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Cancer cells tend to have a high requirement for lipids, including fatty acids, cholesterol and triglyceride, because of their rapid proliferative rate compared to normal cells. In this study, we investigated the effects of inhibition of lipid synthesis on the proliferation and viability of human pancreatic cancer cells. Of the inhibitors of lipid synthesis that were tested, 5-(tetradecyloxy)-2-furoic acid (TOFA), which is an inhibitor of acetyl-CoA carboxylase, and the fatty acid synthase (FAS) inhibitors cerulenin and irgasan, significantly suppressed the proliferation of MiaPaCa-2 and AsPC-1 cells. Treatment of MiaPaCa-2 cells with these inhibitors significantly increased the number of apoptotic cells. In addition, TOFA increased caspase-3 activity and induced cleavage of poly (ADP-ribose) polymerase in MiaPaCa-2 cells. Moreover, addition of palmitate to MiaPaCa-2 cells treated with TOFA rescued cells from apoptotic cell death. These results suggest that TOFA induces apoptosis via depletion of fatty acids and that, among the various aspects of lipid metabolism, inhibition of fatty acid synthesis may be a notable target for the treatment of human pancreatic cancer cells.

Animal experiments cannot predict the probability of idiosyncratic drug toxicity; consequently, an important goal of the pharmaceutical industry is to develop a new methodology for preventing this form of drug reaction. Although the mechanism remains unclear, immune reactions are likely involved in the toxic processes underlying idiosyncratic drug toxicity: the drug is first activated into a chemically reactive metabolite that binds covalently to proteins and then acts as an immunogen. Therefore, screening tests to detect chemically reactive metabolites are conducted early during drug development and typically involve trapping with glutathione. More quantitative methods are then used in a later stage of drug development and frequently employ (14)Cor (3)H-labeled compounds. It has recently been demonstrated that a zone classification system can be used to separate risky drugs from likely safe drugs: by plotting the amount of each protein-bound reactive metabolite in vitro against the dose levels in vivo, the risk associated with each drug candidate can be assessed. A mechanism for idiosyncratic drug-induced hepatotoxicity was proposed by analogy to virus-induced hepatitis, in which cytotoxic T lymphocytes play an important role. This mechanism suggests that polymorphism in human leukocyte antigens is involved in idiosyncrasy, and a strong correlation with a specific genotype of human leukocyte antigens has been found in many cases of idiosyncratic drug toxicity. Therefore, gene biomarkers hold promise for reducing the clinical risk and prolonging the life cycle of otherwise useful drugs.

Analyses using electrophoresis with accelerator mass spectrometry revealed that in vivo bioactivated radiolabeled troglitazone and flutamide, both known to be hepatotoxic in humans, bound nonspecifically to a variety of microsomal and cytosolic proteins in livers from chimeric mice with humanized liver. Unlike those of radiolabeled diazepam (rarely hepatotoxic) and previously reported 5-n-butyl-pyrazolo[1,5-a]pyrimidine (limited hepatotoxicity), some troglitazone and flutamide binding proteins were located in the top right area in a zone analysis, representing high covalent binding contents and high target protein concentrations. Among a variety of liver microsomal proteins bound, the binding target proteins of troglitazone and flutamide with the highest covalent binding contents (in terms of pmol equivalent per μg target protein) were 17β-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase, respectively. Troglitazone and flutamide were activated to reactive metabolites and apparently bound to different target proteins in livers from chimeric mice with humanized liver. The highest covalent binding contents for troglitazone were higher than that for flutamide under the present conditions. These results indicate that the drug metabolism mediated by humanized livers (leading to binding in vivo) in combination with a zone analysis of covalent binding contents/target protein concentration data could be a good tool for evaluating the relationship between the nonspecific protein binding behavior of medicines and potential hepatotoxicity in humans. Thus, testing whether protein binding data of new medicines are unbalanced with respect to deviation from an inverse relationship or the presence of data points in the high covalent binding/high protein concentration zone might be an important concept in evaluating hepatotoxic potential.

