WorldWideScience

Sample records for brain microsomal nasup

  1. Influence of cadmium on ketamine-induced anesthesia and brain microsomal Na[sup +], K[sup +]-ATPase in mice

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Y.; Sangiah, S. (Oklahoma State Univ., Stillwater, OK (United States))

    1994-10-01

    Cadmium is a rare metallic element, present in almost all types of food. Shellfish, wheat and rice accumulate very high amounts. Occupational and environmental pollutants are the main sources of cadmium exposure. Cadmium has a very long biologic half-life. Exposure to Cadmium causes anemia, hypertension, hepatic, renal, pulmonary and cardiovascular disorders as well as being a possible mutagen, teratogen and carcinogen. Acute cadmium treatment increased the hexobarbital sleeping time and inhibited hepatic microsomal drug metabolism due to a decrease in cytochrome P[sub 450] content. Cadmium potentiated ethanol-induced sleep in a dose-dependent manner. Cadmium has been shown to inhibit brain microsomal Na[sup +], K[sup +]-ATPase activity in vitro and in vivo. Cadmium and ethanol additively inhibited brain Na[sup +], K[sup +]-ATPase. This might be a direct interaction between cadmium and ethanol in the central nervous system. Ketamine is an intravenous anesthetic agent. It acts on central nervous system and produces [open quotes]dissociative anaesthesia.[close quotes] Ketamine provides adequate surgical anesthesia and is used alone in humans and/or combination with xylazine, an [alpha][sub 2]-adrenergic agonist in animals. It produces CNS depression, analgesia, amnesia, immobility and a feeling of dissociation from the environment. Ketamine is a non-competitive antagonist of the NMDA subset of the glutamate receptor. This perhaps results in an increase in neuronal activity leading to disorganization of normal neurotransmission and produces dissociative anesthetic state. Because it is different from most other anesthetics, ketamine may be expected to have a unique effect on brain biochemical parameters and enzymes. The purpose of this study was to examine the interactions between cadmium and ketamine on the central nervous system and ATPase, in an attempt to further understand the mechanism of action. 12 refs., 3 figs.

  2. Radiotracer binding to brain microsomes determined by thin-layer chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Zamora, P.O.; Stratesteffan, M.; Guhlke, S.; Sass, K.S.; Cardillo, A.; Bender, H.; Biersack, H.J

    1996-01-01

    A thin-layer chromatography (TLC) assay was developed to monitor the interaction of radiotracers with brain microsomes. Murine brain microsomes were coated onto a zone of a TLC strip, the unreacted sites blocked with gelatin, and the radiotracers chromatographed over the microsomes. Radiotracers bound to the microsomes and were separated from the unreacted materials which migrated at or near the solvent front. Up to 80% of the applied radioactivity bound to the brain microsomes when using {sup 99m}Tc-(d,l) hexamethyl-propyleneamine oxime (HMPAO) and {sup 123}I-(S)-2-hydroxy-3-iodo-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]- benzamide ({sup 123}I-IBZM) as tracers. On the other hand, the presumptive negative control materials p-I-15-phenyl-pentadecanoic acid-{sup 123}I ({sup I}I-IPPA) and {sup 99m}Tc-mercapto-acetyl triglycine (MAG3) bound poorly (7% and 4%, respectively). {sup 99m}Tc-ethyl cysteinate dimer (ECD) interacted poorly (9.9%), a result thought to be consistent with its known inability to be metabolized by nonprimate brain tissue. Radiolabeled octreotide analogues (radiolabeled with {sup 111}In, I-131 or {sup 99m}Tc) also bound, and the binding could be reduced by excess unlabeled octreotide. Also, chemical modification by acylation of Lys{sup 5} in {sup 111}In-labeled octreotide led to decreased binding (approximately 70%) compared to the original radiotracer. Chromatography of the various radiotracers over TLC strips coated only with gelatin was used to monitor nonspecific binding and was low and frequently below 5%. This technique does not require wash steps or centrifugation, and assays are rapidly completed. The assay could be useful in monitoring the interaction of radiotracers with brain microsomes and in evaluating and developing new radiotracers.

  3. Glucosylceramide and glucosylsphingosine modulate calcium mobilization from brain microsomes via different mechanisms.

    Science.gov (United States)

    Lloyd-Evans, Emyr; Pelled, Dori; Riebeling, Christian; Bodennec, Jacques; de-Morgan, Aviv; Waller, Helen; Schiffmann, Raphael; Futerman, Anthony H

    2003-06-27

    We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673-21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca2+ release, although only galactosylsphingosine mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were approximately 10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease.

  4. Lyso-glycosphingolipids mobilize calcium from brain microsomes via multiple mechanisms.

    Science.gov (United States)

    Lloyd-Evans, Emyr; Pelled, Dori; Riebeling, Christian; Futerman, Anthony H

    2003-11-01

    Recently, we demonstrated that the GSL (glycosphingolipid), GlcCer (glucosylceramide), modulates Ca2+ release from intracellular stores and from microsomes by sensitizing the RyaR (ryanodine receptor), a major Ca2+-release channel of the endoplasmic reticulum, whereas the lyso derivative of GlcCer, namely GlcSph (glucosylsphingosine), induced Ca2+ release via a mechanism independent of the RyaR [Lloyd-Evans, Pelled, Riebeling, Bodennec, de-Morgan, Waller, Schiffmann and Futerman (2003) J. Biol. Chem. 278, 23594-23599]. We now systematically examine the mechanism by which GlcSph and other lyso-GSLs modulate Ca2+ mobilization from rat brain cortical and cerebellar microsomes. GlcSph, lactosylsphingosine and galactosylsphingosine all mobilized Ca2+, but at significantly higher concentrations than those required for GlcCer-mediated sensitization of the RyaR. GlcSph-induced Ca2+ mobilization was partially blocked by heparin, an inhibitor of the Ins(1,4,5) P3 receptor, and also partially blocked by thapsigargin or ADP, inhibitors of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), but completely blocked when both acted together. In contrast, neither lactosylsphingosine nor galactosylsphingosine had any effect on Ca2+ release via either the Ins(1,4,5) P3 receptor or SERCA, but acted as agonists of the RyaR. Finally, and surprisingly, all three lyso-GSLs reversed inhibition of SERCA by thapsigargin. We conclude that different lyso-GSLs modulate Ca2+ mobilization via different mechanisms, and discuss the relevance of these findings to the GSL storage diseases in which lyso-GSLs accumulate.

  5. Ammonium ion substitutes for K/sup +/ in ATP-dependent Na/sup +/ transport by basolateral membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Towle, D.W.; Hoelleland, T.

    1987-03-01

    Ion-transporting cells from posterior gills of blue crabs (Callinectes sapidus) acclimated to low salinity were used as starting material for the preparation of microsomal membrane vesicles by density gradient centrifugation. The Na/sup +/-K/sup +/-adenosinetriphosphatase (ATPase)-enriched basolateral vesicles were loaded with KCl- or NH/sub 4//sup +/-containing medium by dilution and centrifugation, and initial rates of /sup 22/Na/sup +/ uptake into the vesicles were measured by a rapid filtration procedure. Varying the extravesicular sucrose concentration altered equilibrium uptake of /sup 22/Na/sup +/, indicating the existence of osmotically sensitive vesicles. Monensin, a sodium-specific ionophore, enhanced passive uptake of /sup 22/Na/sup +/ across the vesicle membrane in the absence of ATP. With 100 mM KCl in the intravesicular medium, addition of ATP to the extravesicular medium increased initial rates of /sup 22/Na/sup +/ uptake 10- to 20-fold over levels measured without ATP. A nonhydrolyzable ATP analog failed to stimulate /sup 22/Na/sup +/ uptake. Intravesicular K/sup +/ could be replaced by NH/sub 4//sup +/ but not by choline. With NH/sub 4//sup +/ as counterion, Na/sup +/ transport was inhibited by digitoxin, but valinomycin had no effect. A study of the kinetic effects of intravesicular K/sup +/ and NH/sub 4//sup +/ on initial rates of /sup 22/Na/sup +/ uptake indicated the existence of two classes of binding sites, one responding to counterion concentrations in the millimolar range and a second class responding to counterion concentrations over 50 mM. The results indicate that ATP-dependent /sup 22/Na/sup +/ uptake by membrane vesicles from Callinectes sapidus gill, mediated by Na/sup +/-K/sup +/-ATPase, can utilize either K/sup +/ or NH/sub 4//sup +/ as counterion.

  6. Investigation of the Association between Genetic Polymorphism of Microsomal Epoxide Hydrolase and Primary Brain Tumor Incidence

    Directory of Open Access Journals (Sweden)

    Ali Aydin

    2013-01-01

    Full Text Available mEH is a critical biotransformation enzyme that catalyzes the conversion of xenobiotic epoxide substrates into more polar diol metabolites: it is also capable of inactivating a large number of structurally different molecules. Two polymorphisms affecting enzyme activity have been described in the exon 3 and 4 of the mEH gene. The hypothesis of this study is that inherent genetic susceptibility to a primary brain tumor is associated with mEH gene polymorphisms. The polymorphisms of the mEH gene were determined with PCR-RFLP techniques and 255 Turkish individuals. Our results indicate that the frequency of the mEH exon 4 polymorphism (in controls is significantly higher than that of primary brain tumor patients (OR = 1.8, 95% CI = 1.0–3.4. This report, however, failed to demonstrate a significant association between mEH exon 3 polymorphism and primary brain tumor susceptibility in this population. Analysis of patients by both histological types of primary brain tumor and gene variants showed no association, although analysis of family history of cancer between cases and controls showed a statistically significant association (χ2=7.0, P=0.01. Our results marginally support the hypothesis that genetic susceptibility to brain tumors may be associated with mEPHX gene polymorphisms.

  7. Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase.

    Science.gov (United States)

    Fry, J P; Li, K Y; Devall, A J; Cockcroft, S; Honour, J W; Lovick, T A

    2014-12-01

    Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

  8. Na/sup +/-H/sup +/ exchange and Na/sup +/-dependent transport systems in streptozotocin diabetic rat kidneys

    Energy Technology Data Exchange (ETDEWEB)

    El-Seifi, S.; Freiberg, J.M.; Kinsella, F.J.; Cheng, L.; Sacktor, B.

    1987-01-01

    The streptozotocin-induced diabetic rat was used to test the hypothesis that Na/sup +/-H/sup +/ exchange activity in the proximal tubule luminal membrane would be increased in association with renal hypertrophy, altered glomerular hemodynamics, enhanced filtered load and tubular reabsorption of /sup 22/Na/sup +/, and stimulated /sup 22/Na= pump activity in the basolateral membrane, previously reported characteristics of this experimental animal model. Amiloride-sensitive H/sup +/ gradient-dependent Na/sup +/ uptake and Na/sup +/ gradient-dependent H/sup +/ flux were increased in brush-border membrane vesicles from the streptozotocin-treated animals. Na/sup +/ gradient-dependent uptakes of phosphate, D-glucose, L-proline, and myoinositol were decreased in the drug-induced diabetic animals. These membrane transport alterations were not found when the streptozotocin-diabetic animals were treated with insulin.

  9. Ionic dependence of active Na-K transport: clamping of cellular Na/sup +/ with monensin

    Energy Technology Data Exchange (ETDEWEB)

    Haber, R.S.; Pressley, T.A.; Loeb, J.N.; Ismail-Beigi, F.

    1987-07-01

    The Na/sup +/ ionophore monensin was used to study the Na/sup +/- and K/sup +/-dependence of ouabain-inhibitable /sup 86/Rb/sup +/ uptake in ARL 15 cells, a rat liver cell line. Graded concentrations of monensin rapidly induced incremental elevations of cellular Na/sup +/ that were stable for up to 2 h. In experiments in which cellular Na/sup +/ was thus clamped at various levels, the activation curve for ouabain-inhibitable /sup 86/Rb/sup +/ uptake as a function of intracellular Na/sup +/ was found to be steepest near basal Na/sup +/ levels (Hill coefficient /congruent/ 2.4), indicating that these cells can respond to relatively large changes in passive Na/sup +/ entry by increasing the rate of Na-K pump function with only minimal increases in cellular Na/sup +/. Exposure of cells to monensin also permitted examination of the extracellular-K/sup +/ dependence of ouabain inhibitable /sup 86/Rb/sup +/ uptake in presence of saturating intracellular Na/sup +/ and yielded a Hill coefficient of approx. 1.5. The rate of ATP hydrolysis calculated from measurements of the maximal rate of ouabain-inhibitable /sup 86/Rb/sup +/ uptake in intact cells was similar to the enzymatic V/sub max/ of the Na/sup +/-K/sup +/-ATPase in cell lysates, suggesting that the Na/sup +/-K/sup +/-ATPase activity in these broken-cell preparations closely reflects the functional transport capacity of the Na-K pump.

  10. Effect of an extract of Aloe vera on the biodistribution of sodium pertechnetate (Na{sup 99m}TcO{sub 4}) in rats

    Energy Technology Data Exchange (ETDEWEB)

    Holanda, Cecilia Maria de Carvalho Xavier [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Dept. of Microbiology and Parasitology. Experimental Radiobiology and Antiparasitic Assays Lab.], e-mail: cechol@ufrnet.br; Costa, Monique Batista da; Silva, Natalia Chilinque Zambao da; Silva Junior, Mauricio Ferreira da; Barbosa, Vanessa Santos de Arruda; Silva, Roseane Pereira da [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil); Medeiros, Aldo da Cunha [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Nucleus of Experimental Surgery

    2009-07-01

    Purpose: Aloe vera is a tropical plant popularly known in Brazil as babosa. We have investigated the effect of aqueous extract of Aloe vera on the biodistribution of Na{sup 99m}TcO{sub 4} and laboratorial parameters in Wistar rats. Methods: twelve animals were divided into treated and control groups. In the treated group, Aloe vera was given by gavage (5mg/mL/day) during 10 days. The control group received sorbitol by the same way and period. One hour after the last dose, we injected 0.1mL of Na{sup 99m}TcO{sub 4} by orbital plexus. After 60 min, all the animals were killed. Samples were harvested from the brain, liver, heart, muscle, pancreas, stomach, femur, kidneys, blood, testis and thyroid and the percentage of radioactivity (% ATI/g) was determined. Biochemical dosages were performed. Results: there was a significant increase of %ATI/g in blood, femur, kidneys, liver, stomach, testis and thyroid and also in blood levels of AST and ALT. A significant decrease in levels of glucose, cholesterol, triglycerides, creatinine and urea occurred. The statistical analyses were performed by Mann-Whitney test and T-Student test (p<0.05). Conclusion: The aqueous extract of Aloe vera facilitated the uptake of Na{sup 99m}TcO{sub 4} in organs of rats and it was responsible to a high increase of levels of AST and ALT. (author)

  11. Monoclonal antibodies that bind the renal Na/sup +//glucose symport system. 1. Identification

    Energy Technology Data Exchange (ETDEWEB)

    Wu, J.S.R.; Lever, J.E.

    1987-09-08

    Phlorizin is a specific, high-affinity ligand that binds the active site of the Na/sup +//glucose symporter by a Na/sup +/-dependent mechanism but is not itself transported across the membrane. The authors have isolated a panel of monoclonal antibodies that influence high-affinity, Na/sup +/-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK/sub 1/, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG/sub 2b/, reproducibly stimulated Na/sup +/-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na/sup +/-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na/sup +//glucose symporter.

  12. Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Sopjani, Mentor [Department of Physiology, University of Tuebingen (Germany); Department of Chemistry, University of Prishtina, Kosovo (Country Unknown); Alesutan, Ioana; Wilmes, Jan [Department of Physiology, University of Tuebingen (Germany); Dermaku-Sopjani, Miribane [Department of Physiology, University of Tuebingen (Germany); Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Lam, Rebecca S. [Department of Physiology, University of Tuebingen (Germany); Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main (Germany); Koutsouki, Evgenia [Department of Physiology, University of Tuebingen (Germany); Jakupi, Muharrem [Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Foeller, Michael [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

    2010-11-19

    Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

  13. Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina [Department of Physiology, University of Tübingen (Germany); Laufer, Stefan [Pharmaceutical Chemistry, University of Tübingen (Germany); Borst, Oliver; Gawaz, Meinrad [Cardiology and Cardiovascular Medicine, University of Tübingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tübingen (Germany)

    2014-09-26

    Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

  14. H{sup +} and Na{sup +} are involved in flagellar rotation of the spirochete Leptospira

    Energy Technology Data Exchange (ETDEWEB)

    Islam, Md. Shafiqul [Department of Applied Physics, Graduate School of Engineering, Tohoku University, 6-6-05 Aoba, Aoba-ku, Sendai, Miyagi 980-8579 (Japan); Morimoto, Yusuke V. [Quantitative Biology Center, RIKEN, 6-2-3 Furuedai, Suita, Osaka 565-0874 (Japan); Graduate School of Frontier BioSciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kudo, Seishi [Department of Applied Physics, Graduate School of Engineering, Tohoku University, 6-6-05 Aoba, Aoba-ku, Sendai, Miyagi 980-8579 (Japan); Nakamura, Shuichi, E-mail: naka@bp.apph.tohoku.ac.jp [Department of Applied Physics, Graduate School of Engineering, Tohoku University, 6-6-05 Aoba, Aoba-ku, Sendai, Miyagi 980-8579 (Japan); Graduate School of Frontier BioSciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2015-10-16

    Leptospira is a spirochete possessing intracellular flagella. Each Leptospira flagellar filament is linked with a flagellar motor composed of a rotor and a dozen stators. For many bacterial species, it is known that the stator functions as an ion channel and that the ion flux through the stator is coupled with flagellar rotation. The coupling ion varies depending on the species; for example, H{sup +} is used in Escherichia coli, and Na{sup +} is used in Vibrio spp. to drive a polar flagellum. Although genetic and structural studies illustrated that the Leptospira flagellar motor also contains a stator, the coupling ion for flagellar rotation remains unknown. In the present study, we analyzed the motility of Leptospira under various pH values and salt concentrations. Leptospira cells displayed motility in acidic to alkaline pH. In the presence of a protonophore, the cells completely lost motility in acidic to neutral pH but displayed extremely slow movement under alkaline conditions. This result suggests that H{sup +} is a major coupling ion for flagellar rotation over a wide pH range; however, we also observed that the motility of Leptospira was significantly enhanced by the addition of Na{sup +}, though it vigorously moved even under Na{sup +}-free conditions. These results suggest that H{sup +} is preferentially used and that Na{sup +} is secondarily involved in flagellar rotation in Leptospira. The flexible ion selectivity in the flagellar system could be advantageous for Leptospira to survive in a wide range of environment. - Highlights: • This is a study on input energy for motility in the spirochete Leptospira. • Leptospira biflexa exhibited active motility in acidic to alkaline pH. • Both H{sup +} and Na{sup +} are involved in flagellar rotation in Leptospira. • H{sup +} is a primary energy source, but Na{sup +} can secondarily enhance motility.

  15. Na/sup +/-independent, phloretin-sensitive monosaccharide transport system in isolated intestinal epithelial cells. [Chickens

    Energy Technology Data Exchange (ETDEWEB)

    Kimmich, G.A.; Randles, J.

    1975-01-01

    A monosaccharide transport system in addition to the active Na/sup +/-dependent system characteristic of the brush border surface of vertebrate intestinal tissue has been identified in isolated chick intestinal epithelial cells. The newly described system differs in several characteristics from the Na/sup +/-dependent process, including function in the absence of Na/sup +/; a high sensitivity to phloretin, relative insensitivity to phlorizin; different substrate specificity; and a very high K/sub T/ and V/sub max/. The system apparently functions only in a facilitated diffusion manner so that it serves to move monosaccharide across the cell membrane down its chemical gradient. An appreciable fraction of total sugar efflux occurs via the Na/sup +/-independent carrier from cells which have accumulated sugar to a steady state. Phloretin selectively blocks this efflux so that a normal steady-state sugar gradient of seven- to eightfold is transformed to a new steady-state gradient which is greater than 14-fold. Locus of the new system is tentatively ascribed to the serosal cell surface where it would serve for monosaccharide transfer between enterocyte and lamina propria of the villus. (auth)

  16. Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

    Directory of Open Access Journals (Sweden)

    Menéndez R.

    2000-01-01

    Full Text Available The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS. When D002 (5-100 mg/kg body weight was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46% occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40% and brain (28-44% microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

  17. Effect of tripanossomicide benznidazole (Rochagan) on the biodistribution of sodium pertechnetate (Na{sup 99m}TcO4) in Wistar rats

    Energy Technology Data Exchange (ETDEWEB)

    Barbosa, Vanessa Santos de Arruda; Holanda, Cecilia Maria de Carvalho Xavier; Silva, Roseane Pereira da; Medeiros, Aldo Cunha [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Centro de Ciencias da Saude]. E-mail: vambio@oi.com.br; Oliveira, Daniel Pereira de; Silva Junior, Mauricio Ferreira da; Oliveira, Elias Herculano de [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Centro de Biociencias. Dept. de Microbiologia e Parasitologia; Spyrides, Maria Helena Constantino [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Dept. de Estatistica

    2008-12-15

    Benznidazole, a drug with specific anti-Trypanosoma cruzi activity, is used in the treatment of Chagas' disease. The radiopharmaceutical sodium pertechnetate (Na{sup 99m}TcO{sub 4}) is used to obtain diagnostic images of the stomach, thyroid, parathyroids, salivary glands, brain and in the study of esophageal reflux and blood flow. This study aimed at evaluating in vivo the influence of benznidazole treatment on the sodium pertechnetate biodistribution in Wistar rats. The percentage of radioactivity per gram (%ATI/g) of various organs (brain, heart, esophagus, stomach, small intestine, large intestine, spleen, liver, muscle and blood) was determined. Comparing the treated rats with the controls, we observed that sodium pertechnetate biodistribution did not change when administered to rats treated for thirty days with benznidazole. (author)

  18. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  19. Microsomal metabolism of trenbolone acetate metabolites ...

    Science.gov (United States)

    Trenbolone acetate (TBA) is a synthetic growth promoter widely used in animal agriculture, and its metabolites are suspected endocrine disrupting compounds in agriculturally impacted receiving waters. However, beyond the three widely recognized TBA metabolites (17-trenbolone, 17-trenbolone and trendione), little is known about other metabolites formed in vivo and subsequently discharged into the environment, with some evidence suggesting these unknown metabolites comprise a majority of the TBA mass dosed to the animal. Here, we explored the metabolism of the three known TBA metabolites using rat liver microsome studies. All TBA metabolites are transformed into a complex mixture of monohydroxylated products. Based on product characterization, the majority are more polar than the parent metabolites but maintain their characteristic trienone backbone. A minor degree of interconversion between known metabolites was also observed, as were higher order hydroxylated products with a greater extent of reaction. Notably, the distribution and yield of products were generally comparable across a series of variably induced rat liver microsomes, as well as during additional studies with human and bovine liver microsomes. Bioassays conducted with mixtures of these transformation products suggest that androgen receptor (AR) binding activity is diminished as a result of the microsomal treatment, suggesting that the transformation products are generally less potent than

  20. The prevalence of Antithyroglobulin and Antithyroid Microsomal ...

    African Journals Online (AJOL)

    Plasma levels of antithyroglobulin {TG} and microsomal thyroid peroxidase {TPO} autoantibodies were determined using the ELISA methods, in 87 euthyroid women. These were made up of 44 control women which included 8{18%} nulligravidae, 18{41%} non pregnant multiparous and 18{41%}, pregnant subjects.

  1. cap alpha. -Methylglucoside satisfies only Na/sup +/-dependent transport system of intestinal epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Kimmich, G.A.; Randles, J.

    1981-01-01

    The unidirectional influx of ..cap alpha..-methylglucoside (..cap alpha..-MG) by isolated chicken intestinal epithelial cells is 98% inhibited by phlorizin. The remaining 2% of the total influx occurs in the absence of Na/sup +/, is not sensitive to phloretin, and is equal to the diffusional entry rate for 2-deoxyglucose. The glucoside is much more strongly accumulated (75-fold) than 3-O-methylglucose (3-OMG) (10-fold). Inhibitors of the serosal sugar carrier (phloretin, cytochalasin B, theophylline, and flavanoids) do not enhance ..cap alpha..-MG accumulation. It is concluded that the glycoside is not a substrate for the intestinal serosal transport system. Steady-state gradients of the sugar can be represented accurately by a concentrative, phlorizin-sensitive system that is opposed by a diffusional efflux process.

  2. Metabolism of bupropion by baboon hepatic and placental microsomes

    Science.gov (United States)

    Wang, Xiaoming; Abdelrahman, Doaa R.; Fokina, Valentina M.; Hankins, Gary D.V.; Ahmed, Mahmoud S.; Nanovskaya, Tatiana N.

    2011-01-01

    The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (Km 36 ± 6 µM, Vmax 258 ± 32 pmol mg protein−1 min−1), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent Km values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB. PMID:21570381

  3. Role of the Na{sup +}/H{sup +} exchanger on the development of diabetes mellitus and its chronic complications

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yan-Ming [Department of Cardiac Care Unit, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China); Su, Ying [Department of Endocrinology, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China); Li, Jia; Tian, Ye [Department of Cardiac Care Unit, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China); Wang, Lan-Feng, E-mail: wlfccu@126.com [Department of Cardiac Care Unit, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer NHE protect against intracellular hydrogen overload. Black-Right-Pointing-Pointer NHE protect {beta}-cells against strong acidification. Black-Right-Pointing-Pointer NHE inhibitors improve myocardial ischemia and reperfusion. -- Abstract: Micro- and macrovascular complications are the main cause of morbidity and mortality in diabetes mellitus. The Na{sup +}/H{sup +} exchanger (NHE) is a family of proteins which exchange Na{sup +} for H{sup +} according to their concentration gradients in an electroneutral manner. The exchanger also plays a key role in several other cellular functions including proliferation, differentiation, apoptosis, migration, and cytoskeletal organization. Since not much is known on the relationship between NHE and diabetes mellitus, this review outlines the contribution of NHE to chronic complications of diabetes mellitus, such as diabetic nephropathy; diabetic cardiomyopathy.

  4. Protein radioiodination in a radioassay laboratory: evaluation of commercial Na/sup 125/I reagents and related biohazards

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, R.G.; Button, T.M.

    1980-01-01

    Three commercial Na/sup 125/I solutions (Amersham, New England Nuclear, and Union Carbide) have been examined with respect to multiple parameters affecting their use in the radioiodination of three representative peptides (insulin, growth hormone, and gastrin): % of radioiodine incorporation in protein; immunoreactivity and non-specific binding properties of the radiolabeled proteins; pH, volatility, and radionuclidic purity of radioiodine solutions; and vial construction with respect to multidose use. All three commercial Na/sup 125/I produced radioiodinated proteins of good quality for use in radioligand assays. The radioiodines differed with respect to the amount of iodine released during initial vial opening as a consequence of different pH levels. Two of the three products were shipped in vials with poor construction with respect to multidose use. Selection of a radioiodine was therefore reduced to the secondary considerations of iodine volatility and vial construction. The volatilized radioiodine observed during the spill of millicuries quantities of unbuffered pH 7.5 Na/sup 125/I was 14 microcuries per millicurie within the first 30 minutes. One thickness of rubber gloves reduced potential skin contamination from an accidental spill to insignificant levels: 20-30 picocuries per microcurie. Common good housekeeping procedures: i.e. rubber gloves, laboratory coat and a fume hood were found to be sufficient protection to eliminate most radioiodine volatility and contamination hazards associated with protein radiolabeling procedures.

  5. Microsomal lipid peroxidation as a mechanism of cellular damage. [Dissertation

    Energy Technology Data Exchange (ETDEWEB)

    Kornbrust, D.J.

    1979-01-01

    The NADPH/iron-dependent peroxidation of lipids in rat liver microsomes was found to be dependent on the presence of free ferrous ion and maintains iron in the reduced Fe/sup 2 +/ state. Chelation of iron by EDTA inhibited peroxidation. Addition of iron, after preincubation of microsomes in the absence of iron, did not enhance the rate of peroxidation suggesting that iron acts by initiating peroxidative decomposition of membrane lipids rather than by catalyzing the breakdown of pre-formed hydroperoxides. Liposomes also underwent peroxidation in the presence of ferrous iron at a rate comparable to intact microsomes and was stimulated by ascorbate. Carbon tetrachloride initiated lipid peroxidation in the absence of free metal ions. Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely between different tissues and species. The effects of paraquat on lipid peroxidation was also studied. (DC)

  6. Effect of chloride substitution on the order–disorder transition in NaBH{sub 4} and Na{sup 11}BD{sub 4}

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, Jørn Eirik [Institute for Energy Technology, Physics Department, P.O. Box 40, NO-2027 Kjeller (Norway); Karen, Pavel [University of Oslo, Department of Chemistry, P.O. Box 1033 Blindern, NO-0315 Oslo (Norway); Sørby, Magnus H. [Institute for Energy Technology, Physics Department, P.O. Box 40, NO-2027 Kjeller (Norway); Hauback, Bjørn C., E-mail: bjorn.hauback@ife.no [Institute for Energy Technology, Physics Department, P.O. Box 40, NO-2027 Kjeller (Norway)

    2014-02-25

    Graphical abstract: Interactions that order the BD{sub 4}{sup -} tetrahedra below the order–disorder transition became increasingly frustrated by the solute in the Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} solid solutions, and the order disappears at x = 0.158. Highlights: • The order–disorder transition temperature for Na(BH{sub 4}){sub 1−x}Cl{sub x} and Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} is highly dependent on the Cl-content, x. • The transition is characterized by DSC for Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} for x = 0, 0.10 and 0.15. • No transition is observed for x ⩾ 0.20 on cooling to 8 K. • The crystal structures are reported for Na{sup 11}BD{sub 4} at room temperature and 8 K and Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} (x = 0.10, 0.15, 0.20 and 0.25) at 8 K from powder neutron diffraction. -- Abstract: Phase transition associated with anion disordering over two orientations in Na{sup 11}BD{sub 4} (NaBH{sub 4}) and its solid solutions with NaCl, Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x}, is investigated with powder diffraction (neutron and synchrotron radiation), differential scanning calorimetry and Raman spectroscopy. Upon heating, the transition temperature extrapolated to zero rate of heating is 192.2 K for Na{sup 11}BD{sub 4}, ΔS = 4.41 J/mol K, hysteresis 1.7 K and the volume increase 0.43%. Thermal parameters of the transition in Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} follow a colligative-property model of an ideal solution, with x = 0.158(1) as the critical concentration at which the ordering interactions and the transition itself are eliminated. On approaching this limit, the tetragonal distortion of the ordered structure decreases somewhat towards the cubic average, and this is associated with a partial disorder of the tetrahedral anions seen by diffraction methods. In fact, a 3% disorder is already present in the pure solvent of the solid solution (Na{sup 11}BD{sub 4}) at 8 K.

  7. Oxidation of an organosulfur xenobiotic by microsomes from soybean cotyledons.

    Science.gov (United States)

    Blee, E; Durst, F

    1986-03-28

    Methiocarb, an aromatic-alkyl sulfide insecticide was enzymatically oxidized into its sulfoxide by microsomes from soybean cotyledons. No further oxidation into sulfone was detected. Distribution of the sulfoxidase activity was studied in soybean seedlings and found maximal in cotyledons. Subcellular fractionation of cotyledons homogenates indicated that the activity was almost entirely associated with the microsomal fraction. Sulfoxidation of methiocarb did not require cofactors such as NAD(P)H. Nevertheless, the sulfoxidase did not act as a peroxidase.

  8. Effect of the dilution factor on {sup 18}FDG and Na{sup 18}F samples for bacterial endotoxin test using PTS (portable test system)

    Energy Technology Data Exchange (ETDEWEB)

    Silveira, Marina B.; Costa, Flavia M.; Ferreira, Soraya Z., E-mail: mbs@cdtn.b [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Unidade de Pesquisa e Producao de Radiofarmacos

    2011-07-01

    {sup 18}FDG and Na{sup 18}F are radiopharmaceuticals produced as sterile solutions suitable for intravenous administration, which must contain no more than 175 EV/V. The most commonly used approach to detect endotoxins is the gelclot technique that requires 60 minutes for results. For radiopharmaceuticals containing short-life radionuclides, such as {sup 18}F, there is an increasing interest for faster quality control methods. FDA licensed the Endosafe, PTS, a kinetic chromogenic endotoxin detection system that takes about 15 minutes for results. As other techniques, PTS test is susceptible to interferences which can be solved by product dilution. The aim of this study was to establish the best dilution of {sup 18}FDG and Na{sup 18}F for PTS analysis. Two different dilution factors for {sup 18}FDG and 1:10 for Na{sup 18}F were essayed: 1:10 and 1:100. {sup 18}FDG and Na{sup 18} solutions were prepared by the addition of LAL reagent water. Considering the assay acceptance criteria, the best dilution factor was 1:100 for {sup 18}FDG and 1:10 for Na{sup 18}F. The recovery of the product positive control was 98-12% for {sup 18}FDG 1:100 and 104-120% for Na{sup 18}F 1:10, which were, in both cases, within the specification (50-200%) and very close to 100%. Results obtained with these dilution studies were important to establish the most appropriate and non-interfering dilution factor for {sup 18}FDG and Na{sup 18}F routine endotoxin test. (author)

  9. Binding of uteroglobin to microsomes and plasmatic membranes.

    Science.gov (United States)

    Diaz González, K; Nieto, A

    1995-03-20

    Microsomes and plasmatic membranes from rat liver bind radioactive uteroglobin (UG) in vitro with high affinity (Kd = 1.7 x 10(-10) M. The binding is saturable and specific and dependent on previous reduction of UG with dithiothreitol. Microsomes from rat spleen or lung or from rabbit endometrium also possess a similar ability. Binding capacity is not affected by previous treatment of microsomes with phospholipase A2 or peptide-N-glycosidase F but is lost after brief treatment with trypsin. The complex formed between UG and the binding component can be solubilized from microsomes with 5 mM CHAPS and it elutes with an apparent Mr of 90,000 in a Sephacryl 200 column. The complex is resistant to 8 M urea but is completely dissociated by Triton X-100. The UG-binding protein(s) has been partially purified from solubilized microsomes and membranes by affinity chromatography. The results are discussed in relation to a possible physiological effect of UG on cellular membranes.

  10. Ethosuximide is primarily metabolized by CYP3A when incubated with isolated rat liver microsomes.

    Science.gov (United States)

    Sarver, J G; Bachmann, K A; Zhu, D; Klis, W A

    1998-01-01

    The cytochrome P450 (CYP) subfamily responsible for ethosuximide metabolism was investigated by HPLC assay of ethosuximide incubations with isolated rat liver microsomes from control rats and from rats treated with inducing agents to enrich hepatic microsomes in selected CYP isoforms. Inducing agents included beta-naphthoflavone (BNF, CYP1A inducer), phenobarbital (PB, CYP2B/2C/3A), isoniazid (INH, CYP2E1), clotrimazole (CTZ, CYP3A), clofibrate (CLO, CYP4A), and an imidazole CTZ-analog known as CDD3543 (CYP3A). Incubations with BNF, INH, CTZ, and control microsomes showed significantly (pCTZ microsomes vs. BNF, INH, and control microsomes at 10, 30, 60, and 120 min incubation. Ethosuximide metabolite levels generated by CTZ microsomes at 120 min were 36.5 times those of control microsomes. Correspondingly, ethosuximide concentrations were significantly (pCTZ microsomes compared with BNF, INH, and control microsomes at 60 and 120 min. Sixty-minute incubations with all microsome groups exhibited significantly (pCTZ (11.8x control) and PB (9.6x control) microsomes vs. all other groups. Antibody inhibition experiments demonstrated ethosuximide metabolite levels for PB microsomes were not affected by CYP2B1 antibodies, whereas CYP3A2 antibodies reduced metabolite levels for both PB and CTZ microsomes by over 80%. These results indicate CYP3A is primarily responsible for ethosuximide metabolism in rats.

  11. Liver microsomal cytochromes P-450 and azoreductase activity.

    Science.gov (United States)

    Fujita, S; Peisach, J

    1978-07-10

    Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is proportional to the levels of microsomal cytochrome P-450 from control or phenobarbital-pretreated rats and mice or cytochrome P-448 from 3-methylchol-anthrene-pretreated animals. In the "inducible" C57B/6J strain of mice, 3-methylcholanthrene and phenobarbital pretreatment cause an increase in cytochrome P-448 and P-450 levels, respectively, which is directly proportional to the increase of azoreductase activity. However, in the "noninducible" DBA/2J strain of mice, only phenobarbital treatment causes the increase both in cytochrome P-450 levels and azoreductase activity, while 3-methylcholanthrene has no effect. These experiments suggest that the P-450 type cytochromes are responsible for azoreductase activity in liver microsomes.

  12. UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes

    Directory of Open Access Journals (Sweden)

    Moayad Saad

    2018-02-01

    Full Text Available This article represents data regarding a study published in Toxicology in vitro entitled “ in vitro CYP-mediated drug metabolism in the zebrafish (embryo using human reference compounds” (Saad et al., 2017 [1]. Data were acquired with ultra-performance liquid chromatography – accurate mass mass spectrometry (UPLC-amMS. A full spectrum scan was conducted for the testosterone (TST metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM and female (FLM livers, whole body homogenates of 96 h post fertilization larvae (EM and a pool of human liver microsomes from 50 donors (HLM. Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

  13. Microsome composition-based model as a mechanistic tool to predict nonspecific binding of drugs in liver microsomes.

    Science.gov (United States)

    Poulin, Patrick; Haddad, Sami

    2011-10-01

    The purpose of this study was to investigate the ability of the microsome composition-based model to predict the unbound fraction determined in vitro in microsomal incubation system (fuinc ). Another objective was to make a comparative assessment between the proposed mechanistic method and three empirical methods published in the literature, namely the models of Austin et al. (2002, Drug Metab Dispos 30:1497-1503), Turner et al. [2007, Drug Metab Rev 38(S1):162], and Halifax and Houston (2006, Drug Metab Rev 34:724-726), which are based solely on physicochemical properties. The assessment was confined by the availability of measured fuinc data in rat and human at diverse microsomal protein concentrations for 132 compounds. The proposed microsome composition-based model can be viewed as a combination of two distinct processes, namely the nonspecific binding to neutral lipids and the ionic binding to acidic phospholipids. Across methods, the maximum success rate in predicting fuinc of all compounds was 98%, 91%, and 84% with predictions falling within threefold, twofold, and 1.5-fold error of the observed fuinc , respectively. The statistical analyses suggest that the prediction models are more effective at computing fuinc (i) for rat as compared with human, and (ii) for acids and neutral drugs as compared with strong basic drugs. In addition, on the basis of the comparisons made using all datasets, the method that made use of microsome composition data compares well with those methods that relied solely on physicochemistry. The sensitivity analysis demonstrated the importance of the compound properties and physiological parameters reflective of specific mechanistic determinants relevant to prediction of fuinc values of drugs. Overall, the results obtained with our proposed model demonstrate a significant step toward the development of a generic and mechanistic model of fuinc for liver microsomes, which should provide rationale extrapolation procedures of hepatic

  14. Effect of thiols on lipid peroxidation in rat liver microsomes

    NARCIS (Netherlands)

    Haenen, G R; Vermeulen, N P; Timmerman, H; Bast, A

    1989-01-01

    The stimulatory or inhibitory effects of various thiol compounds on in vitro lipid peroxidation by iron-ascorbate in rat liver microsomes were determined. Glutathione had no measurable pro-oxidant capacity, in contrast, it protected against lipid peroxidation. N-Acetyl l-cysteine and

  15. Inhibition Of Microsomal Lipid Peroxidation And Protein Oxidation ...

    African Journals Online (AJOL)

    The antioxidant activities of 53 medicinal plants used in Bamun Folk Medicine for the management of jaundice and hepatitis were investigated. The studies were done using rat hepatic microsomes for lipid peroxidation and bovine serum albumin (BSA) for carbonyl group formation. Silymarine was used as reference ...

