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The syntheses of two different molecular species of cytochrome P-450, P-450(PB), and P-450(MC), were examined using normal and drug-treated rats, and the rate of synthesis was correlated with the drug-induced increase of the amounts in the liver microsomes of treated animals. Phenobarbital (PB) and 3-methylcholanthrene (MC) were used as inducers. The syntheses of cytochrome b5 and NADPH-cytochrome P-450 reductase (fpT) were also examined. In vivo incorporation of L-[3H]leucine indicated a large increase of P-450(PB) synthesis in the livers of PB-treated animals, which reached a plateau value about 18-fold higher than the control level at 12 h. The synthesis of fpT was also stimulated by PB showing a peak value of 3 times the control at 12 h after PB administration, but it returned to the control level afterwards. On the other hand, the syntheses of P-450(MC) and cytochrome b5 did not change at all. Similarly, MC administration selectively induced the synthesis of P-450(MC), which was about 24 times the control at 6 h, whereas those of P-450(PB), cytochrome b5, and fpT were not affected by MC. Analysis of nascent peptides and in vitro translation of polysomes and total liver RNA prepared from control and drug-treated animals were also carried out, and the results of these in vitro experiments confirmed the observations in in vivo incorporation studies. It was found that both free and membrane-bound ribosomes participate in the syntheses of P-450(PB) and P-450(MC) in the case of control animals. PB and MC induced the syntheses of P-450(PB) and P-450(MC) by bound ribosomes but not by free ribosomes, and the contribution of bound ribosomes to the syntheses of these two species of cytochrome P-450 was predominant in the case of drug-treated animals. The molecular sizes of in vitro synthesized P-450(PB) and P-450(MC) were the same as those of authentic samples prepared from liver microsomes.

Highly purified divalent and monovalent antibodies against cytochrome b 5 , anti- b 5 immunoglobulin G (IG) and anti- b 5 Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti- b 5 IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti- b 5 Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome b 5 in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline.

A 418 base pair DNA duplex consisting of a gene for human lysozyme with a synthetic ribosome binding site of E. coli was assembled by stepwise ligation of 56 chemically synthesized oligodeoxyribonucleotides. The synthesized gene was expressed under the control of the lambda pRpL promoter and tfie gene product accumulated as several percent of the total cellular proteins in E. coli. The expressed product existed as insoluble and biologically inactive aggregates in the cells, but the biological activity was regenerated by solubilization and renaturation of the aggregated protein. The results of the N-terminal amino acid sequence analysis of the product showed the human lysozyme synthesized in E. coli has an additional methionine residue at the N-terminal of the mature human lysozyme due to the translational initiation codon of E. coli.

Discharge phenomena (nonequilibrium) in an MHD disk generator with potassium-seeded argon as a working gas have been investigated experimentally using a shock tube facility. A detailed study of high-speed photographs shows that an unsteady motion of a strongly constricted discharge occurs, particularly in the disk entrance region, characterized by a negative Hall potential. Responding to a suitable external load resistance, the negative Hall potential is reduced due to the development of a stable region against the ionization instability under full seed ionization; thus, a substantial increase of power output can be achieved. Under this condition, a uniform discharge is observed downstream and the strongly inhomogeneous and unsteady discharge is confined to a narrow region at the entrance. For smaller load resistances, intense spiral arcs with enhanced fluctuations are observed. 18 references.

Four forms of cytochrome P-450, tentatively designated PB-1, PB-2, MC-1, and MC-2, were purified from liver microsomes of rats treated with phenobarbital (PB-1 and PB-2) or 3-methylcholanthrene (MC-1 and MC-2). Each purified form showed a single protein-staining band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56,000 (MC-1), 53,000 (PB-1), 53,000 (MC-2), or 49,000 (PB-2). PB-1 and MC-1 were the major cytochrome P-450 components inducible by phenobarbital (PB) and 3-methylcholanthrene (MC), respectively. Antibodies prepared against each form of purified cytochrome P-450 did not cross-react with heterologous antigens in Ouchterlony double diffusion tests, confirming the immunological distinctness of the four forms. The CO-compounds of reduced PB-1 and PB-2 had an absorption maximum at 450 nm, whereas those of MC-1 and MC-2 had a maximum at 447 nm. Judging from the oxidized absolute spectra, MC-2 was of high spin type and the others were of low spin type. Amino acid analysis revealed considerable differences among the purified four forms of cytochrome P-450, and the amino acid sequences of their NH2-terminal portions confirmed that the four forms were different proteins. In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, PB-1 and PB-2 oxidized benzphetamine at high rates, but their oxidation of benzo(a)pyrene was much slower than that by MC-1, which catalyzed rapid hydroxylation of benzo(a)pyrene but had low activity with benzphetamine. The quantity of each form of cytochrome P-450 in microsomes was determined by quantitative immunoprecipitation, and selective induction of PB-1 and MC-1 by PB and MC, respectively, was confirmed. Some induction of PB-2 and MC-2 by the corresponding inducers was also noticed. PB group P-450's were not increased by MC treatment, nor were MC group P-450's by PB.