Microdose study enables us to understand the pharmacokinetic profiles of drugs in humans prior to the conventional clinical trials. The advantage of microdose study is that the unexpected pharmacological/toxicological effects of drugs caused by drug interactions or genetic polymorphisms of metabolic enzymes/transporters can be avoided due to the limited dose. With a combination use of accelerator mass spectrometry (AMS) and 14 C-labaled compounds, the pharmacokinetics of both parent drug and its metabolites can be sensitively monitored. Thus, to demonstrate the usability of microdose study with AMS for the prediction of the impact of genetic polymorphisms of CYP enzyme on the pharmacokinetics of unchanged drugs and metabolites, we performed microdose pharmacogenetic study using tolbutamide as a CYP2C9 probe drug. A microdose of 14 C-tolbutamide (100 μg) was administered orally to healthy volunteers with the CYP2C9 ∗ 1/ ∗ 1 or CYP2C9 ∗ 1/ ∗ 3 diplotype. Area under the plasma concentration–time curve for the 14 C-radioactivity, determined by AMS, or that for the parent drug, determined by liquid chromatography/mass spectrometry, was about 1.6 times or 1.7 times greater in the CYP2C9 ∗ 1/ ∗ 3 than in the CYP2C9 ∗ 1/ ∗ 1 group, which was comparable to the previous reports at therapeutic dose. In the plasma and urine, tolbutamide, carboxytolbutamide, and 4-hydroxytolbutamide were detected and practically no other metabolites could be found in both diplotype groups. The fraction of metabolites in plasma radioactivity was slightly lower in the CYP2C9 ∗ 1/ ∗ 3 group. Microdose study can be used for the prediction of the effects of genetic polymorphisms of enzymes on the pharmacokinetics and metabolic profiles of drugs with minimal care of their pharmacological/toxicological effects.

The dynamics of flapping motion of a rectangular jet under acoustic excitation is studied experimentally by means of hot-wire measurement and flow visualization with smoke method. The excitation sufficiently enables“phase-lock”, which permitted us to extract the organized wave motion from a background field of finite turbulent fluctuations. The mean and fluctuation velocity are investigated and focused on the excitation frequency and the Reynolds number. As the excitation frequency decreases, it was found that the jet flapping and the jet spread were enhanced. The excitation with sub-harmonic frequency has significant effects on the rectangular jet behavior. The maximum value of the periodic velocity fluctuation strongly depends on the excitation frequency.

Direct numerical simulation of a jet issuing from a nozzle having a rectangular cross-section is conducted. The vortex in cell (VIC) method, of which computational accuracy was heightened by the authors in a prior study, is used for the DNS. The aspect ratio of the nozzle cross-section is 15, and the Reynolds number based on the shorter side length of the nozzle exit is 6700. The turbulence statics, such as the mean velocity and the turbulence intensity, are favorably compared with the experimentally measured results. The behavior of the large-scale eddies as well as the development of the turbulent flow is also confirmed to agree with the measurement. These indicate that the authors’ VIC method is successfully employed for the DNS of rectangular jet.

Many kinds of an atmospheric-pressure plasma jet have been developed and used for widespread applications such as a surface treatment and modified. This study focused on the argon atmospheric-pressure m jet generated by discharging of RF power of 2.45 GHz microwave. The plasma jet shows sensitivity to surrounding environment: pressure, temperature and gaseous species. It is therefore absolutely imperative that a nature of atmospheric-pressure plasma jet should be understood from a point of fluid dynamics. This study, therefore, focused on the interrelationship between the plasma jet and the working gas. Motion of the plasma jet and the working gas was evaluated by velocity measurement and fast photography. As a result, the unsteady sinusoidal waving motion in the radial direction of a torch was observed. Advection velocity of the plasma in just downstream region of the torch exit increases with the supplying flow rate, and the velocity ratio is in the range of 0.75-0.87., Article, SURFACE & COATINGS TECHNOLOGY. 206(6):1449-1453 (2011)

The aim of this investigation was to improve power performance of Savonius hydraulic turbine utilizing small stream for electric generation. An attempt was made to increase the power coefficient of runner by the use of flat shield plate placed upstream of the runner. The difference of the power coefficient is discussed in relation to clearance between the runner and the bottom wall and the rotation direction of the runner. The flow field around the runner was also examined visually to clarify influences of setting conditions on the power performance. From this study it was found that the power coefficient is achieved for 0.47 by only using a flat shield plate, the increase is up to 80% over the runner without the plate. Moreover, it is the proper condition that clearance ratio is 0.73 in this study., Article, JOURNAL OF THERMAL SCIENCE. 20(3):224-228 (2011)