  16. Isoflavones modulate the glucuronidation of estradiol in human liver microsomes

    National Research Council Canada - National Science Library

    Pfeiffer, Erika; Treiling, Christian R; Hoehle, Simone I; Metzler, Manfred

    2005-01-01

    ... by the endogenous hormone 17beta-estradiol (E2). In the present study, we have examined if daidzein and genistein as well as several structurally related isoflavones are able to modulate the in vitro glucuronidation of E2 in human hepatic microsomes...

  17. Ginkgo biloba extract alters the binding of the sodium [{sup 123}I] iodide (Na{sup 123}I) on blood constituents

    Energy Technology Data Exchange (ETDEWEB)

    Aleixo, Luiz Claudio Martins [Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, 28 de Setembro, 87, 20551-030, Rio de Janeiro, RJ (Brazil); Comissao Nacional de Energia Nuclear, Instituto de Engenharia Nuclear, Cidade Universitaria, Ilha do Fundao, Via Cinco s/n, 21945-450 Rio de Janeiro (Brazil); Moreno, Silvana Ramos Farias, E-mail: srfmoreno@hotmail.com [Departamento de Patologia, Universidade Federal Fluminense, 24030-210, Niteroi, RJ (Brazil); Programa de Pos-Graduacao em Ciencias Medicas, Universidade Federal Fluminense, 24030-210, Niteroi, RJ (Brazil); Freitas, Rosimeire de Souza [Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, 28 de Setembro, 87, 20551-030, Rio de Janeiro, RJ (Brazil); Thomaz, Helio [Comissao Nacional de Energia Nuclear, Instituto de Engenharia Nuclear, Cidade Universitaria, Ilha do Fundao, Via Cinco s/n, 21945-450 Rio de Janeiro (Brazil); Santos-Filho, Sebastiao David [Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, 28 de Setembro, 87, 20551-030, Rio de Janeiro, RJ (Brazil)

    2012-01-15

    We evaluated the in vitro effect of an aqueous extract of Ginkgo biloba (EGb) on the distribution in blood cells (BC) and plasma (P) and on the binding of Na{sup 123}I to the blood constituents using precipitation with trichloroacetic acid. The radioactivity percentages insoluble (SF) and insoluble fraction (IF) of blood constituents were determined. The EGb interfered (p<0.05) on the distribution of Na{sup 123}I in the P (from 69.64 to 86.13) and BC (from 30.36 to 13.87) and altered the fixation of the Na{sup 123}I in IF-P and in IF-BC. - Highlights: Black-Right-Pointing-Pointer Interaction between the Ginkgo biloba and blood constituents radiolabeled. Black-Right-Pointing-Pointer Modification of the binding of sodium iodide (Na{sup 123}I) to the blood constituents. Black-Right-Pointing-Pointer This alteration should have influence in a diagnosis of nuclear medicine.

  18. In vitro biotransformation of flavonoids by rat liver microsomes

    DEFF Research Database (Denmark)

    Nielsen, S. E.; Breinholt, V.; Justesen, U.

    1998-01-01

    1. Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes. Naringenin, hesperetin, chrysin, apigenin, tangeretin, kaempferol, galangin and tamarixetin were all metabolized extensively by induced rat liver...... pathway leading to the corresponding 3',4'-dihydroxylated flavonoids either by hydroxylation or demethylation. Structural requirements for microsomal hydroxylation appeared to be a single or no hydroxy group on the B-ring of the flavan nucleus. The presence of two or more hydroxy groups on the B......-ring seemed to prevent further hydroxylation. The results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C-4'-, and not at the C-3'-position. 3. The CYP1A isozymes were found to be the main enzymes involved in flavonoid hydroxylation, whereas other cytochrome P...

  19. Hydroperoxide-dependent sulfoxidation catalyzed by soybean microsomes.

    Science.gov (United States)

    Blee, E; Durst, F

    1987-04-01

    The sulfoxidation of methiocarb, an aromatic-alkyl sulfide pesticide, catalyzed by soybean microsomes was found to be strongly stimulated in the presence of cumene and linoleic acid hydroperoxides. We have shown that this S-oxidation, which does not require cofactors such as NAD(P)H, is an hydroperoxide-dependent reaction: 18O2-labeling experiments demonstrated that the oxygen atom incorporated into the sulfoxide originated from hydroperoxides rather than from molecular oxygen. In the absence of exogenous hydroperoxides, soybean microsomes catalyzed methiocarb sulfoxide formation at a basal rate dependent on their endogenous hydroperoxides, especially those derived from free fatty acids. The nature of the sulfoxidase is discussed. Our results seem to rule out the participation of cytochrome P-450 in this oxidation, whereas the studied sulfoxidase presents some similarities to plant peroxygenase.

  20. Enzymatic denitrification of 2-nitropropane in uninduced mouse liver microsomes.

    Science.gov (United States)

    Marker, E K; Kulkarni, A P

    1985-08-01

    Hepatic microsomes from 5 strains of untreated mice were tested for the ability to enzymatically cleave the nitro group from 2-nitropropane (2NP). All strains showed significant NADPH-dependent nitrite release at pH 7.6 and pH 8.8. Statistical differences in nitrite-releasing activity between strains were found between BALB and PL/J and ATH strains at pH 7.6. At pH 8.8, BIO.M differed from CD-1 and BALB. These results are in contrast to a report of little or no denitrification activity in uninduced rats and suggest that the 2NP microsomal metabolism may be of greater importance than previously thought.

  1. Role of H{sub 2}O{sub 2} on the kinetics of low-affinity high-capacity Na{sup +}-dependent alanine transport in SHR proximal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pinto, Vanda; Pinho, Maria Joao [Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, 4200-319 Porto (Portugal); Jose, Pedro A. [Center for Molecular Physiology Research, Children' s National Medical Center, Department of Pediatrics, George Washington School of Medicine and Health Sciences, Washington, DC (United States); Soares-da-Silva, Patricio, E-mail: pss@med.up.pt [Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, 4200-319 Porto (Portugal)

    2010-07-30

    Research highlights: {yields} H{sub 2}O{sub 2} in excess is required for the presence of a low-affinity high-capacity component for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in SHR PTE cells only. {yields} It is suggested that Na{sup +} binding in renal ASCT2 may be regulated by ROS in SHR PTE cells. -- Abstract: The presence of high and low sodium affinity states for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538-R547). This study evaluated the role of H{sub 2}O{sub 2} on the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na{sup +} dependence of [{sup 14}C]-L-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na{sup +} removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H{sub 2}O{sub 2} levels in the extracellular medium significantly reduced Na{sup +}-K{sub m} and V{sub max} values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake. After removal of apocynin from the culture medium, H{sub 2}O{sub 2} levels returned to basal values within 1 to 3 h in both WKY and SHR PTE cells and these were found stable for the next 24 h. Under these experimental conditions, the Na{sup +}-K{sub m} and V{sub max} of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1 day but not 4 days after apocynin removal. In conclusion, H{sub 2}O{sub 2} in excess is required for the presence of a low-affinity high-capacity component for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in SHR PTE cells only

  2. Free energy profiles for Na{sup +} and Cl{sup -} adsorption onto water/NaCl crystal interfaces evaluated by molecular dynamics simulation

    Energy Technology Data Exchange (ETDEWEB)

    Shinto, H.; Sakakibara, T.; Higashitani, K. [Kyoto Univ. (Japan)

    1998-10-01

    The potential of mean force (PMF) of solute Na{sup plus} and Cl{sup minus} ions approaching water/NaCl(001) and water/NaCl(011) interfaces is calculated by using classical molecular dynamics (MD) simulations. It is found that (1) Na{sup plus} and Cl{sup minus} ions adsorb on the crystal surface either directly (direct adsorption) or with a water molecule interposed between the surface and themselves (solvent-separated adsorption), (2) both ions are adsorbed more stably on the NaCl surfaces under solvent-separated conditions in most cases, and they adsorb on NaCl(001) more easily than on NaCl(011) in the case of direct adsorption, and (3) Na{sup plus} adsorbs on the NaCl surfaces more easily than Cl{sup minus} in the case of direct adsorption, but direct adsorption of Cl{sup minus} on NaCl(011) is impossible. These results indicate that the solute-surface and solvent-surface interactions are strongly affected by the lattice arrangement of the surface such that the PMF profiles largely depend not only on the size of solute ions but also on the lattice arrangement. 28 refs., 13 figs., 3 tabs.

  3. Assignment of the human Na[sup +]/glucose cotransporter gene SGLT1 to chromosome 22q13. 1

    Energy Technology Data Exchange (ETDEWEB)

    Turk, E.; Klisak, I.; Bacallao, R.; Sparkes, R.S.; Wright, E.M. (UCLA School Medicine, Los Angeles, CA (United States))

    1993-09-01

    The Na[sup +]/glucose cotransporter gene SGLT1 encodes the primary carrier protein responsible for the uptake of the dietary sugars glucose and galactose from the intestinal lumen. SGLT1 transport activity is currently exploited in oral rehydration therapy. The 75-kDa glycoprotein is localized in the brush border of the intestinal epithelium and is predicted to comprise 12 membrane spans. In two patients with the autosomal recessive disease glucose/galactose malabsorption, the underlying cause was found to be a missense mutation in SGLT1, and the Asp28 [yields] Asn change was demonstrated in vitro to eliminate SGLT1 transport activity. The SGLT1 gene was previously shown to reside on the distal q arm of chromosome 22(11.2 [yields] qter). The authors have used a cosmid probe for fluorescence in situ hybridization, which refines the localization to 22q13.1, and provide an example of the utility of the SGLT1 probe as a diagnostic for genetic diseases associated with trans-locations of chromosome 22. 18 refs., 2 figs.

  4. Evidence for dual cyclooxygenases in mouse and rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Keeting, P.E.; Zweig, A.; Lysz, T.W.

    1986-05-01

    The existence of dual forms of cyclooxygenase (CO) in the whole brain of rat and mouse was investigated. Using microsomes prepared from tissue homogenized in 10 mM EDTA and 1% BSA, they assayed for prostaglandin (PG) in a medium containing 1-(/sup 14/C)-arachidonic acid (AA: 1 ..mu..g; 300,000 cpm) 1.2 mM epinephrine, and 1 mM glutathione. The mouse microsomal PGE/sub 2/ synthesis rose rapidly and plateaued within 5 minutes while PGF/sub 2..cap alpha../ levels continued to rise through the 60 minute incubation. Evidence for the existence of two forms of the CO in the mouse brain came from the observations that (1) 0.4 ..mu..M indomethacin inhibited PGE/sub 2/ production) by 80% while PGF/sub 2..cap alpha../ synthesis decreased only 20% and (2) a 3 minute preincubation of the mouse microsomes with unlabelled AA (1 ..mu.. g) eliminated PGE/sub 2/ synthesis while PGF/sub 2..cap alpha../ synthesis continued. Similar results were obtained with rat brain microsomes. Rat kidney microsomal preparations appear not to have the two CO forms. From these observations, it is concluded that there are PGE/sub 2/ and PGF/sub 2..cap alpha../ associated CO in mouse and rat brain microsomal preparations. The PGF/sub 2..cap alpha../ associated CO is somewhat resistant to arachidonate induced destruction while the PGE/sub 2/ associated CO undergoes autodestruction readily.

  5. Acridine orange-mediated photodamage of microsomal- and lysosomal fractions.

    Science.gov (United States)

    Olsson, G M; Brunmark, A; Brunk, U T

    1989-01-01

    Irradiation of microsomes with visible light in the presence of externally-added acridine orange results in O2 uptake, malondialdehyde accumulation, and inactivation of the microsomal drug-metabolizing system. The latter effect is reflected by a decrease in NADPH-cytochrome P450- and NADH-cytochrome b5 reductase activities and cytochromes P450 and b5 content by 88-, 85-, 60-, and 34%, respectively, after 5-min irradiation. Anoxia prevented inactivation of both reductases by 70-90%, whereas it prevented completely cytochrome b5 destruction. The presence of reducing equivalents, at the expense of NADPH and NADH, exert a partial protection (40-54% residual activities) against photosensitization damage on both reductase activities, whereas it almost fully protected cytochrome b5. Photosensitization of lipid peroxidation, as well as inactivation of the microsomal drug-metabolizing system, appears to involve both a type I and type II process. Products of lipid peroxidation might also play a role in enzyme inactivation and cytochrome destruction, as suggested by kinetic and time course studies and the redox state of microsomes. The uptake of acridine orange by isolated lysosomes is linearly dependent on the concentration of added dye and the distribution between extra- and intralysosomal acridine orange is strongly dependent on the amount of lysosomes. Irradiation of acridine orange-loaded lysosomes (light intensity at the sample position approximately 320 mW/cm2) produces an impairment of the membrane which leads to a rapid release of enzyme (N-acetyl-beta-glucosaminidase activity) into the medium, accompanied by a loss of activity in the lysosome-containing pellet and a partial photodamage of the enzyme. Concomitantly, thiobarbituric acid-reactive material accumulation increases in the reaction mixture with increasing irradiation time. When light intensity at the position was reduced to approximately 3.6 mW/cm2, photodamage of lysosomes was of a lesser magnitude

  6. Coordinated role of voltage-gated sodium channels and the Na{sup +}/H{sup +} exchanger in sustaining microglial activation during inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Muhammad M. [Department of Environmental and Occupational Medicine and Environmental and Occupational Health Sciences Institute, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Sonsalla, Patricia K. [Department of Neurology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine and Environmental and Occupational Health Sciences Institute, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

    2013-12-01

    Persistent neuroinflammation and microglial activation play an integral role in the pathogenesis of many neurological disorders. We investigated the role of voltage-gated sodium channels (VGSC) and Na{sup +}/H{sup +} exchangers (NHE) in the activation of immortalized microglial cells (BV-2) after lipopolysaccharide (LPS) exposure. LPS (10 and 100 ng/ml) caused a dose- and time-dependent accumulation of intracellular sodium [(Na{sup +}){sub i}] in BV-2 cells. Pre-treatment of cells with the VGSC antagonist tetrodotoxin (TTX, 1 μM) abolished short-term Na{sup +} influx, but was unable to prevent the accumulation of (Na{sup +}){sub i} observed at 6 and 24 h after LPS exposure. The NHE inhibitor cariporide (1 μM) significantly reduced accumulation of (Na{sup +}){sub i} 6 and 24 h after LPS exposure. Furthermore, LPS increased the mRNA expression and protein level of NHE-1 in a dose- and time-dependent manner, which was significantly reduced after co-treatment with TTX and/or cariporide. LPS increased production of TNF-α, ROS, and H{sub 2}O{sub 2} and expression of gp91{sup phox}, an active subunit of NADPH oxidase, in a dose- and time-dependent manner, which was significantly reduced by TTX or TTX + cariporide. Collectively, these data demonstrate a closely-linked temporal relationship between VGSC and NHE-1 in regulating function in activated microglia, which may provide avenues for therapeutic interventions aimed at reducing neuroinflammation. - Highlights: • LPS causes immediate increase in sodium through VGSC and subsequently through the NHE-1. • Inhibition of VGSC reduces increases in NHE-1 and gp91{sup phox}. • Inhibition of VGSC and NHE-1 reduces NADPH oxidase-mediated Tnf-α, ROS, and H{sub 2}O{sub 2} production. • NHE-1 and Na{sub v}1.6 may be viable targets for therapeutic interventions to reduce neuroinflammation in neurodegenerative disease.

  7. Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques

    NARCIS (Netherlands)

    Moonen, H.; Bosch, H. van den

    1979-01-01

    1. 1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate

  8. Solubilisation, purification and reconstitution of hepatic microsomal azoreductase activity.

    Science.gov (United States)

    Mallett, A K; King, L J; Walker, R

    1985-02-01

    Microsomal NADPH-cytochrome c (P-450) reductase and cytochrome P-450 were purified from the livers of phenobarbitone-treated rats. Purified NADPH-cytochrome c (P-450) reductase effected the NADPH-dependent reduction of FMN and FAD under anaerobic conditions in a non-enzymic manner, but was unable to reduce directly the azo dye, amaranth. In the presence of FMN, the purified reductase effected reduction of amaranth through the production of reduced FMN. Incorporation of NADPH-cytochrome c (P-450) reductase into the microsomal fraction increased the azoreductase activity of liver preparations from phenobarbitone-treated rats, but had no effect on azoreductase activity in preparations from control animals. Azoreductase activity was reconstituted into dilauroyl phosphatidylcholine vesicles containing purified cytochrome P-450 and purified NADPH-cytochrome c (P-450) reductase. In the absence of supplementary FMN, amaranth reduction was completely dependent upon all three components, but in the presence of FMN, the omission of any one component failed to abolish completely azoreductase activity.

  9. Inositol trisphosphate and thapsigargin discriminate endoplasmic reticulum stores of calcium in rat brain

    DEFF Research Database (Denmark)

    Verma, A; Hirsch, D J; Hanley, M R

    1990-01-01

    ATP dependent Ca2+ accumulation into oxalate-loaded rat brain microsomes is potently inhibited by thapsigargin with an IC50 of 2 nM and maximal inhibition at 10 nM. Approximately 15% of the total A23187-releasable microsomal calcium store is insensitive to thapsigargin concentrations up to 100...... microM. Inositol-1,4,5-trisphosphate (IP3) maximally inhibits 40% of the net Ca2+ accumulation by whole brain microsomes. Its effects are non-additive with thapsigargin suggesting that the IP3-sensitive Ca2+ pool is a subset of the thapsigargin sensitive Ca2+ pool. Marked regional differences occur...

  10. Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions.

    Science.gov (United States)

    Svobodová, Kateřina; Mikesková, Hana; Petráčková, Denisa

    2013-12-01

    Microsomal fraction of fungal cells grabs the attention of many researchers for it contains enzymes that play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were shown to metabolize a wide range of xenobiotic compounds, including PAHs, PCBs, dioxins, and endocrine disruptors, and take part in other fungal biotransformation reactions. However, little is known about the nature and regulation of these membrane-associated reactions. Advanced proteomic and post-genomic techniques make it possible to identify larger numbers of microsomal proteins and thus add to a deeper study of fungal intracellular processes. In this work, proteins that were identified through a shotgun proteomic approach in fungal microsomes under various culture conditions are reviewed. However, further research is still needed to fully understand the role of microsomes in fungal biodegradation and biotransformation reactions.

  11. Toxico-kinetics, recovery efficiency and microsomal changes following administration of deltamethrin to black Bengal goats.

    Science.gov (United States)

    Juliet, S; Chakraborty, A K; Koley, K M; Mandal, T K; Bhattacharyya, A

    2001-03-01

    A study of the toxico-kinetics, recovery percentage from different substrates, cytotoxicity and role of cytochrome P450 and b5 of liver microsome in the metabolism of deltamethrin were carried out in female black Bengal goat. The ALD50 value of deltamethrin in goat by intravenous route lies between 0.2 and 0.6 mg kg-1. Intravenous disposition kinetics using a dose of 0.2 mg kg-1 showed that the maximum blood concentration of deltamethrin was recorded at 0.5 min, followed by rapid decline, and a minimum concentration was detected at 6 min after administration. The following values were obtained: Vdarea 0.148 (+/- 0.02) litre kg-1; t1/2 (alpha) 0.22 (+/- 0.02) min; t1/2 (beta) 2.17 (+/- 0.37) min; Kel 1.05 (+/- 0.24) min-1; AUC 4.30 (+/- 0.45) micrograms min ml-1; ClB 0.05 (+/- 0.006) litre kg-1 min-1; T-B 1.93 (+/- 0.58); fc 0.40 (+/- 0.05). After 10 min, liver retained the maximum residue, and heart, adrenal gland, kidney, spleen, fat and brain also held the insecticide; liver, fat, heart and spleen retained residue after 30 min, and bone, liver and fat retained residue after 60 min of intravenous administration. Oral absorption of deltamethrin was poor and inconsistent, and approximately 65% of administered dose was recovered from faeces and gastrointestinal contents. The excretion of deltamethrin through urine was meagre, and only 0.01 and 0.013% of the administered dose was recovered after 3 and 5 days of oral administration respectively. All the tissues retained the residue after 3 days; while fat, rumen, reticulum, omasum, abomasum, large and small intestine and bone retained the residue after 5 days of oral administration; and the percentage recoveries were 1.73 and 0.027 respectively. Deltamethrin reduced the level of cytochrome P450 content of liver microsomal pellet of goat after 5 days of oral administration. Histopathological examination of liver, kidney, heart, spleen brain and lung sections of treated goats did not reveal any pathological changes.

  12. Transesterification of a series of 12 parabens by liver and small-intestinal microsomes of rats and humans.

    Science.gov (United States)

    Fujino, Chieri; Watanabe, Yoko; Uramaru, Naoto; Kitamura, Shigeyuki

    2014-02-01

    Hydrolytic transformation of parabens (4-hydroxybenzoic acid esters; used as antibacterial agents) to 4-hydroxybenzoic acid and alcohols by tissue microsomes is well-known both in vitro and in vivo. Here, we investigated transesterification reactions of parabens catalyzed by rat and human microsomes, using a series of 12 parabens with C1-C12 alcohol side chains. Transesterification of parabens by rat liver and small-intestinal microsomes occurred in the presence of alcohols in the microsomal incubation mixture. Among the 12 parabens, propylparaben was most effectively transesterified by rat liver microsomes with methanol or ethanol, followed by butylparaben. Relatively low activity was observed with longer-side-chain parabens. In contrast, small-intestinal microsomes exhibited higher activity towards moderately long side-chain parabens, and showed the highest activity toward octylparaben. When parabens were incubated with liver or small-intestinal microsomes in the presence of C1-C12 alcohols, ethanol and decanol were most effectively transferred to parabens by rat liver microsomes and small-intestinal microsomes, respectively. Human liver and small-intestinal microsomes also exhibited significant transesterification activities with different substrate specificities, like rat microsomes. Carboxylesterase isoforms, CES1b and CES1c, and CES2, exhibited significant transesterification activity toward parabens, and showed similar substrate specificity to human liver and small-intestinal microsomes, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Age-dependent changes in diastolic Ca{sup 2+} and Na{sup +} concentrations in dystrophic cardiomyopathy: Role of Ca{sup 2+} entry and IP{sub 3}

    Energy Technology Data Exchange (ETDEWEB)

    Mijares, Alfredo [Instituto Venezolano de Investigaciones Científicas, Centro de Biofísica y Bioquímica, Caracas (Venezuela, Bolivarian Republic of); Altamirano, Francisco [Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616 (United States); Kolster, Juan [Centro de Investigaciones Biomédicas, México D.F. (Mexico); Adams, José A. [Division of Neonatology, Mount Sinai Medical Center, Miami, FL 33140 (United States); López, José R., E-mail: jrlopez@ucdavis.edu [Instituto Venezolano de Investigaciones Científicas, Centro de Biofísica y Bioquímica, Caracas (Venezuela, Bolivarian Republic of); Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616 (United States)

    2014-10-03

    Highlights: • Age-dependent increase in [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} in mdx cardiomyocytes. • Gadolinium significantly reduced both [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} at all ages. • IP{sub 3}-pathway inhibition reduced cations concentrations in dystrophic cardiomyocytes. - Abstract: Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca{sup 2+} concentration ([Ca{sup 2+}]{sub d}) and diastolic Na{sup +} concentration ([Na{sup +}]{sub d}) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd{sup 3+})-sensitive Ca{sup 2+} entry and inositol triphosphate (IP{sub 3}) signaling pathways in abnormal [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} were investigated. Our results showed an age-dependent increase in both [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd{sup 3+} treatment significantly reduced both [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} at all ages. In addition, blockade of the IP{sub 3}-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd{sup 3+} normalized both [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca{sup 2+} and Na{sup +} overload mediated at least in part by enhanced Ca{sup 2+} entry through Gd{sup 3+} sensitive transient receptor potential channels (TRPC), and by IP{sub 3} receptors.

  14. The Na{sup +}/K{sup +} -pump in rat peritoneal mast cells: Some aspects of regulatio of activity and cellular fusion

    Energy Technology Data Exchange (ETDEWEB)

    Knudsen, T. [Odense Univ., Dept. of Pharmacology, Inst. of Medical Biology, The Faculty of Health Scineces (Denmark)

    1995-12-31

    The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also also have important clinical implications. The aim of the present work has been to document the existence of the Na{sup +}/K{sup +}-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na{sup +}/K{sup +}-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with {sup 86}Rb{sup +} as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE-directed stimulation of the cell was used as a parameter of cellular degranulation. Histamine was measured by spectrofluorometry. Besides describing aspects of the function and regulation of the Na{sup +}/K{sup +}-pump in the rat peritoneal mast cell, this thesis points to the potential role of sodium transport mechanisms in mast cell physiology. Pharmacological manipulations of such transport mechanisms might in the future add to the treatment of allergic diseases. (au) 253 refs.

  15. In Vitro Glucuronidation of Ochratoxin A by Rat Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Zheng Han

    2013-12-01

    Full Text Available Ochratoxin A (OTA, one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS, UHPLC-Orbitrap-high resolution mass spectrometry (HRMS and liquid chromatography-multiple stage mass spectrometry (LC-MSn were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with β-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.

  16. Development of in silico models for human liver microsomal stability

    Science.gov (United States)

    Lee, Pil H.; Cucurull-Sanchez, Lourdes; Lu, Jing; Du, Yuhua J.

    2007-12-01

    We developed highly predictive classification models for human liver microsomal (HLM) stability using the apparent intrinsic clearance (CLint, app) as the end point. HLM stability has been shown to be an important factor related to the metabolic clearance of a compound. Robust in silico models that predict metabolic clearance are very useful in early drug discovery stages to optimize the compound structure and to select promising leads to avoid costly drug development failures in later stages. Using Random Forest and Bayesian classification methods with MOE, E-state descriptors, ADME Keys, and ECFP_6 fingerprints, various highly predictive models were developed. The best performance of the models shows 80 and 75% prediction accuracy for the test and validation sets, respectively. A detailed analysis of results will be shown, including an assessment of the prediction confidence, the significant descriptors, and the application of these models to drug discovery projects.

  17. Coupled motions direct electrons along human microsomal P450 Chains.

    Directory of Open Access Journals (Sweden)

    Christopher R Pudney

    2011-12-01

    Full Text Available Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR to all microsomal cytochrome P450s (CYPs. Our hypothesis is that tight coupling of motion with enzyme chemistry can signal "ready and waiting" states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals "ready and waiting" conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron

  18. Preparation and Characterization of Rodent Intestinal Microsomes: Comparative Assessment of Two Methods

    Science.gov (United States)

    Damre, Anagha; Mallurwar, S. R.; Behera, D.

    2009-01-01

    Small intestine plays an important role in the first-pass metabolism of orally ingested xenobiotics as a result of expression of both Phase I and Phase II metabolic enzymes, together with associated transporters. Intestinal microsomes thus can be used to study susceptibility of compounds to metabolism in vitro. The present study was undertaken to have a comparative assessment between different methods of preparation of rodent intestinal microsomes. Mouse and rat intestinal microsomes were prepared by two methods, in method A intestines were homogenized, while in method B mucosal cells were scrapped followed by homogenization. Further, microsomes were prepared by centrifugation (10000xg) followed by ultra centrifugation (100000×g) of the homogenates. The prepared microsomes were characterized for protein concentration using Bradford's method and CYP450 content using carbon monoxide bubbling method. The protein concentration and CYP450 content in microsomes prepared by method B was significantly higher than method A. In conclusion, superior quality intestinal microsomes can be obtained from rodents by using scrapped intestinal mucosal cells as compared to the intestinal homogenates. PMID:20177465

  19. Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans.

    Science.gov (United States)

    Carlile, D J; Hakooz, N; Bayliss, M K; Houston, J B

    1999-06-01

    To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CLint ) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. V max, Km and CLint (defined as V max/Km ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Microsomal CLint values differed by over two orders of magnitude, with the means ranging from 0.18 (phenytoin) to 40.70 (diclofenac) microl min-1 mg-1 microsomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CLint tended to be underpredicted. While the in vivo unbound Km for phenytoin, 1-5 micron is substantially lower than the value determined in microsomes based on total concentrations (56 micron), correction for the in vitro binding reduces this value to 20 micron and 6 micron in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide Km. An appreciation of the utility of in vitro prediction can be best achieved when the range of CLint values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CLint values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent.

  20. Parathyroid hormone inhibition of Na{sup +}/H{sup +} exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

    Energy Technology Data Exchange (ETDEWEB)

    Neri, Elida Adalgisa; Bezerra, Camila Nogueira Alves, E-mail: camilab@icb.usp.br; Queiroz-Leite, Gabriella Duarte; Polidoro, Juliano Zequini; Rebouças, Nancy Amaral

    2015-06-12

    The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na{sup +}/H{sup +} exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels. - Highlights: • PTH regulation of Nhe3 promoter depends on EGR1 binding. • EGR1, PKA and JAK/STAT are involved in PTH inhibition of the Nhe3 promoter. • PTH alters expression of EGR1 and Sp3. • PTH inhibits the Nhe3 promoter by regulating PKA and JAK/STAT signaling.

  1. Effect of ethanol on CHCl3 metabolism in hepatic microsomes from Osborne-Mendel rats.

    OpenAIRE

    Testai, E.; Gemma, S; Gervasi, P; Menicagli, S; Vittozzi, L

    1994-01-01

    The treatment of Osborne-Mendel rats with ethanol in drinking water for 2 weeks resulted in a 3-fold increase of hepatic microsomal hydroxylation of both p-nitrophenol and aniline, two substrates considered highly selective for P4502E1. No other forms of P450 seemed to be affected. These results, confirmed by the immunoblot analysis of microsomal protein, showed an induction of P4502E1. The levels of total covalent binding to microsomal phospholipid due to 14CHCl3 reactive intermediates in et...

  2. Modification of radiation-induced oxidative damage in liposomal and microsomal membrane by eugenol

    Energy Technology Data Exchange (ETDEWEB)

    Pandey, B.N. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Lathika, K.M. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Mishra, K.P. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India)]. E-mail: kpm@magnum.barc.ernet.in

    2006-03-15

    Radiation-induced membrane oxidative damage, and their modification by eugenol, a natural antioxidant, was investigated in liposomes and microsomes. Liposomes prepared with DPH showed decrease in fluorescence after {gamma}-irradiation, which was prevented significantly by eugenol and correlated with magnitude of oxidation of phospholipids. Presence of eugenol resulted in substantial inhibition in MDA formation in irradiated liposomes/microsomes, which was less effective when added after irradiation. Similarly, the increase in phospholipase C activity observed after irradiation in microsomes was inhibited in samples pre-treated with eugenol. Results suggest association of radio- oxidative membrane damage with alterations in signaling molecules, and eugenol significantly prevented these membrane damaging events.

  3. Modification of radiation-induced oxidative damage in liposomal and microsomal membrane by eugenol

    Science.gov (United States)

    Pandey, B. N.; Lathika, K. M.; Mishra, K. P.

    2006-03-01

    Radiation-induced membrane oxidative damage, and their modification by eugenol, a natural antioxidant, was investigated in liposomes and microsomes. Liposomes prepared with DPH showed decrease in fluorescence after γ-irradiation, which was prevented significantly by eugenol and correlated with magnitude of oxidation of phospholipids. Presence of eugenol resulted in substantial inhibition in MDA formation in irradiated liposomes/microsomes, which was less effective when added after irradiation. Similarly, the increase in phospholipase C activity observed after irradiation in microsomes was inhibited in samples pre-treated with eugenol. Results suggest association of radio- oxidative membrane damage with alterations in signaling molecules, and eugenol significantly prevented these membrane damaging events.

  4. Effects of lisuride on body temperature of rats and rabbits: relation to microsomal biotransformation and dopaminergic receptor stimulation.

    Science.gov (United States)

    Carruba, M O; Ricciardi, S; Negreanu, J; Calogero, M; Mantegazza, P

    1980-01-01

    In rats, lisuride, either administered systemically or intracerebroventricularly induced a dose-related hypothermia. This effect was selectively antagonized by blockade of DA receptors in the CNS but not by inhibition of catecholamine synthesis or blockade of serotoninergic receptors. Also a blocker of "peripheral" DA receptors failed to antagonize the hypothermic effect of lisuride in rats. Induction of rat liver microsomal drug-metabolizing enzymes by phenobarbital counteracted lisuride-induced hypothermia. In rabbits lisuride induced a hyperthermic response which was sensitive to both pimozide and metergoline pretreatment. These findings indicate that stimulation of brain DA receptors involved in thermoregulation is responsible for the changes in body temperature indiced by lisuride in rats and rabbits and that these effects are caused by the drug itself and do not require previous biotransformation into an active metabolite.

  5. Phospholipase A2 in rat-lung microsomes: Substrate specificity towards endogenous phosphatidylcholines

    NARCIS (Netherlands)

    Longmore, W.J.; Oldenburg, V.; Golde, L.M.G. van

    1979-01-01

    1. 1. Isolated rat lungs were perfused with a variety of radioactive precursors to label the phosphatidylcholines of the microsomal and lamellar body fractions. These endogenously labelled phosphatidylcholines were used as substrates in experiments to identify and characterize phospholipase A

  6. Enantioselective metabolism of hydroxychloroquine employing rats and mice hepatic microsomes

    Directory of Open Access Journals (Sweden)

    Carmem Dickow Cardoso

    2009-12-01

    Full Text Available Hydroxychloroquine (HCQ is an important chiral drug used, mainly, in the treatment of rheumatoid arthritis, systemic lupus erythematosus and malaria, and whose pharmacokinetic and pharmacodynamic properties look to be stereoselective. Respecting the pharmacokinetic properties, some previous studies indicate that the stereoselectivity could express itself in the processes of metabolism, distribution and excretion and that the stereoselective metabolism looks to be a function of the studied species. So, the in vitro metabolism of HCQ was investigated using hepatic microsomes of rats and mice. The microsomal fraction of livers of Wistar rats and Balb-C mice was separated by ultracentrifugation and 500 μL were incubated for 180 minutes with 10 μL of racemic HCQ 1000 μg mL-1. Two stereospecific analytical methods, high performance liquid chromatography (HPLC and capillary electrophoresis (CE, were used to separate and quantify the formed metabolites. It was verified that the main formed metabolite is the (--(R-desethyl hydroxychloroquine for both animal species.A hidroxicloroquina (HCQ é um importante fármaco quiral usado, principalmente, no tratamento de artrite reumatóide, lupus eritematoso sistêmico e malária e cujas propriedades farmacocinéticas e farmacodinâmicas parecem ser estereosseletivas. Em relação às propriedades farmacocinéticas, alguns estudos prévios indicam que a estereosseletividade pode se expressar nos processos de metabolismo, distribuição e excreção e que o metabolismo estereosseletivo parece ser função da espécie estudada. Sendo assim, o metabolismo in vitro da HCQ foi investigado usando microssomas de fígado de ratos e de camundongos. A fração microssômica de fígados de ratos Wistar e de camundongos Balb-C foi isolada por ultracentrifugação e 500 μL foram incubados por 180 minutos com 10 μL de HCQ racêmica 1000 μg mL-1. Dois métodos analíticos estereoespecíficos, por cromatografia líquida de

  7. Effect of ethanol on CHCl3 metabolism in hepatic microsomes from Osborne-Mendel rats.

    Science.gov (United States)

    Testai, E; Gemma, S; Gervasi, P; Menicagli, S; Vittozzi, L

    1994-11-01

    The treatment of Osborne-Mendel rats with ethanol in drinking water for 2 weeks resulted in a 3-fold increase of hepatic microsomal hydroxylation of both p-nitrophenol and aniline, two substrates considered highly selective for P4502E1. No other forms of P450 seemed to be affected. These results, confirmed by the immunoblot analysis of microsomal protein, showed an induction of P4502E1. The levels of total covalent binding to microsomal phospholipid due to 14CHCl3 reactive intermediates in ethanol-pretreated microsomes were identical to those measured in microsomes from untreated rats at any pO2. The distribution of radioactivity obtained after transmethylation of the adducts of 14CHCl3 intermediates with microsomal phospholipids (PL) indicated that binding to fatty acyl chains (due to .CHCl2 radicals) increased with decreasing pO2. On the contrary, the binding to polar heads due to phosgene decreased. The ethanol treatment did not affect binding to either PL moieties. These results indicated that, in our experimental conditions, the in vitro production of both oxidative and reductive intermediates of CHCl3 in the liver of Osborne-Mendel rats were not influenced by ethanol consumption.