Relaxation of a stationarily flowing jet-like plasma, which penetrates into a neutral gas with temperature and kinetic energy much lower than the ionization potential, is investigated experimentally by measuring spatial distributions of hydrodynamic quantities such as plasma flow velocity, electron temperature and density in the plasma-neutral gas interaction zone. The experimental results agree with calculations. The particle, momentum and energy conservations of the plasma in the interaction zone are dominated by diffusion, ion elastic collision with neutral atoms and ionization, respectively.

Summary A complete cDNA clone encoding a human aromatase was isolated from a human placental cDNA library in λgtll. An antibody to the polypeptide specified by the isolated clone was prepared, and Western blot analysis and antibody inhibition experiments of human placental aromatase activity confirmed the identification of the clone as aromatase cDNA. The isolated aromatase cDNA clone of 3030 bp with two unique Eco RI sites contained a 3′-noncoding region of 1397 bp, an open reading frame of 1509 bp encoding 503 amino acid residues, and a 5′-noncoding region of 124 bp. Analysis of the amino acid sequence of aromatase and comparison of aromatase with other forms of cytochrome P-450 indicated that this enzyme is a unique form of the cytochrome P-450 superfamily.

The ionization relaxation region in an Ar/K-driven disk MHD generator and its effects have been experimentally investigated. Experimental results have shown that a decrease in the stagnation temperature of a working gas increases the area covered by the negative Hall field near the anode, which is strongly affected by ionization relaxation, and results in reducing output power. It is also observed that the power output increases with an increase in seed fraction. The unfavorable effects of ionization relaxation can be reduced by increasing applied magnetic field strength. Comparison between the results of one-dimension al calculations and experimental results has also been performed. Calculations can predict the Hall potential distribution along the flow direction when we assume an adequate electron temperature at the MHD channel inlet and fluctuations of a plasma at the inlet of the channel are assumed to be insignificant.

The last two steps of de novo synthesis of UMP are catalyzed by the sequential actions of orotate phosphoribosyltransferase (orotate PRTase; pyrophosphate phosphoribosyltransferase; EC 2.4.2.10) and orotidine 5′-monophosphate decarboxylase (OMP decarboxylase; orotidine 5′-phosphate carboxy-lyase; EC 4.1.1.23)

Two forms of cytochrome P-450, P-450PB and P-450MC, were purified to homogeneity from the liver microsomes of phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated rats, respectively. Rabbit antibodies against P-450PB and P-450MC were prepared and the monospecificity of each antibody preparation was confirmed by various lines of evidence. By the use of these antibodies, the contents of P-450PB and P-450MC in microsomes could be determined separately by quantitative immunoprecipitation. P-450PB and P-450MC each amounted to about 10% of total cytochrome P-450 in the microsomes of normal rats. However, each of them was selectively and substantially increased by the appropriate inducer, and became a predominant component of cytochrome P-450 in the microsomes of drug-treated animals. The increase of each specific molecular species of cytochrome P-450 was about 10- and 20-fold within 48 h, whereas the increase of total cytochrome P-450 was only about 2- to 3-fold even after maximal induction by the drugs. The drug oxidation activities of P-450PB and P-450MC were also significantly altered by the drug treatments. The administration of MC to PB-treated rats induced a drastic decrease in the P-450PB-dependent oxidations of benzo(a)pyrene and 7-ethoxycoumarin, while the corresponding activities of P-450MC increased sharply, and these changes were much more rapid than the change of the amount of each form of cytochrome P-450. However, the oxidation of benzphetamine was almost exclusively P-450PB-dependent even after extensive induction by MC. These observations suggest that the specific activities of p-450PB and P-450MC in the oxidations of various drugs are quite differently affected by the induced states of animals.