A large size botulinum toxin complex (L-TC) is composed of a single neurotoxin (BoNT), a single nontoxic nonhaemagglutinin (NTNHA) and a haemagglutinin (HA) complex. The HA complex is comprised of three HA-70 molecules and three arm structures of HA-33/HA-17 that consist of two HA-33 and a single HA-17. In addition to the mature L-TC, smaller TCs are present in cultures: M-TC (BoNT/NTNHA), M-TC/HA-70 and immature L-TCs with fewer HA-33/HA-17 arms than mature L-TC. Because L-TC displays higher oral toxicity than pure BoNT, it was presumed that nontoxic proteins are critical for food poisoning. In this study, the absorption of TCs across intestinal epithelial cells was assessed by examining the cell binding and monolayer transport of serotype D toxins in the rat intestinal epithelial cell line IEC-6. All TCs, including pure BoNT, displayed binding and transport, with mature L-TC showing the greatest potency. Inhibition experiments using antibodies revealed that BoNT, HA-70 and HA-33 could be responsible for the binding and transport. The findings here indicate that all TCs can transport across the cell layer via a sialic acid-dependent process. Nonetheless, binding and transport markedly increased with number of HA-33/HA-17 arms in the TC. We therefore conclude that the HA-33/HA-17 arm is not necessarily required for, but facilitates, transport of botulinum toxin complexes.

Ecotype hydraulic turbines can be utilized small and distributed water resources with little civil engineering works and without environmental burdens. In Japan, network of agricultural waterway extends 400 thousands kilometers, this abundance of waterpower has a high expectations for electric power generation to supply to agricultural facilities. This promotes local production for local consumption of electricity. Four types of turbine, Savonius turbine, waterfall turbine, jet turbine and undershot turbine, were investigated and demonstrated in some fields. We have been also investigated the standalone smart grid system which can harness a several types of ecotype turbines. It can control the amount of generation power and supplying electricity. In this manuscript, we summarize our results of the investigation of ecotype hydraulic turbines.

Troglitazone induced an idiosyncratic, hepatocellular injury-type hepatotoxicity in humans. Statistically, double null genotype of glutathione S-transferase isoforms, GSTT1 and GSTM1, was a risk factor, indicating a low activity of the susceptible patients in scavenging chemically reactive metabolites. CYP3A4 and CYP2C8 were involved in the metabolic activation and CYP3A4 was inducible by repeated administrations of troglitazone. The genotype analysis, however, indicated that the metabolic idiosyncrasy resides in the degradation of but not in the production of the toxic metabolites of troglitazone. Antibody against hepatic aldolase B was detected in the case patients, suggesting involvement of immune reaction in the toxic mechanism. Troglitazone induced apoptotic cell death in human hepatocytes at a high concentration, and this property may have served as the immunological danger signal, which is thought to play an important role in activating immune reactions. Hypothesis is proposed in analogy to the virus-induced hepatitis. After the troglitazone-case, pharmaceutical companies implemented screening systems for chemically reactive metabolites at early stage of drug development, taking both the amount of covalent binding to the proteins in vitro and the assumed clinical dose level into consideration. At the post-marketing stage, gene analyses of the case patients, if any, to find pharmacogenetic biomarkers could be a powerful tool for contraindicating to the risky patients.

Objectives Our aim was to investigate the effect of PEGylation on the uptake of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) into rat liver, kidney and spleen, and human liver. Methods Copolymer of polyethyleneglycol allylmethylether and maleamic acid sodium salt with OCIF (poly(PEG)-OCIF) (0.5 mg/kg) was administered to rats and the concentrations of poly(PEG)-OCIF in the liver, kidney and spleen at 15 min after administration were measured by ELISA. For human liver uptake, the liver perfusion of OCIF and 3H-labelled poly(PEG)-OCIF was conducted using fresh human liver block. Key findings The tissue uptake of poly(PEG)-OCIF in rats was significantly lower compared with that of OCIF. In fresh human liver perfusion, 3H-poly(PEG)-OCIF was rarely taken up into the liver. On the other hand, more than 50% of the perfused OCIF was taken up. Conclusions PEGylation of OCIF using poly(PEG) dramatically suppressed the uptake of OCIF into human liver as well as into rat liver and could be a promising approach for improving the pharmacokinetic and pharmacological effects of OCIF in the clinical setting.