  8. Assessment of in silico models for fraction of unbound drug in human liver microsomes.

    Science.gov (United States)

    Gao, Hua; Steyn, Stefanus J; Chang, George; Lin, Jing

    2010-05-01

    Fraction of unbound drug in human liver microsome (fu(mic)) incubation media is an important parameter for accurate assessment of hepatic intrinsic clearance and drug-drug interactions. In recent years, there have been considerable advances in understanding structure-microsomal binding relationships. This review highlights the in silico modeling techniques for fu(mic) including physicochemical properties-based modeling, pharmacophore feature-based classification modeling and more complex statistical method-based modeling. The application of these modeling techniques to the understanding of the structure-binding relationships is also discussed. The reader will gain an understanding of the modeling techniques used for prediction of binding to human liver microsomes (fu(mic)). The reader will also understand the molecular structure-microsomal protein binding relationships. In all of these models, lipophilicity is the most important molecular property underlying the structure-binding relationship. Other molecular properties such as charge type (positive vs negative) and hydrogen bonding are also important factors for microsomal binding. The predictive accuracy of fu(mic) models in the high lipophilicity and tight microsomal binding ranges still needs to be further improved. However, in silico models are still valuable tools to aid chemical library design and prioritize multiple chemical series, which could provide efficiency and decrease knowledge cycle times in drug discovery.

  9. Saponin permeabilization of rough microsomes from rat liver reveals a novel prothrombin pool.

    Science.gov (United States)

    Tollefsen, S; Wierød, L; Skotte, A; Rob, J A; Helgeland, L

    2001-06-15

    Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following (14)C-gamma-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.

  10. Alteration of rat liver microsomal monooxygenase activities by gasoline treatment

    Energy Technology Data Exchange (ETDEWEB)

    Brady, J.F.; Xiao Fang; Gapac, J.M.; Ning, S.M.; Yang, C.S. (Rutgers - the State Univ., Piscataway, NJ (USA). Dept. of Chemical Biology and Pharmacognosy)

    1990-11-01

    Previous work has shown an increase in rat liver enzyme activities after chronic exposure to gasoline vapor. In the present study, male Sprague-Dawley rats were pretreated with unleaded gasoline at 1 and 5 ml/kg, i.p., and selected hepatic microsomal monooxygenase activities were determined at 18, 48, and 72 h. At 18 h, moderate increases were observed in P450 content (1.3-fold), cytochrome c-reductase activity (1.25-fold), and in N-nitrosodimethylamine demethylation rate (1.25- to 1.6-fold). Pentoxyresorufin dealkylase activity (an activity displayed primarily by P450IIB1) was significantly elevated at 18 and 48 h (30- to 60-fold), and ethoxyresorufin dealkylase activity (an activity displayed by P450 IA1) was elevated (2- to 4-fold). Immunoblot analysis revealed no change in P450IIE1 at these time points, but an elevation in P450IIB1 in agreement with the pentoxyresorufin dealkylase activity measurements. (orig.).

  11. Stereoselective degradation of tebuconazole in rat liver microsomes.

    Science.gov (United States)

    Shen, Zhigang; Zhu, Wentao; Liu, Donghui; Xu, Xinyuan; Zhang, Ping; Zhou, Zhiqiang

    2012-01-01

    The aim of this study was to assess the stereoselectivity of two tebuconazole [(RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol] enantiomers in in vitro system (rat liver microsomes). The analytes were extracted with acetic ether and concentrations were determined by high performance liquid chromatography (HPLC) with a cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase. The degradation of rac-tebuconazole (15 μM) followed first-order kinetics, and the degradation of the S-tebuconazole (t(1/2) = 22.31 min) was faster than that of the R-tebuconazole (t(1/2) = 48.76 min), but no significant difference between the enantiomers was found in the respective incubation (7.5 μM for each). Kinetic assays showed that the K(m) was different between the two enantiomers (K(mR) = 14.83 ± 2.19, K(mS) = 12.23 ± 2.72). The interaction results revealed that there was competitive inhibition between S- and R-form, and there was a significant difference between the IC(50) of R- to S-tebuconazole and S- to R-tebuconazole (IC(50R/S)/IC(50S/R) = 4.98). Copyright © 2011 Wiley Periodicals, Inc.

  12. Duration of hexobarbital-induced sleep and monoamine oxidase activities in rat brain: Focus on the behavioral activity and on the free-radical oxidation.

    Science.gov (United States)

    Tseilikman, Vadim E; Kozochkin, Denis A; Manukhina, Eugenia B; Downey, H Fred; Tseilikman, Olga B; Misharina, Maria E; Nikitina, Anna A; Komelkova, Maria V; Lapshin, Maxim S; Kondashevskaya, Marina V; Lazuko, Svetlana S; Kusina, Oxana V; Sahabutdinov, Marat V

    2016-04-01

    The present study is focused on the relationship between monoamine oxidase (MAO) activity and hepatic content of cytochrome P450 (CYP), which reflects the status of microsomal oxidation. For vital integrative evaluation of hepatic microsomal oxidation in rats, the hexobarbital sleep test was used, and content of CYP was measured in hepatic microsomes. Rats with short hexobarbital sleep time (SHST) had higher content of microsomal CYP than rats with long hexobarbital sleep time (LHST). Whole brain MAO-A and MAO-B activities, serotonin and carbonylated protein levels were higher in SHST than in LHST rats. MAO-A and MAO-B activities were higher in brain cortex of SHST rats; MAO-A activity was higher only in hypothalamus and medulla of LHST. The same brain regions of LHST rats had higher concentrations of carbonylated proteins and lipid peroxidation products than in SHST rats. MAO activity was correlated with microsomal oxidation phenotype. Rats with higher hepatic content of CYP had higher activities of MAO-A and MAO-B in the brain and higher plasma serotonin levels than rats with lower microsomal oxidation. In conclusion, data obtained in this study showed a correlation between MAO activity and microsomal oxidation phenotype.

  13. Inhibition of microsomal oxidation of alcohols and of hydroxyl-radical-scavenging agents by the iron-chelating agent desferrioxamine.

    OpenAIRE

    Cederbaum, A I; Dicker, E

    1983-01-01

    Rat liver microsomes (microsomal fractions) catalyse the oxidation of straight-chain aliphatic alcohols and of hydroxyl-radical-scavenging agents during NADPH-dependent electron transfer. The iron-chelating agent desferrioxamine, which blocks the generation of hydroxyl radicals in other systems, was found to inhibit the following microsomal reactions: production of formaldehyde from either dimethyl sulphoxide or 2-methylpropan-2-ol (t-butylalcohol); generation of ethylene from 4-oxothiomethyl...

  14. Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane.

    Science.gov (United States)

    Canova-Davis, E; Chiang, J Y; Waskell, L

    1985-06-01

    Cytochrome b5 has recently been shown to be required in the reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane [E. Canova-Davis and L. A. Waskell, J. biol. Chem. 259, 2541 (1984)]. To determine whether this observation in the reconstituted system was merely dependent on the particular ratios of the various components or some other fortuitous, unknown factor, or whether cytochrome b5 plays a role in the liver microsomal metabolism of methoxyflurane, the following studies were undertaken. Antibody to rabbit holocytochrome b5 was raised in guinea pigs. The antibody to cytochrome b5 was able to inhibit 75% of the microsomal metabolism of methoxyflurane. This same antibody also inhibited methoxyflurane metabolism in the reconstituted system. When the antibody to cytochrome b5 was treated with purified cytochrome b5 before addition to the microsomes, it did not inhibit methoxyflurane metabolism. Furthermore, the antibody to cytochrome b5 did not inhibit the microsomal metabolism of benzphetamine. This suggests that cytochrome b5 was required for the microsomal metabolism of methoxyflurane. It is possible that cytochrome b5 functioned in the metabolism of methoxyflurane by retaining a specific conformation of cytochrome P-450 and not by transferring the second electron to cytochrome P-450. To explore this possibility, cytochrome b5 was reconstituted with Mn3+-protoporphyrin IX. The Mn3+-protoporphyrin IX derivative retained the conformation of cytochrome b5 but not its electron transfer properties. This manganese derivative of cytochrome b5 was unable to stimulate the metabolism of methoxyflurane. The study demonstrated that cytochrome b5 was obligatory for the microsomal metabolism of methoxyflurane, whereas it was not required for the microsomal N-demethylation of benzphetamine. Moreover, the heme moiety of cytochrome b5 functioned to transfer electrons in this reaction.

  15. Noninvasive assessment of microsomal enzyme activity in occupational medicine: present state of knowledge and future perspectives.

    Science.gov (United States)

    Døssing, M

    1984-01-01

    The activity of the hepatic microsomal enzyme system, which may be of great importance for metabolic activation and deactivation of hepatotoxic agents and carcinogens, is changed by exposure to commonly used industrial chemicals. The antipyrine test is the most widely used method for assessing microsomal enzyme activity in man. The clearance of antipyrine can be accurately calculated from one sample of saliva obtained about 24 h after an oral dose of the drug. By measuring antipyrine metabolism during exposure to industrial chemicals and at the end of 3-4 weeks free from exposure, the impact of industrial chemicals on antipyrine metabolism can be estimated, provided the chemicals are eliminated within 3-4 weeks. This test can be performed by skilled and unskilled workers using written instructions. This has broadened the application of the test. Other noninvasive indices of microsomal enzyme activity include the aminopyrine and caffeine breath tests and the urinary excretion of 6-beta-hydroxycortisol and D-glucaric acid. These tests probably reflect the activity of different but overlapping parts of the microsomal enzyme system and may be of value in research in occupational medicine. Previous studies indicate that chlorinated hydrocarbon insecticides, phenoxyacids, chlorophenols, polychlorinated biphenyles, some organic solvents and high concentrations of inhalation anaesthetics may stimulate microsomal enzyme activity, while styrene, toluene and inhalation anaesthetics in concentrations at about the allowed safety limit values have no effect. Lead, chemicals used by spray painters, and carbon disulphide probably inhibit the activity. While the short-term consequences of these changes include altered metabolism of hormones, vitamins, drugs, and other microsomally metabolized compounds, the possible impact on health on a long-term scale is unknown. It is now possible to study this with the use of the available noninvasive simple indices of microsomal enzyme

  16. Metabolism of diazepam and related benzodiazepines by human liver microsomes.

    Science.gov (United States)

    Hooper, W D; Watt, J A; McKinnon, G E; Reilly, P E

    1992-01-01

    The metabolism of diazepam has been studied in vitro using microsomal preparations from five human livers. An HPLC method was developed for the assay of diazepam, its congeners and its metabolites. Various methods for the incorporation of diazepam into the incubation medium were explored. It was shown that the use of organic solvents or small quantities of hydrochloric acid enhanced the solubility of this substrate. However all of the organic solvents tested were associated with substantial (around 50%) inhibition of metabolism of diazepam by both major pathways (N-demethylation and C3-hydroxylation). The use of hydrochloric acid gave satisfactory solubilization of diazepam, but not of pinazepam, prazepam or halazepam. Detailed metabolic studies were conducted only for diazepam, using neither hydrochloric acid nor organic solvents in the incubation medium. Formation of N-desmethyl-diazepam increased approximately linearly with diazepam concentration to 200 microM, and did not show saturation. Formation of temazepam gave a curved profile over the same range of diazepam concentrations, suggestive of a sigmoidal relationship. Michaelis-Menten parameters could not be determined for either reaction, but intrinsic clearances for N-demethylation varied over a 6-fold range. Diazepam N-demethylation was apparently promoted by the inclusion of temazepam in the incubation medium, while C3-hydroxylation of diazepam was enhanced in the presence of N-desmethyldiazepam. Mephenytoin in the incubation mixture had no effect on diazepam metabolism by either pathway. The present studies have defined some of the methodological problems inherent in in vitro metabolic studies with benzodiazepines, and have shed further light on the metabolism of diazepam in vitro by human liver.

  17. Overexpression of Catalase Enhances Benzo(a)pyrene Detoxification in Endothelial Microsomes.

    Science.gov (United States)

    Yang, Fang; Yang, Hong; Ramesh, Aramandla; Goodwin, J Shawn; Okoro, Emmanuel U; Guo, ZhongMao

    2016-01-01

    We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1μM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites, and

  18. A two-dimensional protein map of Pleurotus ostreatus microsomes-proteome dynamics.

    Science.gov (United States)

    Petráčková, Denisa; Halada, Petr; Bezoušková, Silvia; Křesinová, Zdena; Svobodová, Kateřina

    2016-01-01

    Recent studies documented that several processes in filamentous fungi are connected with microsomal enzyme activities. In this work, microsomal subproteomes of Pleurotus ostreatus were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. To assess proteome dynamics, microsomal proteins were isolated from fungal cultures after 7 and 12 days of cultivation. Additionally, 10 mg/L of 17α-ethinylestradiol (EE2) was treated with the cultures during 2 days. Despite the EE2 degradation by the fungus reached 97 and 76.3 % in 7- and 12-day-old cultures, respectively, only a minor effect on the composition of microsomal proteins was observed. The changes in protein maps related to ageing prevailed over those induced by EE2. Epoxide hydrolase, known to metabolize EE2, was detected in 12-day-old cultures only which suggests differences in EE2 degradation pathways utilized by fungal cultures of different age. The majority (32 %) of identified microsomal proteins were parts of mitochondrial energy metabolism.

  19. Molecular characterization of the microsomal tamoxifen binding site.

    Science.gov (United States)

    Kedjouar, Blandine; de Médina, Philippe; Oulad-Abdelghani, Mustapha; Payré, Bruno; Silvente-Poirot, Sandrine; Favre, Gilles; Faye, Jean-Charles; Poirot, Marc

    2004-08-06

    Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ER-independent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration- and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5alpha-cholest-8-en-3beta-ol for tamoxifen treatment and 5alpha-cholest-8-en-3beta-ol and cholesta-5,7-dien-3beta-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase. Steroidal antiestrogens such as ICI 182,780 and RU 58,668 did not affect these enzymatic steps, because they do not bind to the AEBS. Transient co-expression of human 3beta-hydroxysterol-Delta8-Delta7-isomerase and 3beta-hydroxysterol-Delta7-reductase and immunoprecipitation experiments showed that both enzymes were required to reconstitute the AEBS in mammalian cells. Altogether, these data provide strong evidence that the AEBS is a hetero-oligomeric complex including 3beta-hydroxysterol-Delta8-Delta7-isomerase and the 3beta-hydroxysterol-Delta7-reductase as subunits that are necessary and sufficient for tamoxifen binding in mammary cells. Furthermore, because selective AEBS ligands are antitumoral compounds, these data suggest a link between cholesterol metabolism at a post-lanosterol step and tumor growth control. These data afford both the identification

  20. Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

    DEFF Research Database (Denmark)

    Danielsen, E M; Norén, Ove; Sjöström, H

    1983-01-01

    that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence......-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000...

  1. Biosynthesis of very long chain fatty acids in microsomes from epidermal cells of Allium porrum L.

    Science.gov (United States)

    Agrawal, V P; Lessire, R; Stumpf, P K

    1984-05-01

    The elongation system present in leek epidermal cells functions to synthesize very long chain fatty acids which, in turn, are the precursors to alkanes. The elongation system is microsomal, employs only saturated acyl components of the endogenous lipid pool as acceptors, utilizes malonyl-CoA as the C2 donor, has an absolute requirement for ATP, and is markedly inhibited by acetyl-ACP. Only saturated acyl-CoAs are readily elongated to very long chain fatty acids by malonyl-CoA in the absence of ATP. ACP is not required by the microsomal system.

  2. Induction of liver microsomal UDP-glucuronyltransferase in the rat administered with a plant phenol, eugenol.

    Science.gov (United States)

    Yokota, H; Yuasa, A

    1990-02-01

    UDP-glucuronyltransferase activity toward xenobiotics in rat liver microsomes was increased about 2.6-fold by administration of a eugenol (4-allyl-2-methoxyphenol). Km value of the induced enzyme toward UDP-glucuronic acid, however, did not change. Immunoblotting analysis revealed that the amount of UDP-glucuronyltransferase protein was increased in the microsomes of eugenol-treated rat liver. In vitro translation assay showed that the level of translatable mRNA encoding this enzyme increased in the liver. These results indicate that mRNA specific for production of UDP-glucuronyltransferase has accumulated, presumably by de novo synthesis in response to a plant phenol, eugenol.

  3. Cytotoxicity of MEIC chemicals Nos. 11-30 in 3T3 mouse fibroblasts with and without microsomal activation

    DEFF Research Database (Denmark)

    Rasmussen, Eva

    1999-01-01

    in other studies on microsomal modulation of the cytotoxicity of the test substances. Moderate to good correlations were found between the cytotoxicity data and rodent lethality data, and the addition of microsomes slightly improved the in vitro/in vivo concordance. The evidence to support the relevance......The cytotoxicity of MEIC chemicals Nos, 11-30 was evaluated by determination of neutral red uptake in Balb/c 3T3 mouse fibroblasts with and without the addition of a microsomal activation mixture. The use of microsomes significantly decreased the cytotoxicity of malathion, 2,4-dichlorophenoxyacetic...... acid, propranolol, thioridazine, lithium sulfate, copper sulfate and thallium sulfate, whereas the cytotoxicity of 1,1,1-trichloroethylene, phenol, nicotine, and paraquat was significantly increased by use of the microsomal activation mixture. These cytotoxicity data are in line with observations...

  4. Metabolism and covalent binding of [14C]toluene by human and rat liver microsomal fractions and liver slices.

    Science.gov (United States)

    Chapman, D E; Moore, T J; Michener, S R; Powis, G

    1990-01-01

    The in vitro metabolism of [14C]toluene by liver microsomes and liver slices from male Fischer F344 rats and human subjects has been compared. Rat liver microsomes produced only benzyl alcohol from toluene. Liver microsomes from human subjects metabolized toluene to benzyl alcohol, benzaldehyde, and benzoic acid. Liver microsomes from one human donor also produced p-cresol and o-cresol. The overall rate of toluene metabolism by human liver microsomes was 9-fold greater than by rat liver microsomes. Human liver microsomal metabolism of benzyl alcohol to benzaldehyde required NADPH and was inhibited by carbon monoxide and high pH (pH 10). but was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P-450, rather than alcohol dehydrogenase, was responsible for the metabolism of benzyl alcohol to benzaldehyde. Human and rat liver slices metabolized toluene to hippuric acid and benzoic acid. The overall rate of toluene metabolism by human liver slices was 1.3-fold greater than by rat liver slices. Cresols and cresol conjugates were not detected in human or rat liver slice incubations. Covalent binding of [14C]toluene to human liver microsomes and slices was 21-fold and 4-fold greater than to the comparable rat liver preparations. Covalent binding did not occur in the absence of NADPH, was significantly decreased by coincubation with cysteine, glutathione, or superoxide dismutase, and was unaffected by coincubation with lysine. Protease and ribonuclease digestion decreased the amount of toluene covalently bound to human liver microsomes by 78% and 27% respectively. Acid washing of human liver microsomes had no effect on covalent binding.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Diffusion of Tritiated Water (HTO) and {sup 22}Na{sup +}-Ions through Non-Degraded Hardened Cement Pastes - II. Modelling Results

    Energy Technology Data Exchange (ETDEWEB)

    Jakob, A

    2002-12-01

    In this report, the procedure and the results of an inverse modelling study on the through-diffusion of tritiated water (HTO) and {sup 2}2Na{sup +}-ions are presented using high-porous hardened cement pastes with a water/cement ratio of 1.3 in the first stage of the cement degradation. For the analysis two alternative models were applied: 1) a diffusion model where a possible sorption of the tracer was entirely neglected, and 2) a diffusion model with linear sorption. The analysis of the through-diffusion phase allowed extracting values for the effective diffusion coefficient (D{sub e}) and the rock-capacity factor ({alpha}). Both models could fit the breakthrough curves equally well, and also mass-balance considerations did not allow to clearly preferring one of the two competing models to the other. But blind-predictions for tracer out-diffusion using the best-fit parameter values deduced from analysing the former through-diffusion phase gave a clear indication that linear sorption had to be included in the diffusion model. The extracted K{sub d} values for HTO are in excellent agreement with values from batch sorption experiments and are of the order of 0.8. 10{sup -3} m{sup 3}/kg. Those for {sup 2}2Na{sup +} are of the order of 1.0. 10{sup -3} m{sup 3}/kg and are by a factor of two larger than values from batch sorption experiments. The values for the effective diffusion coefficients for HTO are of the order of (2-3).10{sup -1}0 m{sup 2}/s, and those for sodium are roughly by a factor of two smaller than values for HTO. On the one hand, the observed tracer uptake could only partially be addressed to isotope exchange; the most obvious process which could account for the remaining part of the uptaken tracer mass is diffusion into a second type of porosity, the dead-end pores. On the other hand, the results and conclusions drawn are encouraging for future investigations; therefore no major deficiency concerning the applied equipment and the modelling methodology

  6. Activation of K{sup +} channels and Na{sup +}/K{sup +} ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

    Energy Technology Data Exchange (ETDEWEB)

    Fiorim, Jonaina, E-mail: nanafiorim@hotmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Ribeiro Júnior, Rogério Faustino, E-mail: faustino43@oi.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Azevedo, Bruna Fernades, E-mail: brunafernandes.azevedo@gmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Simões, Maylla Ronacher, E-mail: yllars@hotmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Padilha, Alessandra Simão, E-mail: ale_spadilha@yahoo.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Stefanon, Ivanita, E-mail: ivanita@pq.cnpq.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Alonso, Maria Jesus, E-mail: mariajesus.alonso@urjc.es [Departamento de Ciencias de la Salud III, Universidad Rey Juan Carlos, Alcorcón (Spain); Salaices, Mercedes, E-mail: mercedes.salaices@uam.es [Departamento de Farmacología, Universidad Autónoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPaz) (Spain); Vassallo, Dalton Valentim, E-mail: daltonv2@terra.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil)

    2012-07-01

    Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K{sup +} channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K{sup +} channels and Na{sup +}/K{sup +}-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O{sub 2}{sup −} production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K{sup +}-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K{sup +}-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K{sup +} channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress. -- Highlights: ► Increased free radicals production ► Increased Na{sup +}/K{sup +} ATPase activity ► Promotes activation of the K{sup +} channels and reduced vascular reactivity ► These effects preserve endothelial function against oxidative

  7. HNF-1B specifically regulates the transcription of the {gamma}a-subunit of the Na{sup +}/K{sup +}-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Ferre, Silvia [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands); Veenstra, Gert Jan C. [Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen (Netherlands); Bouwmeester, Rianne; Hoenderop, Joost G.J. [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands); Bindels, Rene J.M., E-mail: r.bindels@fysiol.umcn.nl [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands)

    2011-01-07

    Research highlights: {yields} Defects in HNF-1B transcription factor affect Mg{sup 2+} handling in the distal kidney. {yields} {gamma}a- and {gamma}b- subunits of the Na{sup +}/K{sup +}-ATPase colocalize in the distal convoluted tubule of the nephron. {yields} HNF-1B specifically activates {gamma}a expression. {yields} HNF-1B mutants have a dominant negative effect on wild type HNF-1B activity. {yields} Defective transcription of {gamma}a may promote renal Mg{sup 2+} wasting. -- Abstract: Hepatocyte nuclear factor-1B (HNF-1B) is a transcription factor involved in embryonic development and tissue-specific gene expression in several organs, including the kidney. Recently heterozygous mutations in the HNF1B gene have been identified in patients with hypomagnesemia due to renal Mg{sup 2+} wasting. Interestingly, ChIP-chip data revealed HNF-1B binding sites in the FXYD2 gene, encoding the {gamma}-subunit of the Na{sup +}/K{sup +}-ATPase. The {gamma}-subunit has been described as one of the molecular players in the renal Mg{sup 2+} reabsorption in the distal convoluted tubule (DCT). Of note, the FXYD2 gene can be alternatively transcribed into two main variants, namely {gamma}a and {gamma}b. In the present study, we demonstrated via two different reporter gene assays that HNF-1B specifically acts as an activator of the {gamma}a-subunit, whereas the {gamma}b-subunit expression was not affected. Moreover, the HNF-1B mutations H69fsdelAC, H324S325fsdelCA, Y352finsA and K156E, previously identified in patients with hypomagnesemia, prevented transcription activation of {gamma}a-subunit via a dominant negative effect on wild type HNF1-B. By immunohistochemistry, it was shown that the {gamma}a- and {gamma}b-subunits colocalize at the basolateral membrane of the DCT segment of mouse kidney. On the basis of these data, we suggest that abnormalities involving the HNF-1B gene may impair the relative abundance of {gamma}a and {gamma}b, thus affecting the transcellular Mg{sup 2

  8. Genetically lowered microsomal epoxide hydrolase activity and tobacco-related cancer in 47,000 individuals

    DEFF Research Database (Denmark)

    Lee, Julie; Dahl, Morten; Nordestgaard, Børge G

    2011-01-01

    Two functional polymorphisms of the microsomal epoxide hydrolase (mEH) gene (EPHX1), Tyr113His (rs1051740) and His139Arg (rs2234922), have variably been found to influence susceptibility to various cancer forms. We tested whether genetically lowered mEH activity affects risk of developing cancer...

  9. ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT.

    Science.gov (United States)

    Chao, Hsu; Zhou, Minglong; McIntosh, Avery; Schroeder, Friedhelm; Kier, Ann B

    2003-01-01

    Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.

  10. Evidence for two enzymatic pathways for omega-oxidation of docosanoic acid in rat liver microsomes

    NARCIS (Netherlands)

    Sanders, Robert-Jan; Ofman, Rob; Valianpour, Fredoen; Kemp, Stephan; Wanders, Ronald J. A.

    2005-01-01

    We studied the omega-oxidation of docosanoic acid (C22:0) in rat liver microsomes. C22:0 and 22-hydroxy-docosanoic acid (omega-hydroxy-C22:0) were used as substrates, and the reaction products were analyzed by electrospray ionization mass spectrometry. In the presence of NADPH, omega-oxidation of

  11. Rat liver microsomal cytochrome P450-dependent oxidation of 3,5-disubstituted analogues of paracetamol

    NARCIS (Netherlands)

    Bessems, J.G.M.; Koppele, J.M. te; Dijk, P.A. van; Stee, L.L.P. van; Commandeur, J.N.M.; Vermeulen, N.P.E.

    1996-01-01

    1. The cytochrome P450-dependent binding of paracetamol and a series of 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -C(H)3, -C2H5, -iC3H7) have been determined with β-naphthoflavone (βNF)-induced rat liver microsomes and produced reverse type I spectral changes. K(s,app) varied

  12. Structure-based prediction of the nonspecific binding of drugs to hepatic microsomes.

    Science.gov (United States)

    Li, Haiyan; Sun, Jin; Sui, Xiaofan; Yan, Zhongtian; Sun, Yinghua; Liu, Xiaohong; Wang, Yongjun; He, Zhonggui

    2009-06-01

    For the accurate prediction of in vivo hepatic clearance or drug-drug interaction potential through in vitro microsomal metabolic data, it is essential to evaluate the fraction unbound in hepatic microsomal incubation media. Here, a structure-based in silico predictive model of the nonspecific binding (fu(mic), fraction unbound in hepatic microsomes) for 86 drugs was successfully developed based on seven selected molecular descriptors. The R(2) of the predicted and observed log((1 - fu(mic))/fu(mic)) for the training set (n = 64) and test set (n = 22) were 0.82 and 0.85, respectively. The average fold error (AFE, calculated by fu(mic) rather than log((1 - fu(mic))/fu(mic))) of the in silico model was 1.33 (n = 86). The predictive capability of fu(mic) for neutral drugs compared well to that for basic compounds (R(2) = 0.82, AFE = 1.18 and fold error values were all below 2, except for felodipine and progesterone) in our model. This model appears to perform better for neutral compounds when compared to models previously published in the literature. Therefore, this in silico model may be used as an additional tool to estimate fu(mic) and for predicting in vivo hepatic clearance and inhibition potential from in vitro hepatic microsomal studies.

  13. Monitoring by HPLC of Chamomile Flavonoids Exposed to Rat Liver Microsomal Metabolism

    Science.gov (United States)

    Petroianu, Georg; Szőke, Éva; Kalász, Huba; Szegi, Péter; Laufer, Rudolf; Benkő, Bernadett; Darvas, Ferenc; Tekes, Kornélia

    2009-01-01

    Three major flavonoid chamomile components (quercetin, apigenin-7-O-glucoside and rutin) were subjected to oxidative metabolism by cytochrome P-450 of rat liver microsomal preparations. Changes over time in their respective concentrations were followed using reversed-phase HPLC with UV detection. No clean-up had to be applied as only the specific flavonoid had to be separated from the background components originating from the rat liver microsome. Neither the concentration of apigenin-7-O-glucoside nor that of the diglycoside rutin decreased during one hour of exposure to rat microsomal treatment. In contrast, the concentration of quercetin, a lipophilic aglycon, decreased. Our analytical HPLC results complement the in silico calculated lipophilicity (logP) of these compounds; the relatively high lipophilicity of quercetin appears to predispose it to oxidative metabolism in order to decrease its fat solubility. In contrast the much less lipophilic compounds apigenin-7-O-glucoside and rutin were resistant in vitro to microsomal treatment. PMID:19707521

  14. Microsomal epoxide hydrolase genotypes and the risk for head and neck cancer.

    NARCIS (Netherlands)

    Lacko, M.; Roelofs, H.M.J.; Morsche, R.H.M. te; Voogd, A.C.; Ophuis, MB Oude; Peters, W.H.M.; Manni, J.J.

    2008-01-01

    BACKGROUND: Microsomal epoxide hydrolase (mEH) is an enzyme involved in the metabolism of (pre)carcinogens in tobacco smoke. We investigated whether functional genetic polymorphisms in mEH may have a risk-modifying effect on head and neck carcinogenesis. METHODS: Blood from 429 patients with oral,

  15. Effect of an extract of Centella asiatica on the biodistribution of sodium pertechnetate (Na{sup 99m}TcO{sub 4}) and on the fixation of radioactivity on blood constituents

    Energy Technology Data Exchange (ETDEWEB)

    Diniz, Claudia Leite; Carmo, Fernanda Santos do; Almeida, Danielle Santos de; Santos-Filho, Sebastiao David; Fonseca, Adenilson de Souza da; Bernardo Filho, Mario [Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Dept. de Biofisica e Biometria. Lab. de Radiofarmacia Experimental]. E-mail: claudiadiniz@uerj.br; Missailidis, Sotiris [The Open University, Milton Keynes (United Kingdom). Dept. of Chemistry and Analytical Sciences

    2008-12-15

    This study evaluates the effects of an acute treatment with a Centella asiatica (CA) extract on the biodistribution of the radiopharmaceutical Na{sup 99}'mTcO{sub 4} and on the fixation of technetium-99m on blood constituents. Wistar rats were treated with CA extract and, 1 hour after, Na{sup 99m}TcO{sub 4} was administered; organs/tissues were withdrawn and weighted. The radioactivity was counted to calculate the percentage of activity per gram (%ATI/g). Also, blood samples were withdrawn, plasma (P), blood cells (BC), insoluble fraction (IF) and soluble fractions of P and BC were isolated and the radioactivity was counted to calculate the percentage of activity (%ATI). Data indicated that the acute treatment with CA extract changed significantly (p<0.05) the %ATI/g in several organs/tissues (spleen, heart, duodenum, stomach, liver, muscle, kidney, testis and blood) and the %ATI on the blood constituents (P, BC, IF-P and IF-BC). These results indicate that the substances or metabolites of the CA extract would change the biodistribution of Na{sup 99m}TcO{sub 4} and the fixation of the technetium-99m on blood constituents in an acute treatment. (author)

  16. Poling-induced structural change and second-order nonlinearity of Na{sup +}-doped Nb{sub 2}O{sub 5}-TeO{sub 2} glass

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Katsuhisa [Department of Chemistry and Materials Technology, Faculty of Engineering and Design, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto (Japan); Narazaki, Aiko [National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki (Japan); Yonezaki, Yoshinori; Hirao, Kazuyuki [Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto (Japan)

    2000-07-31

    The effect of thermal/electrical poling on surface structure and poling-induced second-harmonic generation have been examined for 15Nb{sub 2}O{sub 5}{center_dot}85TeO{sub 2} glass. Since the poling was carried out with the 15Nb{sub 2}O{sub 5}{center_dot}85TeO{sub 2} glass sandwiched in between two commercial borosilicate glasses containing Na{sup +}, penetration of Na{sup +} into the anode-side surface of 15Nb{sub 2}O{sub 5}{center_dot}85TeO{sub 2} glass took place, as revealed by x-ray photoelectron spectroscopy (XPS). XPS studies also suggest that Nb{sup 5+} migrates from the anode-side surface into the bulk by the poling. It is thought that the migration of Na{sup +} and Nb{sup 5+} forms an internal dc electric field, leading to the second-harmonic generation. (author). Letter-to-the-editor.

  17. Beluga whale liver microsomal cytochrome P4501A (CYP1A) enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bullock, P.L.; Addison, R. [Inst. of Ocean Sciences, Sidney, British Columbia (Canada); Lockhart, L.; Metner, D. [Dept. of Fisheries and Oceans, Winnipeg, Manitoba (Canada)

    1995-12-31

    Beluga whale (Delphinapterus leucas) liver from the Canadian arctic was analyzed for the presence of CYP1A enzymes, as part of current studies on biomarkers for environmental contamination. CYP1A1-associated 7-ethoxyresorufin O-dealkylase activity (EROD) varied 13 fold among sixteen male whale liver microsomal samples and 31 fold among five females. Similarly, the rate of 7-methoxyresorufin O-dealkylation (MROD) varied 7 fold and 3 fold in microsomal samples from males and females, respectively. Furthermore, 7-pentoxyresorufin O-dealkylase activity (PROD) varied 10 fold in both sexes. None of these enzyme activities were sexually differentiated, and EROD and MROD were inhibited by {alpha}-naphthoflavone. There was very good correlation between EROD and MROD (r{sup 2} = .894), EROD and PROD (r{sup 2} = .909), but MROD and PROD were not as well correlated (r{sup 2} = 785). On Western immunoblots, a single band was recognized in Beluga whale liver microsomes by a polygonal antibody raised against an oligopeptide related to trout CYP1A1. This antibody also recognized purified rat CYP1A1 (56 kDa) and stained only one band (56 kDa) in liver microsomes isolated from male rats treated with {beta}-naphthoflavone. The interindividual variation in EROD paralleled differences in the amount of whale liver microsomal protein that cross-reacted with the anti-peptide antibody. The results suggest that Beluga whale liver contains at least one CYP1A enzyme which catalyzes the 0-dealkylation of 7-ethoxy, 7-methoxy and 7-pentoxyresorufin and has a molecular weight less than that of rat CYP1A1, but similar to rat CYP1A2 (52 kDa).

  18. Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane.

    Science.gov (United States)

    Li, Xue-Qing; Hayes, Martin A; Grönberg, Gunnar; Berggren, Kristina; Castagnoli, Neal; Weidolf, Lars

    2016-08-01

    Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of (18)O from H2 (18)O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  19. A Toxoplasma gondii protein with homology to intracellular type Na{sup +}/H{sup +} exchangers is important for osmoregulation and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Francia, Maria E.; Wicher, Sarah [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States); Pace, Douglas A. [Center for Tropical and Emerging Global Diseases and Department of Cellular Biology University of Georgia, Athens, GA 30602 (United States); Sullivan, Jack [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States); Moreno, Silvia N.J. [Center for Tropical and Emerging Global Diseases and Department of Cellular Biology University of Georgia, Athens, GA 30602 (United States); Arrizabalaga, Gustavo, E-mail: gustavo@uidaho.edu [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States)

    2011-06-10

    The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na{sup +}/H{sup +} exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca{sup 2+} concentration [Ca{sup 2+}]{sub i}, and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

  20. Synthesis, conductivity behaviour and second-order nonlinear optics of partially substituted double KDP containing As{sup 5+} and Na{sup +}

    Energy Technology Data Exchange (ETDEWEB)

    Ennaceur, Nasreddine, E-mail: nennaceu@ens-cachan.fr [Laboratoire Physico-chimie de l' Etat Solide, Faculte des Sciences BP 1171, 3000 Sfax, Universite de Sfax (Tunisia); Laboratoire de Photonique Quantique Moleculaire, Institut d' Alembert-Ecole Normale Superieure, 94230 Cachan (France); Ledoux-Rak, Isabelle [Laboratoire de Photonique Quantique Moleculaire, Institut d' Alembert-Ecole Normale Superieure, 94230 Cachan (France); Mhiri, Tahar [Laboratoire Physico-chimie de l' Etat Solide, Faculte des Sciences BP 1171, 3000 Sfax, Universite de Sfax (Tunisia)

    2013-02-01

    A new solid solution K{sub 0.95}Na{sub 0.05}H{sub 2}(PO{sub 4}){sub 0.5}(AsO{sub 4}){sub 0.5} abbreviated (KNDAP) has been prepared by slow evaporation of an aqueous solution at room temperature. Its crystal structure which was solved by the direct method from single crystal X-ray diffraction data can give birth to two kinds of disorder. The first one is statistical or dynamical in the shortest O---H{sup Horizontal-Ellipsis }O hydrogen bond that can facilitate the migration of proton. As for the second kind, it is related to the effect of the substitution of Na{sup +} and As{sup 5+} in the same site occupancy as that of the K{sup +} and P{sup 5+}, respectively. This disorder could promote hard defects in the structure, which can enhance the physical properties, especially, the conductivity.

  1. NaOH-rich corner of the (Na/sup +/, Ca/sup 2 +/) (OH/sup -/, CO/sub 3//sup 2 -/) reciprocal system. [LMFBR

    Energy Technology Data Exchange (ETDEWEB)

    Sallach, R.A.

    1983-08-01

    The sodium-rich portion of the binary system (NaOH)/sub 2/ - Ca(OH)/sub 2/ and the reciprocal system (Na/sup +/, Ca/sup 2 +/) (OH/sup -/, CO/sub 3//sup 2 -/) are described. The (NaOH)/sub 2/ - Ca(OH)/sub 2/ system has a peritectic decomposition at 330 +- 5/sup 0/C and a mole fraction Ca(OH)/sub 2/ of 0.0055 +- 0.0020. A compound with the apparent stoichiometry Na/sub 6/Ca(OH)/sub 8/ is formed in the system. The compound melts at 560 +- 10/sup 0/C. X-ray diffraction data for this compound are presented. Solid state solubility of the compound in ..beta..-NaOH causes a depression in the temperature of the ..cap alpha.. ..-->.. ..beta.. phase change. Liquidus data for 27 compositions in the NaOH-rich portion of the reciprocal system are reported. These data may be interpreted in terms of a ternary peritectic or a semi-eutectic. Ternary invariant temperatures were not measured. An apparent invariant at 285 +- 5/sup 0/C is attributed to a disequilibrium eutectic reaction between NaOH and Na/sub 2/CO/sub 3/.