Cytochrome P-450 (I-P-450(16) alpha), which is associated with phenobarbital-induced testosterone 16 alpha-hydroxylation activity, was purified from livers of phenobarbital-treated female 129/J mice on the basis of the specific hydroxylation activity in fractions eluted from columns of octylamino-Sepharose 4B, hydroxylapatite, DEAE-Bio-Gel A, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified I-P-450(16) alpha fraction was 12.4 nmol/mg of protein, and it had an apparent molecular weight of 54K. The specific activity of reconstituted testosterone 16 alpha-hydroxylation activity with the purified I-P-450(16) alpha fraction was 6-8 nmol min-1 (nmol of cytochrome P-450)-1. Rabbit antibody raised against the purified I-P-450(16) alpha fraction inhibited nearly 100% of the 16 alpha-hydroxylation activity in liver microsomes of phenobarbital-treated female 129/J mice but did not affect hepatic microsomal 16 alpha-hydroxylation activity of untreated male and female 129/J mice at all. In hepatic microsomes of phenobarbital-treated male 129/J mice, 70% of the 16 alpha-hydroxylation activity, at most, was catalyzed by I-P-450(16) alpha, and the residual 30% of the activity was catalyzed by C-P-450(16) alpha. The increase of I-P-450(16) alpha by phenobarbital was due to de novo synthesis of I-P-450(16) alpha, and this induction was not sexually regulated in 129/J mice. Anti-C-P-450(16) alpha [Harada, N., & Negishi, M. (1984) J. Biol. Chem. 259, 12285-12290] did not inhibit the 16 alpha-hydroxylation catalyzed by I-P-450(16) alpha; thus, I-P-450(16) alpha and C-P-450(16) alpha are immunochemically distinct isozymes of testosterone 16 alpha-hydroxylase.

A multicopy plasmid was constructed to direct the synthesis and secretion of human lysozyme (HLY) in Saccharomyces cerevisiae. This plasmid contains a synthetic chicken-lysozyme signal sequence (SIG) and a synthetic HLY structural gene, both inserted between the yeast GAL10 promoter and 2 mu plasmid FLP (flip-flop recombination gene) terminator. The resulting plasmid directed the expression of the hybrid pre-lysozyme, with most of the HLY activity secreted into the culture medium and extracellular periplasmic space. The HLY activity in the culture medium increased with cell growth. The yeast accurately processed the hybrid precursor at the junction between the chicken SIG and the coding sequence downstream, yielding mature HLY. HLY purified from the culture medium was homogeneous and displayed specific activity identical to that of authentic HLY.

Monooxygenase reactions catalyzed by cytochrome P-450 are mainly responsible for the oxidative transformation of xenobiotics in animal tissues. Cytochrome P-450 is always found associated with a reductase system which supplies reducing equivalents for the monooxygenase reactions from NADH or NADPH. We find two distinct types of the reductase system; one is membrane-bound microsomes type and the other is soluble mitochondria-bacteria type. A remarkable common property of various forms of cytochrome P-450 participating in the metabolism of xenobiotics is their inducibility by their own substrates and related chemical compounds. The induction is characterized by highly selective increases of inducer-specific forms of cytochrome P-450 concomitant with great alterations in the activity and substrate specificity of the monooxygenase reactions.

Poly(ADP-ribosyl)ation has been proposed to regulate a variety of cellular functions, such as DNA repair, chromatin condensation, cell differentiation, and gene expression. Involvement of poly(ADP- ribosyl)ation in cell differentiation has been reported in a variety of cells, but a consensus with respect to these results has not yet been obtained. In particular, the change in the enzyme activity during erythroleukemia cell differentiation has been contradictory (1–3). Enzyme activity has been reported to increase 10- to 20-fold due to the fragmentation of DNA in cells (4). The physiological enzyme activity was determined by mildly permeabilizing cells, and the total potential enzyme activity was assayed by treating cells with detergent and DNase I. In muscle cell differentiation (5) the total potential enzyme activity did not change but the physiological enzyme activity increased, probably because DNA strand breaks formed during the differentiation. In contrast, both enzyme activities decreased during murine teratocarcinoma cell differentiation (6) and rooster spermatogenesis (7). Enzyme activity does not provide a definitive method for changes in the level of enzyme. Thus, we attempted to isolate a cDNA encoding the enzyme in order to evaluate the critical change in the level of mRNA for the enzyme during the cellular processes.