Vortex levitation can achieve non-contact handling by blowing air into a vortex cup through a tangential nozzle to generate a swirling flow. In this paper, we focused on the relationship between the sucking pressure and the flow dynamics when gap distance from the cup to a work piece changes. Then simultaneous measurement of a pressure and a flow field in the cup was performed. As a result, the mean pressure changes and the pressure fluctuation inside the cup enhances with increasing gap height. Especially, periodic pressure perturbation is observed with wide gap height and it synchronizes with an eccentric rotation of the swirling flow. It is also found that the rotation axis of swirling flow steadily inclines against the central axis of the cup for appropriate gap height., Article, JOURNAL OF VISUALIZATION. 13(4):319-326 (2010)

Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys(132) of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics.

The aim of this investigation was to develop an environmentally friendly nano-hydraulic turbine utilizing waterfalls. A model of an impulse type hydraulic turbine constructed and tested with an indoor type waterfall to arrive at an optimum installation condition. Effects of an installation parameter, namely distance between the rotor and the waterfall on the power performance were studied. The flow field around the rotor was examined visually to clarify influences of installation conditions on the flow field. The flow visualization showed differences of flow pattern around the rotor by the change of flow rate and rotational speed of the rotor. From this study it was found that the power performances of the rotor were changed with the distance between the rotor and the waterfalls. The maximum power coefficient of this turbine is approximately 60%. Also, to respond to changes in the waterfall flow rate, we placed a flat plate on the upper side of the rotor to control the water flow direction. As a result, we found that the coefficient of this turbine is increased with the flow rate and power could be obtained even when the flow rate changed by 3.5 times if the plate was placed on the upper side of the rotor. Although the power coefficient decreased when the plate was installed, the power coefficient still is from 53 to 58%., Article, Renewable Energy. 35(1):293-300 (2010)

Clostridium botulinum produces a large toxin complex (L-TC) composed of neurotoxin (BoNT) and non-toxic proteins. In animal botulism, BoNT or L-TC is absorbed via the intestinal epithelium. To establish the cellular mechanisms of botulinum toxin absorption, we used cultured rat intestinal epithelial cells to test the binding and transport of serotype C l BoNT and L-TC through the cell layers. BoNT and L-TC bound to and passed through the cell layers, with L-TC exhibiting larger binding and transport. Binding and transport of these toxins were inhibited by N-acetyl neuraminic acid or neuraminidase treatment of the cells. These results suggest that binding of serotype C1 BoNT and L-TC to sialic acid on the cells promoted their transport through intestinal epithelial cell layers.

Background and purpose: We investigated the immunogenicity of a humanized anti-human Fas monoclonal antibody, R-125224, in cynomolgus monkeys to estimate its efficacy, as well as its toxicity in clinical situations. Experimental approach: R-125224 was intravenously administered to cynomolgus monkeys at single doses of 0.4, 1.2, 6 and 30 mg·kg−1, and the plasma concentrations of R-125224 and anti-R-125224 antibody (ARA) were measured. We conducted a competitive enzyme-linked immunosorbent assay to determine which part of R-125224 was recognized by ARA. We also examined the retention of radioactivity in mononuclear cells and granulocytes after the injection of [125I]-R-125224 to a collagen-induced arthritis monkey model. Key results: After i.v. administration of R-125224, the elimination of the plasma R-125224 concentrations was accelerated at around 10 days post-dose, and 10 of 12 monkeys were ARA positive. From an epitope analysis of ARA, the ARA produced in monkeys recognized the mouse-derived regions located in complementarity determining regions, but could not recognize the human IgG. After the injection of [125I]-R-125224 to a collagen-induced arthritis monkey model, a significantly longer retention of the radioactivity in mononuclear cells compared to granulocytes was observed. Conclusions and implications: In monkeys, the development of antibodies against R-125224 is rapid and highly frequent. Our hypothesis is that this highly frequent development of ARA might be due to the binding of R-125224 to immune cells, and its circulation in monkey blood might contribute to an increase in its chances of being recognized as an immunogen.

