DNA digested with various restriction enzymes. To our surprise, the probe detected a hypervariable polymorphism of autosomal origin in human DNA when digested with TaqI. None of other 12 restriction enzymes revealed polymorphic patterns. Codominant segregation of the polymorphism was established in family studies. This probe has been widely used in the detection of the Y chromosome. Its ease of availability as well as highly discriminating polymorphic pattern makes it potentially very useful for forensic and human genetic purposes.

A dynamic mathematical simulation model has been developed in order to study the dynamic behavior of a blast furnace. The model can predict the change of the internal state of the furnace by the numerical integration of a set of first order partial differential equations of the gas and solid state variables in the longitudinal direction. The procedure for the solution of this model is the method of characteristics to change the partial deferential equations to the ordinary differential equations. Ten kinds of chemical reactions including gaseous reduction of ore based on two-interfaces unreacted core model, the heat loss through the wall dependent on the heat capacity of the furnace body, and the heat exchange between gas and solid are considered in this model.The blow-in operation of a blast furnace has been simulated by the model. The good agreement of the simulated results of the change in the temperature of the furnace refractories as well as the top gas temperature and composition has verified the usefulness of the application of the model to the planning of a blast furnace blow-in operation.

A antigens of red blood cells and body fluids such as saliva, semen and sweat could be serologically distinguished using rabbit or guinea pig immune anti-A. As for antisera specific for red blood cell A, A+ rabbits were intravenously immunized with A group red blood cells. The resulting antisera were absorbed with O and B red cells and with A. Se saliva. The absorbed anti-A reacted with A red cells (titer 1:32) and was not inhibited with A. Se saliva. Guinea pigs were intramuscularly injected with A. Se saliva. Crude antisera contained agglutinins to human red cells which were abolished by absorption with A red cells. After absorption with O. Se saliva, the antisera were proved to have agglutinin activity with A group saliva using latex coated with A. Se saliva. A antigens from blood or body fluid stains could be distinguished by the elution method with these anti-A sera.

SAGISAKA, K., YAMASHITA, H., IWASA, M. and YOKOI, T. Human Specific Antigens and Species Difference in Non-Hemoglobin Proteins of Red Cell Lysate. Tohoku J. exp. Med., 1981, 135 (2), 197-204- Non-hemoglobin proteins (NHPs) of 12 species of mammalian were effectively isolated from red cell lysates with CM-Sephadex chromatography. Polyacrylamide gel disc electrophoresis demonstrated that human and monkey NHPs had 13 and 11 bands, respectively, but guinea pig and swine two to three bands. Rabbit anti-human NHP reacted with monkey, bovine, canine and badger as well as human. Absorption of the anti-NHP with monkey NHP resulted in preparing human specific anti-NHP. The specific antiserum gave two bands locating near the origin in immunoelectrophoresis. Human specific antigens were prepared by affinity chromatography with the anti-NHP. The antigens were composed of three bands, two of which had slightly faster mobility than carbonic anhydrase and the other migrated fastly on disc electrophoresis. These bands were proved to have neither LDH nor esterase activity.

To examine common antigens between human and monkey sera, sera from 19 kinds of monkey were investigated with crossed and rocket immunoelectrophoresis using antisera to human serum proteins. In the crossed immunoelectrophoresis, the number of precipitation lines, 40 in human and 8 in the lower primates, increased according to the order of phylogenetic classification, and the lines due to albumin and gamma-globulin were observed in all the monkey sera examined. Quantitative investigation was performed by rocket immunoelectrophoresis with 8 kinds of mono-specific anti-serum protein. Alpha 2-Macroglobulin, orosomucoid, transferrin, IgG and IgM were not observed in monkeys of Loricidae family. Relatively large amounts of orosomucoid, albumin, IgG and transferrin were detected in monkeys of Cebidae family. It was considered that the distribution of serum proteins possessing antigens common to human is not related with the order of phylogenetic classification.

IWASA, M., YOKOI, T. and SAGISAKA, K. Individual Difference of Seminal Plasma Detected with Crossed Immunoelectrophoresis. Tohoku J. exp. Med., 1983, 140 (2), 187-192 - Seminal plasma from 120 subjects was examined by crossed immunoelectrophoresis using anti-seminal plasma. The samples were divided into two groups depending on a precipitation line which migrated fast in the first dimension electrophoresis. This precipitation line showed an incidence of 48 percent and was proved to have no acid phosphatase, esterase or lactate dehydrogenase activity and was not stained with Schiff's reagent. Analysis with column chromatography, Sephadex G-200 and Sephadex G-50, revealed that molecular weight of the protein was about 10, 000 dolton. The difference was not identified with the other methods; polyacrylamide gel electrophoresis and agarose immunoelectrophoresis.