The intrinsic primary deuterium isotope effect for the O-deethylation of 7-ethoxycoumarin has been estimated by the Northrop method [D. B. Northrop (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., and Northrop, D. B., eds.), pp. 122-152, University Park Press, Baltimore] for the microsomal cytochrome P-448 system from 3-methylcholanthrene-induced hamster livers. The intrinsic isotope effect (Dk = 5.5) was found to be equivalent to the observed deuterium isotope effect, demonstrating that the isotope effect for this reaction was fully expressed by this cytochrome P-448 system. These data unequivocally demonstrate that C-H bond cleavage is the rate-limiting step in the overall reaction catalyzed by this system. The decrease in the rate of product formation, occurring as a consequence of deuterium substitution, resulted in a reduction in the quantity of substrate metabolized but was not accompanied by the change in regiospecificity observed in previous studies with a hepatic cytochrome P-448 isozyme purified from 3-methylcholanthrene-induced rats. These data demonstrate that the catalytic site of the hamster isozyme(s) offers more constraints to 7-ethoxycoumarin reorientation than does the catalytic site of the rat liver isozyme.

A 2.7-kb cDNA clone coding for bovine poly(ADP-ribose) synthetase was isolated from a lambda gt11 expression library by direct immunological screening with an antiserum to the enzyme. The cDNA hybridizes to an approximately 3.8-kb bovine thymus polyadenylated RNA, which translates an immunoprecipitable 120-kDa protein with the antibody to the enzyme. The partial DNA sequence of the cDNA was determined and portions of the predicted amino acid sequence matched the sequence of 26 amino acids at the N terminal of the 41-kDa alpha-chymotryptic fragment and two cyanogen-bromide-cleaved peptides of the enzyme. A subcloned fragment from the coding region of the cDNA was used as a probe to estimate the level of mRNA for the enzyme during the interferon-gamma-induced activation process of the murine macrophage tumor P388D1 cell line. The amount of mRNA for the enzyme decreased nearly completely within 24 h after incubation in a medium containing interferon-gamma, while mRNA of the Ia antigen, one of the major histocompatibility gene products, was increased in the macrophage tumor cells by interferon-gamma as confirmed by the I-A beta cDNA as a probe. These results suggest that the gene expression for poly(ADP-ribose) synthetase is depressed during the interferon-gamma-induced activation process of macrophage tumor cells.

Publisher Summary This chapter describes the distribution of electron transfer components on the surface of microsomal membrane. It is now generally agreed that NADPH-cytochrome P-450 reductase, NADH-cytochrome reductase, and cytochrome b5 are exposed on the outside surface of microsomal vesicles. Evidence suggests that these microsomal electron transfer components, which constitute the microsomal drug-oxidizing enzyme system together with cytochrome P-450, are not uniformly distributed on the entire surface of the endoplasmic reticulum. The subfractionation of liver microsomes by density-gradient centrifugation have often showed uneven distribution of these components among the subfractions. The subfractionation of microsomes by immunoprecipitation of microsomal vesicles by specific antibodies also showed some tendency of separation of fpT and P-450 from fpD and b5. An electronmicroscopic examination of the distribution of fpT and P-450 by the use of specific antibodies indicated their clustering on the surface of microsomal membranes. Kinetical studies on the electron transfer between microsomal flavoproteins and cytochromes suggested clustering of fpT and P-450 or fpD and b5. Tight association of fpT and P-450 in microsomal membrane was also suggested by a spin labeling study on the fluidity of the membrane lipids.

This paper describes the results of three-dimensional numerical simulation on MHD accelerator. The main stress falls on the comprehension of three-dimensional phenomena. As a result of this study, we found that local Hall current is enhanced under the high Hall parameter condition on Faraday type accelerator. This local Hall current promotes the Joule dissipation and induces the secondary flow. In Diagonal type accelerator, the Faraday current exists in upstream region effectively. It is quiet in downstream region in contrast. Also it is important that Diagonal system could obtain almost the same acceleration performance to Faraday case in Hall current neutralized condition. We found that transforming to generator mode occurs easily in upstream region according to decreased diagonal angle. It is serviceable that we could possess qualitative view about threedimensional behavior in MHD accelerator.

In many applications of Magnetohydrodynamic (MHD) process, there is a great interest to consider MHD acceleration for advanced propulsion. The essential requirement for using this acceleration technique is that the propulsion system should have minimum weight, size, and complexity. In this paper, fundamental performance of a diagonal type MHD accelerator under chemical and thermal equilibrium conditions is described. The 1dimensional numerical simulation is performed to characterize acceleration along the specified channel which has been designed and developed by NASA Marshall Space Flight Center. In order to solve a set of differential MHD equations, MacCormack scheme is employed. In the present calculation, working gas of aero-plasma, consists of diatomic molecule of nitrogen, oxygen, and hydrogen, is considered in order to actualize application of MHD accelerator propulsion system. Numerical results show basic characteristic of gasdynamic, electrical properties, and electrical efficiency of MHD accelerator. Moreover, 3D analysis is newly carried out and comparison with 1-D analysis is performed. The almost equivalent result is obtained with special information on boundary layers and secondary flows.