The aim of this investigation was to develop an environmentally friendly nano-hydraulic turbine. A model of a two-bucket Savonius type hydraulic turbine was constructed and tested in a water tunnel to arrive at an optimum installation condition. Effects of two installation parameters, namely a distance between a rotor and a bottom wall of the tunnel, a rotation direction of the rotor, on the power performance were studied. A flow field around the rotor was examined visually to clarify influences of installation conditions on the flow field. The flow visualization showed differences of flow pattern around the rotor by the change of these parameters. From this study it was found that the power performances of Savonius hydraulic turbine were changed with the distance between the rotor and the bottom wall of the tunnel and with a rotation direction of the rotor., Article, Journal of Fluid Science and Technology. 3(3):420-429 (2008)

For revealing the transition process in a flat plate boundary layer subjected to a weak free stream turbulence, flow visualization and hot-wire measurements were performed. A weak free stream turbulence was generated by a turbulence grid mounted upstream of the contraction. The flow visualization clearly displayed a transition scenario in which a local two-dimensional wave packet rapidly forms a Λ shape structure and then breaks down to turbulence, resulting in the generation of a turbulent spot. Quantitative measurements performed by using a hot-wire anemometer also confirmed the existence of local Tollmien-Schlichting waves that agreed with the parallel linear theory in terms of their frequency, phase velocity, and the wall-normal distribution of band-pass-filtered fluctuations. For comparison, a boundary layer subjected to a moderate-intensity free stream turbulence was investigated. This investigation showed that streaky structures play an important role in the boundary layer transition, as shown by Matsubara et al. [J. Fluid Mech., 430, (2001), 149-168.] A drastic change occurred in the transition process and this change could be sensitively determined by employing the intensity and/or spectra of the free stream turbulence.

Differences in the inhibition of cytochrome P450 activities among thienopyridine antiplatelet agents, ticlopidine, clopidogrel, prasugrel, and the metabolites, 2-oxo-clopidogrel, clopidogrel acid metabolite, deacetylated metabolite of prasugrel (R-95913) and the pharmacologically active metabolites of clopidogrel and prasugrel, were examined using recombinant cytochromes P450 and fluorescent probe substrates. Ticlopidine and clopidogrel inhibited CYP2B6 with IC(50) values of 0.0517+/-0.0323 microM and 0.0182+/-0.0069 microM, respectively, and inhibited CYP2C19 with IC(50) values of 0.203+/-0.124 microM and 0.524+/-0.160 microM, respectively. Ticlopidine also inhibited CYP2D6 (IC(50) of 0.354+/-0.158 microM). In contrast, 2-oxo-clopidogrel, prasugrel and R-95913 were much weaker inhibitors of CYP2B6, CYP2C19 and CYP2D6. The inhibitory effects of all the compounds tested were much weaker on the isoforms other than those indicated above. The active metabolites of clopidogrel and prasugrel and clopidogrel acid metabolite also did not affect the activities of the P450s examined.

The liver of a chimeric urokinase-type plasminogen activator (uPA)(+/+)/severe combined immunodeficient (SCID) mouse line recently established in Japan could be replaced by more than 80% with human hepatocytes. We previously reported that the chimeric mice with humanized liver could be useful as a human model in studies on drug metabolism and pharmacokinetics. In the present study, the humanization of an excretory pathway was investigated in the chimeric mice. Cefmetazole (CMZ) was used as a probe drug. The CMZ excretions in urine and feces were 81.0 and 5.9% of the dose, respectively, in chimeric mice and were 23.7 and 59.4% of the dose, respectively, in control uPA(-/-)/SCID mice. Because CMZ is mainly excreted in urine in humans, the excretory profile of chimeric mice was demonstrated to be similar to that of humans. In the chimeric mice, the hepatic mRNA expression of human drug transporters could be quantified. On the other hand, the hepatic mRNA expression of mouse drug transporters in the chimeric mice was significantly lower than in the control uPA(-/-)/SCID mice. In conclusion, chimeric mice exhibited a humanized profile of drug excretion, suggesting that this chimeric mouse line would be a useful animal model in excretory studies.

To characterize the renal handling of CS-023 (RO4908463), a novel parenteral carbapenem antibiotic, and meropenem in humans, we examined their affinities as substrates to human renal transporters. In vitro studies on the uptake of [14C]CS-023 and [14C]meropenem were conducted using HEK293 cells expressing human organic anion transporters (hOAT) 1, hOAT3, hOAT4, and the human organic cation transporters (hOCT) 1 and hOCT2. CS-023 did not serve as the substrate for any of the transporters tested. On the other hand, meropenem was transported by hOAT1 and hOAT3. The Km value of the hOAT3-mediated transport was 847 microM, and the uptake was inhibited by probenecid, p-aminohippurate and benzylpenicillin with Ki values of 3.76, 712, and 202 microM, respectively. One of the reasons why CS-023 is not a substrate of hOATs, and vice versa for meropenem, would be that a very small proportion of CS-023 exists as the anionic form at the physiological pH, whereas 50% of meropenem exists as the anionic form. These findings indicate that the lack of recognition of CS-023 by renal transporters is one of the reasons for its long plasma half-life in humans compared with meropenem which undergoes renal tubular secretion mediated by hOAT1 and hOAT3.