  2. Some properties of the fatty alcohol oxidation system and reconstitution of microsomal oxidation activity in intestinal mucosa.

    Science.gov (United States)

    Ichihara, K; Kusunose, E; Noda, Y; Kusunose, M

    1986-10-03

    This paper describes the metabolism of fatty alcohols by microsomal and cytosolic fractions from intestinal mucosa. Microsomes of rabbit intestinal mucosa had a high activity of [1-14C]dodecanol oxidation as did those of liver. The intestinal cytosolic fraction also exhibited oxidation activity to a lesser extent than the microsomes did. The reaction product was determined as lauric acid using thin-layer chromatography. Laurylaldehyde was detected as another product, when semicarbazide was added to the incubation system. Cyclodextrins exhibited a stimulation effect similarly to bovine serum albumin on the microsomal activity. We have compared the stimulatory effects of dimethyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and alpha-cyclodextrin, which decrease in that order. Effects of NAD+ and dodecanol concentrations, pH and pyrazole on microsomal activity were compared with those on cytosolic activity. Dodecanol oxidation activity was solubilized and reconstituted with a fatty alcohol dehydrogenase and a fatty aldehyde dehydrogenase separated from the intestinal microsomes. These findings indicate that both the dehydrogenases participate in microsomal oxidation of fatty alcohols to fatty acids with fatty aldehydes as intermediates in the reaction.

  3. Gender and Species-Mediated Differences in the In Vitro Metabolism of Triadimefon by Rodent Hepatic Microsomes

    Science.gov (United States)

    Understanding how metabolism kinetics differ between genders and species is important in developing informative pharmacokinetic models and accurately assessing risk. Metabolism of the conazole fungicide Triadimefon (TDN) was studied in hepatic microsomes of SD rats and CD-1 mice...

  4. Microsomal EROD data of fish liver sample assay from species collected in the Salt and Gila Rivers, Arizona

    Science.gov (United States)

    Nicks, Diane

    2017-01-01

    This dataset includes microsomal ERDO data from an assay done with liver samples from several fish species that are found in Arizona at sites that are being assessed for PBDE contamination. The data was created in September and October 2016.

  5. Soy isoflavone metabolism in cats compared with other species: Urinary metabolite concentrations and glucuronidation by liver microsomes

    OpenAIRE

    Redmon, Joanna M.; Shrestha, Binu; Cerundolo, Rosario; Court, Michael H.

    2015-01-01

    Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here we evaluated species’ differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species.Significant concentrations of conjugated (but not unconjugated) genistein, daidzein, and gl...

  6. Specific metabolic pathway in vitro of pinazepam and diazepam by liver microsomal enzymes of different animal species.

    Science.gov (United States)

    Comi, V; Fossati, A; Gervasi, G B

    1977-04-01

    The metabolic pathway of Pinazepam and Diazepam in vitro was studied with rat, guinea pig and dog liver microsomes using a chromatographic and spectrophotometric technique. Two main pathways were observed, N1-dealkylation and C3-hydroxylation. N1-dealkylation was shown to be the predominant reaction for Pinazepam in all the animal species studied, while C3-hydroxylation was the major metabolic pathway for Diazepam in the rat. No oxazepam was found when Pinazepam and Diazepam were incubated with liver microsomes.

  7. Determination of species-difference in microsomal metabolism of amitriptyline using a predictive MRM-IDA-EPI method.

    Science.gov (United States)

    Lee, Ji-Yoon; Lee, Sang Yoon; Lee, KiHo; Oh, Soo Jin; Kim, Sang Kyum

    2015-03-05

    We investigated to compare species differences in amitriptyline (AMI) metabolism among mouse, rat, dog, and human liver microsomes. We developed a method for simultaneous determination of metabolic stability and metabolite profiling using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) scanning. In the cofactor-dependent microsomal metabolism study, AMI was metabolized more rapidly in rat and human liver microsomes incubated with NADPH than UDPGA. AMI incubated with NADPH+UDPGA in rat, dog, or mouse liver microsomes disappeared rapidly with a half-life of 3.5, 8.4, or 9.2 min, respectively, but slowly in human liver microsomes with a half-life of 96 min. In total, 9, 10, 11, and 6 putative metabolites of AMI were detected in mouse, rat, dog, and human liver microsomes, respectively, based on mass spectrometric analyses. Kinetic analysis of metabolites in liver microsomes from each species over 120 min showed common metabolic routes of AMI, such as N-demethylation, hydroxylation, and glucuronidation, and subtle interspecies differences in AMI metabolism. The main metabolic routes in mouse, rat, dog, and human liver microsomes were hydroxylation followed by glucuronide conjugation, methyl hydroxylation, and N-demethylation, respectively. The MRM-IDA-EPI method can provide quantitative and qualitative information about metabolic stability and metabolite profiling simultaneously. Moreover, time course analysis of metabolites can not only eliminate false identification of metabolites, but also provide a rationale for proposed metabolic pathways. The MRM-IDA-EPI method combined with time course analysis of metabolites is useful for investigating drug metabolism at the early drug discovery stage. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice.

    Science.gov (United States)

    Lewandowski, M; Chui, Y C; Levi, P; Hodgson, E

    1991-02-01

    A simple and sensitive method for the separation of 14C-labelled acetanilide, 4-hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBA2/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [14C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.

  9. The influence of ouabain and alpha angelica lactone on calcium metabolism of dog cardiac microsomes

    Science.gov (United States)

    Entman, Mark L.; Cook, Joseph W.; Bressler, Rubin

    1969-01-01

    The influence of ouabain and alpha angelica lactone on 45calcium accumulation in cardiac microsomes was studied. Calcium binding (accumulation in the absence of excess oxalate or phosphate) was augmented by both ouabain and alpha angelica lactone in the presence of adenosine triphosphate (ATP) but unaffected in its absence. Calcium turnover (defined as the change in 45Ca++ bound to the microsomes after the specific activity is changed) was studied to determine if the augmented bound pool was freely exchangeable at equilibrium. Ouabain and alpha angelica lactone augmented calcium turnover in both the presence and absence of ATP. Calcium-stimulated ATPase was increased by both agents. It is proposed that these two unsaturated lactones, with known cardiotonic activity, may exert their effects by providing an increased contraction-dependent calcium pool to be released upon systolic depolarization. PMID:4236805

  10. Assessment of mutagenic potential of pyrolysis biochars by Ames Salmonella/mammalian-microsomal mutagenicity test.

    Science.gov (United States)

    Anjum, Reshma; Krakat, Niclas; Toufiq Reza, M; Klocke, Michael

    2014-09-01

    Biochar is of raising interest in sustainable biomass utilization concepts. Particularly biochar derived from pyrolysis attaches important agricultural capacities mandatory for an improved carbon sequestration, soil fertility and amelioration, respectively. In fact, large scale field trials and commercial business with biochar materials have already been started but still only few are known about the mutagenic potential of biochars produced. In this study hemp bedding and wood pellet biomass were used for biochar production by pyrolysis. The total concentrations of polycyclic aromatic hydrocarbons (PAHs) were 34.9µgg(-1) of dry mass and 33.7µgg(-1) of dry mass for hemp biochar and wood biochar, respectively. The concentration of PAHs in tar produced during wood carbonization was 17.4µgg(-1). The concentrations of phenolic compounds were 55µgg(-1) and 8.3µgg(-1) for hemp and wood biochar, respectively. Salmonella/microsomal mutagenicity tests (i.e. Ames test) revealed a maximum mutagenicity for hemp biochar extracts with strains TA97, TA98 and TA100 in the presence and absence of liver microsomal fractions, respectively. Wood biochar and tar extract exhibited maximum mutagenicity with strains TA98 and T100 both in the presence and absence of liver microsomal fraction. The reversion of the applied tester strains increased in the presence and absence of liver microsomal fractions with an increasing dose of hemp biochar extract up to 2µl per plate and decreased at a concentration of 2.5µl per plate. For wood biochar and tar extracts, reversion of tester strains increased both in the presence and absence of S9 at extract concentrations of 4µl per plate and declined at a dose of 8µl per plate. By this study a significant higher mutagenic potential for hemp biochar compared to wood biochar and tar could be observed suggesting careful application in soil melioration. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Altered lauric acid metabolism in renal microsomes from spontaneously hypertensive rats (SHR)

    Energy Technology Data Exchange (ETDEWEB)

    Shiverick, K.T.; Applewhite, J.; Okita, R.

    1986-03-01

    Studies investigated whether changes in omega- and (omega-1)-hydroxylation (OH) of lauric acid (LA) occurred in renal microsomes prepared from SHR compared to Wistar-Kyoto (WK) control rats. Systolic blood pressure in age-matched adult SHR and WKR were 189 +/- 3 and 123 +/- 4 mm Hg(anti X +/- SE) respectively (p < 0.001). No significant differences between SHR and WKR were seen in body weight, kidney weight or renal microsomal protein content. Renal microsomes, prepared from whole kidneys, were incubated with 10 mM NADPH and (/sup 14/C)LA at concentrations between 5-50 ..mu..M. The 11- and 12-OH metabolites of LA were separated by HPLC using a reverse phase column with a methanol:water:acetic acid (62:37.8:0.2) mobile phase. Apparent (app) V/sub max/ values for 12-OH in WKR and SHR were 0.87 +/- 0.19 vs 1.48 +/- .11 nmoles/mg protein/min (p < 0.05), respectively, while values for 11-OH were 0.51 +/- 0.12 vs 0.60 +/- .07, respectively. No significant differences were found in app K/sub m/ values for either 11- or 12-OH between the two strains. SDS-polyacrylamide gel electrophoresis of renal microsomes showed the increased prominence of a 52,000 dalton protein in SHR preparations. Thus data suggest that selective alterations in renal cytochrome P-450 monooxygenase reactions may be associated with the endogenous biochemical processes underlying hypertension.

  12. CHARACTERIZATION OF HUMAN LIVER MICROSOMAL UDP-GLYCOSYLTRANSFERASES USING PHOTOAFFINITY ANALOGS

    NARCIS (Netherlands)

    LITTLE, JM; DRAKE, RR; VONK, R; KUIPERS, F; LESTER, R; RADOMINSKA, A

    The photoaffinity analogs [beta-P-32]5-azido-UDP-glucuronic acid ([P-32]5N3UDP-GlcUA) and [beta-P-32]5-azido-UDP-glucose ([P-32]5N(3)UDP-Glc) were used to characterize UDP-glycosyl-transferases of microsomes prepared from human liver. Photoincorporation of both probes into proteins in the 50- to

  13. Cumene hydroperoxide-supported denitrification of 2-nitropropane in uninduced mouse liver microsomes.

    Science.gov (United States)

    Marker, E K; Kulkarni, A P

    1986-01-01

    Cumene hydroperoxide supported oxidative denitrification of 2-nitropropane was investigated in uninduced mouse liver microsomes. The cytochrome P-450 peroxygenase catalyzed reaction resulted in the production of nitrite and acetone. Several lines of evidence suggested the involvement of multiple forms of cytochrome P-450. Acetone production was at least two times greater than nitrite release possibly due to sequestration of nitrite in the reaction mixtures.

  14. Cytochrome P-450-mediated denitrification of 2-nitropropane in mouse liver microsomes.

    Science.gov (United States)

    Marker, E K; Kulkarni, A P

    1986-09-01

    Enzymatic denitrification of 2-nitropropane (2NP) was investigated in an NADPH-dependent hepatic microsomal system from male CD1 mice. The involvement of cytochrome P-450 (P-450) as the catalyst in 2NP denitrification was revealed by the induction of nitrite-releasing activity following phenobarbital (PB) pretreatment, by a decrease in activity with carbon tetrachloride pretreatment, by the inhibition of the reaction with classical P-450 inhibitors, and by the observation of a type I binding spectrum. Under optimal conditions, two pH-dependent peaks of activity were observed at pH 7.6 and pH 8.8, each with its own optimal substrate concentration. Inhibition of the reaction by metyrapone and carbon monoxide (CO) (among others) produced differential responses dependent on pH. These results, along with two pH optima and two substrate optima, suggested the involvement of multiple P-450 isozymes. Average specific activities were 8.05 nmoles of nitrite released per minute per milligram microsomal protein at pH 7.6 and 6.44 nmoles of nitrite released per minute per milligram microsomal protein at pH 8.8. Acetone was identified as the second product of the reaction by gas chromatography/mass spectrometry (GC/MS). Stoichiometry studies indicated that the acetone production was slightly less than expected (about 70%) from nitrite release. Up to 25% residual activity was observed under anaerobic conditions. These results suggested that though the predominant reaction mechanism was oxidative, oxygen-independent metabolism of 2NP also occurred to some extent. In contrast to the reported lack of activity in untreated rat, the observed denitrification in uninduced mouse liver microsomes was significant and suggested that major species-specific differences exist in the in vitro metabolism of 2NP.

  15. Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam

    Energy Technology Data Exchange (ETDEWEB)

    Thomassin, J.; Tephly, T.R. (Univ. of Iowa, Iowa City (USA))

    1990-09-01

    Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of (3H)flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with (3H) flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000).

  16. Inducible bilirubin-degrading system in the microsomal fraction of rat liver.

    Science.gov (United States)

    De Matteis, F; Trenti, T; Gibbs, A H; Greig, J B

    1989-06-01

    The hypothesis that treatment of Gunn rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates an alternative pathway of bilirubin disposal, involving an induced form of cytochrome P-450 [Proc. Natl. Acad. Sci. USA 75:682-685 (1978)], has been investigated by studying the mechanisms of bilirubin oxidation in chemical model systems and in liver microsomal systems in vitro. Hematin, copper sulfate, and the iron chelate of EDTA were all active in promoting degradation of bilirubin in the presence of hydrogen peroxide. Evidence was obtained for a microsomal bilirubin-degrading system that could be induced in the liver by treating either rats or chick embryos with TCDD, beta-naphthoflavone, or 3,4,3',4'-tetrachlorobiphenyl (3,4-TCB) in vivo. The activity of this system required NADPH and oxygen and was markedly stimulated by addition of 3,4-TCB (a planar polyhalogenated biphenyl) and much less markedly by the nonplanar analogue 2,4,2',4'-tetrachlorobiphenyl. These two biphenyls were also inhibitory towards the 7-ethoxyresorufin O-deethylase activity of the induced microsomes and here again the nonplanar analogue was markedly less active. Dose-response experiments for stimulation of bilirubin breakdown and inhibition of 7-ethoxyresorufin O-deethylase activity after addition of 3,4-TCB in vitro showed both effects to be caused by similar concentrations of the biphenyl. These results suggest that a polyhalogenated chemical may interact with TCDD-induced microsomes, inhibit their monooxygenase activity, and stimulate production of a bilirubin-degrading species.

  17. Inhibition of in vitro metabolism of testosterone in human, dog and horse liver microsomes to investigate species differences.

    Science.gov (United States)

    Zielinski, Jana; Mevissen, Meike

    2015-04-01

    Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subfamily in all three species. While CYP2C9 did not metabolise testosterone, the canine ortholog CYP2C21 produced androstenedione. Quercetin significantly inhibited 6β- and 11β-hydroxytestosterone in all species investigated, suggesting that CYP2C8 is involved in testosterone metabolism, whereas sulfaphenazole significantly inhibited the formation of 6β- and 11β-hydroxytestosterone in human microsomes, at 60 min in equine microsomes, but not in canine microsomes. A contribution of CYP2B6 in testosterone metabolism was only found in human and equine microsomes. Inhibition of 17β-hydroxysteroid dehydrogenase 2 indicated its involvement in androstenedione formation in humans, increased androstenedione formation was found in equines and no involvement in canines. These findings provide improved understanding of differences in testosterone biotransformation in animal species. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Investigation of glycosylation processes in mitochondria and microsomal membranes from human skeletal muscle.

    Science.gov (United States)

    Gasnier, F; Lerme, F; Rousson, R; Roussouly, P; Vaganay, E; Louisot, P; Gateau-Roesch, O

    1991-05-31

    Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined.

  19. Biotransformation of Flavokawains A, B, and C, Chalcones from Kava (Piper methysticum), by Human Liver Microsomes.

    Science.gov (United States)

    Zenger, Katharina; Agnolet, Sara; Schneider, Bernd; Kraus, Birgit

    2015-07-22

    The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4' and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2'-O-glucuronide, FKB-2'-O-glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo.

  20. Prediction of hepatic microsomal intrinsic clearance and human clearance values for drugs.

    Science.gov (United States)

    Nikolic, Katarina; Agababa, Danica

    2009-10-01

    Twenty-nine drugs of different structures were used in theoretical QSAR analysis of human hepatic microsomal intrinsic clearance (in vitro T(1/2) and in vitro CL'(int)) and whole body clearance (CL(blood)). The examined compounds demonstrated a wide range of scaled intrinsic clearance values. Constitutional, geometrical, physico-chemical and electronic descriptors were computed for the examined structures by use of the Marvin Sketch 5.1.3_2, the Chem3D Ultra 7.0.0 and the Dragon 5.4 program. Partial least squares regression (PLSR), has been applied for selection of the most relevant molecular descriptors and development of quantitative structure-activity relationship (QSAR) model for human hepatic microsomal intrinsic clearance (in vitro T(1/2)). Optimal QSAR models with nine and ten variables, R(2)>0.808 and cross-validation parameter q(pre)(2)>0.623, were selected and compared. Since the microsomal in vitro T(1/2) data can be used for calculation of in vitro CL'(int) and in vivo CL(blood), the developed QSAR model will enable one to analyze the kinetics of cytochrome P450-mediated reactions in term of intrinsic clearance and whole body clearance. A comparison is made between predictions produced from the QSAR analysis and experimental data, and there appears to be generally satisfactory correlations with the literature values for intrinsic clearance data.

  1. Effect of chronic alcohol drinking on rat liver microsomal nitroreductive metabolism of nifurtimox and benznidazole.

    Science.gov (United States)

    de Mecca, M M; Bartel, L C; Castro, J A

    2013-12-01

    Nifurtimox (Nfx) and benznidazole (Bz) have serious toxic side effects. Manufacturers warn about significant adverse effects when simultaneous alcohol consumption is being made, but its mechanism is not known. The levels and toxicity of these drugs are linked to their liver microsomal nitroreduction to reactive metabolites. In this study, we analyzed whether alcohol drinking enhanced those nitroreductive processes. Male and female Sprague-Dawley rats, 5-6 weeks old (125-150 g body weight) were used. They were fed ad libitum for 28 days with Lieber and De Carli control or alcohol regular liquid diets. The rats were separated into two dietary groups: ethanol and control group. Both were pair fed with the respective diet. Their liver microsomes were isolated and the nicotinamide adenine dinucleotide phosphate-dependent nitroreduction of Nfx and Bz were determined. Alcohol drinking significantly induced microsomal nitroreduction of these drugs in male rats (11% for Nfx and 41% for Bz) but not in females. The activity observed in the alcohol-induced male rats was 100% inhibited by diphenyleneiodonium and attributable to P450 reductase. Inductive effects of alcohol drinking on nitroreductive activation of both drugs might be only partially involved in the harmful interactions described.

  2. Subterminal hydroxylation of lauric acid by microsomes from a marine fish.

    Science.gov (United States)

    Lemaire, P; Lafaurie, M; Weissbart, D; Durst, F; Pflieger, P; Mioskowski, C; Salaün, J P

    1992-03-01

    Microsomes from the liver of sea bass (Dicentrarchus labrax) were shown to hydroxylate lauric acid at subterminal positions. The cytochrome P-450 system converted lauric acid to several mono-hydroxylated metabolites including omega-1 hydroxylaurate, which was the major metabolite (44% of total products). In addition, omega-2, omega-3, omega-4 and a small amount (2.3%) of omega hydroxylaurates were found. Reaction products were identified using thin-layer chromatography (TLC) and gas chromatography/mass spectrometry (GC/MS). Oxidation reactions were dependent upon O2 and NADPH, and did not occur with boiled microsomes or in the presence of a mixture of CO/O2. Hydroxylation proceeded linearly up to 20 min at 28 degrees C for protein concentrations below 380 micrograms. Treatment of fish with benzo(a)pyrene (BP) (20 mg/kg) drastically increased xenobiotic metabolism (ECOD, EROD and BPMO activities), but no difference in laurate hydroxylase activity was observed between untreated and treated fish. Starvation strongly enhanced laurate hydroxylase activity, and resumption of feeding reduced by half this increase of activity. In all of the experiments we did not observe any modification of the regioselectivity of lauric acid hydroxylation by this microsomal in-chain hydroxylating system. We suggest that cytochrome P-450 enzymes involved in lauric acid and xenobiotics metabolism are regulated independently.

  3. The mechanism of microsomal azoreduction: predictions based on electronic aspects of structure-activity relationships.

    Science.gov (United States)

    Zbaida, S

    1995-01-01

    The mechanism of microsomal azoreductase is regulated by the overall Hammett sigma substituent values on each ring. A substrate dye must exhibit an overall Hammett sigma substituent value either equal or more negative than -0.37 on either ring. Dyes with Hammett sigma substituent constants less negative than -0.37 will not be reduced by microsomal cytochrome P450. Microsomal reduction of azo dyes containing only electron-donating substituents on either ring is insensitive to both oxygen and carbon monoxide. The required Hammett sigma substituent value on the opposite benzene (prime) ring for I-substrates is therefore, sigma' P 0 (Table 3). Redox Potentials. Anaerobic cyclic voltammograms of azobenzene derivatives verify the following points: A nonsubstrate azo dye will not exhibit a positive potential. (Several nonsubstrate hydrazobenzenes exhibited positive potentials, but in a low range 0.41-0.48 V. Consequently, cyclic voltammetry can distinguish between nonsubstrate azobenzenes and their nonsubstrate half-reduced hydrazo analogs.) A substrate azo dye will exhibit a positive potential in the range +1.00 to +1.50 V. I-substrate: Both negative potentials are stable in air. S-substrate: The first negative potential will immediately quench upon exposure to air. I-substrates exhibit on average potentials which are approximately 0.6 V more negative than those for S-substrates. A comparison between the oxidative and the reductive pathway of microsomal cytochrome P450 indicates a similarity in the first two steps in the reaction cycle, for example, substrate binding and uptake of the first electron by the cytochrome [76, 109, 110]. Upon reduction of the iron, ferrous cytochrome P450 may bind oxygen or carbon monoxide in a competitive manner in the oxidative cycle or may directly transfer the electrons to the substrate in a stepwise fashion in the reductive cycle [76]. Estabrook et al. [111] suggested that carbon monoxide insensitivity can occur when the formation of

  4. Effects of quinolones on liver microsome cytochrome P450 in rats

    Directory of Open Access Journals (Sweden)

    Yi ZHANG

    2012-11-01

    Full Text Available Objective  To study and compare the effects of fluoroquinolones (levofloxacin, gatifloxacin, moxifloxacin and pazufloxacin on the enzyme system of liver microsome cytochrome P450 in rat. Methods  Thirty male Wistar rats were equally assigned into five groups: control group, levofloxacin (LV group, gatifloxacin (GT group, moxifloxacin (MX group and pazufloxacin (PZ group. Each drug was consecutively administered by tail vein injection for 7 days in a dosage of 120 mg/(kg•d. Liver microsomes were prepared by differential centrifugation, the concentration of protein in the liver microsome was measured by Lowry method, the content and activity of cy tochrome P450 were detected by spectrophotometric determination, and the results were analyzed by one-way ANOVA. Results  Compared with control group, the weight of liver in MX group and GT group was significantly reduced (P 0.05. Assay of aminopyrine-N-demethylase activity showed that the difference in enzyme activity was statistically significant between the control group and groups LV, GT and MX (P < 0.01. Erythromycin-N-demethylase activity measurement revealed that the enzyme activity was lowered in GT group and slightly elevated in MX group, and the difference was statistically significant compared with that of control group (P < 0.01. Measurement of activity of rat liver microsomal CYP450 enzyme system subfamily showed that the BROD activity increased in LV, MX and PZ groups (P < 0.01, and slightly decreased in GT group as compared with control group (P < 0.05. The PROD activity increased in GT group, but decreased in PZ group (P < 0.01. The EROD activity increased in all the four groups (P < 0.01. Conclusions  The four fluoroquinolones have some effects on the enzyme system of liver microsome cytochrome P450 in rats, but the effects may be different (enhancement or attenuation of the enzymatic activity depending on the enzymes, and the extent of the decrease of effect is in the

  5. Nonadiabatic ionic--covalent transitions. Exponential-linear model for the charge exchange and neutralization reactions Na+H arrow-right-left Na/sup +/+H/sup -/

    Energy Technology Data Exchange (ETDEWEB)

    Errea, L.F.; Mendez, L.; Mo, O.; Riera, A.

    1986-01-01

    A previous study of charge exchange processes taking place through ionic--covalent transitions is extended to the case of Na+H and Na/sup +/+H/sup -/ collisions. A five-state molecular expansion, with the inclusion of two-electron translation factors, is employed to calculate the charge exchange and neutralization cross sections. Transitions at the first two pseudocrossings between the energy curves, practically determine the cross sections in the energy range 0.16--5 keV amu/sup -1/. We also show that the widely used multichannel Landau--Zener theory is totally inadequate, to treat these transitions.

  6. Temporal evolution of {sup 137}Cs{sup +}, K{sup +} and Na{sup +} in fruits of South American tropical species

    Energy Technology Data Exchange (ETDEWEB)

    Cid, A.S. [LARA — Laboratório de Radioecologia, Instituto de Física, Universidade Federal Fluminense, Av. Gal Milton Tavares de Souza, s/no, Gragoatá, 24210-340, Niterói, RJ (Brazil); Anjos, R.M., E-mail: meigikos@if.uff.br [LARA — Laboratório de Radioecologia, Instituto de Física, Universidade Federal Fluminense, Av. Gal Milton Tavares de Souza, s/no, Gragoatá, 24210-340, Niterói, RJ (Brazil); Zamboni, C.B. [Instituto de Pesquisas Energéticas e Nucleares (IPEN/CNEN), Av. Lineu Prestes 2242, Cidade Universitária, 05508-000, Paulo, SP (Brazil); Velasco, H. [GEA, Instituto de Matemática Aplicada San Luis (IMASL), Universidad Nacional de San Luis, Consejo Nacional de Investigaciones Científicas y Técnicas. Ej. de los Andes 950, D5700HHW San Luis (Argentina); Macario, K. [LARA — Laboratório de Radioecologia, Instituto de Física, Universidade Federal Fluminense, Av. Gal Milton Tavares de Souza, s/no, Gragoatá, 24210-340, Niterói, RJ (Brazil); Rizzotto, M. [GEA, Instituto de Matemática Aplicada San Luis (IMASL), Universidad Nacional de San Luis, Consejo Nacional de Investigaciones Científicas y Técnicas. Ej. de los Andes 950, D5700HHW San Luis (Argentina); and others

    2013-02-01

    Concentrations of {sup 137}Cs, K and Na in fruits of lemon (Citrus limon B.) and of K and Na in fruits of coconut (Cocos nucifera L.) trees were measured by both gamma spectrometry and neutron activation analysis, with the aim to understand the behaviour of monovalent inorganic cations in tropical plants as well as the plant ability to store these elements. Similar amounts of K{sup +} were incorporated by lemon and coconut trees during the growth and ripening processes of its fruits. The K concentration decreased exponentially during the growth of lemons and coconuts, ranging from 13 to 25 g kg{sup −1} dry weight. The incorporation of Na{sup +} differed considerably between the plant species studied. The Na concentration increased linearly during the lemon growth period (0.04 to 0.70 g kg{sup −1} d.w.) and decreased exponentially during the coconut growth period (1.4 to 0.5 g kg{sup −1} d.w.). Even though radiocaesium is not an essential element to plants, our results have shown that {sup 137}Cs incorporation to vegetable tissues is positively correlated to K distribution within the studied tropical plant species, suggesting that the two elements might be assimilated in a similar way, going through the biological cycle together. A mathematical model was developed from the experimental data allowing simulating the incorporation process of monovalent inorganic cations by the fruits of such tropical species. The agreement between the theoretical approach and the experimental values is satisfactory along fruit development. - Highlights: ► Concentrations of {sup 137}Cs, K and Na in fruits of lemon (Citrus limon B.) are presented. ► Concentrations of K and Na in fruits of coconut (Cocos nucifera L.) are also showed. ► We investigated the use of {sup 137}Cs as a tracer for the plant absorption of macronutrients. ► A model was developed to simulate the temporal evolution of {sup 137}Cs, K and Na by fruits. ► This model exhibited close agreement with our

  7. Effects of salinity stress on Bufo balearicus and Bufo bufo tadpoles: Tolerance, morphological gill alterations and Na{sup +}/K{sup +}-ATPase localization

    Energy Technology Data Exchange (ETDEWEB)

    Bernabò, Ilaria; Bonacci, Antonella; Coscarelli, Francesca [Department of Ecology, University of Calabria, Via P. Bucci, 87036 Rende (Cosenza) (Italy); Tripepi, Manuela [University of Pennsylvania, Department of Biology, 201 Leidy Laboratories, Philadelphia, PA 19104 (United States); Brunelli, Elvira, E-mail: brunelli@unical.it [Department of Ecology, University of Calabria, Via P. Bucci, 87036 Rende (Cosenza) (Italy)

    2013-05-15

    Freshwater habitats are globally threatened by human-induced secondary salinization. Amphibians are generally poorly adapted to survive in saline environments. We experimentally investigated the effects of chronic exposure to various salinities (5%, 10%, 15%, 20%, 25%, 30% and 35% seawater, SW) on survival, larval growth and metamorphosis of tadpoles from two amphibian populations belonging to two species: the green toad Bufo balearicus and the common toad Bufo bufo. In addition, gill morphology of tadpoles of both species after acute exposure to hypertonic conditions (20%, 25%, and 30% SW) was examined by light and electron microscopy. Tadpoles experienced 100% mortality above 20% SW in B. balearicus while above 15% SW in B. bufo. We detected also sublethal effects of salinity stress on growth and metamorphosis. B. bufo cannot withstand chronic exposure to salinity above 5% SW, tadpoles grew slower and were significantly smaller than those in control at metamorphosis. B. balearicus tolerated salinity up to 20% SW without apparent effects during larval development, but starting from 15% SW tadpoles metamorphosed later and at a smaller size compared with control. We also revealed a negative relation between increasing salt concentration and gill integrity. The main modifications were increased mucous secretion, detachment of external layer, alteration of epithelial surface, degeneration phenomena, appearance of residual bodies, and macrophage immigration. These morphological alterations of gill epithelium can interfere with respiratory function and both osmotic and acid-base regulation. Significant variations in branchial Na{sup +}/K{sup +}-ATPase activity were also observed between two species; moreover an increase in enzyme activity was evident in response to SW exposure. Epithelial responses to increasing salt concentration were different in the populations belonging to two species: the intensity of histological and ultrastructural pathology in B. bufo was

  8. Production of gaseous radiotracers CH{sub 3}I and I{sub 2} through Na{sup 123}I salt

    Energy Technology Data Exchange (ETDEWEB)

    Candeiro, R.E.M., E-mail: ricardocandeiro@cnen.gov.b [Comissao Nacional de Energia Nuclear (DIFOR/CNEN-CE), Fortaleza, CE (Brazil). Distrito de Fortaleza; Brandao, L.B. [Instituto de Engenharia Nuclear (IEN/CNEN-RJ), Rio de Janeiro, RJ (Brazil); Pereira, W.P. [Universidade Federal Fluminense (UFF), Niteroi, RJ (Brazil)

    2011-07-01

    The objective of the present work was to develop, separately, methodology for production of two gaseous tracers through the sodium iodide NaI marked with {sup 123}I. Found in the nature in form different, the iodine has been used in diverse works in the area of the industry and health. These two forms of the gaseous iodine, the methyl iodide, CH{sub 3}I, and molecular iodine, I{sub 2}, are very unstable and volatile in the ambient temperature and presents different problems in clean-up and monitoring systems. The syntheses were processed with sodium iodide (NaI) 1M aqueous solution marked with 1{sup 23I}. The production of gas I{sub 2} was realized with in chlorine acid (HCl) and sodium iodate salt (NaIO{sub 3}) and the CH{sub 3}I was used, the salt of NaI and the reagent (CH{sub 3}){sub 2}SO{sub 4}. The production of gases was initially realized through in unit in glass with an inert material and the purpose was to study the kinetic of reaction and to determine the efficiency of production. The two synthesis occurs in the reaction bottle and after of produced, the gas is stored in the collect bottle that contains a starch solution for fixed the I{sub 2}, and in syntheses of CH{sub 3}I contains a silver nitrate solution for your fixation. To determine the efficiency of production of gases, analytic tests were realized, where the consumption of iodide ions of the bottle of reaction are measured. The optimization of production of the each gaseous tracer was studied varying parameter as: concentration of iodide, concentration of acid and temperature. After, the syntheses of the radiotracers were realized in the compact unit, having been used as main reagent the salt radiated of sodium iodide, Na{sup 123}I. The transportation of elementary iodine and methyl iodine was studied by a scintillation detector NaI (2 x 2)' positioned in the reaction bottle. (author)

  9. Brachytherapy model with sodium pertechnetate-{sup 99m}Tc balloon (Na{sup 99m}TcO{sub 4}{sup -}) for breast cancer: evaluation of dosimetry and cell response; Modelo de braquiterapia com balao de pertecnetato de sodio-{sup 99m}Tc (Na{sup 99m}TcO{sub 4}{sup -}) para cancer de mama: avaliacao da dosimetria e resposta celular

    Energy Technology Data Exchange (ETDEWEB)

    Lima, Carla Flavia de

    2016-07-01

    Breast cancer is the most common type of cancer that affects more women worldwide. Among various treatment options, radiotherapy which is often used as a treatment for locoregional recurrences control or to decrease tumor size. In patients with breast cancer at an early stage, a booster dose (boost) in the primary tumor area can be applied after conventional radiation therapy. There are several drawbacks to applying this technique. In this work we aimed to perform a dosimetric analysis in a breast model, where it put a balloon filled with sodium pertechnetate-{sup 99m}Tc (Na{sup 99m}TcO{sub 4}{sup -}) which in future could be used in preference to other possible therapies. The methodology involved the development of dosimetry in water based on radiochromic films and in a computational voxel thorax model. Calibration protocol achieved a mathematical relation between absorbed dose versus optical density (OD) measured at a set of radiochromic sample films placed at the surface of the balloon plus 1 cm up to 10 cm far, in which theoretical dose values were provided by MCNP modeling, reproducing the water equivalent physical simulator. A voxel model of a female thorax, developed at the SISCODES/MCNP codes, received a filled balloon inside. Spatial dose distribution was generated, illustrating the dose received in the chest wall, glandular tissue, breast skin and lung. The dosimetric findings contribute to present the Na{sup 99m}TcO{sub 4}{sup -} balloon modality which provides a suitable spatial dose distribution in the tumor bed preserving adjacent health tissues. We also studied the radiobiological response radio resistant mammary adenocarcinoma cells (MDAMB231) by exposure of these cells to Na{sup 99m}TcO{sub 4}{sup -} balloon. The findings include the presence of apoptotic cells in the balloon around point out a favorable response. In conclusion, the balloon may represent a viable option in the supplementary therapy of breast cancer in patients who have appropriate

  10. Human microsomal carbonyl reducing enzymes in the metabolism of xenobiotics: well-known and promising members of the SDR superfamily.

    Science.gov (United States)

    Skarydová, Lucie; Wsól, Vladimír

    2012-05-01

    The best known, most widely studied enzyme system in phase I biotransformation is cytochrome P450 (CYP), which participates in the metabolism of roughly 9 of 10 drugs in use today. The main biotransformation isoforms of CYP are associated with the membrane of the endoplasmatic reticulum (ER). Other enzymes that are also active in phase I biotransformation are carbonyl reducing enzymes. Much is known about the role of cytosolic forms of carbonyl reducing enzymes in the metabolism of xenobiotics, but their microsomal forms have been mostly poorly studied. The only well-known microsomal carbonyl reducing enzyme taking part in the biotransformation of xenobiotics is 11β-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase superfamily. Physiological roles of microsomal carbonyl reducing enzymes are better known than their participation in the metabolism of xenobiotics. This review is a summary of the fragmentary information known about the roles of the microsomal forms. Besides 11β-hydroxysteroid dehydrogenase 1, it has been reported, so far, that retinol dehydrogenase 12 participates only in the detoxification of unsaturated aldehydes formed upon oxidative stress. Another promising group of microsomal biotransformation carbonyl reducing enzymes are some members of 17β-hydroxysteroid dehydrogenases. Generally, it is clear that this area is, overall, quite unexplored, but carbonyl reducing enzymes located in the ER have proven very interesting. The study of these enzymes could shed new light on the metabolism of several clinically used drugs or they could become an important target in connection with some diseases.

  11. Cadmium exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, H. [Kumamoto Univ. (Japan). Faculty of Education; Yamaguchi, S.; Murata, H.; Otagiri, M.; Imamura, Y. [Kumamoto Univ. (Japan). Faculty of Pharmaceutical Sciences

    2002-02-01

    Administration of cadmium (Cd) at a dose of 1.23 mg/kg (2.0 mg/kg as CdCl2) markedly decreased the activity of an enzyme (acetohexamide reductase) catalysing the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of male rats. However, the decreased enzyme activity was increased by repeated treatment with testosterone propionate (TP). When male rats were castrated and TP was given to the castrated ones, a similar decrease and increase, as described above, were observed in the microsomal enzyme activity. Cd exposure to male rats induced haemorrhage and atrophy of the testes and significantly diminished serum testosterone levels. There was no possibility that Cd accumulated in liver microsomes of male rats causing direct inhibition of the microsomal enzyme activity. We conclude that Cd exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity. Cd exposure had no effect on acetohexamide reductase activity in liver cytosol of male rats. (orig.)

  12. Neonicotinoid insecticides: reduction and cleavage of imidacloprid nitroimine substituent by liver microsomal and cytosolic enzymes.