To elucidate immunochemical properties of Rh-Hr antigens, influence of various solubilizers on the activity, and isolation of the antigens were investigated. As a very sensitive method to detect Rh-Hr antigens, the agglutination inhibition test was recommended in which diluted antisera (1:400-1:1,600), papainized red cells and microtitrate plates were used. Extraction of D antigen with Brij 35 and Triton X-100 was effective, but SDS was not successful. However, D activity of SDS solubilized stroma was recovered by adding reductants. The activity was slightly demonstrated at the preparation from D negative red cells. The influence of the reductants was discussed. SDS solubilized stroma was fractionated with gel filtrations and activated thiol Sepharose 4B. The final preparation with D activity was proved to correspond with Band 3 which was not stained with PAS reagent. Deoxycholic acid solubilized stroma was fractionated with gel filtrations and affinity chromatography, resulting in the preparation corresponding to Band 3 with potent Rh-Hr blood type activities. The activities were detected without reductants and the preparation from D negative red cells had no D activity. Rh-Hr blood type activities other than D were observed in the same fraction.

A series of eighteen consecutive overlapping synthetic peptides, of uniform size and overlaps, which encompass the entire extracellular part (residues 1-210) of the Torpedo californica acetylcholine receptor alpha chain were examined in vitro for their ability to stimulate lymph node cells from acetylcholine receptor-primed C57BL/6 (H-2b), C3H/He (H-2k), SWR (H-2q) and SJL (H-2s) mice. The recognition sites (T sites) by acetylcholine receptor-primed lymph node cells from these mouse strains resided within six regions on the extracellular part of the alpha chain. Three of the regions recognized by T cells coincided with regions recognized by antibodies (i.e. B cells) and one of these three regions also coincided with an alpha-neurotoxin-binding region. It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has at least two sites which are recognized exclusively by T cells and to which no detectable antibody responses are directed.

IWASA, M., YOKOI, T. and SAGISAKA, K. An Interaction between Human Seminal Acid Phosphatase and Canavalia gladiata DC Lectin. Tohoku J. exp. Med., 1982, 137 (4), 415-421 - Interactions between human seminal acid phosphatase (AcP) and five kinds of lectins were studied. Seminal plasma was mixed with the lectins at various ratios. The mixtures were centrifuged and the supernatants were assayed for AcP activity. The activity was effectively reduced only with Canavalia gladiata DC (CG) lectin. CG lectin purified by Sephadex G-200 gel filtration was added to two fractions of AcP separated by Sephadex G-200 gel filtration. There was no difference in the rate of reduction of the two fractions. To isolate seminal AcP by affinity chromatography, CG lectin was coupled to agarose gel. AcP bound with the agarose gel could be eluted with α-methyl-D-mannoside. Specific activity of the purified AcP was elevated to as much as 54-fold. - lectin; seminal plasma; acid phosphatase

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1-210) of the alpha-subunit of human acetylcholine receptor were studied for their binding activity of 125I-labeled alpha-bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide alpha 122-138. In addition, low-binding activities were obtained with peptides alpha 34-49 and alpha 194-210. It is concluded that the region within residues alpha 122-138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.

A recombinant DNA probe hybridizing specifically to human repeat DNA sequence (pHY10) of which about 3000 copies are present on the Y chromosome was used for sex determination of degraded DNA samples of blood stains. Human blood stains of male and female origin were readily differentiated with the pHY10 DNA probe. This radioactive technique enabled reliable and sensitive sex determination from blood or dried blood stains greater than 20 years old. Less than 1 microliter of blood or 1 piece of 0.5 cm length thread of blood stain from cotton fabric was sufficient for the test using dot blot hybridization. Compared with the radioactive labeling method, the photobiotin labeling method showed one thirtieth to one fiftieth lower sensitivity and presented some problems which are expected to be resolvable.