Many industrial instruments use rubber; NBR(Nitrile Butadiene Rubber) is especially used for the packing attached to pneumatic equipment. Recently, with the spread of dry pneumatic pumps, the deterioration of NBR by ozone has become a serious problem. However, the process of the ozone degradation of NBR has not been sufficiently clarified. When examining the ozone degradation of NBR, it is very important to control the ozone concentration. In general, it is difficult to get a low ozone concentration using an ozonizer with an ultraviolet ray lamp. Therefore, we attempted the optimum control of the ozone concentration with dilution by adjusting the air flow rate through the ozonizer using the flow proportional valve. As a result, the ozone concentration in the testing chamber is controlled with greater accuracy than the method based on supplying voltage to the ozonizer.

Article, JSME INTERNATIONAL JOURNAL SERIES B-FLUIDS AND THERMAL ENGINEERING. 49(4): 988-994 (2006)

Idiosyncratic drug toxicity is a rare, toxic drug reaction found only in the drug-treated patients clinically, and is not reproducible in animal experiments. In many cases, this toxicity is initiated quite likely by a production of chemically reactive metabolites, which covalently bind to macromolecules such as proteins and nucleic acids unless otherwise detoxified by glutathione S-transferases or other scavenging enzymes. Drug-modified proteins, particularly those present in the supernatant fraction, are generally processed to peptide fragments by a proteasome. These peptides are transported to the cell surface after binding to the major histocompatibility complex class I (MHC-I), and presented to the immune system. It is believed that abnormal peptides, when recognized by cytotoxic T-lymphocytes as non-self peptides, induce the toxic immune reaction finally leading to the cell death. Idiosyncrasy would reside on 1) production of the chemically reactive metabolites, 2) degradation of the reactive metabolites, and 3) recognition of drug-modified proteins as non-self proteins by the immune system. In order to prevent the idiosyncratic drug toxicity, organic chemists need to establish a strategy donating the structural nature being hardly converted to the reactive forms to the drug molecules. To detect the chemically reactive metabolites, highly sensitive detection of glutathione conjugate, produced by incubating the parent drug with liver microsomes in the presence of NADPH and glutathione, by a neutral loss scan set at 129 using LC/MS/MS could be utilized. A high pharmacological potency of the drug that eventually leads to a reduced dose level not higher than 10 mg/body would be another avenue to circumvent the idiosyncratic drug toxicity.

To obtain basic data for ballast water management, we examined the lethal effects of shock waves on a marine Vibrio. In the present experiment, shock waves were generated by hitting a projectile, which had been accelerated with a single-stage light gas gun, in an aluminum container having a cylindrical well where cell suspension was tightly maintained. We compared the shock wave effects to the isolate by use of two types of target containers with 5 and 25 mm well depths respectively. In the case of the shallow well, a lethal effect of shock compression was readily observed, but this did not occur for the deep well. Detailed pressure histories showed completely different peak patterns for the two well types. The propagation process of the shock wave was also analyzed theoretically and numerically. Results suggested that the lethal effects of shock waves are closely related to a narrow, rapid pressure change that occurs initially or a broad pressure change after the initial peak, rather than to the maximum pressure value.

The development of a large vortex structure in fluid flow is responsible for some of the most fascinating aspects of fluid dynamics, such as mixing, transport, and instability. This paper describes an experimental study on the vortex nature of a pulsating jet issuing from a rectangular nozzle of an aspect ratio of 5 which has short parallel section upstream of the jet exit. The visualized images by hydrogen bubble method revealed the influence of the bounded plates upon the vortex nature.