    Science.gov (United States)

    Schulz-Jander, Daniel A; Leimkuehler, William M; Casida, John E

    2002-09-01

    The major insecticide imidacloprid (IMI) is known to be metabolized by human cytochrome P450 3A4 with NADPH by imidazolidine hydroxylation and dehydrogenation to give 5-hydroxy-imidacloprid and the olefin, respectively, and by nitroimine reduction and cleavage to yield the nitrosoimine, guanidine, and urea derivatives. More extensive metabolism by human or rabbit liver microsomes with NADPH or rabbit liver cytosol without added cofactor reduces the IMI N-nitro group to an N-amino substituent, i.e., the corresponding hydrazone. A major metabolite on incubation of IMI in the human microsome-NADPH system is tentatively assigned by LC/MS as a 1,2,4-triazol-3-one derived from the hydrazone; the same product is obtained on reaction of the hydrazone with ethyl chloroformate. The hydrazone and proposed triazolone are considered here together (referred to as the hydrazone) for quantitation. Only a portion of the microsomal reduction and cleavage of the nitroimine substituent is attributable to a CYP450 enzyme. The cytosolic enzyme conversion to the hydrazone is inhibited by added cofactors (NAD > NADH > NADP > NADPH) and enhanced by an argon instead of an air atmosphere. The responsible cytosolic enzyme(s) does not appear to be DT-diaphorase (which is inhibited by several neonicotinoids), aldose reductase, aldehyde reductase, or xanthine oxidase. However, the cytosolic metabolism of IMI is inhibited by several aldo-keto-reductase inhibitors (i.e., alrestatin, EBPC, Ponalrestat, phenobarbital, and quercetin). Other neonicotinoids with nitroimine, nitrosoimine, and nitromethylene substituents are probably also metabolized by "neonicotinoid nitro reductase(s)" since they serve as competitive substrates for [(3)H]IMI metabolism.

  13. Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes.

    Science.gov (United States)

    Puccini, P; Menicagli, S; Longo, V; Santucci, A; Gervasi, P G

    1992-11-01

    A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activities towards 7-ethoxycoumarin, 7-ethoxyresorufin, aminopyrine, ethylmorphine and benzphetamine, whereas it catalysed the oxidation of aniline, acetone and thiobenzamide with a high catalytic-centre activity. In addition, ha P-450j catalysed at a high rate the high-affinity component of dimethylnitrosamine N-demethylase; in contrast, only the low-affinity component of diethylnitrosamine N-de-ethylase was efficiently catalysed. The addition of cytochrome b5 to the reconstitution system decreased the Km value for dimethylnitrosamine N-demethylase by a factor of 5 and increased the Vmax. value, and slightly enhanced the other activities. Thiobenzamide and diethyldithiocarbamate were found to be the most effective inhibitors of the ha-P-450j-dependent aniline hydroxylation. Polyclonal antibodies against rat P-450j recognized ha P-450j in immunoblots of control and treated hamster liver microsomes. Treatment of hamsters with acetone increased the apparent abundance of ha P-450j in microsomes, whereas phenobarbital and beta-naphthoflavone did not induce it. Analysis of N-terminal amino acid sequences demonstrated that ha P-450j has a high degree of sequence identity with rat P-450j. All the evidence presented in this study indicates that ha P-450j could represent the hamster orthologue of the previously described CYP2E1(s) of other species.

  14. Effects of Eupatilin and Jaceosidin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Ji Hyun Jeong

    2010-09-01

    Full Text Available Eupatilin and jaceosidin are bioactive flavones found in the medicinal herbs of the genus Artemisia. These bioactive flavones exhibit various antioxidant, antiinflammatory, antiallergic, and antitumor activities. The inhibitory potentials of eupatilin and jaceosidin on the activities of seven major human cytochrome P450 enzymes in human liver microsomes were investigated using a cocktail probe assay. Eupatilin and jaceosidin potently inhibited CYP1A2-catalyzed phenacetin O-deethylation with 50% inhibitory concentration (IC50 values of 9.4 mM and 5.3 mM, respectively, and CYP2C9-catalyzed diclofenac 4-hydroxylation with IC50 values of 4.1 mM and 10.2 mM, respectively. Eupatilin and jaceosidin were also found to moderately inhibit CYP2C19-catalyzed [S]-mephenytoin 4¢-hydroxylation, CYP2D6-catalyzed bufuralol 1¢-hydroxylation, and CYP2C8-catalyzed amodiaquine N-deethylation. Kinetic analysis of human liver microsomes showed that eupatilin is a competitive inhibitor of CYP1A2 with a Ki value of 2.3 mM and a mixed-type inhibitor of CYP2C9 with a Ki value of 1.6 mM. Jaceosidin was shown to be a competitive inhibitor of CYP1A2 with a Ki value of 3.8 mM and a mixed-type inhibitor of CYP2C9 with Ki value of 6.4 mM in human liver microsomes. These in vitro results suggest that eupatilin and jaceosidin should be further examined for potential pharmacokinetic drug interactions in vivo due to inhibition of CYP1A2 and CYP2C9.

  15. Metabolism of Ginger Component [6]-Shogaol in Liver Microsomes from Mouse, Rat, Dog, Monkey, and Human

    Science.gov (United States)

    Chen, Huadong; Soroka, Dominique; Zhu, Yingdong; Sang, Shengmin

    2013-01-01

    Scope There are limited data on the metabolism of [6]-shogaol, a major bioactive component of ginger. This study demonstrates metabolism of [6]-shogaol in liver microsomes from mouse, rat, dog, monkey, and human. Methods and results The in vitro metabolism of [6]-shogaol was compared among five species using liver microsomes from mouse, rat, dog, monkey, and human. Following incubations with [6]-shogaol, three major reductive metabolites 1-(4'-hydroxy-3'-methoxyphenyl)-4-decen-3-ol (M6), 1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-ol (M9), and 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M11), as well as two new oxidative metabolites (1E, 4E)-1-(4'-hydroxy-3'-methoxyphenyl)-deca-1,4-dien-3-one (M14) and (E)-1-(4'-hydroxy-3'-methoxyphenyl)-dec-1-en-3-one (M15) were found in all species. The kinetic parameters of M6 in liver microsomes from each respective species were quantified using Michaelis-Menten theory. A broad CYP-450 inhibitor, 1-aminobenzotriazole, precluded the formation of oxidative metabolites M14 and M15, and 18β-glycyrrhetinic acid, an aldo-keto reductase inhibitor, eradicated the formation of the reductive metabolites M6, M9, and M11 in all species. Metabolites M14 and M15 were tested for cancer cell growth inhibition and induction of apoptosis and both showed substantial activity, with M14 displaying greater potency than [6]-shogaol. Conclusion We conclude that [6]-shogaol is metabolized extensively in mammalian species mouse, rat, dog, monkey, and human, and that there are significant interspecies differences to consider when planning pre-clinical trials towards [6]-shogaol chemoprevention. PMID:23322474

  16. Lucigenin as a substrate of microsomal NAD(P)H-oxidoreductases.

    Science.gov (United States)

    Schepetkin, I A

    1999-01-01

    NADPH oxidation and cytochrome c reduction with and without lucigenin as well as NAD(P)H/lucigenin-dependent chemiluminescence of rat liver microsomes were studied. An increased rate of NADPH oxidation and cytochrome c reduction in the presence of lucigenin was related to one-electron lucigenin reduction by microsomal NADPH reductases. The apparent Michaelis constant values for lucigenin (Km appLuc) were 3.6 and 5.0 microM in normoxygenic (pO2 = 150 +/- 5 mm Hg) and 8.7 and 8.3 microM in hypoxygenic (pO2 = 45 +/- 4 mm Hg) media in the reactions of lucigenin-dependent NADPH oxidation and cytochrome c reduction, respectively. The maximal level of NADPH/lucigenin-dependent chemiluminescence was registered at lucigenin concentration close to the mean K Luc/m app in the lucigenin-reductase reaction. Increasing the lucigenin concentration from 5 to 100 microM was associated with a decrease in the chemiluminescence intensity; this could be due to the inactivation of cytochrome P450. In the presence of superoxide dismutase (SOD), the rate of lucigenin-dependent cytochrome c reduction and NADPH/lucigenin-dependent chemiluminescence were decreased by 10 and 30%, respectively. The addition of lucigenin to microsomes which contain the reduced hemoprotein--CO complex was followed by the disappearance of the differential absorption spectrum specific for the carboxy complex and by increase in chemiluminescence intensity versus the control (without carboxy complex). Thus, lucigenin-dependent chemiluminescence of microsomes may be due to some enzymes including lucigenin reductase (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase), generation of O2-. in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of the cytochrome P450 heme on dioxetane decomposition followed by light quantum emission. Thus, lucigenin cannot be used to measure the basal O2-. formation in tissue homogenates with high levels of NAD(P)H-oxidoreductases.

  17. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cawley, George F.; Ardoin, Taylor G. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Backes, Wayne L. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

  18. Microsomal drug-metabolizing enzymes in the olive baboon (papio anabis)

    DEFF Research Database (Denmark)

    Autrup, Herman; Thurlow, Brenda J.; Wakhisi, Johnston

    1975-01-01

    1.1. The activity of microsomal drug-metabolizing enzymes—azo reductase, nitroreductase, p-hydroxylation, N-demethylation, O-demethylation, NADPH cytochrome c reductase and cytochrome P P-450—in the olive baboon are lower than in other animal species, e.g. mouse, rat, guinea-pig. 2. 2. The level...... is comparable with the level in man for N-demethylation, azo reductase, O-demethylation and NADPH cytochrome c reductase. 3. 3. The ratio of the activity of the p-hydroxylation as compared with cytochrome P-450 is very similar in baboon and man. 4. 4. Arylsulfatase is localized in the lysosomal fraction...

  19. Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes.

    OpenAIRE

    Puccini, P; Menicagli, S; Longo, V.; Santucci, A.; Gervasi, P. G.

    1992-01-01

    A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activitie...

  20. Differences in metabolite-mediated toxicity of tamoxifen in rodents versus humans elucidated with DNA/microsome electro-optical arrays and nanoreactors.

    Science.gov (United States)

    Zhao, Linlin; Krishnan, Sadagopan; Zhang, Yun; Schenkman, John B; Rusling, James F

    2009-02-01

    Tamoxifen, a therapeutic and chemopreventive breast cancer drug, was chosen as a model compound because of acknowledged species specific toxicity differences. Emerging approaches utilizing electro-optical arrays and nanoreactors based on DNA/microsome films were used to compare metabolite-mediated toxicity differences of tamoxifen in rodents versus humans. Hits triggered by liver enzyme metabolism were first provided by arrays utilizing a DNA damage end point. The arrays feature thin-film spots containing an electrochemiluminescent (ECL) ruthenium polymer ([Ru(bpy)(2)PVP(10)](2+); PVP, polyvinylpyridine), DNA, and liver microsomes. When DNA damage resulted from reactions with tamoxifen metabolites, it was detected by an increase in light from the oxidation of the damaged DNA by the ECL metallopolymer. The slope of ECL generation versus enzyme reaction time correlated with the rate of DNA damage. An approximate 2-fold greater ECL turnover rate was observed for spots with rat liver microsomes compared to that with human liver microsomes. These results were supported by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of reaction products using nanoreactors featuring analogous films on silica nanoparticles, allowing the direct measurement of the relative formation rate for alpha-(N(2)-deoxyguanosinyl)tamoxifen. We observed 2-5-fold more rapid formation rates for three major metabolites, i.e., alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and tamoxifen N-oxide, catalyzed by rat liver microsomes compared to human liver microsomes. Comparable formation rates were observed for N-desmethyl tamoxifen with rat and human liver microsomes. A better detoxifying capacity for human liver microsomes than rat liver microsomes was confirmed utilizing glucuronyltransferase in microsomes together with UDP-glucuronic acid. Taken together, lower genotoxicity and higher detoxication rates presented by human liver microsomes correlate with the lower risk of tamoxifen in

  1. Fluorescence polarization study of lipids and membranes prepared from brain hemispheres of a hibernating mammal.

    Science.gov (United States)

    Montaudon, D; Robert, J; Canguilhem, B

    1984-02-29

    The physical behavior of total lipids, microsomes and microsomal lipids prepared from brain hemispheres of European Hamsters (Cricetus cricetus) was approached by the measure of the fluorescence polarization of the probe 1,6-diphenyl 1,3,5-hexatriene. We compare in this study the results obtained for two critical periods for a hibernator: winter (torpid state) and summer (active state). An increase in fluidity was noticed in the winter lipid and membrane preparations. The difference was however of very low magnitude, suggesting that only the microenvironment of some proteins was involved, rather than the bulk membrane fluidity.

  2. Activation and detoxification metabolism of urban air pollutants 2-nitrobenzanthrone and carcinogenic 3-nitrobenzanthrone by rat and mouse hepatic microsomes.

    Science.gov (United States)

    Stiborova, Marie; Cechova, Tereza; Borek-Dohalska, Lucie; Moserova, Michaela; Frei, Eva; Schmeiser, Heinz H; Paca, Jan; Arlt, Volker M

    2012-01-01

    2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. Understanding which enzymes are involved in metabolism of these toxicants is important in the assessment of individual susceptibility. Here, metabolism of 2-NBA and 3-NBA by rat and mouse hepatic microsomes containing cytochromes P450 (CYPs), their reductase (NADPH:CYP reductase), and NADH:cytochrome b5 reductase was investigated under anaerobic and aerobic conditions. In addition, using the same microsomal systems, 2-NBA and 3-NBA were evaluated to be enzymatically activated under anaerobic conditions to species generating 2-NBA- and 3-NBA-derived DNA adducts. High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed for the separation and characterization of 2-NBA and 3-NBA metabolites formed by hepatic microsomes of rats and mice under the anaerobic and aerobic conditions. Microsomal systems isolated from the liver of the control (untreated) rats and rats pretreated with Sudan I, β-naphthoflavone (β-NF), phenobarbital (PB), ethanol and pregnenolon 16α-carbonitrile (PCN), the inducers of cytochromes P450 (CYP) 1A1, 1A1/2, 2B, 2E1 and 3A, respectively, were used in this study. Microsomes of mouse models, a control mouse line (wild-type, WT) and Hepatic Cytochrome P450 Reductase Null (HRN) mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in their livers, were also employed. To detect and quantify the 2-NBA- and 3-NBA-derived DNA adducts, the 32P postlabeling technique was used. Both reductive metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), found to be formed predominantly under the anaerobic conditions, and two 3-NBA oxidative metabolites, whose structures have not yet been investigated, were formed by several microsomal systems used in the study. Whereas a 3-NBA reductive metabolite

  3. Inhibition of microsomal oxidation of alcohols and of hydroxyl-radical-scavenging agents by the iron-chelating agent desferrioxamine.

    Science.gov (United States)

    Cederbaum, A I; Dicker, E

    1983-01-15

    Rat liver microsomes (microsomal fractions) catalyse the oxidation of straight-chain aliphatic alcohols and of hydroxyl-radical-scavenging agents during NADPH-dependent electron transfer. The iron-chelating agent desferrioxamine, which blocks the generation of hydroxyl radicals in other systems, was found to inhibit the following microsomal reactions: production of formaldehyde from either dimethyl sulphoxide or 2-methylpropan-2-ol (t-butylalcohol); generation of ethylene from 4-oxothiomethylbutyric acid; release of 14CO2 from [I-14C]benzoate; production of acetaldehyde from ethanol or butanal (butyraldehyde) from butan-1-ol. Desferrioxamine also blocked the increase in the oxidation of all these substrates produced by the addition of iron-EDTA to the microsomes. Desferrioxamine had no effect on a typical mixed-function-oxidase activity, the N-demethylation of aminopyrine, nor on the peroxidatic activity of catalase/H2O2 with ethanol. H2O2 appears to be the precursor of the oxidizing radical responsible for the oxidation of the alcohols and the other hydroxyl-radical scavengers. Chelation of microsomal iron by desferrioxamine most likely decreases the generation of hydroxyl radicals, which results in an inhibition of the oxidation of the alcohols and the hydroxyl-radical scavengers. Whereas desferrioxamine inhibited the oxidation of 2-methylpropan-2-ol, dimethyl sulphoxide, 4-oxothiomethylbutyrate and benzoate by more than 90%, the oxidation of ethanol and butanol could not be decreased by more than 45-60%. Higher concentrations of desferrioxamine were required to block the metabolism of the primary alcohols than to inhibit the metabolism of the other substrates. The desferrioxamine-insensitive rate of oxidation of ethanol was not inhibited by competitive hydroxyl-radical scavengers. These results suggest that primary alcohols may be oxidized by two pathways in microsomes, one dependent on the interaction of the alcohols with hydroxyl radicals (desferrioxamine

  4. Origanum majorana Essential Oil Lacks Mutagenic Activity in the Salmonella/Microsome and Micronucleus Assays

    Directory of Open Access Journals (Sweden)

    Andrea dos Santos Dantas

    2016-01-01

    Full Text Available The present study aimed to investigate the in vitro mutagenic activity of Origanum majorana essential oil. The most abundant compounds identified by GC-MS were γ-terpinene (25.73%, α-terpinene (17.35%, terpinen-4-ol (17.24%, and sabinene (10.8%. Mutagenicity was evaluated by the Salmonella/microsome test using the preincubation procedure on TA98, TA97a, TA100, TA102, and TA1535 Salmonella typhimurium strains, in the absence or in the presence of metabolic activation. Cytotoxicity was detected at concentrations higher than 0.04 μL/plate in the absence of S9 mix and higher than 0.08 μL/plate in the presence of S9 mix and no gene mutation increase was observed. For the in vitro mammalian cell micronucleus test, V79 Chinese hamster lung fibroblasts were used. Cytotoxicity was only observed at concentrations higher than or equal to 0.05 μg/mL. Moreover, when tested in noncytotoxic concentrations, O. majorana essential oil was not able to induce chromosome mutation. The results from this study therefore suggest that O. majorana essential oil is not mutagenic at the concentrations tested in the Salmonella/microsome and micronucleus assays.

  5. Effect of repeated exposure to aniline, nitrobenzene, and benzene on liver microsomal metabolism in the rat.

    Science.gov (United States)

    Wiśniewska-Knypl, J M; Jablońska, J K; Piotrowski, J K

    1975-02-01

    Exposure of rats to aniline at daily doses of 50 mg/kg of body weight over a month stimulated the microsomal metabolism as manifested by (1) acceleration of p-hydroxylation of anilin and N-demethylation of aminopyrine in 9-000 times g postmitochondrial supernatant of the liver, (2) shortening the sleeping time after hexobarbital, and (3) reduction of the antipyretic effect of phenacetin. In the rats exposed to nitrobenzene in a similar manner to aniline, nitroreduction of nitrobenzene and p-hydroxylation of aniline remained unaffected; the antipyretic effect of phenacetin was decreased, whereas hexobarbital sleeping time remained unchanged. Exposure of rats to benzene (50 mg/kg of body weight daily for a month) had no effect on the rate of hydroxylation of benzene and N-demethylation of aminopyrine. In benzene-exposed rats hexobarbital sleeping time was prolonged whereas the antipyretic effect of phenacetin was unaffected. Microsomal metabolism of aniline, nitrobenzene, and benzene was stimulated and inhibited when the rats were pretreated with phenobarbital and SKF 525-A, respectively.

  6. Metabolism of arachidonic acid in hamster lung microsomes is not completely inhibited by aspirin and indomethacin

    Energy Technology Data Exchange (ETDEWEB)

    Uotila, P.; Paajanen, H.; Schalin, M.; Simberg, N.

    1983-10-01

    Aspirin (100 microM or 1 mM) or indomethacin (10 microM or 100 microM) was incubated with a microsomal preparation of hamster lungs in the presence of NADPH for 10 min. Then 14C-arachidonic acid (20 microM) was added and the incubation was continued for an additional 20 min. The metabolites were extracted with ethyl acetate first at pH 7.4 and then at pH 3.5 and analysed by thin layer chromatography. Both aspirin and indomethacin inhibited dose dependently the formation of all identified prostaglandins, including PGF2 alpha, 6-keto-PGF1 alpha, PGE2 and PGD2. The rate of formation of some unidentified metabolites extracted at pH 7.4 and 3.5 was, however, not changed by aspirin or indomethacin. We have earlier reported that in isolated perfused hamster lungs the formation of all arachidonate metabolites is inhibited by both aspirin and indomethacin. As the present study indicates that in the microsomes of hamster lungs all metabolic pathways of arachidonic acid are not inhibited by aspirin or indomethacin, it is possible that in isolated tissues and in vivo aspirin-like drugs have some other inhibitory effects on arachidonate metabolism than the inhibition of the cyclo-oxygenase enzyme.

  7. Origanum majorana Essential Oil Lacks Mutagenic Activity in the Salmonella/Microsome and Micronucleus Assays

    Science.gov (United States)

    Klein-Júnior, Luiz Carlos; Guecheva, Temenouga N.; dos Santos, Luciana D.; Zanette, Régis A.; de Mello, Fernanda B.; de Mello, João Roberto Braga

    2016-01-01

    The present study aimed to investigate the in vitro mutagenic activity of Origanum majorana essential oil. The most abundant compounds identified by GC-MS were γ-terpinene (25.73%), α-terpinene (17.35%), terpinen-4-ol (17.24%), and sabinene (10.8%). Mutagenicity was evaluated by the Salmonella/microsome test using the preincubation procedure on TA98, TA97a, TA100, TA102, and TA1535 Salmonella typhimurium strains, in the absence or in the presence of metabolic activation. Cytotoxicity was detected at concentrations higher than 0.04 μL/plate in the absence of S9 mix and higher than 0.08 μL/plate in the presence of S9 mix and no gene mutation increase was observed. For the in vitro mammalian cell micronucleus test, V79 Chinese hamster lung fibroblasts were used. Cytotoxicity was only observed at concentrations higher than or equal to 0.05 μg/mL. Moreover, when tested in noncytotoxic concentrations, O. majorana essential oil was not able to induce chromosome mutation. The results from this study therefore suggest that O. majorana essential oil is not mutagenic at the concentrations tested in the Salmonella/microsome and micronucleus assays. PMID:27891531

  8. Localization of the Proton Pump of Corn Coleoptile Microsomal Membranes by Density Gradient Centrifugation 1

    Science.gov (United States)

    Mandala, Suzanne; Mettler, Irvin J.; Taiz, Lincoln

    1982-01-01

    Previous studies characterizing an ATP-dependent proton pump in microsomal membrane vesicles of corn coleoptiles led to the conclusion that the proton pump was neither mitochondrial nor plasma membrane in origin (Mettler, Mandala, Taiz 1982 Plant Physiol 70: 1738-1742). To facilitate positive identification of the vesicles, corn coleoptile microsomal membranes were fractionated on linear sucrose and dextran gradients, with ATP-dependent [14C]methylamine uptake as a probe for proton pumping. On sucrose gradients, proton pumping activity exhibited a density of 1.11 grams/cubic centimeter and was coincident with the endoplasmic reticulum (ER). In the presence of high magnesium, the ER shifted to a heavier density, while proton pumping activity showed no density shift. On linear dextran gradients, proton pumping activity peaked at a lighter density than the ER. The proton pump appears to be electrogenic since both [14C]SCN− uptake and 36Cl− uptake activities coincided with [14C] methylamine uptake on dextran gradients. On the basis of density and transport properties, we conclude that the proton pumping vesicles are probably derived from the tonoplast. Nigericin-stimulated ATPase activity showed a broad distribution which did not coincide with any one membrane marker. PMID:16662755

  9. A Microsomal Proteomics View of H2O2- and ABA-Dependent Responses

    KAUST Repository

    Alquraishi, May Majed

    2017-08-21

    The plant hormone abscisic acid (ABA) modulates a number of plant developmental processes and responses to stress. In planta, ABA has been shown to induce reactive oxygen species (ROS) production through the action of plasma membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases. Although quantitative proteomics studies have been performed to identify ABA- or hydrogen peroxide (H₂O₂)-dependent proteins, little is known about the ABA- and H₂O₂-dependent microsomal proteome changes. Here, we examined the effect of 50 µM of either H₂O₂ or ABA on the Arabidopsis microsomal proteome using tandem mass spectrometry and identified 86 specifically H₂O₂-dependent, and 52 specifically ABA-dependent proteins that are differentially expressed. We observed differential accumulation of proteins involved in the tricarboxylic acid (TCA) cycle notably in response to H₂O₂. Of these, aconitase 3 responded to both H₂O₂ and ABA. Additionally, over 30 proteins linked to RNA biology responded significantly to both treatments. Gene ontology categories such as \\'response to stress\\' and \\'transport\\' were enriched, suggesting that H₂O₂ or ABA directly and/or indirectly cause complex and partly overlapping cellular responses. Data are available via ProteomeXchange with identifier PXD006513.

  10. Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

    Science.gov (United States)

    Wu, Pingzhi; Zhang, Sheng; Zhang, Lin; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2013-10-15

    Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

  11. A Microsomal Proteomics View of H₂O₂- and ABA-Dependent Responses.

    Science.gov (United States)

    Alqurashi, May; Thomas, Ludivine; Gehring, Chris; Marondedze, Claudius

    2017-08-18

    The plant hormone abscisic acid (ABA) modulates a number of plant developmental processes and responses to stress. In planta, ABA has been shown to induce reactive oxygen species (ROS) production through the action of plasma membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases. Although quantitative proteomics studies have been performed to identify ABA- or hydrogen peroxide (H₂O₂)-dependent proteins, little is known about the ABA- and H₂O₂-dependent microsomal proteome changes. Here, we examined the effect of 50 µM of either H₂O₂ or ABA on the Arabidopsis microsomal proteome using tandem mass spectrometry and identified 86 specifically H₂O₂-dependent, and 52 specifically ABA-dependent proteins that are differentially expressed. We observed differential accumulation of proteins involved in the tricarboxylic acid (TCA) cycle notably in response to H₂O₂. Of these, aconitase 3 responded to both H₂O₂ and ABA. Additionally, over 30 proteins linked to RNA biology responded significantly to both treatments. Gene ontology categories such as 'response to stress' and 'transport' were enriched, suggesting that H₂O₂ or ABA directly and/or indirectly cause complex and partly overlapping cellular responses. Data are available via ProteomeXchange with identifier PXD006513.

  12. Participation of cytochrome P-450 in the reduction of nitro compounds by rat liver microsomes.

    Science.gov (United States)

    Harada, N; Omura, T

    1980-05-01

    1. The subcellular distribution of nitrobenzene reduction activity in rat liver cells indicated the existence of two different enzyme systems, one localized in microsomes and the other localized in cytosol. The activity in the cytosol was mainly attributable to xanthine oxidase, judging from its substrate specificity and the inhibition by allopurinol. 2. The participation of the microsomal electron transport system in nitrobenzene reduction was examined by using antibodies against four components of the system, NADPH-cytochrome c reductase (fpT), NADH-cytochrome b5 reductase (fpD), cytochrome b5, and cytochrome P-450. Both NADH- and NADPH-dependent nitrobenzene reduction activities were strongly inhibited by anti-fpT IG and also by anti-P450 IG, but not inhibited by anti-fpD IG or anti-b5 IG. The reduction of nitrosobenzene and phenylhydroxylamine, which are supposed to be the intermediates of nitrobenzene reduction, was also examined, and it was found that NADH- and NADPH-dependent reduction of both compounds were strongly inhibited by anti-fpT IG and anti-P450 IG, but not by anti-fpD IG or anti-b5 IG. 3. Reconstruction experiments using purified NADPH-cytochrome P-450 reductase and cytochrome P-450 were also carried out and it was confirmed that the reduction of nitrobenzene, nitrosobenzene, and phenylhydroxylamine to aniline could be effected by these two components. 4. Nitrobenzene reduction by microsomes exhibited a short initial time lag and was activated by the addition of purified NADPH-cytochrome c reductase, whereas nitrosobenzene and phenylhydroxylamine reductions did not show any initial time lag and were not activated by the reductase. These observations suggest that the reduction of nitrobenzene to an intermediate, possibly nitrosobenzene or phenylhydroxylamine, limits the rate of aniline formation, and such an initial step of nitrobenzene reduction can be catalyzed by NADPH-cytochrome c reductase alone. Cytochrome P-450 is essential at least in the

  13. Effect of cholesterol feeding on tissue lipid perioxidation, glutathione peroxidase activity and liver microsomal functions in rats and guinea pigs

    NARCIS (Netherlands)

    TSAI, A. C.; THIE, G. M.; Lin, C. R.

    1977-01-01

    The effect of cholesterol feeding on liver and aortic nonenzymatic lipid peroxidation and glutathione peroxidase activities, and on liver microsomal NADPH-dependent lipid peroxidation, codeine hydroxylation and cytochrome P-450 levels was examined in rats and guinea pigs. One percent cholesterol was

  14. Activation of the microsomal glutathione-S-transferase and reduction of the glutathione dependent protection against lipid peroxidation by acrolein

    NARCIS (Netherlands)

    Haenen, G R; Vermeulen, N P; Tai Tin Tsoi, J N; Ragetli, H M; Timmerman, H; Blast, A

    1988-01-01

    Allyl alcohol is hepatotoxic. It is generally believed that acrolein, generated out of allyl alcohol by cytosolic alcohol dehydrogenase, is responsible for this toxicity. The effect of acrolein in vitro and in vivo on the glutathione (GSH) dependent protection of liver microsomes against lipid

  15. Expression of microsomal prostaglandin E synthase-1 in intestinal type gastric adenocarcinoma and in gastric cancer cell lines

    NARCIS (Netherlands)

    van Rees, Bastiaan P.; Sivula, Anna; Thorén, Staffan; Yokozaki, Hiroshi; Jakobsson, Per-Johan; Offerhaus, G. Johan A.; Ristimäki, Ari

    2003-01-01

    Gastrointestinal carcinomas synthesize elevated levels of prostaglandin E(2) (PGE(2)), which has been mechanistically linked to carcinogenesis. Recently, microsomal prostaglandin E synthase-1 (mPGES-1) was cloned, which seems to be inducible and linked to cyclooxygenase-2 (Cox-2) in the biosynthesis

  16. Lysosomal degradation of cell organelles. II. Ultrastructural analysis of uptake and digestion of intravenously injected microsomes and ribosomes by Kupffer cells.

    Science.gov (United States)

    Glaumann, H; Berezesky, I K; Ericsson, J L; Trump, B F

    1975-09-01

    Rough and smooth microsomes, "mixed" or total microsomes, and ribosomes were isolated from one single rat liver and subsequently injected intravenously into a series of inbred rats. The uptake and the degradation of the injected organelles by Kupffer cells were followed by means of electron microscopic analysis. By 1 minute after injection, microsomes were seen attached to the surface of Kupffer cells separated by a gap of 200 to 300 A. No attachment to hepatocytes, fat-storing cells, or endothelial cells was seen. By 5 and 10 minutes, most microsomes were phagocytosed and sequestered in large numbers within single membrane-enclosed vacuoles or phagosomes. The engulfment proceeded by two mechanisms: (1) most frequently, flaplike processes of cytoplasm embraced aggregates of microsomes, concomitant with the formation of indention of the cytoplasm; (2) occasionally, single microsomal profiles were taken up by bristle-coated endocytic vacuoles. Ribosomes were also seen penetrating into the wormlike structures (micropinocytosis vermiformis) at the cell surface. At 30 minutes after injection, clear signs of alteration were noted starting with vesicle aggregation, clumping, and elongation of the microsomal profiles. The ribosomes were quickly stripped from their microsomal membranes and marginated to the inside of the vacuoles but separated from the limiting membrane by a distance of 200 to 300 A. By 1 and 2 hours, disruption of the vesicles into membrane fragments and formation of dense material in and between the profiles occurred. By 8 hours it was difficult to recognize the degradation products as membrane derivatives. The digestive vacuoles retained their size at this time interval. Typical pentalaminar structures were observed. By 14 to 24 hours the digestive vacuoles became electron lucent and appeared to shrink, and in addition to containing various types of granular material, many were laden with lipid-like droplets presumed to be conglomerates of phospholipid

  17. A comparative study of 19-iodocholesterol-{sup 125}I 3-acetate and Na{sup 125}I in liquid scintillation measurements; Estudio comparativo del acetato de 19-iodocolesterol- ``125 I con Na ``125I en medidas por centelleo liquido

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez Barquero, L.; Grau Malonda, A.; Los Arcos Merino, J.M.; Grau Carles, A.

    1994-08-01

    A comparative study of performance of 19-iodocholesterol-{sup 125}I 3-acetate and sodium iodine samples labelled with {sup 125}I is presented for liquid scintillation counting measurements. Quench effect, count rate stability and spectral evolution of samples have been followed for several weeks in Toluene, Hisafe II, Instagel, Dioxane-naphthalene and Toluene-alcohol scintillators. Organic samples have negligible quench effect in the interval of I{sup -} concentration of 0-90 ug and inorganic samples only show a very small variation, lower than 12%, for Dioxane-naphthalene, in the same range of concentration. Satisfactory stability is obtained in general for both, organic and inorganic samples, but small counting losses, 0.03% for 19-iodocholesterol-{sup 125}I 3-acetate samples in Toluene-alcohol and 0.04% for Na{sup 125}I samples in Dioxane-naphthalene and Toluene-alcohol, have been reported. (Author) 8 refs.

  18. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Yoshimura, Kazunori [Department of Physiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kobayashi, Nobuharu; Sugiyama, Kazuo [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Sawada, Jun-ichi; Saito, Yoshiro [Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

  19. Phytoecdysteroid C2-hydroxylase is microsomal in spinach, Spinacia oleracea L.

    Science.gov (United States)

    Bakrim, Ahmed; Guittard, Emilie; Maria, Annick; De Virville, Jacques Davy; Lafont, René; Takvorian, Najat

    2009-12-01

    An enzyme involved in the biosynthesis of phytoecdysteroids, the C2-hydroxylase, has been investigated in spinach, Spinacia oleracea. This enzyme is microsomal and its K(m) has been determined using 2-deoxy-20-hydroxyecdysone as substrate (K(m)=3.72 microM). It is much more efficient with 2-deoxy-20-hydroxyecdysone than with 2-deoxyecdysone and, conversely, the C20-hydroxylase is more active on 2-deoxyecdysone than on ecdysone. These data support the conclusion that C20-hydroxylation precedes C2-hydroxylation. The C2-hydroxylase is inhibited by high concentrations of 20E. Substrate specificity and subcellular localization of C2-hydroxylase differ between plants and insects, and these data, as well as those previously reported on other biosynthetic steps, show the great difference between plant and insect ecdysteroid biosynthetic pathways and suggest an independent origin for the pathways in both kingdoms. (c) 2009 Wiley Periodicals, Inc.

  20. Differential inhibition of aflatoxin B1 oxidation by gestodene action on human liver microsomes.

    Science.gov (United States)

    Kim, B R; Oh, H S; Kim, D H

    1997-11-01

    Human cytochrome P450 (P450) 3A is known to be involved in the formation of both aflatoxin B1-exo-8,9-epoxide (exo-epoxidation) and aflatoxin Q1 (3 alpha-hydroxylation). Gestodene, a known inactivator of P450 3A4, inhibited the formation of AFB1 metabolites in a variety of ways depending on the incubation condition. Preincubation of gestodene with human liver microsomes prior to the addition of AFB1 inhibited both exo-epoxidation and 3 alpha-hydroxylation whereas simultaneous incubation of gestodene with AFB1 only inhibited 3 alpha-hydroxylation. These results suggest that two independent substrate binding sites exist in P450 3A4, and AFB1 binds to both of the binding sites. Gestodene selectively binds to one of the binding sites leading to the formation of AFQ1, whereas it does not affect the formation of exo-epoxide via the other binding site.

  1. Effect of heating oxymyoglobin and metmyoglobin on the oxidation of muscle microsomes.

    Science.gov (United States)

    Bou, Ricard; Guardiola, Francesc; Codony, Rafael; Faustman, Cameron; Elias, Ryan J; Decker, Eric A

    2008-10-22

    Myoglobin (Mb) and its iron have been proposed to be major prooxidants in cooked meats. To understand the mechanisms and differentiate between the prooxidant and antioxidant potential of oxymyoglobin (OxyMb) and metmyoglobin (MetMb), their prooxidant activity, iron content, solubility, free radical scavenging activity, and iron binding capacity were determined as a function of thermal processing. The ability of native and heat denatured OxyMb and MetMb to promote the oxidation of muscle microsomes was different. MetMb promoted lipid oxidation in both its native and denatured states. Conversely, OxyMb became antioxidative when the protein was heated to temperatures >or=75 degrees C. The increased antioxidant activity of heat denatured OxyMb was likely due to a decrease in its prooxidative activity due to its loss of solubility. These data show that the impact on oxidative reactions of Mb is the result of the balance between its antioxidant and prooxidant activities.

  2. Purification and characterization of a 4-hydroxybiphenyl UDP-glucuronosyltransferase from rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Styczynski, B.; Green, M.; Coffman, B.; Puig, J.; Tephly, T. (Univ. of Iowa, Iowa City (United States))

    1991-03-11

    A phenobarbital-inducible rat liver microsomal UDP-glucuronosyltransferase (4-HBP UDPGT) which catalyzes the glucuronidation of 4-hydroxybiphenyl has been purified to homogeneity. The apparent subunit molecular weight of this protein is 52,500 as determined by SDS-PAGE. 4-HBP UDPGT was shown to react with 4-hydroxybiphenyl, p-nitrophenol and 4-methylumbelliferone, but did not react with morphine, testosteron or chloramphenicol. Upon treatment with Endoglycosidase H, the 4-HBP UDPGT underwent about a 2,000 dalton decrease in subunit molecular weight, suggesting that this protein in N-glycosylated. Western blot analysis has revealed immunorecognition of 4-HBP UDPGT by antibodies raised in rabbit against rat 3{alpha}- and 17{beta}-hydroxysteroid UDPGTs. Additionally, the authors have obtained the N-terminal amino acid sequence of 4-HBP UDPGT. These data provide evidence which suggests that this protein is different from another UDPGT previously shown to react with 4-hydroxybiphenyl, testosterone and chloramphenicol.

  3. Transporter-mediated uptake of UDP-glucuronic acid by human liver microsomes: assay conditions, kinetics, and inhibition.