Presentation of a protein antigen to T cells is believed to follow its intracellular breakdown by the antigen-presenting cell, with the fragments constituting the trigger of immune recognition. It should then be expected that T-cell recognition of protein antigens in vitro will be independent of protein conformation. Three T-cell lines were made by passage in vitro with native lysozyme of T cells from two mouse strains (B10.BR and DBA/1) that had been primed with the same protein. These cell lines responded well to native lysozyme and very poorly to unfolded (S-sulphopropyl) lysozyme. The response of the T-cell lines to the antigen was major histocompatibility complex (MHC)-restricted. A line from B10.BR was selected for further studies. This line responded to the three surface-simulation synthetic sites of lysozyme (representing the discontinuous antigenic, i.e. antibody binding, sites) and analogues that were extended to a uniform size by a nonsense sequence. T-cell clones prepared from this line were specific to native lysozyme and did not respond to the unfolded derivative. Furthermore, several of these clones showed specificity to a given surface-simulation synthetic site. The exquisite dependency of the recognition by the clones on the conformation of the protein antigen and their ability to recognize the surface-simulation synthetic sites indicate that the native (unprocessed) protein was the trigger of MHC-restricted T-cell recognition.

SAGISAKA, K., YAMASHITA, H., IWASA, M. and YOKOI, T. Serological Distinction of H Antigen between Saliva and Red Cell in Forensic Practice. Tohoku J. exp. Med., 1983, 141 (2), 211-215-Rabbit antisera were prepared by immunization with O red cells or O.Se saliva. Anti-O red cells (anti-Hr) were diluted with O.Se saliva and each of diluents was adopted for elution method on blood or body fluid stains. Anti-Hr gave positive reactions only with red cell stains. On the other hand, anti-O.Se (anti-Hs) absorbed with O red cells kept activity reacting with latex coated with O.Se saliva. Anti-Hs reacted with various body fluid stains but not with red cell stain. It is considered that anti-Hr and -Hs are useful tools for individualization of blood or body fluid stains in forensic practice.

Lea substance or immune complex which was prepared with anti-D and D-active Band 3 was trapped in autologous red cell ghost. The trapped immunogens were administered intravenously to rabbits or guinea pigs. Rabbits immunized with Lea substance loading ghost produced antisera of relatively low titers, but they contained specific incomplete anti-Lea antibody (titer 1:128) after absorption procedure, which were higher than that of antisera prepared by the ordinary method. Guinea pig antiserum to the immune complex contained specific incomplete anti-D (titer 1:128). Immunoglobulin class analyses revealed that the anti-Lea consisted mainly of IgM and the anti-D of IgG. It was considered that intravenous injection of the immunogen trapped in ghost is useful for preparing hemagglutinin of sufficient titers.

Rabbit antisera were raised for red cell stroma extracted with each of the following solutions; 5 mM phosphate buffer pH 8.0 (A antigen). 5 mM EDTA containing 5 mM 2-mercaptoethanol (B), 1/15 M phosphate buffered saline (C), 0.05% SDS (D), 50 mM Tris-HCl buffer pH 8.0 containing 1% SDS, 1 mM EDTA and 1% 2-mercaptoethanol (E) and 1% Triton X-100 (F). Crossed immunoelectrophoresis showed that E antigen produced relatively numerous precipitation lines, 7-9, against each of the antisera, suggesting that E solution was superior to the others for membrane solubilization. However, the Ouchterlony test demonstrated potent precipitin activity in anti-C, and the antiserum absorbed with monkey stroma which was extracted with F solution was proved to have strict human specificity. By adopting the antiserum to affinity chromatography, human specific antigens were isolated. The antigens gave four bands moving faster than Band 5 at SDS-polyacrylamide gel electrophoresis none of which was stained by Schiff's reagent. Using absorbed anti-C, human specificity of blood stains kept us to for two years could be identified.

H and A antigens expressed only in the RBC were quantitated using specific antisera, and the amount of the antigens was compared with that of total H and A antigens shared by the RBC and saliva. Total H antigen decreased in the order of O, A, B and AB blood groups. However, H antigen specific for the RBC distributed similarly among four blood groups. In addition, the amount of total A antigen in A group was more than that of AB group, but the difference in the amount of A antigen specific for the RBC was not significant between the two blood groups. B antigen specific for the RBC was not examined because of failure in preparing specific anti-B serum. In conclusion, the quantitative difference of the total H or A between A and AB groups might be due to the difference in the amount of antigen common to the RBC and saliva but not one specific for the RBC.