Androgens (androsterone, dihydrotestosterone and testosterone) and estrogens (estradiol, estriol and estrone) were incubated with liver microsomes from rats, dogs, monkeys and humans in the presence of uridine diphosphoglucuronic acid (UDPGA), and the glucuronides produced were structurally characterized by liquid chromatography-tandem mass spectrometry. After 2-h incubation with dog liver microsomes, all substrates tested were converted (approximately 2-10%) to structurally novel diglucuronides, where two glucuronosyl groups are bound to a single hydroxyl group in tandem. Two-dimensional nuclear magnetic resonance spectroscopy unambiguously elucidated the chemical structures of the 3-O-diglucuronide of estrone and the 17-O-diglucuronide of testosterone isolated from the incubation mixture. Monkey and human liver microsomes were also found to have the activity to form this type of diglucuronide, albeit more slowly than the dog liver microsomes, but rat liver microsomes produced no detectable diglucuronides. The rate of formation of estrone 3-O-diglucuronide from the corresponding monoglucuronide in dog liver microsomes followed classical Michaelis-Menten kinetics at substrate concentrations from 50 to 1000 microM, with a K(m) value of 127.1 microM and a V(max) value of 47.0 pmol/min/mg protein.

Phase control of a jet impinging on a wedge has been attempt using an external sound wave. The sound wave irradiating the flow around the exit of the nozzle enables 'phase-lock' which means the synchronization between the flow oscillation and the external sound. Hot-wire measurements with phase locking directly provide a part of the phase averaged flow fields and they illustrate appearance of the jet swing in front of the wedge and collision of the jet on one side of the wedge. These are in good agreement with flow visualization in the water. At the collision amplitude of the periodic velocity component abruptly increases. This phenomenon may contribute the feedback mechanism of the flow oscillation.

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/nuclear magnetic resonance spectroscopy (LC/NMR) were applied to the identification of the positional isomers of gemfibrozil metabolites produced in vitro by human liver microsomes. Gemfibrozil was metabolized to two major oxidative metabolites (M1 and M2) by human liver microsomes in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-generating system. M1 and M2 were characterized as metabolites formed by the oxidation of the 2′,5′-dimethylphenoxy moiety, which has potentially 5 different positions for hydroxylation, based on the results of negative-ion tandem mass spectrometry in combination with those of accurate mass measurements by quadrupole time-of-flight mass spectrometry. Separation and identification of M1 and M2 were conducted by LC/NMR analyses at 500 MHz. An on-flow 1H NMR analysis demonstrated that M1 was produced by hydroxylation at either the 2′-methyl group or the 5′-methyl group and M2 by hydroxylation at either the 3′-C or the 4′-C position of the aromatic ring. A stop-flow nuclear Overhauser effect spectroscopy (NOESY) analysis demonstrated that M1 and M2 were metabolites hydroxylated at the 5′-methyl group and the 4′-C position of the aromatic ring, respectively.

Summary: RS-7897, a novel organic nitrate, structurally contains aminoethylnitrate (AEN) and L-2oxothiazolidine-4-carboxylic acid (L-OTCA), which are linked together via an amide bond. Vasodilating activity of RS-7897 was 130 times weaker than that of AEN in vitro , while in vivo it was comparable to but longer lasting than those of AEN and nitroglycerin in anesthetized dogs. Intravenous administration of RS-7897 to dogs resulted in the appearance in plasma of AEN, which decreased with about 2.5 times longer t 1/2 (0.49 h) than that after administration of AEN itself. The T max value of AEN (0.25 h) after RS-7897 dosing agreed with the time showing the maximum vasodilating effect, indicating that RS-7897 serves as a prodrug releasing AEN slowly in vivo . The activity to hydrolyze RS-7897 to AEN and L-OTCA was localized in the cytosolic fractions of dog tissues, inhibited by thiol-blocking agents and was strongly inhibited by thyrotropin-releasing hormone, a substrate of pyroglutamyl aminopeptidase I (PAP-I). Furthermore, the RS-7897-hydrolyzing activity in dog liver cytosol was completely inhibited by an antibody against rat PAP-I. Therefore, it was found that PAP-I is involved in bioactivation of RS-7897 by amide bond hydrolysis, recognizing the sulfur-substituted L-pyroglutamyl moiety (L-OTCA) of this xenobiotic substrate.