    Science.gov (United States)

    Rowland, Andrew; Mackenzie, Peter I; Miners, John O

    2015-01-01

    This study characterized the kinetics, variability, and factors that affect UDP-glucuronic acid (UDP-GlcUA) uptake by human liver microsomes (HLM). Biphasic kinetics were observed for UDP-GlcUA uptake by HLM. Uptake affinities (assessed as Kd) of the high- and low-affinity components differed by more than an order of magnitude (13 ± 6 vs. 374 ± 175 µM), but were comparable in terms of the maximal rate of uptake, with mean Vmax values differing less than 2.3-fold (56 ± 26 vs. 131 ± 35 pmol/min per mg). Variability in total intrinsic transporter activity (Uint) for microsomal UDP-GlcUA uptake across 12 livers was less than 4-fold. Experiments performed to optimize the conditions for microsomal UDP-GlcUA uptake demonstrated that both components were trans-stimulated by preloading (luminal addition) with an alternate UDP-sugar, and essentially abolished by the thiol-alkylating agent N-ethylmaleimide. Furthermore, interaction studies undertaken with a panel of drugs, alternate UDP-sugars, and glucuronide conjugates, at low (2.5 μM) and high (1000 μM) UDP-GlcUA concentrations, demonstrated that both components were inhibited to varying extents. Notably, the nucleoside analogs zidovudine, stavudine, lamivudine, and acyclovir inhibited both the high- and low- affinity components of microsomal UDP-GlcUA uptake by >45% at an inhibitor concentration of 100 μM. Taken together, these data demonstrate that human liver microsomal UDP-GlcUA uptake involves multiple protein-mediated components, and raises the possibility of impaired in vivo glucuronidation activity resulting from inhibition of UDP-GlcUA uptake into the endoplasmic reticulum membrane by drugs and other compounds. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Cytochrome P450 isoenzymes in rat and human liver microsomes associate with the metabolism of total coumarins in Fructus Cnidii.

    Science.gov (United States)

    Hu, Xiao; Huang, Wei; Yang, Yuan

    2015-12-01

    Fructus Cnidii (Cnidium) is isolated from the dry and ripe fruit of Cnidium monnier (L.) Cuss (umbelifera), an annual herb. It is demonstrated that the active constituents of Fructus Cnidii are coumarins, known as Total Coumarins of Cnidium Monnier (TCCM). Osthole (Ost) and imperatorin (Imp) are the most active constituents of TCCM which are usually regarded as the quality indicators of medicinal Fructus Cnidii. The aim is to study the metabolism of Fructus Cnidii effective monomer osthole and imperatorin in vitro by liver microsomes. CYP3A4 inhibitor ketoconazole, CYP2D6 inhibitor qunidine, CYP2C8 inhibitor trimethoprim, CYP2C9 inhibitor sulfaphenazole, and CYP1A2 inhibitor α-naphthoflavone were used to investigate the metabolism from incubation time, substrate concentration and liver microsomal concentration, respectively. The concentration of liver microsomes was 0.2 mg/ml. Ost (0.8/3.2/12.8 uM) was incubated at 37 °C for 20 min while Imp (1.6/6.4/19.2 uM) was incubated for 30 min. Qunidine, trimethoprim and α-naphthoflavone could significantly inhibit the disappearance of Imp; meanwhile ketoconazole, sulfaphenazole and qunidine could inhibit the disappearance of Ost. CYP1A, CYP2C are involved in the metabolism of Imp and CYP3A mediates the metabolism of Ost in rat liver microsomes. In human liver microsomes, CYP1A2, CYP2C8, CYP2D6 are involved in the metabolism of Imp; CYP3A4 is involved in the metabolism of Ost at all the tested concentrations of Ost, while CYP2C9, CYP2D6 mediate the metabolism at high concentration of Ost.

  5. Diffusion of HTO, {sup 36}Cl{sup -}, {sup 125}I{sup -} and {sup 22}Na{sup +} in Opalinus Clay: Effect of Confining Pressure, Sample Orientation, Sample Depth and Temperature

    Energy Technology Data Exchange (ETDEWEB)

    Van Loon, L.R.; Soler, J.M

    2004-02-01

    Effective diffusion coefficients (D{sub e}), rock capacity factors ({alpha}) and diffusion-accessible porosities ({epsilon}) were measured using the through-diffusion technique. Transport (diffusion) was measured both perpendicular and parallel to the bedding. Special cells that allowed the application of an axial confining pressure were designed. The pressures applied ranged from 1 to 5 MPa for Mont Terri samples and between 4 and 15 MPa for Benken samples, the upper values representing the in-situ confining pressure at both locations. The test solutions used in the experiments were synthetic Opalinus Clay pore water, which has Na and Cl as main components (Mont Terri: I = 0.39 M; Benken: I = 0.20 M). Pressure only had a small effect on the value of the effective diffusion coefficients. In the case of Mont Terri samples, increasing the pressure from 1 to 5 MPa resulted in a decrease of the effective diffusion coefficient of 20% for HTO, 27% for {sup 36}Cl{sup -}, 29% for {sup 125}I{sup -} and 17 % for {sup 22}Na{sup +}. In the case of Benken samples, increasing the pressure from 4 to 15 MPa resulted in a decrease of D{sub e} of 17% for HTO, 22% for {sup 36}Cl{sup -}, 32% for {sup 125}I{sup -} and 17 % for {sup 22}Na{sup +}. Moreover, the effective diffusion coefficients for for {sup 36}Cl{sup -}are smaller than for HTO, which is consistent with an effect arising from anion exclusion. This ion exclusion effect is smaller in samples from Mont Terri than in samples from Benken, which can be explained by the higher ionic strength of the Mont Terri water used in the experiments. The diffusion of {sup 22}Na{sup +} is similar to that of HTO in the case of Mont Terri OPA. For Benken OPA, the D{sub e} value of {sup 22}Na{sup +} is a factor of 2 higher than that of HTO. This last observation cannot be explained so far but is comparable to experimental data from ANDRA (1999) on Callovo-Oxfordian claystones from the Meuse/Haute Same site. {sup 125}I{sup -} is retarded with

  6. Identification of three genes encoding microsomal oleate desaturases (FAD2) from the oilseed crop Camelina sativa.

    Science.gov (United States)

    Kang, Jinling; Snapp, Anna R; Lu, Chaofu

    2011-02-01

    Camelina sativa is a re-emerging low-input oilseed crop that may provide economical vegetable oils for industrial applications. It is desirable to increase the monounsaturated oleic acid (cis-9-octadecenoic acid, 18:1), and to decrease polyunsaturated fatty acids (PUFA), linoleic (cis, cis-9,12-octadecadienoic acid, 18:2) and α-linolenic (all-cis-9,12,15-octadecatrienoic acid, 18:3) acids, in camelina oils to improve oxidative stability. 18:1 desaturation is mainly controlled by the microsomal oleate desaturase (FAD2; EC 1.3.1.35) encoded by the FAD2 gene. Three FAD2 genes, designated CsFAD2-1 to 3, were identified in camelina. Functional expression of these genes in yeast confirmed that they all encode microsomal oleate desaturases. Although the three CsFAD2 genes share very high sequence similarity, they showed different expression patterns. Expression of CsFAD2-1 was detected in all tissues examined, including developing seed, flower, as well as in vegetable tissues such as leaf, root, and stem. Transcripts of CsFAD2-2 and CsFAD2-3 were mainly detected in developing seeds, suggesting their major roles in storage oil desaturation in seed. The introns of the three CsFAD2 genes, which showed greater sequence variations, may provide additional resources for designing molecular markers in breeding. Furthermore, the roles of CsFAD2 in PUFA synthesis were demonstrated by mutant analysis and by antisense gene expression in camelina seed. Published by Elsevier Masson SAS.

  7. Activity of liver microsomal enzymes during the chronic phase of murine schistosomiasis

    Directory of Open Access Journals (Sweden)

    F.P. Conte

    2007-05-01

    Full Text Available The effects of schistosomiasis on microsomal enzymes were studied on post-infection day 90 when accumulated damage and fibrosis are most intense but granulomatous reaction around the eggs harbored in the liver is smaller than during the earlier phases. Swiss Webster (SW and DBA/2 mice of either sex (N = 12 per sex per group were infected with 100 Schistosoma mansoni cercariae on postnatal day 10 and killed on post-infection day 90. Cytochrome P-450 (CYP concentration and alkoxyresorufin-O-dealkylases (EROD, MROD, BROD, and PROD, p-nitrophenol-hydroxylase (PNPH, coumarin-7-hydroxylase (COH, and UDP-glucuronosyltransferase (UGT activities were measured in hepatic microsomes. Age-matched mice of the same sex and strain were used as controls. In S. mansoni-infected mice, CYP1A- and 2B-mediated activities (control = 100% were reduced in SW (EROD: male (M 36%, female (F 38%; MROD: M 38%, F 39%; BROD: M 46%, F 19%; PROD: M 50%, F 28% and DBA/2 mice (EROD: M 64%, F 58%; MROD: M 60%; BROD: F 49%; PROD: M 73% while PNPH (CYP2E1 was decreased in SW (M 31%, F 38% but not in DBA/2 mice. COH did not differ between infected and control DBA/2 and UGT, a phase-2 enzyme, was not altered by infection. In conclusion, chronic S. mansoni infection reduced total CYP content and all CYP-mediated activities evaluated in SW mice, including those catalyzed by CYP2E1 (PNPH, CYP1A (EROD, MROD and 2B (BROD, PROD. In DBA/2 mice, however, CYP2A5- and 2E1-mediated activities remained unchanged while total CYP content and activities mediated by other CYP isoforms were depressed during chronic schistosomiasis.

  8. Brain Basics

    Medline Plus

    Full Text Available ... About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain Brain ... called the hypothalamic-pituitary-adrenal (HPA) axis. Brain Basics in Real Life Brain Basics in Real Life— ...

  9. The effect of ghee (clarified butter) on serum lipid levels and microsomal lipid peroxidation

    Science.gov (United States)

    Sharma, Hari; Zhang, Xiaoying; Dwivedi, Chandradhar

    2010-01-01

    Ghee, also known as clarified butter, has been utilized for thousands of years in Ayurveda as a therapeutic agent. In ancient India, ghee was the preferred cooking oil. In the last several decades, ghee has been implicated in the increased prevalence of coronary artery disease (CAD) in Asian Indians due to its content of saturated fatty acids and cholesterol and, in heated ghee, cholesterol oxidation products. Our previous research on Sprague-Dawley outbred rats, which serve as a model for the general population, showed no effect of 5 and 10% ghee-supplemented diets on serum cholesterol and triglycerides. However, in Fischer inbred rats, which serve as a model for genetic predisposition to diseases, results of our previous research showed an increase in serum total cholesterol and triglyceride levels when fed a 10% ghee-supplemented diet. In the present study, we investigated the effect of 10% dietary ghee on microsomal lipid peroxidation, as well as serum lipid levels in Fischer inbred rats to assess the effect of ghee on free radical mediated processes that are implicated in many chronic diseases including cardiovascular disease. Results showed that 10% dietary ghee fed for 4 weeks did not have any significant effect on levels of serum total cholesterol, but did increase triglyceride levels in Fischer inbred rats. Ghee at a level of 10% in the diet did not increase liver microsomal lipid peroxidation or liver microsomal lipid peroxide levels. Animal studies have demonstrated many beneficial effects of ghee, including dose-dependent decreases in serum total cholesterol, low density lipoprotein (LDL), very low density lipoprotein (VLDL), and triglycerides; decreased liver total cholesterol, triglycerides, and cholesterol esters; and a lower level of nonenzymatic-induced lipid peroxidation in liver homogenate. Similar results were seen with heated (oxidized) ghee which contains cholesterol oxidation products. A preliminary clinical study showed that high doses of

  10. The effect of ghee (clarified butter) on serum lipid levels and microsomal lipid peroxidation.

    Science.gov (United States)

    Sharma, Hari; Zhang, Xiaoying; Dwivedi, Chandradhar

    2010-04-01

    Ghee, also known as clarified butter, has been utilized for thousands of years in Ayurveda as a therapeutic agent. In ancient India, ghee was the preferred cooking oil. In the last several decades, ghee has been implicated in the increased prevalence of coronary artery disease (CAD) in Asian Indians due to its content of saturated fatty acids and cholesterol and, in heated ghee, cholesterol oxidation products. Our previous research on Sprague-Dawley outbred rats, which serve as a model for the general population, showed no effect of 5 and 10% ghee-supplemented diets on serum cholesterol and triglycerides. However, in Fischer inbred rats, which serve as a model for genetic predisposition to diseases, results of our previous research showed an increase in serum total cholesterol and triglyceride levels when fed a 10% ghee-supplemented diet. In the present study, we investigated the effect of 10% dietary ghee on microsomal lipid peroxidation, as well as serum lipid levels in Fischer inbred rats to assess the effect of ghee on free radical mediated processes that are implicated in many chronic diseases including cardiovascular disease. Results showed that 10% dietary ghee fed for 4 weeks did not have any significant effect on levels of serum total cholesterol, but did increase triglyceride levels in Fischer inbred rats. Ghee at a level of 10% in the diet did not increase liver microsomal lipid peroxidation or liver microsomal lipid peroxide levels. Animal studies have demonstrated many beneficial effects of ghee, including dose-dependent decreases in serum total cholesterol, low density lipoprotein (LDL), very low density lipoprotein (VLDL), and triglycerides; decreased liver total cholesterol, triglycerides, and cholesterol esters; and a lower level of nonenzymatic-induced lipid peroxidation in liver homogenate. Similar results were seen with heated (oxidized) ghee which contains cholesterol oxidation products. A preliminary clinical study showed that high doses of

  11. Changes in the plasmatic membrane characteristics during microsomal monooxygenase induction in the liver of adult and old rats.

    Science.gov (United States)

    Frolkis, V V; Kobzar, A L; Paramonova, G I

    1995-05-12

    The experiments on adult (6-8 months) and old (24-26 months) male Wistar rats have shown that treatment of animals with phenobarbital results in a significant increase in hepatic microsomal enzyme content, plasmatic membrane Na+, K(+)-ATPase activities and the elevation of hepatocyte membrane potential value. It is presumed that the changes in plasmatic membrane characteristics during microsomal monooxygenase induction are related to the synthesis of specific intracellular factors (invertors). This assumption was verified by the experiments with 'cellular hybrid' system (cytosol--plasmatic membranes). Using this cross-systems, it was shown that the hepatocyte cytosol of rats treated with phenobarbital produced Na+, K(+)-ATPase activity. The extent of Na+, K(+)-ATPase activation was essentially lower when cytosol derived from old rat hepatocytes was used. The presence of specific factors that activated Na+, K(+)-ATPase in hepatocyte plasmatic membrane was also discovered in blood serum of induced adult and old rats.

  12. Determination of fluoxetine and its metabolite norfluoxetine in human liver microsomes by reversed-phase HPLC in vitro.

    Science.gov (United States)

    Liu, Z Q; Cheng, Z N; Wang, W; Tan, Z R; Ou-Yang, D S; Zhou, H H

    2000-11-01

    A high-performance liquid chromatography (HPLC) method was developed for the determination of fluoxetine (FLU) and its metabolite norfluoxetine (N-FLU) in human liver microsomes in vitro. An incubation buffer containing human liver microsomes, NADPH-generating system, and FLU, after termination of enzyme reaction and addition of nortriptyline (NOR) as internal standard (IS), was extracted with n-hexane/acetonitrile, and separated on a reversed-phase ODS column. Detection was achieved at 226 nm by ultraviolet detector (UV). The limit of detection was 5 micrograms/L for both FLU and N-FLU. No potential interference was found. The method provides recoveries of up to 94%-104% and acceptable coefficients of variation were found for both within-run (< 7.8%) and day to day (< 9.1%) assays. This method is rapid, sensitive, and simple for studying the metabolism of FLU and N-FLU.

  13. SUPRESSION OF MICROSOMAL OXIDATION WEAKENS HISTOCHROME’S DIURETIC EFFECT AT RATS

    Directory of Open Access Journals (Sweden)

    O. S. Talalaeva

    2013-01-01

    Full Text Available Histochrome is the medicinal form of echinochrome (2, 3, 5, 6, 8-pentahydroxy-7-ethyl-1,4-naphthoquinone. Arisen during clinical application of the drug questions concerning its biotransformation have predetermined the aim of this research: to study participation liver monooxygenase system in maintenance of histochrome’s pharmacological activity.Simple and informative method of the lifetime control of liver monooxygenase systems influence on a metabolism of a medical product is the estimation of changes of pharmacological effect of a r esearched preparation on a background microsomal oxidations i nhibitor. In experiments on rats chloramphenicol action on diuretic effect of histochrome, as the most convenient for screening, was i nvestigated.To control group of animals during 10 days were hypodermically entered by histochrome in a doze of 10 mg/kg (n = 15. Experimental animals preliminary oral received 50 mg/kg of chloramphenicol before three hours of histochrome introduction (n = 16. In both groups of animals measured volume daily excretion of water, creathinin, sodium and potassium ions excretions in experimental rats each two days. The initial level of parameters of excretory kidneys functions were estimated before introduction of preparations at animals.Long-term histochrome’s injection was followed by a fivefold increasing of water excretion and simultaneously creathinin growth one. Allocation of ions of sodium was statistically significantly increased by 11-th day of experiment, and potassium ions – since the ninth day of histochrome injection. In conditions preliminary chloramphenicol applications volume daily daily urine output and creathinin excretion were essentially less control parameters. Allocation with urine of ions of sodium was decreased almost twice in comparison with the values, fixed at introduction histochrome. Excretion potassium ions ware corresponded to an initial level during all period of supervision.Taking into

  14. Oxidative denitrification of 2-nitropropane and propane-2-nitronate by mouse liver microsomes: lack of correlation with hepatocytotoxic potential.

    Science.gov (United States)

    Dayal, R; Goodwin, B; Linhart, I; Mynett, K; Gescher, A

    1991-01-01

    2-Nitropropane (2-NP) is an industrial chemical with hepatotoxic and genotoxic properties. It exists in chemical equilibrium with propane-2-nitronate, which is much more genotoxic than 2-NP. In this work the link between toxicity and metabolism of 2-NP and its nitronate was investigated. To that end 2-NP or propane-2-nitronate were incubated with murine hepatic microsomes at concentrations of up to 10 mM, and generation of nitrite was measured as product of metabolic oxidation of the two species. Under the acidic reaction conditions of the colorimetric nitrite assay propane-2-nitronate decomposed chemically to nitrite. Therefore an ion-pair HPLC assay at neutral pH was developed which enabled determination of nitrite formed from the nitronate. The rate of metabolic nitrite generation from propane-2-nitronate was 5-10-fold that obtained with 2-NP. Metabolism of either species to nitrite was dependent on the presence in the incubate of viable microsomes and of NADPH, and it was inhibited in the presence of carbon monoxide or the cytochrome P-450 inhibitor SKF525A. Acetone could also be measured as a metabolite of 2-NP. Optical difference spectra were recorded in mixtures of propane-2-nitronate with liver microsomes from phenobarbital-pretreated rats. The spectral dissociation constant was found to be 30 mM, which compares with 10 mM reported for 2-NP. 2-NP and propane 2-nitronate were incubated with mouse hepatocytes in suspension and cytotoxicity was determined by measurement of leakage of cellular lactate dehydrogenase into the medium. Both species were hardly toxic, as concentrations of 20 mM were required to elicit significant damage to the cells. The results demonstrate that propane-2-nitronate, like 2-NP, undergoes microsomal oxidative denitrification, probably catalysed by cytochrome P-450. Metabolism of both species occurs at markedly different rates, but the difference in metabolism is not reflected by a difference in hepatocytotoxic potential.

  15. Metabolism of aildenafil in vivo in rats and in vitro in mouse, rat, dog, and human liver microsomes.

    Science.gov (United States)

    Li, Yan; Wu, Linan; Gu, Yuan; Si, Duanyun; Liu, Changxiao

    2014-06-01

    Aildenafil, 1-{[3-(6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4, 3-d] primidin-5-yl)-4-ethoxyphenyl] sulfonyl}-cis-3, 5-dimethylpiperazine, a phosphodiesterase type V enzyme inhibitor (PDE5I), is under development for treatment of erectile dysfunction (ED). The purpose of this study was to elucidate metabolism of aildenafil in vivo in rats and in vitro in mouse, rat, dog, and human liver microsomes. Thirty-one phase I metabolites have been found by LTQ/Orbitrap hybrid mass spectrometry in rat urine, faeces, and bile after oral administration. Major biotransformation pathways of aildenafil included N-dealkylation of the piperazine ring, hydroxylation and dehydrogenation, aliphatic hydroxylation and loss of alkyl group of piperazine ring. Minor pathways involved hydroxylation on the phenyl ring, pyrazole N-demethylation, O-deethylation, loss of piperazine ring (cleavage of N-S bond) and dehydrogenation on the piperazine ring. Similar metabolic pathways of aildenafil were observed in the incubations of liver microsomes from mouse, rat, and dog as well as from human. The depletion rate of parent drug in mouse and rat liver microsomes was significantly different from that in human liver microsomes. The cytochrome P450 reaction phenotyping analysis was conducted using isozyme-specific inhibitors. The results indicated that CYP3A was the main isoenzyme involved in oxidative metabolism of aildenafil. Overall, these in vitro and in vivo findings should provide valuable information on possible metabolic behaviours of aildenafil in humans. Copyright © 2013 John Wiley & Sons, Ltd.

  16. Metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver microsomes and cryopreserved human hepatocytes.

    Science.gov (United States)

    Klette, K L; Anderson, C J; Poch, G K; Nimrod, A C; ElSohly, M A

    2000-10-01

    The metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) was investigated in liver microsomes and cyropreserved hepatocytes from humans. Previous studies have demonstrated that O-H-LSD is present in human urine at concentrations 16-43 times greater than LSD, the parent compound. Additionally, these studies have determined that O-H-LSD is not generated during the specimen extraction and analytical processes or due to parent compound degradation in aqueous urine samples. However, these studies have not been conclusive in demonstrating that O-H-LSD is uniquely produced during in vivo metabolism. Phase I drug metabolism was investigated by incubating human liver microsomes and cryopreserved human hepatocytes with LSD. The reaction was quenched at various time points, and the aliquots were extracted using liquid partitioning and analyzed by liquid chromatography-mass spectrometry. O-H-LSD was positively identified in all human liver microsomal and human hepatocyte fractions incubated with LSD. In addition, O-H-LSD was not detected in any microsomal or hepatocyte fraction not treated with LSD nor in LSD specimens devoid of microsomes or hepatocytes. This study provides definitive evidence that O-H-LSD is produced as a metabolic product following incubation of human liver microsomes and hepatocytes with LSD.

  17. Microsomal glucosidases of rat liver. Partial purification and inhibition by disaccharides.

    Science.gov (United States)

    Ugalde, R A; Staneloni, R J; Leloir, L F

    1980-12-01

    Further work on microsomal glucosidases of rat liver has confirmed that at least two enzymes are involved in the removal of glucose from the glucose-containing oligosaccharide. One acts on the oligosaccharide containing three glucose residues and another on the oligosaccharide which has one or two glucoses. The glucosidase which acts on (Glc)2(Man)9(GlcNAc)2 could be purified with a Concanavalin-A--Sepharose column following by electrofocusing. This purified preparation was active on the oligosaccharide containing one or two glucoses. Heat inactivation and inhibition by disaccharides was parallel for both activities. Inhibition of the glucosidase active on (Glc)3(Man)9(GlcNAC)2 was obtained with kojibiose which has an alpha 1-2 linkage, while the glucosidase acting on (Glc)1-2(Man)9-(GlcNAc)2 was inhibited by nigerose (alpha 1-3 linkage), maltose (alpha 1-4 linkage) and glucose at a higher concentration. None of the beta anomers inhibited. These results are consistent with an alpha configuration of the three glucoses of the dolichyl-diphosphate-linked oligosaccharide. Kojibiose was found to inhibit glucosidase action not only on the free oligosaccharide but also on protein-bound one.

  18. Microsomal epoxide hydrolase genotypes and the risk for head and neck cancer.

    Science.gov (United States)

    Lacko, Martin; Roelofs, Hennie M J; Te Morsche, Rene H M; Voogd, Adri C; Oude Ophuis, Michael B; Peters, Wilbert H M; Manni, Johannes J

    2008-07-01

    Microsomal epoxide hydrolase (mEH) is an enzyme involved in the metabolism of (pre)carcinogens in tobacco smoke. We investigated whether functional genetic polymorphisms in mEH may have a risk-modifying effect on head and neck carcinogenesis. Blood from 429 patients with oral, pharyngeal, and laryngeal carcinoma and 419 healthy subjects was investigated for mEH polymorphisms. Logistic regression analysis did not show differences in mEH genotype distributions between patients and controls, when categorized according to predicted mEH enzyme activity. Also no differences were found when evaluated according to tumor localization, sex, or tobacco consumption. A significantly higher incidence of the 139Arg/Arg variant was found in patients with hypopharyngeal carcinoma compared with controls (OR = 4.39, 95% CI = 1.45 to 13.35). In contrast to earlier reports, we could not demonstrate a risk-modifying effect of genetic polymorphisms in mEH on head and neck carcinogenesis, except for the predicted high activity variant in patients with hypopharyngeal carcinoma.

  19. Liver and lung microsomal metabolism of the tobacco alkaloid beta-nicotyrine.

    Science.gov (United States)

    Shigenaga, M K; Kim, B H; Caldera-Munoz, P; Cairns, T; Jacob, P; Trevor, A J; Castagnoli, N

    1989-01-01

    The in vitro metabolic fate of beta-nicotyrine has been examined in rabbit lung and liver microsomal preparations as part of an effort to characterize the formation of potentially reactive metabolic species that may contribute to the toxic properties of tobacco products. HPLC analysis revealed the formation of an unstable metabolite which displayed HPLC-MS/MS characteristics consistent with the structure 1-methyl-5-(3-pyridyl)-3-pyrrolin-2-one. Attempted synthesis of this pyrrolinone, however, resulted in the isolation of the isomeric 1-methyl-5-(3-pyridyl)-2-pyrrolin-2-one. The HPLC, diode array UV, and mass spectral characteristics of this delta 4,5-isomer proved to be identical with those of the metabolite derived from beta-nicotyrine. Studies in D2O suggest that the 2- and 3-pyrrolinones are in equilibrium in aqueous solution. The metabolite undergoes autoxidation, possibly via radical intermediates, to yield 1-methyl-5-(3-pyridyl)-5-hydroxy-3-pyrrolin-2-one.

  20. Characterization of the metabolic interaction between trihalomethanes and chloroacetic acids using rat liver microsomes.

    Science.gov (United States)

    St-Pierre, Annie; Krishnan, Kannan; Tardif, Robert

    2005-02-27

    The aim of this study was to investigate the in vitro metabolism of trihalomethanes (THMs) in the presence of trichloroacetic acid (TCA), dichloracetic acid (DCA), monochloroacetic acid (MCA), and 4-methylpyrazole (4-MP) using liver microsomes from male Sprague-Dawley rats. Using the vial equilibration technique, initial experiments were carried out with starting concentrations of approximately 40 ppm THMs and 12-22 mM chloroacetic acids. The results indicated a mutual metabolic inhibition between THMs present as binary or quaternary mixtures. Although DCA and MCA had no influence on THMs, TCA produced a marked inhibition of the metabolism of all THMs: chloroform (CHCl3) (55%), bromodichloromethane (BDCM) (34%), dibromochloromethane (DBCM) (30%), and bromoform (TBM) (23%). The presence of 4-MP also reduced THM metabolism, the importance of which decreased in the following order: CHCl3 > BDCM > DBCM = TBM. In further vial equilibration experiments, using 9-140 ppm as starting concentrations of THMs, enzyme kinetic parameters (i.e., Michaelis constant, K(m), and maximum velocity, V(max)) were determined both in the absence and in the presence of TCA (12.2 mM). Results are consistent with a competitive inhibition between TCA and CHCl3, whereas the metabolic inhibition of BDCM and TMB by TCA was non-competitive. As for DBCM, results suggest a more complex pattern of inhibition. These results suggest that CYP2E1 is involved in the metabolism of THMs as well as in the metabolic interaction between THMs and TCA.

  1. Aromatic hydroxylation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) by rabbit liver microsomes.

    Science.gov (United States)

    Kumagai, Y; Schmitz, D A; Cho, A K

    1992-04-01

    1. Metabolites formed during incubation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) with rabbit liver microsomes were examined by h.p.l.c.-electrochemical detection and g.l.c.-mass spectrometry. 2. The trifluoroacetyl derivative of metabolite M-1, obtained from MDB, had a molecular ion at m/z 234 and was identified as 3,4-methylenedioxy-6-hydroxybenzene (sesamol) by comparison with authentic material. 3. The trifluoroacetyl derivative of metabolite M-2, obtained from MDMA, exhibited a molecular ion at m/z 401. Experiments with the deuterium substituted variants of MDMA indicated that the product was hydroxylated on the aromatic ring. 4. The formation of these hydroxylated metabolites required NADPH and was inhibited by carbon monoxide, indicating the possible participation of cytochrome P-450. Phenobarbital (PB) induction caused a marked enhancement of MDP hydroxylase activity whereas MDMA hydroxylation was not affected. 5. The aromatic hydroxylation of MDB and MDMA was also observed in a reconstituted system with cytochrome P-450 isozyme IIB4.

  2. Formation of the Thiol Conjugates and Active Metabolite of Clopidogrel by Human Liver Microsomes

    Science.gov (United States)

    Lau, Wei C.; Hollenberg, Paul F.

    2012-01-01

    We reported previously the formation of a glutathionyl conjugate of the active metabolite (AM) of clopidogrel and the covalent modification of a cysteinyl residue of human cytochrome P450 2B6 in a reconstituted system (Mol Pharmacol 80:839–847, 2011). In this work, we extended our studies of the metabolism of clopidogrel to human liver microsomes in the presence of four reductants, namely, GSH, l-Cys, N-acetyl-l-cysteine (NAC), and ascorbic acid. Our results demonstrated that formation of the AM was greatly affected by the reductant used and the relative amounts of the AM formed were increased in the following order: NAC (17%) clopidogrel. It was observed that the AM was slowly converted to the thiol conjugate, with a half-life of ∼10 h. Addition of dithiothreitol to the reaction mixture reversed the conversion, which resulted in a decrease in AM-thiol conjugate levels and a concomitant increase in AM levels, whereas addition of NAC led to the formation of AM-NAC and a concomitant decrease in AM-GSH levels. These results not only confirm that the AM is formed through oxidative opening of the thiolactone ring but also suggest the existence of an equilibrium between the AM, the thiol conjugates, and the reductants. These factors may affect the effective concentrations of the AM in vivo. PMID:22584220

  3. Brain Basics

    Medline Plus

    Full Text Available ... Brain Research Glossary Brain Basics (PDF, 10 pages) Introduction Watch the Brain Basics video Welcome. Brain Basics provides information on how the brain works, how mental illnesses ...

  4. Brain Basics

    Science.gov (United States)

    ... Events About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain ... to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... Events About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain ... to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... Brain Basics provides information on how the brain works, how mental illnesses are disorders of the brain, ... learning more about how the brain grows and works in healthy people, and how normal brain development ...

  7. Effect of rociverine on P450-dependent monooxygenases and its N-deethylation metabolism in rat liver microsomes.

    Science.gov (United States)

    Menicagli, S; Lippi, A; Criscuoli, M; Gervasi, P G

    1993-03-09

    Rociverine [2-(diethylamino)-1-methylethyl cis-1-hydroxy [bicyclohexyl]-2-carboxylate] citrate (ROC) is an antispasmodic agent therapeutically active in humans at doses of 0.5-1 mg/kg. This study investigated the effect of acute administration of the drug on hepatic microsomal cytochrome P450 (P450)-catalysed drug metabolism. Only high doses (> or = 100 mg/kg) of ROC were able to induce in rats the hepatic microsomal pentoxyresorufin O-depenthylase (PROD) and 16 beta-testosterone hydroxylase activities both associated with P4502B1/2 and the erythromycin N-dimethylase (ErD) and 2 beta-testosterone hydroxylase activities both dependent on P4503A1/2. However, at 100 and 200 mg/kg of ROC, the 16 beta-testosterone hydroxylase and PROD were the most induced activities, suggesting that P4502B1/2 are the isoforms most sensitive to ROC induction. Accordingly, ROC treatment enhanced, in a dose-dependent manner, the amount of P4502B1/2 and 3A1/2 in microsomes as assayed by western blotting. The northern blot analysis of ROC-treated rat liver showed that the P4502B1/2 induction appears to be regulated at the mRNA level as in the induction by phenobarbital (PB). The oxidative metabolism of ROC with hepatic microsomes from control or PB- and ROC-induced rats resulted in a N-deethyl ROC derivative (major metabolite) and an unknown minor ROC derivative. The kinetic parameters for the N-deethylation of ROC were studied with purified P4502B1 and with microsomes from control or rats treated with various inducers (phenobarbital, ethanol, beta-naphthoflavone, dexamethasone and rociverine). It was found that phenobarbital-, dexamethasone- and rociverine-induced microsomes deethylated ROC with a Vmax about five times higher than that (0.9 nmol/min/mg protein) of control microsomes, although with a similar affinity (Km approximately 0.3 mM). In a reconstituted system, the purified P4502B1 metabolized ROC with a high deethylation rate (22 nmol/min/nmol P450). Moreover, the ROC deethylation

  8. Regulation of putative Ca/sup 2 +/ channels in the brain

    Energy Technology Data Exchange (ETDEWEB)

    Skattebol, A.

    1986-01-01

    Ca/sup 2 +/ entry mechanisms were studied in a variety of neuronal preparations. In synaptosomes and intact brain cells K/sup +/-induced Ca/sup 2 +/ entry is a biphasic process. Highest density of both processes is found in the hippocampus and the lowest in the cerebellum. The slow second phase shows no regional distribution, is abolished in Na/sup +/ free medium and is antagonized by 3,4-dichlorobenzamil. It therefore likely represents Ca/sup 2 +/ influx through the Na/sup +//Ca/sup 2 +/ exchanger. The chronic treatment of rats with atropine or diisopropyl phosphororfluoridate (DFP) led to an increase or decrease of 30% and 50% respectively, in the number of muscarinic receptors in the brain. In a similar fashion chronic administration of propranolol increased ..beta..-adrenoceptors 69% and 50% in brain and heart respectively. Isoproterenol administration decreased ..beta..-adrenoceptors by 50% in heart, but had no effect in the brain. None of these protocols had an effects on the number of Ca/sup 2 +/ channel binding sites as detected by (/sup 3/H)nimodipine. Ethanol inhibits the first phase of synaptosomal /sup 45/Ca/sup 2 +/ uptake to a greater extent than the second phase. /sup 45/Ca/sup 2 +/ uptake in PC12 cells is inhibited in a similar fashion to that of the first phase. Chronic exposure to ethanol led to a 44% increase in /sup 45/Ca/sup 2 +/ uptake and a 38% increase in binding sites. This suggests that long-term antagonism of Ca/sup 2 +/ channels may lead to an increase in their numbers.

  9. Luminescence and thermoluminescence of Sr{sub 2}B{sub 5}O{sub 9}X:Ce{sup 3+},A{sup +} (X = Cl, Br, A = Na{sup +}, K{sup +}) phosphors

    Energy Technology Data Exchange (ETDEWEB)

    Sidorenko, A V [Interfaculty Reactor Institute, Delft University of Technology, Mekelweg 15, 2629 JB Delft (Netherlands); Bos, A J J [Interfaculty Reactor Institute, Delft University of Technology, Mekelweg 15, 2629 JB Delft (Netherlands); Dorenbos, P [Interfaculty Reactor Institute, Delft University of Technology, Mekelweg 15, 2629 JB Delft (Netherlands); Rodnyi, P A [St Petersburg State Technical University, Polytekhnicheskaya 29, 195251 St Petersburg (Russian Federation); Eijk, C W E van [Interfaculty Reactor Institute, Delft University of Technology, Mekelweg 15, 2629 JB Delft (Netherlands); Berezovskaya, I V [A V Bogatsky Physico-Chemical Institute, Ukranian Academy of Science, 65080 Odessa (Ukraine); Dotsenko, V P [A V Bogatsky Physico-Chemical Institute, Ukranian Academy of Science, 65080 Odessa (Ukraine)

    2003-05-28

    The luminescence properties of Ce{sup 3+} activated strontium haloborates with and without K{sup +} or Na{sup +} co-doping have been studied under optical and x-ray excitation. Three types of Ce{sup 3+} emission centre have been found as the result of different methods of charge compensation in non-co-doped samples. In the co-doped compounds only one type of Ce{sup 3+} emission centre has been observed. The influence of Ce{sup 3+} concentration, anion type and co-doping ion on thermoluminescence (TL) has been analysed. The TL glow curves of all the studied materials contain two peaks. The emission corresponding to the low temperature peak is associated with charge uncompensated Ce{sup 3+} centres in non-co-doped compounds. The emission of the high temperature peak represents the superposition of both charge compensated and uncompensated Ce{sup 3+} centres. The emission corresponding to both TL peaks in co-doped samples originates from one type of Ce{sup 3+} centre.

  10. Mechanism of secretion of biliary lipids. I. Role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lecithin and cholesterol.

    Science.gov (United States)

    Gregory, D H; Vlahcevic, Z R; Schatzki, P; Swell, L

    1975-01-01

    The role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lipids was investigated by using the isolated perfused rat liver model. Labeled lecithin precursors ((3H)-palmitic acid, (14C)linoleic acid, (3H)choline, and 32PO4) and a cholesterol precursor ((3H)mevalonic acid) were administered with and without sodium taurocholate. The incorporation pattern of these labeled precursors into linoleyl and arachidonyl lecithins and cholesterol fractions of microsomes, bile canaliculi, and bile were examined at 30-min intervals up to 90 min. Marker enzymes and electron microscopy indicated that isolated subfractions of plasma membranes were enriched with bile canaliculi (less than 10 percent microsomal contamination). Taurocholate significantly stimulated the incorporation of 32PO4, (3H)choline, (3H)palmitic acid, and (14C)linoleic acid into linoleyl and arachidonyl lecithin with parallel incorporation curves for microsomal and bile canalicular membranes throughout the 90-min study period. During the 30-60-min period, however, these same lecithin fractions in bile significantly exceeded the specific activity of the membrane lecithins. The enzyme CDP-choline diglyceride transferase was virtually absent from canaliculi relative to microsomes, indicating that canaliculi lack the capacity for de novo lecithin synthesis. Incorporation of (3H)mevalonic acid into membranous and biliary cholesterol followed a pattern similar to that for lecithin. These data provide evidence that (a) biliary lecithin and cholesterol are derived from a microsomal subpool regulated by the flux of enterohepatic bile acids, (b) the role of the bile canalicular membranes with respect to biliary lipids is primarily transport rather than synthesis, and (c) lecithin and cholesterol are transported together from microsomes to bile. The findings are consistent with the existence of a cytoplasmic lipid complex within the hepatocyte which is actively involved in the

  11. Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene.