To avoid the damage of the electrodes, the capacitor coupled MHD generator has been proposed. Because the capacitor coupled MHD generator relies on the conventional MHD generator, the method of generating electromotive force is almost same without the extraction of electric power. To obtain the same output power of the conventional MHD generator, the capacitor coupled MHD generator is required to be higher AC magnetic field frequency or the larger capacitance of the channel. To demonstrate the capacitor coupled MHD generator, the transient MHD generator based on the model rocket engine was constructed. We can see that the capacitor coupled MHD generator extracts the electric energy. To understand the noise from the preionization discharge, we compared the capacitor coupled MHD generator with applied magnetic field to that without applied magnetic field. The result indicates that the extraction of electromotive force is not observed the capacitor coupled MHD generator without applied magnetic field.

Introduction There are several variations in the morphologic characteristics of the extrahepatic biliary system. A hepaticocystic duct is one of the rare variations. Case presentation A 69-year-old Asian man underwent a cholecystectomy for cholelithiasis. His cystic duct was not detected during surgery. An intraoperative cholangiography revealed that his common hepatic ducts drained directly into the neck of his gallbladder. There was no common bile duct, as evidenced by the union of the common hepatic and cystic ducts. Conclusion Knowledge of anomalies related to the extrahepatic biliary system is important for decreasing the severe morbidity and mortality that may result from a failure to recognize the anomaly.

Cimetidine (CIM), a potent anti-ulcer drug, is known to have an anti-androgenic effect, presumably through competitive binding to the dihydrotestosterone receptor. CIM given to men can cause hypoandrogenemia and sexual dysfunction. A single injection of CIM (160 mg/kg) to SD rats 3 days old significantly delayed the increase of body and testes weights. The blood testosterone levels were 30, 90, and 100 mg/dl at 4,6 and 8 weeks after treatment, respectively. These levels were 5 to 7 times lower than those in non-treated rats at corresponding times. Developmental expression of hepatic microsomal testosterone 16α-hydroxylase, which is known to be a malespecific activity and is imprinted by neonatal testosterone, was not significantly affected by this experiment. Treatment of adult male rats with CIM (160 mg/kg/day) for 5 days decreased testosterone levels to one-forth those of nontreated rats. The testosterone metabolites formed by hepatic microsomes from CIM treated adult rats were analyzed by thin layer chromatography; only formation of 16α-hydroxytestosterone was inhibited. CIM in vitro was a better inhibitor of 16α-hydroxylase than of other hydroxylases. Direct binding of CIM or its metabolites to the cytochrome P-450 isozyme specific for 16α-hydroxylation is presumed to be the mechanism of inhibition.

HTLV-1 is the causative retrovirus of ATL. Eighty percent of ATL patients develop hypercalcemia, a severe complication resulting from bone resorption. PTHrP is known as the major pathogenic factor to hypercalcemia associated malignancies. Recently, we observed hypercalcemia in rabbits with antibody against central region of HTLV-1 Env Gp46 (gp46-197), which shows a homology with osteoprotegerin (OPG), an inhibitor to the maturation of osteoclasts. To observe the effect of gp46197 synthetic peptide on osteogenesis, the peptide was intraperitoneally administered to mice in the presence or absence of co-administration of E2. Seven days later, serum E2 level and bone mineral density (BMD) were measured. Both of males and females exhibited a clear reduction in E2 and BMD by the gp46-197 peptide. Histomorphological analyses revealed that the peptide induced the loss of Ob.S/BS and increase in ES/BS, N. Oc/B.Pm, and Oc.S/BS. These results suggest that the structural mimicry of gp46-197 inhibits the OPG function and promote the maturation and stimulation of osteoclasts in vivo. Co-administration of E2 abrogated the adverse effects by gp46-197. This is a novel mechanism of osteolysis directly induced by HTLV-1 structural protein. We presume the therapeutic potential of E2 for the treatment of hypercalcemia in ATL patients.

Abstract 289 Junko Kurokawa, Tokyo Medical & Dental University, MRI, Tokyo, Japan; Masaji Tamagawa, Chiba University Graduate School of Medicine, Chiba, Japan; Nobuhiro Harada, Shinichiro Honda, School of Med, Fujita Health University, Toyoake, Japan; Haruaki Nakaya, Chiba University Graduate School of Medicine, Chiba, Japan; Tetsushi Furukawa, Tokyo Medical & Dental University, MRI, Tokyo, Japan Junko Kurokawa, 2007 Finalist and Presenting Author