Pyroglutamyl aminopeptidase I (PAP-I) is a cytosolic cysteine peptidase, which hydrolytically removes the L-pyroglutamate residue from the amino terminus of endogenous proteins and peptides. L-Pyroglutamyl p-nitroanilide serves as the synthetic substrate of this enzyme, while there is a possibility of other hydrolases being involved in the hydrolysis of this xenobiotic substrate. We cloned a full-length cDNA encoding rat PAP-I from a rat liver cDNA library and expressed this cDNA in Escherichia coli to obtain a recombinant PAP-I as a single protein. The cDNA encoded a sequence of 209 amino acids with a calculated molecular weight of 22913 Da. The homology of the deduced amino acid sequence of rat PAP-I was 98.6 and 94.3% to mouse and human PAP-Is, respectively. The biochemical properties of the recombinant rat PAP-I were almost identical to those of the recombinant mouse and human PAP-Is and the purified rat liver cytosolic PAP-I in terms of the molecular weight, subunit structure, affinity to the substrate, inhibitor profile and pH optimum. Immunoblot analysis using an antibody raised against recombinant rat PAP-I showed that rat PAP-I is present almost exclusively in the cytosolic fraction of the rat liver. Moreover, the hydrolyzing activity for L-pyroglutamyl p-nitroanilide in rat liver cytosolic fraction was completely inhibited by the antibody, strongly suggesting that this xenobiotic substrate is hydrolyzed solely by PAP-I.

Control of a cavity tone has been done using triangle projections attached at an exit of two-dimensional jet. Experimental results of sound pressure level show that the projections obviously reduce both total sound pressure level and sound level of the dominant frequency of the cavity tone. The sound dumping effect is increasing with increasing number and/or height of projections though this dumping effect saturates with the certain number of projections at the nearly same sound level as that in the wall jet case. The spanwise distribution of pressure fluctuation at the downstream cavity edge has the minima at the right downstream position of the projections and at the other spanwise positions pressure fluctuation is homogeneously reduced. This result suggests that the mechanism of sound dumping is not only breaking two-dimensionality of flows.

Protocols for evaluating drug interaction caused by enzyme inhibition were established by the Drug Interaction Database Working Group of the Human and Animal Bridge Discussion Group (HAB). Basically, procedures for measurements of the inhibition constant (Ki) of the test article were methodically investigated. 7-Ethoxyresorufin (CYP1A1&2), coumarin (CYP2A6), 7-benzyloxyresorufin (CYP2B6), tolbutamide (CYP2C8&9), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), chlorzoxazone (CYP2E1), nifedipine (CYP3A4) and testosterone (CYP3A4) were used as specific substrates for various isoforms of human liver cytochrome P450 (P450). At least 3 concentrations of the test article were appropriately selected in experiment on concentration-dependency of enzyme inhibition, and used in measurement of apparent Ki value by Dixon plot. In Ki measurements, the P450 substrate was added to the assay system at 3 different concentrations (Km, 1/2 Km and 2 Km). To correct the apparent Ki value of the test article by the binding to the liver microsomes, free concentration of the test article in the system for Ki measurements was determined most conveniently by ultracentrifugation. In this HAB-protocol, method to judge the mechanismbased inhibitor by detecting increased inhibition by the test article after preincubation in the presence of NADPH was also included. Magnitude of drug interaction (R) was estimated according to the equation: R=1+I/Ki, where I represents the concentration of the test article, most preferably the Cmax value either in the peripheral or the portal blood.

14C-Troglitazone was administered to male cynomolgus monkeys (monkey) orally or intraduodenally, and the time course of the plasma concentrations of the radioactivity and of the biliary excretions of the metabolites were investigated. The same was investigated for male marmosets after oral administration of 14C-troglitazone.1. The main metabolite in the plasma after oral administration to monkeys was the sulfoconjugate of troglitazone (M1). The quinone form metabolite (M3) and glucuronide (M2) of troglitazone were also detected in the plasma.2. The ratio of the biliary excretion of the radioactivity over a 24-hr period after intraduodenal administration to a bile-fistula cynomolgus monkey was 54.4%, which was as high as that observed in rats and dogs. The main metabolites in the bile were M1 and M2, each accounting for about 40% of the biliary radioactivity. An unknown metabolite, U3, was also detected at a ratio of about 6%. U3 was also detected in the bile after administration of M3, and was show to contain the quinone-like structure.3. The plasma metabolites after oral administration to marmosets were almost the same as those in the plasma of monkeys. U3 was also observed in the plasma of marmosets.4. U3 was isolated from the bile and urine of monkey and marmoset after administration of 14C-M3 or M3. According to the MS and NMR spectra, the chemical structure of U3 was determined as the glucuronide of the hydroquinone form of troglitazone, and its was conjugated at position 8"a.