    Science.gov (United States)

    Rubin, Diana; Schneider-Muntau, Alexandra; Klapper, Maja; Nitz, Inke; Helwig, Ulf; Fölsch, Ulrich R; Schrezenmeir, Jürgen; Döring, Frank

    2008-01-01

    The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function, polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol and related traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoter polymorphisms rs1800804:T>C (-164T>C), rs1800803:A>T (-400A>T), and rs1800591:G>T (-493G>T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results were obtained in Huh-7 cells. The common MTTP promoter haplotype -164T/-400A/-493G showed about two-fold lower activity than the rare haplotype -164C/-400T/-493T. MTTP promoter mutant constructs -164T/-400A/-493T and -164T/-400T/-493T exhibited similar activity than the common haplotype. Activities of mutants -164C/-400A/-493G and -164C/-400A/-493T resembled the rare MTTP promoter haplotype. Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factor Sterol regulatory element binding protein1a (SREBP1a) to the -164T probe in comparison to the -164C probe. In conclusion, our study indicates that the polymorphism -164T>C mediates different activities of common MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in -164T than in -164C carriers. (c) 2007 Wiley-Liss, Inc.

  12. Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1.

    Science.gov (United States)

    Shimoji, Miyuki; Figueroa, Ricardo A; Neve, Etienne; Maksel, Danuta; Imreh, Gabriela; Morgenstern, Ralf; Hallberg, Einar

    2017-02-01

    Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Alterations of testosterone metabolism in microsomes from rats with experimental colitis induced by dextran sulfate sodium.

    Science.gov (United States)

    Huang, Yanjuan; Hu, Nan; Gao, Xuejiao; Yan, Zhixiang; Li, Sai; Jing, Wanghui; Yan, Ru

    2015-05-05

    Down-regulation of some hepatic cytochrome P450s (CYP450s) was observed in patients and animals with ulcerative colitis (UC). This study examined changes of CYP450s activities in microsomes of liver (RLMs), intestine (RIMs) and kidney (RRMs) from rats with experimental acute colitis induced by 5% dextran sulfate sodium (DSS) for 7days and those receiving DSS treatment followed by 7-d cessation through measuring 6α-(CYP1A1), 7α-(CYP2A1), 16α-(CYP2C11) and 2β-/6β-(CYP3A2) hydroxytestosterone (OHT) formed from testosterone. Both pro-(IL-1β, IL-6, TNF-α) and anti-(IL-4, IL-10) inflammatory cytokines were elevated in acute colitis, while the production of the former was enhanced and that of the latter declined by DSS withdrawal. In RLMs, the CYP2A1 activity was significantly increased at DSS stimulation and partially returned to normal level when DSS treatment was terminated. Activity of other CYP450s were decreased by acute colitis and remained after DSS withdrawal. In RRMs, formations of 6α-, 16α- and 2β-OHT significantly declined in acute colitis and DSS termination further potentiated the down-regulation, while 7α-OHT formation was suppressed at DSS stimulation and remained after DSS withdrawal. The formation of 6β-OHT only showed significant decrease after DSS withdrawal. Two metabolites (6α- and 6β-OHT) formed in RIMs and 6β-OHT formation was significantly decreased by DSS stimulation and continued after DSS treatment halted. These findings indicate that the alterations of CYP450s activities vary with organ, CYP isoforms and colitis status, which arouse cautions on efficacy and toxicity of drug therapy during disease progression. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. Identification of the metabolites of episesamin in rat bile and human liver microsomes.

    Science.gov (United States)

    Tomimori, Namino; Nakai, Masaaki; Ono, Yoshiko; Kitagawa, Yoshinori; Kiso, Yoshinobu; Shibata, Hiroshi

    2012-01-01

    Episesamin is an isomer of sesamin, resulting from the refining process of non-roasted sesame seed oil. Episesamin has two methylendioxyphenyl groups on exo and endo faces of the bicyclic skeleton. The side methylendioxyphenyl group was metabolized by cytochrome-P450. Seven metabolites of episesamin were found in rat bile after treatment with glucuronidase/arylsulfatase and were identified using NMR and MS. The seven metabolites were (7α,7'β,8α,8'α)-3,4-dihydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane (EC-1-1), (7α,7'β,8α,8'α)-3,4-methylenedioxy-3',4'-dihydroxy-7,9':7',9-diepoxylignane (EC-1-2) and (7α,7'β,8α,8'α)-3,4:3',4'-bis(dihydroxy)-7,9':7',9-diepoxylignane (EC-2), (7α,7'β,8α,8'α)-3-methoxy-4-hydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane (EC-1m-1), (7α,7'β,8α,8'α)-3,4-methylenedioxy-3'-methoxy-4'-hydroxy-7,9':7',9-diepoxylignane (EC-1m-2), (7α,7'β,8α,8'α)-3-methoxy-4-hydroxy-3',4'-dihydroxy-7,9':7',9-diepoxylignane (EC-2m-1) and (7α,7'β,8α,8'α)-3,4-dihydroxy-3'-methoxy-4'-hydroxy-7,9':7',9-diepoxylignane (EC-2m-2). EC-1-1, EC-1-2 and EC-2 were also identified as metabolites of episesamin in human liver microsomes. These results suggested that similar metabolic pathways of episesamin could be proposed in rats and humans.

  15. Carnosol and carnosic acids from Salvia officinalis inhibit microsomal prostaglandin E2 synthase-1.

    Science.gov (United States)

    Bauer, Julia; Kuehnl, Susanne; Rollinger, Judith M; Scherer, Olga; Northoff, Hinnak; Stuppner, Hermann; Werz, Oliver; Koeberle, Andreas

    2012-07-01

    Prostaglandin E(2) (PGE(2)), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE(2) synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE(2) in a cell-free assay by direct interference with microsomal PGE(2) synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC(50) values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC(50) values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE(2) generation upon stimulation with lipopolysaccharide (IC(50) = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF(1α), 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B(2)] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE(2) formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE(2) formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis.

  16. Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders.

    Science.gov (United States)

    Koeberle, Andreas; Werz, Oliver

    2015-11-01

    Prostaglandin (PG)E2 encompasses crucial roles in pain, fever, inflammation and diseases with inflammatory component, such as cancer, but is also essential for gastric, renal, cardiovascular and immune homeostasis. Cyclooxygenases (COX) convert arachidonic acid to the intermediate PGH2 which is isomerized to PGE2 by at least three different PGE2 synthases. Inhibitors of COX - non-steroidal anti-inflammatory drugs (NSAIDs) - are currently the only available therapeutics that target PGE2 biosynthesis. Due to adverse effects of COX inhibitors on the cardiovascular system (COX-2-selective), stomach and kidney (COX-1/2-unselective), novel pharmacological strategies are in demand. The inducible microsomal PGE2 synthase (mPGES)-1 is considered mainly responsible for the excessive PGE2 synthesis during inflammation and was suggested as promising drug target for suppressing PGE2 biosynthesis. However, 15 years after intensive research on the biology and pharmacology of mPGES-1, the therapeutic value of mPGES-1 as drug target is still vague and mPGES-1 inhibitors did not enter the market so far. This commentary will first shed light on the structure, mechanism and regulation of mPGES-1 and will then discuss its biological function and the consequence of its inhibition for the dynamic network of eicosanoids. Moreover, we (i) present current strategies for interfering with mPGES-1-mediated PGE2 synthesis, (ii) summarize bioanalytical approaches for mPGES-1 drug discovery and (iii) describe preclinical test systems for the characterization of mPGES-1 inhibitors. The pharmacological potential of selective mPGES-1 inhibitor classes as well as dual mPGES-1/5-lipoxygenase inhibitors is reviewed and pitfalls in their development, including species discrepancies and loss of in vivo activity, are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Calcium uptake in brain synaptosomes: a pharmacologic study

    Energy Technology Data Exchange (ETDEWEB)

    Rampe, D.E.

    1986-01-01

    Pinched-off nerve endings (synaptosomes) from rat and guinea pig brain were used as a model to study Ca/sup 2 +/ entry mechanisms in neuronal tissue. Synaptosomes contain high affinity binding sites for both, 1,4-dihydropyridine Ca/sup 2 +/ channel antagonists, and activators. The thermodynamic characteristics of (/sup 3/H)nitrendipine building in synaptosomes were similar to those seen in both cardiac and smooth muscle preparations. Synaptosomes display two distinct K/sup +/-induced Ca/sup 2 +/ entry mechanisms. These are kinetically distinct with the faster of the two terminating in approx. 1 second while the slower persists for approx. minute. The slow phase uptake process is abolished in Na/sup +/-free media, is sensitive to antagonism by 3,4-dichlorobenzamil and displays a more rapid ontogenic appearance relative to the fast phase. It is likely that the slow phase represents Ca/sup 2 +/ entry via Na/sup +//Ca/sup 2 +/ exchange. The rapid inactivation of the fast phase coupled with its voltage dependence suggest that it represents Ca/sup 2 +/ entry via one or more types of voltage dependent Ca/sup 2 +/ channels. These channels may not be dihydropyridin sensitive since neither nitrendipine nor Bay K 8644 were shown to modulate synaptosomal Ca/sup 2 +/ uptake. The benzodiazepine receptor ligands Ro 5-4864, PK 11195 and diazepam all selectively inhibited fast phase Ca/sup 2 +/ entry relative to slow phase entry. In addition, these compounds altered (/sup 3/H)nitrendipine binding affinity. It is concluded that certain benzodiazepine receptor ligands can interact specifically with voltage dependent Ca/sup 2 +/ channels.

  18. Brain herniation

    Science.gov (United States)

    ... herniation; Uncal herniation; Subfalcine herniation; Tonsillar herniation; Herniation - brain ... Brain herniation occurs when something inside the skull produces pressure that moves brain tissues. This is most ...

  19. Cytochrome P-450-Dependent Hydroxylation of Lauric Acid at the Subterminal Position and Oxidation of Unsaturated Analogs in Wheat Microsomes 1

    Science.gov (United States)

    Zimmerlin, Alfred; Salaün, Jean-Pierre; Durst, Francis; Mioskowski, Charles

    1992-01-01

    Microsomes from etiolated wheat (Triticum aestivum L. cv Etoile de Choisy) shoots catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation of lauric acid predominantly at the subterminal or (ω-1) position (65%). Minor amounts of 10-hydroxy- (31%) and 9-hydroxylaurate (4%) were also formed. The reaction was catalyzed by cytochrome P-450, since enzyme activity was strongly inhibited by tetcyclacis, carbon monoxide, and antibodies against NADPH-cytochrome c (P-450)-reductase. The apparent Km for lauric acid was estimated to be 8.5 ± 2.0 μm. Seed treatment with the safener naphthalic acid anhydride or treatment of seedlings with phenobarbital increased cytochrome P-450 content and lauric acid hydroxylase (LAH) activity of the microsomes. A combination of both treatments further stimulated LAH activity. A series of radiolabeled unsaturated lauric acid analogs (8-, 9-, 10-, and 11-dodecenoic acids) was used to explore the regioselectivity and catalytic capabilities of induced wheat microsomes. It has been found that wheat microsomes catalyzed the reduced nicotinamide adenine dinucleotide phosphate-dependent epoxidation of sp2 carbons concurrently with hydroxylation at saturated positions. The regioselectivity of oxidation of the unsaturated substrates and that of lauric acid were similar. Preincubation of wheat microsomes with reduced nicotinamide adenine dinucleotide phosphate and 11-dodecenoic acid resulted in a partial loss of LAH activity. PMID:16653069

  20. Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9.

    Science.gov (United States)

    Kishi, Naoki; Takasuka, Akane; Kokawa, Yuki; Isobe, Takashi; Taguchi, Maho; Shigeyama, Masato; Murata, Mikio; Suno, Manabu; Hanioka, Nobumitsu

    2016-01-01

    1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9). 2. Although the K(m) and CL(int) values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans  UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4'-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys. 4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.

  1. A Fragment-Based Approach for the Computational Prediction of the Nonspecific Binding of Drugs to Hepatic Microsomes.

    Science.gov (United States)

    Nair, Pramod C; McKinnon, Ross A; Miners, John O

    2016-11-01

    Correction for the nonspecific binding (NSB) of drugs to liver microsomes is essential for the accurate measurement of the kinetic parameters Km and Ki, and hence in vitro-in vivo extrapolation to predict hepatic clearance and drug-drug interaction potential. Although a number of computational approaches for the estimation of drug microsomal NSB have been published, they generally rely on compound lipophilicity and charge state at the expense of other physicochemical and chemical properties. In this work, we report the development of a fragment-based hologram quantitative structure activity relationship (HQSAR) approach for the prediction of NSB using a database of 132 compounds. The model has excellent predictivity, with a noncross-validated r2 of 0.966 and cross-validated r2 of 0.680, with a predictive r2 of 0.748 for an external test set comprising 34 drugs. The HQSAR method reliably predicted the fraction unbound in incubations of 95% of the training and test set drugs, excluding compounds with a steroid or morphinan 4,5-epoxide nucleus. Using the same data set of compounds, performance of the HQSAR method was superior to a model based on logP/D as the sole descriptor (predictive r2 for the test set compounds, 0.534). Thus, the HQSAR method provides an alternative approach to laboratory-based procedures for the prediction of the NSB of drugs to liver microsomes, irrespective of the drug charge state (acid, base, or neutral). Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  2. Correlation of serum antithyroid microsomal antibody and autologous serum skin test in patients with chronic idiopathic urticaria

    Directory of Open Access Journals (Sweden)

    Snehal Balvant Lunge

    2015-01-01

    Full Text Available Background: About 25-45% of patients of chronic urticaria (CU have been stated to have histamine releasing autoantibodies in their blood. The term autoimmune urticaria is increasingly being accepted for this subgroup of patients. Review of the literature suggests high autologous serum skin test (ASST positivity and presence of antithyroid microsomal antibodies in patients with autoimmune urticaria. Aims: To study prevalence of ASST positivity and antithyroid microsomal antibodies in chronic "idiopathic" urticaria and to study the correlation between the two parameters. Methods: All patients of chronic idiopathic urticaria satisfying inclusion/exclusion criteria were enrolled in the study after written informed consent. Patients of CU secondary to infections and infestations, physical urticaria including dermatographism, mastocytosis, urticarial vasculitis and those on treatment with immunosuppressive drugs for urticaria were excluded from the study. In all of these patients, complete blood count; ASST, serum T3/T4/thyroid stimulating hormone levels, antithyroid microsomal antibody (AMA levels were done. Statistical analysis was done by Chi-square test, Fisher exact test and Kappa statistics. Results: Study included 24 males and 26 females with mean age of 39.54 years. Majority of patients belonged to 20-40 years of age. Females showed more ASST positivity. A total of 12 out of 50 (24% patients showed positive ASST. A total of four out of 12 (33.33% had positive ASST and raised AMA levels. Conclusion: Only 25% of patients of chronic idiopathic urticaria had positive ASST. ASST and AMA levels were positively correlated in our study. Further studies are required to authenticate this association.

  3. Correlation of serum antithyroid microsomal antibody and autologous serum skin test in patients with chronic idiopathic urticaria

    Science.gov (United States)

    Lunge, Snehal Balvant; Borkar, Milind; Pande, Sushil

    2015-01-01

    Background: About 25–45% of patients of chronic urticaria (CU) have been stated to have histamine releasing autoantibodies in their blood. The term autoimmune urticaria is increasingly being accepted for this subgroup of patients. Review of the literature suggests high autologous serum skin test (ASST) positivity and presence of antithyroid microsomal antibodies in patients with autoimmune urticaria. Aims: To study prevalence of ASST positivity and antithyroid microsomal antibodies in chronic “idiopathic” urticaria and to study the correlation between the two parameters. Methods: All patients of chronic idiopathic urticaria satisfying inclusion/exclusion criteria were enrolled in the study after written informed consent. Patients of CU secondary to infections and infestations, physical urticaria including dermatographism, mastocytosis, urticarial vasculitis and those on treatment with immunosuppressive drugs for urticaria were excluded from the study. In all of these patients, complete blood count; ASST, serum T3/T4/thyroid stimulating hormone levels, antithyroid microsomal antibody (AMA) levels were done. Statistical analysis was done by Chi-square test, Fisher exact test and Kappa statistics. Results: Study included 24 males and 26 females with mean age of 39.54 years. Majority of patients belonged to 20–40 years of age. Females showed more ASST positivity. A total of 12 out of 50 (24%) patients showed positive ASST. A total of four out of 12 (33.33%) had positive ASST and raised AMA levels. Conclusion: Only 25% of patients of chronic idiopathic urticaria had positive ASST. ASST and AMA levels were positively correlated in our study. Further studies are required to authenticate this association. PMID:26225328

  4. Benznidazole biotransformation in rat heart microsomal fraction without observable ultrastructural alterations: comparison to Nifurtimox-induced cardiac effects.

    Science.gov (United States)

    Mecca, María Montalto de; Bartel, Laura C; Castro, Carmen Rodríguez de; Castro, José A

    2008-09-01

    Benznidazole (Bz) and Nifurtimox (Nfx) have been used to treat Chagas disease. As recent studies have de-monstrated cardiotoxic effects of Nfx, we attempted to determine whether Bz behaves similarly. Bz reached the heart tissue of male rats after intragastric administration. No cytosolic Bz nitroreductases were detected, although microsomal NADPH-dependent Bz nitroreductase activity was observed, and appeared to be mediated by P450 reductase. No ultrastructurally observable deleterious effects of Bz were detected, in contrast to the overt cardiac effects previously reported for Nfx. In conclusion, when these drugs are used in chagasic patients, Bz may pose a lesser risk to heart function than Nfx when any cardiopathy is present.

  5. Identification of Three New N-Demethylated and O-Demethyled Bisbenzylisoquinoline Alkaloid Metabolites of Isoliensinine from Dog Hepatic Microsomes

    Directory of Open Access Journals (Sweden)

    Su Zeng

    2012-10-01

    Full Text Available Isoliensinine, a natural phenolic bisbenzyltetrahydroisoquinoline alkaloid, has received considerable attention for its potential biological effects such as antioxidant and anti-HIV activities. From the dog hepatic microsomes of isoliensinine, three new N-demethylated and O-demethylated metabolites, 2-N-desmethyl-isoliensinine (M1, 2'-N-desmethylisoliensinine (M2, and 2'-N-6-O-didesmethylisoliensinine (M3, were identified by high-performance liquid chromatography and data-dependent electrospray ionization tandem mass spectrometry. Possible metabolic pathways for isoliensinine have been proposed. The result should prove very helpful for evaluation of the drug-like properties of isoliensinine and other bisbenzylisoquinoline alkaloids.

  6. Brain Basics

    Medline Plus

    Full Text Available ... PDF, 10 pages) Introduction Watch the Brain Basics video Welcome. Brain Basics provides information on how the brain works, how mental illnesses are disorders of the brain, and ongoing research that helps ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... Brain Research Glossary Brain Basics (PDF, 10 pages) Introduction Watch the Brain Basics video Welcome. Brain Basics ... depression experience when starting treatment. Gene Studies ... medication. This information may someday make it possible to predict who ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... Brain Research Glossary Brain Basics (PDF, 10 pages) Introduction Watch the Brain Basics video Welcome. Brain Basics ... fear hub," which activates our natural "fight-or-flight" response to confront or escape from a dangerous ...

  9. Brain Lesions

    Science.gov (United States)

    Symptoms Brain lesions By Mayo Clinic Staff A brain lesion is an abnormality seen on a brain-imaging test, such as ... tomography (CT). On CT or MRI scans, brain lesions appear as dark or light spots that don' ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a middle- ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses ...

  11. Gas chromatographic-mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes.

    Science.gov (United States)

    Peng, S X; Strojnowski, M J; Hu, J K; Smith, B J; Eichhold, T H; Wehmeyer, K R; Pikul, S; Almstead, N G

    1999-03-05

    A gas chromatographic-mass spectrometric (GC-MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC-MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.

  12. Brain Basics

    Medline Plus

    Full Text Available ... Mental Illnesses Clinical Trials Outreach Research Priorities Funding Labs at NIMH News & Events About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain Brain Basics in Real Life Brain Research Glossary Brain Basics (PDF, 10 pages) ...

  13. Studies on the effects of acetylcholine and antiepileptic drugs on /sup 32/P incorporation into phospholipids of rat brain synaptosomes

    Energy Technology Data Exchange (ETDEWEB)

    Aly, M.I.; Abdel-Latif, A.A.

    1982-02-01

    Studies were conducted on the effects of antiepileptic drugs on the acetylcholine-stimulated /sup 32/P labeling of phospholipids in rat brain synaptosomes. Of the four antiepileptic drugs investigated in the present study, namely phenytoin, carbamazepine, phenobarbital, and valproate, only phenytoin blocked the acetylcholine-stimulated /sup 32/P labeling of phosphatidylinositol and phosphatidic acid, and the acetylcholine-stimulated breakdown of polyphosphoinositides. Phenytoin alone, like atropine alone, had no effect on the /sup 32/P labeling of phospholipids nor on the specific radioactivity of (/sup 32/P)ATP. Omission of Na/sup +/ drastically reduced both the /sup 32/P labeling of synaptosomal phospholipids and the specific radioactivity of (/sup 32/P)ATP and furthermore it significantly decreased the phosphoinositide effect. It was concluded that certain antiepileptic drugs, such as phenytoin, could exert their pharmacological actions through their antimuscarinic effects. In addition the finding that phenytoin, which acts to regulate NA/sup +/ and Ca/sup 2 +/ permeability of neuronal membranes, also inhibited the phosphoinositide effects in synaptosomes, support the conclusions that Ca2+ and Na+ are probably involved in the molecular mechanism underlying this phenomenon in excitable tissues.

  14. Acute effects of 2-nitropropane on rat liver and brain.

    Science.gov (United States)

    Zitting, A; Savolainen, H; Nickels, J

    1981-11-01

    Intraperitoneal injection (50 mg/kg) of 2-nitropropane (2-NP) induced lipid accumulation, centrilobular necrosis, degranulation of rough endoplasmic reticulum, proliferation of smooth endoplasmic reticulum and mitochondrial abnormalities in rat liver 24 h after exposure. These pathological changes were accompanied by elevated serum alanine aminotransferase (ALAT) levels. Hepatic glutathione content increased rapidly in exposed rats. 2-NP depressed markedly hepatic cytochrome P-450 and microsomal monooxygenase activity while the enzyme, epoxide hydratase, UDP-glucuronosyltransferase and cytosolic glutathione peroxidase were enhanced. 2-NP caused an increase of acetylcholine esterase activity in the brain. This effect was also detected in synaptosomes isolated from exposed rats. The results suggest peroxidative damage in the cells.

  15. P-NITROPHENOL METABOLISM BY JAPANESE MEDAKA (ORYZIAS LATIPES) LIVER MICROSOMES AND S-9 FRACTION: ADDITIONAL EVIDENCE FOR THE EXISTENCE OF A CYP2E1-LIKE ISOFORM IN TELEOSTS

    Science.gov (United States)

    Liver microsomes and S-9 fraction of Japanese medaka (Oryzias latipes) metabolized the CYP2E1 specific substrate, p-nitrophenol (PNP), to a single hydroxylated product, 4-nitrocatechol. The use of liver S-9 fraction proved to be a viable alternative to liver microsomes and allowe...

  16. Soy isoflavone metabolism in cats compared with other species: Urinary metabolite concentrations and glucuronidation by liver microsomes

    Science.gov (United States)

    Redmon, Joanna M.; Shrestha, Binu; Cerundolo, Rosario; Court, Michael H.

    2016-01-01

    Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here we evaluated species’ differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species.Significant concentrations of conjugated (but not unconjugated) genistein, daidzein, and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine.β-glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats.Glucuronidation rates of genistein, daidzein, and equol by cat livers were consistently ranked within the lowest three out of 13 species’ livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species.Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species. PMID:26366946

  17. Human Liver Microsomal Cytochrome P450 3A Enzymes Involved in Thalidomide 5-Hydroxylation and Formation of a Glutathione Conjugate

    Science.gov (United States)

    Chowdhury, Goutam; Murayama, Norie; Okada, Yusuke; Uno, Yasuhiro; Shimizu, Makiko; Shibata, Norio; Guengerich, F. Peter; Yamazaki, Hiroshi

    2013-01-01

    (R)-Thalidomide was oxidized to 5-hydroxythalidomide and 5’-hydroxythalidomide by NADPH-fortified liver microsomes from humans and monkeys. (R)-Thalidomide was hydroxylated more efficiently than (S)-thalidomide. Recombinant human P450s 3A4, 3A5, and 3A7 and monkey P450s 3A8 and 3A5 (co-expressed with NADPH-P450 reductase in bacterial membranes) also catalyzed (R)-thalidomide 5-hydroxylation. Purified human P450s 2C19, 3A4, and 3A5 mediated (R)-thalidomide 5-hydroxylation at similar rates in reconstituted systems. P450 2C19 showed a rather non-saturable substrate-velocity curve; however, P450s 3A4 and 3A5 showed sigmoidal curves. P450 also oxidized 5-hydroxythalidomide to an epoxide or dihydroxy compound. Liquid chromatography-mass spectrometry analysis revealed formation of a glutathione conjugate from (R)- and (S)-5-hydroxythalidomide, catalyzed by liver microsomal P450s 3A4 and 3A5 in the presence of glutathione (assigned as a conjugate of 5-hydroxythalidomide formed on the phenyl ring). These results indicate that human P450s 3A4 and 3A5 mediate thalidomide 5-hydroxylation and further oxidation leading to a glutathione conjugate, which may be of relevance in the pharmacological and toxicological actions of thalidomide. PMID:20443640

  18. Oxidative one-carbon cleavage of the octyl side chain of olanexidine, a novel antimicrobial agent, in dog liver microsomes.

    Science.gov (United States)

    Umehara, K; Shimokawa, Y; Koga, T; Ohtani, T; Miyamoto, G

    2004-01-01

    1. The oxidative one-carbon cleavage reaction in the octyl side chain of olanexidine [1-(3,4-dichlorobenzyl)-5-octylbiguanide], a new potent biguanide antiseptic, was characterized in dog liver microsomes. 2. Olanexidine was initially biotransformed to a monohydroxylated metabolite, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-2-octanol (DM-215), and DM-215 was subsequently oxidized to the diol derivative, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-1,2-octandiol (DM-220). DM-220 was further biotransformed to 2-hydroxy aldehyde derivative, 2-hydroxy carboxylic acid derivative, and an oxidative C-1-C-2 bond cleavage metabolite, 7-[5-(3,4-dichlorobenzyl)-1-biguanidino] heptanoic acid [DM-223 (C7), a seven-carbon chain derivative], after incubation with dog liver microsomes. 3. DM-223 formation required NADPH as a cofactor and was inhibited by quinidine and quinine, relatively selective inhibitors of CYP2D subfamilies in dogs. 4. The results suggest that the one-carbon fragment of the octyl side chain of olanexidine could be removed by the oxidative C-C bond cleavage with the possible involvement of cytochrome P450 systems such as CYP2D subfamily. This oxidative C-C bond cleavage reaction by cytochrome P450s could play an important role in the removal of one-carbon fragment of other drugs or endogenous compounds containing aliphatic chains.

  19. Stereoselective Metabolism of Bupropion by Cytochrome P4502B6 (CYP2B6) and Human Liver Microsomes

    Science.gov (United States)

    Coles, Rebecka; Kharasch, Evan D.

    2013-01-01

    Purpose Hydroxylation of the antidepressant and smoking deterrent drug bupropion is a clinically important bioactivation and elimination pathway. Bupropion hydroxylation is catalyzed selectively by cytochrome P4502B6 (CYP2B6). CYP2B6-catalyzed bupropion hydroxylation has been used as an in vitro and in vivo phenotypic probe for CYP2B6 activity and CYP2B6 drug interactions. Bupropion is chiral, used clinically as a racemate, and disposition is stereoselective. Nevertheless, it is unknown whether CYP2B6-catalyzed bupropion hydroxylation is stereoselective. Methods Hydroxylation of racemic bupropion by recombinant CYP2B6 and human liver microsomes was evaluated using a stereoselective assay. Results At therapeutic concentrations, hydroxylation of (S)-bupropion was 3-fold and 1.5-greater than (R)-bupropion, respectively, by recombinant CYP2B6 and human liver microsomes. In vitro intrinsic clearances were likewise different for bupropion enantiomers. Conclusions Stereoselective bupropion hydroxylation may have implications for the therapeutic efficacy of bupropion as an antidepressant or smoking cessation therapy, and for the use of bupropion as an in vivo phenotypic probe for CYP2B6 activity. PMID:18219560

  20. Studies on calcium uptake by myometrial microsomes with particular reference to the dependence on inorganic phosphate and oxalate.

    Science.gov (United States)

    Batra, S

    1978-09-01

    Ca uptake by microsomes isolated from non-pregnant rabbit myometrium was potentiated by both inorganic phosphate (Pi) and oxalate anions. Both Pi and oxalate had little effect on the initial rate of uptake but a pronounced effect on the capacity of Ca uptake measured after 20 min which was greater in the presence of oxalate than that of Pi (5 mM each). The presence or absence of sucrose in the uptake medium had a significant effect on oxalate-induced potentiation of Ca uptake but not on that potentiated by Pi or that measured in the absence of either potentiating anion. A part of Ca accumulated additionally under the influence of sucrose could be removed by washing microsomes with KCl. Another significant difference between the pontentiating effect of oxalate and Pi was observed when the pH of the incubation medium was varied. In the presence of oxalate the pH optimum was between 6.4--6.8, whereas that in its absence or in the presence of Pi the optimal pH was around 7.2. Reduction in pH from 7.2 to 6.8 along with the substitution of KCl by sucrose resulted in 3-fold increase in Ca uptake when oxalate was used as the potentiating anion. The results suggest that Ca is taken up by a different mechanism in the presence of oxalate than that in its absence or when oxalate anion is substituted with inorganic phosphate.

  1. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol.

    Science.gov (United States)

    Lehner, R; Vance, D E

    1999-01-01

    A microsomal triacylglycerol hydrolase (TGH) was recently purified from porcine liver [Lehner and Verger (1997) Biochemistry 36, 1861-1868]. To gain further insight into the function of TGH, we have cloned a cDNA encoding TGH from a rat liver cDNA library and generated McArdle RH7777 rat hepatoma cell lines that stably express the rat TGH. The putative protein derived from the cDNA sequence contains a cleavable signal sequence and a catalytic site serine residue present within a pentapeptide motif (GXSXG) that is conserved in all known lipases. TGH-transfected cells showed a 2-fold increase, compared with control cells, in the rate of depletion of prelabelled triacylglycerol stores. Thus, TGH is capable of hydrolysis of stored triacylglycerol. In contrast, the rate of turnover of labelled phosphatidylcholine was similar in both the vector- and TGH-transfected cells. Studies in TGH-transfected cells demonstrated that utilization of intracellular triacylglycerol pools for secretion was approx. 30% higher than in vector-transfected cells. Whereas phosphatidylcholine secretion was essentially the same in control and TGH-transfected cells, TGH-transfected cells also secreted an approx. 25% greater mass of triacylglycerol into the medium and had increased levels of apolipoprotein B100 in the very-low-density lipoprotein density range compared with control cells. The results suggest that the microsomal TGH actively participates in the mobilization of cytoplasmic triacylglycerol stores, some of which can be used for lipoprotein assembly. PMID:10493905

  2. Effects of acetone, acetonitrile, ethanol, methanol and DMSO on cytochrome P450 in rainbow trout (Oncorhynchus mykiss) hepatic microsomes.

    Science.gov (United States)

    Sakalli, Sidika; Burkina, Viktoriia; Zlabek, Vladimir; Zamaratskaia, Galia

    2015-01-01

    In vitro impacts of five organic solvents on cytochrome P450 (CYP450) enzyme activity were investigated using hepatic microsomes of rainbow trout. The rates of several CYP450-mediated reactions were investigated at solvent concentrations ranging from 0.01% to 3%. The solvents greatly affected all tested reactions. In at least 0.8% ethanol, 2% methanol or acetone, 1% acetonitrile or 3% dimethyl sulfoxide (DMSO), 7-ethoxyresorufin-O-deethylase (EROD) activity decreased and at 3% acetonitrile or ethanol, it was undetected. At 3%, all tested solvents except methanol reduced 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylase (BFCOD) activity, but at low concentrations of ethanol (2% and lower) or DMSO (1% and lower), it was induced. This was not seen with the inclusion of a pre-incubation step. p-Nitrophenolhydroxylase (PNPH) activity was not affected at concentrations below 1% DMSO, and at 2% acetonitrile it was reduced, as it was above 1% methanol or 0.5% ethanol. Acetone did not affect PNPH activity with or without a pre-incubation step. In general, the degree of inhibition was similar with and without the pre-incubation step. We conclude that the concentration of organic solvent for solubilizing the substrate and inhibitor in in vitro microsomal studies should be minimized.

  3. Distribution of the intracellular Ca(2+)-ATPase isoform 2b in pig brain subcellular fractions and cross-reaction with a monoclonal antibody raised against the enzyme isoform.

    Science.gov (United States)

    Salvador, J M; Berengena, M; Sepúlveda, M R; Mata, A M

    2001-04-01

    The presence and distribution of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) isoform 2b in microsomes and other subcellular fractions isolated from pig brain has been demonstrated by the combined use of a specific antibody raised against the SERCA2b isoform and ATP phosphorylation experiments. All subcellular fractions show an approximately 110 kDa phosphorylated protein, the band intensity being stronger in microsomes. Preliminary treatment of the samples with trypsin generates two phosphorylated fragments of about 57 and 33 kDa in the presence of Ca(2+). The observed fragments are typical trypsinized products of the SERCA2b isoform. The monoclonal antibody Y/1F4 raised against the sarcoplasmic reticulum Ca(2+)-ATPase (isoform 1) binds to the 110 kDa band in membranes isolated from brain. The binding was stronger in microsomes than in other fractions. Furthermore, this antibody also recognizes a clear band at around 115 kDa. This band is always stronger in plasma membrane than in synaptosomes or microsomes and is unaffected by trypsin. Phosphorylation studies in the absence of Ca(2+) suggest that the 115 kDa protein is not a Ca(2+)-ATPase.

  4. Brain Tumors

    Science.gov (United States)

    A brain tumor is a growth of abnormal cells in the tissues of the brain. Brain tumors can be benign, with no cancer cells, ... cancer cells that grow quickly. Some are primary brain tumors, which start in the brain. Others are ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... Brain Research Glossary Brain Basics (PDF, 10 pages) Introduction Watch the Brain Basics video Welcome. Brain Basics ... basic, working unit of the brain and nervous system, which processes and transmits information. neurotransmitter —A chemical produced by neurons that carries ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... learning more about how the brain grows and works in healthy people, and how normal brain development and function can go awry, leading to mental illnesses. Brain Basics will introduce you to some of this science, such as: ... of the brain communicate and work with each other How changes in the brain ...

  7. 2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is Enantioselectively Oxidized to Hydroxylated Metabolites by Rat Liver Microsomes

    Science.gov (United States)

    Wu, Xianai; Pramanik, Ananya; Duffel, Michael W.; Hrycay, Eugene G.; Bandiera, Stelvio M.; Lehmler, Hans-Joachim; Kania-Korwel, Izabela

    2011-01-01

    Developmental exposure to multiple-ortho substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB-), dexamethasone (DEX-) and corn oil (VEH-)treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzymes. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer didn't affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of

  8. Dependence of microsomal methoxyflurane O-demethylation on cytochrome P-450 reductase and the stoichiometry of fluoride ion and formaldehyde release.

    Science.gov (United States)

    Waskell, L; Gonzales, J

    1982-07-01

    In order to characterize further the in vitro liver microsomal O-demethylation and defluorination of the volatile anesthetic methoxyflurane, and obtain additional information regarding the participation of cytochrome P-450 in the oxidation, the stoichiometry of the reaction was determined and the effect of antibody to cytochrome P-450 reductase on this unique biotransformation was examined. Liver microsomes were isolated from rabbits and rats in which enzyme induction had previously been produced by phenobarbital. The O-demethylation of methoxyflurane by phenobarbital-induced microsomes results in the production of 1 mol of formaldehyde for every 2 mol of fluoride ion produced. Dichloroacetic acid is also a product of methoxyflurane O-demethylation. Antibody to cytochrome P-450 reductase inhibits by 85% the amount of fluoride ion produced by the microsomal metabolism of methoxyflurane. Thus critical indirect supportive data are contributed to the hypothesis that at least one, but perhaps more, cytochrome P-450 is indeed responsible for methoxyflurane O-demethylation and defluorination.

  9. Role of hepatic microsomal and purified cytochrome P-450 in one-electron reduction of two quinone imines and concomitant reduction of molecular oxygen

    NARCIS (Netherlands)

    Van de Straat, R; de Vries, J; Vermeulen, N P

    1987-01-01

    The possible role of cytochrome P-450 in one-electron reduction of quinoid compounds as well as in the formation of reduced oxygen species was investigated in hepatic microsomal and reconstituted systems of purified cytochrome P-450 and purified NADPH-cytochrome P-450 reductase using electron spin

  10. Microsomal activation of thioacetamide-S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides.

    Science.gov (United States)

    Vadi, H V; Neal, R A

    1981-04-01

    In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [3H]thioacetamide-S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O2 or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide-S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide-S-oxide. Only background levels of radio-activity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [3H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide-S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.

  11. GLUCURONIDATION OF LABETALOL AT THE 2 HYDROXY POSITIONS BY BOVINE LIVER-MICROSOMES - ISOLATION, PURIFICATION, AND STRUCTURE ELUCIDATION OF THE GLUCURONIDES OF LABETALOL

    NARCIS (Netherlands)

    NIEMEIJER, NR; GERDING, TK; DEZEEUW, RA

    1991-01-01

    Glucuronidation is known to be a major metabolic pathway for labetalol. As the drug contains a phenolic and an alcoholic hydroxy group, in principle two regio isomeric glucuronides can be formed. By incubating the substrate labetalol with bovine liver microsomes, in the presence of the co-substrate

  12. Metabolism of UV-filter benzophenone-3 by rat and human liver microsomes and its effect on endocrine-disrupting activity

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Yoko, E-mail: y-watanabe@nichiyaku.ac.jp [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan); Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Kojima, Hiroyuki; Takeuchi, Shinji [Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819 (Japan); Uramaru, Naoto [Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Sanoh, Seigo [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan); Sugihara, Kazumi [Faculty of Pharmaceutical Science, Hiroshima International University, Koshingai 5-1-1, Kure, Hiroshima 737-0112 (Japan); Kitamura, Shigeyuki [Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Ohta, Shigeru [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan)

    2015-01-15

    Benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is widely used as sunscreen for protection of human skin and hair from damage by ultraviolet (UV) radiation. In this study, we examined the metabolism of BP-3 by rat and human liver microsomes, and the estrogenic and anti-androgenic activities of the metabolites. When BP-3 was incubated with rat liver microsomes in the presence of NADPH, 2,4,5-trihydroxybenzophenone (2,4,5-triOH BP) and 3-hydroxylated BP-3 (3-OH BP-3) were newly identified as metabolites, together with previously detected metabolites 5-hydroxylated BP-3 (5-OH BP-3), a 4-desmethylated metabolite (2,4-diOH BP) and 2,3,4-trihydroxybenzophenone (2,3,4-triOH BP). In studies with recombinant rat cytochrome P450, 3-OH BP-3 and 2,4,5-triOH BP were mainly formed by CYP1A1. BP-3 was also metabolized by human liver microsomes and CYP isoforms. In estrogen reporter (ER) assays using estrogen-responsive CHO cells, 2,4-diOH BP exhibited stronger estrogenic activity, 2,3,4-triOH BP exhibited similar activity, and 5-OH BP-3, 2,4,5-triOH BP and 3-OH BP-3 showed lower activity as compared to BP-3. Structural requirements for activity were investigated in a series of 14 BP-3 derivatives. When BP-3 was incubated with liver microsomes from untreated rats or phenobarbital-, 3-methylcholanthrene-, or acetone-treated rats in the presence of NADPH, estrogenic activity was increased. However, liver microsomes from dexamethasone-treated rats showed decreased estrogenic activity due to formation of inactive 5-OH BP-3 and reduced formation of active 2,4-diOH BP. Anti-androgenic activity of BP-3 was decreased after incubation with liver microsomes. - Highlights: • Metabolic modification of the endocrine-disrupting activity of BP-3 was examined. • 2,4,5-TriOH BP and 3-OH BP-3 were identified as new BP-3 metabolites. • 2,4-DiOH BP and 2,3,4-triOH BP exhibited high or similar estrogenic activities. • Estrogenic activity of BP-3 was enhanced by incubation with rat liver

  13. Brain Malformations

    Science.gov (United States)

    Most brain malformations begin long before a baby is born. Something damages the developing nervous system or causes it ... medicines, infections, or radiation during pregnancy interferes with brain development. Parts of the brain may be missing, ...

  14. Brain surgery

    Science.gov (United States)

    Craniotomy; Surgery - brain; Neurosurgery; Craniectomy; Stereotactic craniotomy; Stereotactic brain biopsy; Endoscopic craniotomy ... cut depends on where the problem in the brain is located. The surgeon creates a hole in ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a ... brain. DNA —The "recipe of life," containing inherited genetic information that helps to define physical and some ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... brain's structure, studies show that brain growth in children with autism appears to peak early. And as ... grow there are differences in brain development in children who develop bipolar disorder than children who do ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... brain's structure, studies show that brain growth in children with autism appears to peak early. And as they grow there are differences in brain development in children who develop bipolar disorder than children ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... imaging (MRI), which uses magnetic fields to take pictures of the brain's structure, studies show that brain ... imaging technique that uses magnetic fields to take pictures of the brain's structure. mutation —A change in ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit of the brain ... specialized for the function of conducting messages. A neuron has three basic parts: Cell body which includes ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... of the brain's structure, studies show that brain growth in children with autism appears to peak early. And as they grow there are differences in brain development in children who develop bipolar disorder than children ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... science, such as: How the brain develops How genes and the environment affect the brain The basic ... that with brain development in people mental disorders. Genes and environmental cues both help to direct this ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... the Brain Meet Sarah Sarah is a middle-aged woman who seemed to have it all. She ... brain's structure, studies show that brain growth in children with autism appears to peak early. And as ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... about how the brain grows and works in healthy people, and how normal brain development and function ... chart how the brain develops over time in healthy people and are working to compare that with ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a middle- ... unit of the brain and nervous system, which processes and transmits information. neurotransmitter —A chemical produced by ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... Mental Illnesses Clinical Trials Outreach Outreach Home Stakeholder Engagement Outreach Partnership Program Alliance for Research Progress Coalition ... Brain Basics provides information on how the brain works, how mental illnesses are disorders of the brain, ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... in Real Life Brain Research Glossary Brain Basics (PDF, 10 pages) Introduction Watch the Brain Basics video ... and epigenetic changes can be passed on to future generations. Further understanding of genes and epigenetics may ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... can lead to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits ... tailored treatments, and possibly prevention of such illnesses. The Working Brain Neurotransmitters Everything we do relies on ...

  8. Age-related changes in O-deethylase and aldrin epoxidase activity in mouse skin and liver microsomes.

    Science.gov (United States)

    Williams, D; Woodhouse, K

    1996-09-01

    The metabolism of three model substrates for the cytochrome P450 dependent mono-oxygenase enzyme system (P450-MMO) was studied in microsomes isolated from skin and liver of young adult and senescent C57B1/6J mice. The substrates chosen were aldrin (AE), 7-ethoxycoumarin (EOC), and 7-ethoxyresorufin (EOR). Both EOC and EOR activities were lower in senescent skin. By contrast, no-age related changes were seen in senescent liver. AE was similar in young and old, in both tissues. We suggest that some important age-related differences in cutaneous xenobiotic metabolism do occur, but that these are not mirrored by hepatic differences, and are substrate specific. Previous work from these laboratories would also suggest significant species differences.

  9. Gas chromatography-mass spectrometric study of 19-oxygenation of the aromatase inhibitor 19-methylandrostenedione with human placental microsomes.

    Science.gov (United States)

    Numazawa, Mitsuteru; Nagaoka, Masao; Handa, Wakako; Yamada, Akane

    2006-06-01

    To gain insight into the catalytic function of aromatase, we studied 19-oxygenation of 19-methyl-substituted derivative of the natural substrate androstenedione (AD), compound 1, with human placental aromatase by use of gas chromatography-mass spectrometry (GC-MS). Incubation of the 19-methyl derivative 1 with human placental microsomes in the presence of NADPH under an aerobic condition did not yield a detectable amount of [19S]19-hydroxy product 2 or its [19R]-isomer 3 when the product was analyzed as the bis-methoxime-trimethylsilyl (TMS) derivative by GC-MS; moreover, the production of estrogen was not detected as the bis-TMS derivative of estradiol (detection limit: about 3 ng and 10 pg per injection for the 19-ol and estradiol, respectively). The results reveal that the 19-methyl steroid 1 does not serve as a substrate of aromatase, although it does serve as a powerful inhibitor of the enzyme.

  10. Electron spin resonance studies and theoretical quantum calculations of free radicals generated from anthracenetrione by electrochemical and microsomal reduction

    Science.gov (United States)

    Olea-Azar, Claudio; Mendizábal, Fernando; Alarcón, Jaime; Briones, Rodolfo; Cassels, Bruce K.; Delgado-Castro, Tomás; Araya-Maturana, Ramiro

    2001-08-01

    The ESR spectra of radicals obtained by electrolytic reduction of 4,4-dimethylanthracene-1,9,10 (4H)-trione ( 1) and the regioisomeric quinones 8-acetyloxymethyl-4,4,5-trimethyl- ( 2), and 5-acetyloxy-methyl-4,4,8-trimethyl-(4H)-1,9,10-anthracenetrione ( 3) were measured in DMSO and analyzed by quantum chemical calculations. The electrochemistry of these compounds was characterized using cyclic voltammetry, in DMSO and DMF solvents and compared with nifurtimox. The quinones were also reduced by microsomal NADPH-cytochrome P-450 reductase and the corresponding radicals species were also detected by ESR spectroscopy. AM1, INDO, and ADF calculations were performed to obtain the optimized geometries, theoretical hyperfine constants, and spin distributions, respectively. Density functional theory was used to rationalize the reduction potential of these compounds.

  11. Benznidazole biotransformation in rat heart microsomal fraction without observable ultrastructural alterations: comparison to Nifurtimox-induced cardiac effects

    Directory of Open Access Journals (Sweden)

    María Montalto de Mecca

    2008-09-01

    Full Text Available Benznidazole (Bz and Nifurtimox (Nfx have been used to treat Chagas disease. As recent studies have de-monstrated cardiotoxic effects of Nfx, we attempted to determine whether Bz behaves similarly. Bz reached the heart tissue of male rats after intragastric administration. No cytosolic Bz nitroreductases were detected, although microsomal NADPH-dependent Bz nitroreductase activity was observed, and appeared to be mediated by P450 reductase. No ultrastructurally observable deleterious effects of Bz were detected, in contrast to the overt cardiac effects previously reported for Nfx. In conclusion, when these drugs are used in chagasic patients, Bz may pose a lesser risk to heart function than Nfx when any cardiopathy is present.

  12. In vitro metabolism of isoline, a pyrrolizidine alkaloid from Ligularia duciformis, by rodent liver microsomal esterase and enhanced hepatotoxicity by esterase inhibitors.

    Science.gov (United States)

    Tang, Jun; Akao, Teruaki; Nakamura, Norio; Wang, Zheng-Tao; Takagawa, Kiyoshi; Sasahara, Masakiyo; Hattori, Masao

    2007-10-01

    Isoline, a major retronecine-type pyrrolizidine alkaloid (PA) from the Chinese medicinal herb Ligularia duciformis, was suggested to be the most toxic known PA. Its in vitro metabolism was thus examined in rat and mouse liver microsomes, and its toxicity was compared with that of clivorine and monocrotaline after i.p. injection in mice. Isoline was more rapidly metabolized by both microsomes than clivorine and monocrotaline and converted to two polar metabolites M1 and M2, which were spectroscopically determined to be bisline (a deacetylated metabolite of isoline) and bisline lactone, respectively. Both metabolites were formed in the presence or absence of an NADPH-generating system with liver microsomes but not cytosol. Their formation was completely inhibited by the esterase inhibitors, triorthocresyl phosphate (TOCP) and phenylmethylsulfonyl fluoride, but not at all or partially by cytochrome P450 (P450) inhibitors, alpha-naphthoflavone and proadifen (SKF 525A), respectively. These results demonstrated that both metabolites were produced by microsomal esterase(s) but not P450 isozymes. The esterase(s) involved showed not only quite different activities but also responses to different inhibitors in rat and mouse liver microsomes, suggesting that different key isozyme(s) or combinations might be responsible for the deacetylation of isoline. Isoline injected i.p. into mice induced liver-specific toxicity that was much greater than that with either clivorine or monocrotaline, as judged by histopathology as well as serum alanine aminotransferase and aspartate aminotransferase levels. Isoline-induced hepatotoxicity was remarkably enhanced by the esterase inhibitor TOCP but was reduced by the P450 inhibitor SKF 525A, indicating that rodent hepatic esterase(s) played a principal role in the detoxification of isoline via rapid deacetylation in vivo.

  13. Metabolism-mediated interaction potential of standardized extract of Tinospora cordifolia through rat and human liver microsomes.

    Science.gov (United States)

    Bahadur, Shiv; Mukherjee, Pulok K; Milan Ahmmed, S K; Kar, Amit; Harwansh, Ranjit K; Pandit, Subrata

    2016-01-01

    Tinospora cordifolia is used for treatment of several diseases in Indian system of medicine. In the present study, the inhibition potential of T. cordifolia extracts and its constituent tinosporaside to cause herb-drug interactions through rat and human liver cytochrome enzymes was evaluated. Bioactive compound was quantified through reverse phase high-performance liquid chromatography, to standardize the plant extracts and interaction potential of standardized extract. Interaction potential of the test sample was evaluated through cytochrome P450-carbon monoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human liver microsomes such as CYP3A4, CYP2D6, CYP2C9, and CYP1A2 isozymes was analyzed through fluorescence microplate assay, and respective IC50 values were determined. The content of tinosporaside was found to be 1.64% (w/w) in T. cordifolia extract. Concentration-dependent inhibition was observed through T. cordifolia extract. Observed IC50 (μg/ml) value was 136.45 (CYP3A4), 144.37 (CYP2D6), 127.55 (CYP2C9), and 141.82 (CYP1A2). Tinosporaside and extract showed higher IC50 (μg/ml) value than the known inhibitors. T. cordifolia extract showed significantly less interaction potential and indicates that the selected plant has not significant herb-drug interactions relating to the inhibition of major CYP450 isozymes. Plant extract showed significantly higher IC50 value than respective positive inhibitors against CYP3A4, 2D6, 2C9, and 1A2 isozymes. Consumption of T. cordifolia may not cause any adverse effects when consumed along with other xenobiotics.

  14. Effects of hypo- and hyperthyroidism on rat liver microsomal long-chain fatty acyl-CoA synthetase and hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Dang, A.Q.; Faas, F.H.; Carter, W.J.

    1986-05-01

    The effects of hyperthyroidism (hyperT/sub 3/), (tri-iodothryonine (T/sub 3/) injected rats), and hypothyroidism (hypoT/sub 3/) (thyroidectomized rats) on the activation of fatty acids by a microsomal long-chain fatty acyl-CoA (LCA-CoA) synthetase and the degradation of LCA-CoA by a microsomal LCA-CoA hydrolase was determined. MAS was assayed by measuring the (1-/sup 14/C)-palmitate or -1-/sup 14/C) oleate incorporated into its water soluble CoA ester. MAH was assayed spectrophotomerically by following the reduction of 5',5'-dithiobis-(2-nitrobenzoic acid) by the CoA released from palmitoyl-CoA or oleoyl-CoA. Enzyme activities are given as mean (nmoles/mg/min) +/- SEM. MAS activities were decreased 36-44% (p < 0.01) in both hypoT/sub 3/ and hyperT/sub 3/ (controls = 101 +/- 4 (n = 11, (1-/sup 14/C)-palmitate) of 72 +/- 2 (n = 5,(1-/sup 14/C)oleate)). These decreases may contribute to the decreased triacelyglycerol (TG) and phospholipid contents in the hyperT/sub 3/ liver and the decreased clearance rate of plasma TG in the hypoT/sub 3/. MAH was decreased 27-42% (p<0.01) only in hypoT/sub 3/ (controls = 77 +/- 3 (n = 11, palmitoyl-CoA) or 45 +/- 1 (n = 5, oleoyl-CoA)). This decrease was corrected by T/sub 3/ treatment. Since the decreased MAH would increase the availability of LCA-CoA, it may contribute to the increased TG synthesis in hypoT/sub 3/.

  15. Effects of Vernonia cinerea Compounds on Drug-metabolizing Cytochrome P450s in Human Liver Microsomes.

    Science.gov (United States)

    Pouyfung, Phisit; Sarapusit, Songklod; Rongnoparut, Pornpimol

    2017-12-01

    Vernonia cinerea has been widely used in traditional medicines for various diseases and shown to aid in smoking abstinence and has anticancer properties. V. cinerea bioactive compounds, including flavonoids and hirsutinolide-type sesquiterpene lactones, have shown an inhibition effect on the nicotine-metabolizing cytochrome P450 2A6 (CYP2A6) enzyme and hirsutinolides reported suppressing cancer growth. In this study, V. cinerea ethanol extract and its bioactive compounds, including four flavonoids and four hirsutinolides, were investigated for an inhibitory effect on human liver microsomal CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 using cocktail inhibition assays combined with LC-MS/MS analysis. Among tested flavonoids, chrysoeriol was more potent in inhibition on CYP2A6 and CYP1A2 than other liver CYPs, with better binding efficiency toward CYP2A6 than CYP1A2 (Ki values in competitive mode of 1.93 ± 0.05 versus 3.39 ± 0.21 μM, respectively). Hirsutinolides were prominent inhibitors of CYP2A6 and CYP2D6, with IC50 values of 12-23 and 15-41 μM, respectively. These hirsutinolides demonstrated time-dependent inhibition, an indication of mechanism-based inactivation, toward CYP2A6. Quantitative prediction of microsomal metabolism of these flavonoids and hirsutinolides, including half-lives and hepatic clearance rate, was examined. These findings may have implications for further in vivo studies of V. cinerea. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  16. AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Kim

    2017-03-01

    Full Text Available AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP or uridine 5′-diphospho-glucuronosyltransferase (UGT enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7 enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

  17. Catalytic activity and quantitation of cytochrome P-450 2E1 in prenatal human brain.

    Science.gov (United States)

    Brzezinski, M R; Boutelet-Bochan, H; Person, R E; Fantel, A G; Juchau, M R

    1999-06-01

    Cytochrome P-450 2E1 (CYP2E1) is a readily inducible hemoprotein that catalyzes the oxidation of endogenous compounds and many low molecular weight xenobiotics. As the major component of the microsomal ethanol oxidizing system, it contributes significantly to ethanol metabolism and the formation of the highly reactive metabolite acetaldehyde. The leaky property of this enzyme results in the generation of reactive oxygen species that can induce oxidative stress and cytotoxic conditions deleterious to development. To further investigate the proposed role of CYP2E1 in the etiology of alcohol teratogenesis, the current study focused on the quantification of CYP2E1 in prenatal human brain, a tissue that is highly vulnerable to the damaging effects of ethanol throughout gestation. In microsomal samples prepared from pools of brain tissues, immunoreactive protein was detected by Western blot analysis using enhanced chemiluminescence, whereas functional protein was estimated with an enzymatic assay using p-nitrophenol and an electrochemical detection system. CYP2E1 transcript was consistently detected in RNA samples prepared from individual brain tissues using the ribonuclease protection assay. Quantitative data were collected by scanning densitometry and phosphorimaging technology. There was a dramatic increase in human brain CYP2E1 content around gestational day 50 and a fairly constant level was maintained throughout the early fetal period, until at least day 113. The relatively low levels of the P-450 isoform present in conceptal brain may be sufficient to generate reactive intermediates that elicit neuroembryotoxicity following maternal alcohol consumption.

  18. Brain Basics

    Medline Plus

    Full Text Available ... pituitary-adrenal (HPA) axis. Brain Basics in Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah ... axis —A brain-body circuit which plays a critical role in the body's response to stress. impulse — ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... time in healthy people and are working to compare that with brain development in people mental disorders. Genes and environmental ... the brain than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... of cells in the body, the results can affect many aspects of life. Scientists are continually learning more about how the brain grows and works in healthy people, and how normal brain development and function can go awry, leading ... the environment affect the brain The basic structure of the brain ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... basic working unit of the brain and nervous system. These cells are highly specialized for the function of conducting messages. ... specialized brain systems. We have many specialized brain systems that work ... research are listed below. Amygdala —The brain's "fear hub," which ...

  2. Anticuerpos anti 21 hidroxilasa séricos en pacientes con anticuerpos antifracción microsomal: Síndrome poliendocrino autoinmune Seric 21- hydroxilase antibodies in patients with anti-microsomal fraction antibodies: Autoimmune polyendocrine syndrome

    Directory of Open Access Journals (Sweden)

    Silvia Botta

    2007-04-01

    Full Text Available El síndrome poliendocrino autoinmune (SPA es la asociación de enfermedades endocrinas autoinmunes con otros desórdenes autoinmunes no endocrinos. Los tipos 1, 2 y 4 presentan adrenalitis autoinmune, esto indica la presencia de autoanticuerpos, y su marcador serológico específico es el anti 21 hidroxilasa (a21-OH. El SPA tipo 2 es la asociación de adrenalitis, enfermedad tiroidea y/o diabetes mellitus inducidas por autoanticuerpos. Como componentes menores, pueden estar asociados entre otros, vitiligo, alopecia y miastenia. Nuestros objetivos fueron: establecer la prevalencia de a21-OH séricos en pacientes con anticuerpos anti fracción microsomal (AFM positivos, enfermedad tiroidea autoinmune y/o afecciones endocrinas y no endocrinas autoinmunes; diagnosticar formas incompletas de SPA y estudiar individuos con probable riesgo de progresión a un SPA completo. Estudiamos 72 pacientes AFM positivos y 60 sujetos tomados como grupo control, AFM negativos. Hallamos a21-OH elevados en dos pacientes: A= 47 U/ml, hipotiroidismo autoinmune y miastenia; y B= 8.75 U/ml, hipotiroidismo autoinmune y vitiligo; ambos con ausencia de insuficiencia adrenal. La prevalencia de a21-OH encontrada fue del 2.8%. Las pacientes A y B corresponden a un SPA tipo 2 incompleto y latente en relación al componente adrenal. Considerando a los a21-OH marcadores de enfermedad autoinmune latente, el eventual riesgo de evolución hacia la afección clínica sugiere la necesidad de estrechos controles clínicos y bioquímicos periódicos.Autoimmune polyendocrine syndrome (APS is the association of autoimmune endocrine diseases, with other autoimmune nonendocrine disorders. APS types 1, 2 and 4 include autoimmune adrenalitis; this suggests the presence of autoantibodies. A specific serological marker for these is the anti 21- hydroxilase autoantibody (a21-OH. APS type 2 is the association of autoimmune adrenalitis, to autoimmune thyroid disease and/or diabetes mellitus, all

  3. Effect of oxygen tension on the generation of F2-isoprostanes and malondialdehyde in peroxidizing rat liver microsomes.

    Science.gov (United States)

    Longmire, A W; Swift, L L; Roberts, L J; Awad, J A; Burk, R F; Morrow, J D

    1994-03-29

    Although numerous methods have been developed for the detection of lipid peroxidation, it is generally recognized that most of these lack specificity and/or sensitivity, particularly when applied to in vivo situations. We have reported recently that a series of prostaglandin F2-like compounds, termed F2-isoprostanes, are formed in vivo from the free radical catalyzed peroxidation of arachidonic acid and appear to be a useful marker of oxidant stress. Because of formation of other products of lipid peroxidation, such as alkanes and malondialdehyde (MDA), are affected by oxygen tension, which may influence their usefulness as markers of oxidant stress, we carried out a systematic study of the generation of F2-isoprostanes at various oxygen concentrations and compared these changes with the generation of MDA. The disappearance of the F2-isoprostane precursor, arachidonic acid, was used as a reference measure. Rat liver microsomes were peroxidized using an iron-ascorbate system. The incubations were carried out in sealed flasks at 37 degrees under N2 and various concentrations of O2 up to 100%. F2-isoprostanes were quantified by mass spectrometry and MDA by the thiobarbituric acid reaction. Microsomal fatty acids were measured by gas chromatography. Both MDA and F2-isoprostane formation increased in a time-dependent manner up to 15 min. Their formation correlated with a loss of polyunsaturated fatty acid and with an increase in O2 tension up to 21% O2. At oxygen tensions above 21%, MDA generation continued to increase, while F2-isoprostane generation and arachidonic acid loss did not. Levels of MDA and F2-isoprostanes increased a maximum of 65 and 9.4 times baseline values, respectively. These studies, therefore, define factors that influence the formation of F2-isoprostanes in an in vitro model of lipid peroxidation. Further, they demonstrate that higher O2 tensions do not block formation of F2-isoprostanes and validate their usefulness for assessing lipid

  4. [Changes in the activity of enzymes of heme synthesis and catabolism, and level of microsomal hemoproteins during the liver acute intoxication by thioacetamide].

    Science.gov (United States)

    Kharimov, Kh Ia; Inoiatova, F Kh; Dolimova, M A

    2001-01-01

    Thioacetamide administration to rats (20 mg/100 g) caused the development of toxic hepatitis which was accompanied by the increase of hepatic ALA-synthase and D-ALA that led to accumulating free porphyrines in the liver. At the same time an increase in activity of heme oxigenase was also found. A decrease in heme synthesis correlated with a decrease in content of cytochrome P450 and b5 in microsomal hepatic fraction of experimental animals.

  5. Enzymatic activity in turkey, duck, quail and chicken liver microsomes against four human cytochrome P450 prototype substrates and aflatoxin B1

    Directory of Open Access Journals (Sweden)

    Hansen W. Murcia

    2011-10-01

    Full Text Available Cytochrome P450 enzymes (CYP are a group of monooxygenases able to biotransform several kinds of xenobiotics including aflatoxin B1 (AFB1, a highly toxic mycotoxin. These enzymes have been widely studied in humans and others mammals, but there is not enough information in commercial poultry species about their biochemical characteristics or substrate specificity. The aim of the present study was to identify CYPs from avian liver microsomes with the use of prototype substrates specific for human CYP enzymes and AFB1. Biochemical characterization was carried out in vitro and biotransformation products were detected by high-performance liquid chromatography (HPLC. Enzymatic constants were calculated and comparisons between turkey, duck, quail and chicken activities were done. The results demonstrate the presence of four avian ortholog enzyme activities possibly related with a CYP1A1, CYP1A2, CYP2A6 (activity not previously identified and CYP3A4 poultry orthologs, respectively. Large differences in enzyme kinetics specific for prototype substrates were found among the poultry species studied. Turkey liver microsomes had the highest affinity and catalytic rate for AFB1 whereas chicken enzymes had the lowest affinity and catalytic rate for the same substrate. Quail and duck microsomes showed intermediate values. These results correlate well with the known in vivo sensitivity for AFB1 except for the duck. A high correlation coefficient between 7-ethoxyresorufin-Odeethylase (EROD and 7-methoxyresorufin- O-deethylase (MROD activities was found in the four poultry species, suggesting that these two enzymatic activities might be carried out by the same enzyme. The results of the present study indicate that four prototype enzyme activities are present in poultry liver microsomes, possibly related with the presence of three CYP avian orthologs. More studies are needed in order to further characterize these enzymes.

  6. Sigmoidal kinetics of CYP3A substrates: an approach for scaling dextromethorphan metabolism in hepatic microsomes and isolated hepatocytes to predict in vivo clearance in rat.

    Science.gov (United States)

    Witherow, L E; Houston, J B

    1999-07-01

    The metabolism of a number of compounds by the cytochrome P-450 subfamily CYP3A does not exhibit classic Michaelis-Menten kinetics but displays a sigmoidal rate-substrate concentration relationship. Intrinsic clearance (CLint) cannot be calculated for these drugs due to the lack of a first order region in their kinetic profiles, and a suitable parameter has yet to be identified to allow such data to be scaled to predict in vivo clearance. As sigmoidal kinetics have only been observed with microsomal systems, we have investigated whether this behavior is demonstrable in freshly isolated hepatocytes. We have also evaluated the term maximum clearance (CLmax), which refers to the in vitro clearance when the enzyme is fully activated, to predict in vivo clearance. To these ends we have studied the metabolism of dextromethorphan to methoxymorphinan and dextrorphan; methoxymorphinan production is best described by sigmoidal kinetics in both hepatocytes and microsomes, dextrorphan production is best described by a two site Michaelis-Menten model in microsomes but is sigmoidal in hepatocytes. Total clearance, estimated from the CLmax and CLint terms, was scaled to give mean predictions of 127 to 319 ml/min/standard rat weight of 250 g. In vivo CLint, determined after infusion via the hepatic portal vein to steady state and correcting for plasma protein binding and blood-to-plasma concentration ratio, was 259 +/- 59.2 ml/min/standard rat weight of 250 g. These investigations show that sigmoidal kinetics is not unique to microsomes and that CLmax is a useful parameter for scaling to the in vivo situation.

  7. Stimulation of tolbutamide hydroxylation by acetone and acetonitrile in human liver microsomes and in a cytochrome P-450 2C9-reconstituted system.

    Science.gov (United States)

    Palamanda, J; Feng, W W; Lin, C C; Nomeir, A A

    2000-01-01

    Organic solvents are often used to solubilize lipophilic new chemical entities before their addition to in vitro test systems such as microsomal stability or cytochrome P-450 (CYP) inhibition. However, the effect of these organic solvents on the test systems is not usually characterized. This study was initiated to evaluate the effect of acetonitrile and acetone, in addition to other organic solvents, on the tolbutamide hydroxylation activity of CYP2C9 in both human liver microsomes and a CYP2C9-reconstituted system. Both acetonitrile and acetone significantly stimulated the NADPH-dependent tolbutamide hydroxylation by nearly 2- to 3-fold in human liver microsomes and CYP2C9-reconstituted system when incubated at 2 and 4% final solvent concentrations. When cumene hydroperoxide was used instead of NADPH, both acetone and acetonitrile significantly inhibited tolbutamide hydroxylation. This NADPH-dependent stimulatory effect was further evaluated by examining the effect of a series of other organic solvents with different carbon chain lengths and various functional groups, including hydroxyl, ketone, and aldehyde. Unlike acetone, two other ketone-containing solvents, methyl ethyl ketone (2-butanone) and diethyl ketone (3-pentanone) failed to significantly enhance tolbutamide hydroxylation. Other solvents tested, including methanol, ethanol, propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, acetaldehyde, and dimethyl sulfoxide significantly inhibited NADPH-dependent tolbutamide hydroxylation. Overall, the stimulatory effect of both acetonitrile and acetone on tolbutamide hydroxylation was found to be primarily due to a consistent increase in V(max), whereas K(m) was unchanged in both human liver microsomes and the reconstituted CYP2C9 system. These data suggest that acetone and acetonitrile stimulate NADPH-mediated tolbutamide hydroxylation via the CYP reductase and not by modifying the affinity of tolbutamide for the CYP2C9 enzyme.

  8. The effect of alcoholic cirrhosis on the activities of microsomal aldrin epoxidase, 7-ethoxycoumarin O-de-ethylase and epoxide hydrolase, and on the concentrations of reduced glutathione in human liver.

    Science.gov (United States)

    Woodhouse, K W; Williams, F M; Mutch, E; Wright, P; James, O F; Rawlins, M D

    1983-01-01

    Activities of the microsomal mono-oxygenases 7-ethoxycoumarin O-de-ethylase (EOC) and aldrin epoxidase (AE), together with microsomal epoxide hydrolase (EH) activity and concentrations of reduced glutathione (GSH) have been measured in liver from patients with alcoholic cirrhosis and in normals. Activities of both mono-oxygenases were significantly reduced in alcoholic cirrhosis. EOC activity (pmol 7-OH coumarin formed/mg microsomal protein/min) was 108.0 +/- 10.6 (n = 8) in normals and 60.9 +/- 11.6 (n = 8) in alcoholic cirrhosis (P less than 0.01). AE activity (pmol dieldrin formed/mg microsomal protein/min) was 58.9 +/- 9.5 (n = 11) in normal liver biopsies and 29.9 +/- 8.6 (n = 9) in alcoholic cirrhosis (P less than 0.05). Microsomal EH activity (nmol styrene glycol formed/mg microsomal protein/min) was similar in normals (39.2 +/- 4.4, n = 11) and alcoholic cirrhosis (40.5 +/- 9.1, n = 6). GSH concentrations (microgram GSH/g liver tissue) were lower (P less than 0.01) in alcoholic cirrhosis (792 +/- 73, n = 10) compared to normals (1182 +/- 76, n = 6). PMID:6603231

  9. Characterization of the intracellular and the plasma membrane Ca2+-ATPases in fractionated pig brain membranes using calcium pump inhibitors.

    Science.gov (United States)

    Salvador, J M; Mata, A M

    1998-03-15

    The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions. Copyright 1998 Academic Press.

  10. In maize, glutelin-2 and low molecular weight zeins are synthesized by membrane-bound polyribosomes and translocated into microsomal membranes.

    Science.gov (United States)

    Torrent, M; Poca, E; Campos, N; Ludevid, M D; Palau, J

    1986-09-01

    Experiments to establish the site of biosynthesis and the possible translocation into microsomes of glutelins-2 (28 kD G2) and low molecular weight zeins (10, 14, 15 kD Z2) have been carried out. Free and membrane-bound polyribosomes as well as microsomal membranes were isolated from immature endosperms of W64A Zea mays L. In vitro translation studies were carried out in the presence and in the absence of membranes using [(35)S]-methionine or [(35)S]-cysteine as precursors. Cell-free translation products were characterized by electrophoretic mobility, solubility and antigenic properties. The results obtained indicate that 28 kD G2 and low molecular weight zeins are primarily synthesized on membrane-bound polysomes. From experiments using proteinase K as a probe, we also conclude that these proteins are translocated into microsomes where they accumulate. The translocated and pre-28 kD G2 proteins do not present changes in the apparent molecular weight. However we show that there are differences in their isoelectric points, a fact that indicates the existence of 28 kD G2 processing.

  11. Aromatization of androstenedione and 16alpha-hydroxyandrostenedione in human placental microsomes. Kinetic analysis of inhibition by the 19-oxygenated and 3-deoxy analogs.

    Science.gov (United States)

    Numazawa, Mitsuteru; Watari, Yoko; Komatsu, Sachiko; Yamashita, Kouwa; Nagaoka, Masao

    2008-11-01

    Inhibition of aromatase activity in human placental microsomes with androstenedione (AD) (1a) and its 19-oxygenated derivatives 1b and 1c, their 16alpha-hydroxy compounds 2 and 3, and 3-deoxyandrost-4-ene compounds 5 and 6 was studied using [1beta-(3)H]AD as a substrate and compared to that with [1beta-(3)H]16alpha-hydroxyandrostenedione (16-OHAD). AD series of steroids, compounds 1, inhibited competitively [1beta-(3)H]AD aromatization whereas other 16alpha-hydroxy steroids 2, 3, 5, and 6 inhibited AD aromatization in a non-competitive manner. On the other hand, all of 16-OHAD series, compounds 2, blocked the [1beta-(3)H]16-OHAD aromatization in a competitive manner whereas the AD series steroids 1 as well as the 3-deoxy-16alpha-hydroxy-17-one steroids 5 and 3-deoxy-16alpha,17beta-diol steroids 6 inhibited 16-OHAD aromatization non-competitively. 3-carbonyl and 16alpha-hydroxy functions of 16-OHAD play a critical role of selection of the 16-OHAD binding site. The results suggest that the AD derivatives 1 are kinetically aromatized at a different site from the 16-OHAD derivatives 2. Physical and/or chemical environments around the aromatase protein in the microsomal membrane may play a significant role in the expression of the substrate specificity, and the present results do not exclude the idea that the placental microsomes have a single binding site.

  12. Brain Basics

    Medline Plus

    Full Text Available ... are sent from one neuron to another. Share Science News New BRAIN Grants BRAIN Cell Census Launched ... human volunteers PubMed Central: An archive of life sciences journals NIH Research Fact Sheets NIH Office of ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... to change the way she thinks about and reacts to things that may trigger her depression. Several ... early brain development, and may also assist in learning and memory. hippocampus —A portion of the brain ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... Careers at NIMH Staff Directories Getting to NIMH Transforming the understanding and treatment of mental illnesses. Search ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... early brain development, and may also assist in learning and memory. hippocampus —A portion of the brain involved in creating and filing new memories. hypothalmic-pituitary-adrenal (HPA) ...

  16. Brain Diseases

    Science.gov (United States)

    The brain is the control center of the body. It controls thoughts, memory, speech, and movement. It regulates the function of many organs. When the brain is healthy, it works quickly and automatically. However, ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... may help improve treatments for anxiety disorders like phobias or post-traumatic stress disorder (PTSD) . Prefrontal cortex ( ... brain. Using MEG, some scientists have found a specific pattern of brain activity that may help predict ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... how the brain works, how mental illnesses are disorders of the brain, and ongoing research that helps us better understand and treat disorders. Mental disorders are common. You may have a ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... doctor that she had experienced long periods of deep sadness throughout her teenage years, but had never ... early brain development, and may also assist in learning and memory. hippocampus —A portion of the brain ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... but can still remember past events and learned skills, and carry on a conversation, all which rely ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of the brain's ... resonance imaging (MRI) mdash;An imaging technique that uses magnetic fields to take pictures of the brain's ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... go awry, leading to mental illnesses. Brain Basics will introduce you to some of this science, such ... released it increases the chance that the neuron will fire. This enhances the electrical flow among brain ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... Functional magnetic resonance imaging (fMRI) is another important research tool in understanding how the brain functions. Another type of brain scan called magnetoencephalography, or MEG, can ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... front of the brain, which is linked to thought and emotion. It is also linked to reward ... little dopamine or problems using dopamine in the thinking and feeling regions of the brain may play ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a middle- ... symptoms and family medical history. Epigenetic changes from stress or early-life experiences may have made it ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... Evidence shows that they can be related to changes in the anatomy, physiology, and chemistry of the ... brain communicate and work with each other How changes in the brain can lead to mental disorders, ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... Brain Basics will introduce you to some of this science, such as: How the brain develops How ... cell, and responds to signals from the environment; this all helps the cell maintain its balance with ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... all. She was happily married and successful in business. Then, after a serious setback at work, she ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... The basic structure of the brain How different parts of the brain communicate and work with each ... of conducting messages. A neuron has three basic parts: Cell body which includes the nucleus, cytoplasm, and ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... and the environment affect the brain The basic structure of the brain How different parts of the ... for the cell to work properly including small structures called cell organelles. Dendrites branch off from the ...

  11. Brain Basics

    Medline Plus

    Full Text Available ... Opportunities & Announcements Funding Strategy for Grants Application Process Managing Grants Clinical Research Training Labs at NIMH Labs ... normal brain development and function can go awry, leading to mental illnesses. Brain Basics will introduce you ...

  12. Brain Basics

    Medline Plus

    Full Text Available ... the brain How different parts of the brain communicate and work with each other How changes in ... communication signal sent between neurons by which neurons communicate with each other. magnetic resonance imaging (MRI) mdash; ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... at the front of the brain that, in humans, plays a role in executive functions such as ... Grants BRAIN Cell Census Launched How DNA Shapes Human Gene Expression More General Health Information from NIH ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... have been linked to many mental disorders, including autism , obsessive compulsive disorder (OCD) , schizophrenia , and depression . Brain ... studies show that brain growth in children with autism appears to peak early. And as they grow ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... disorder and to tailor the treatment for a person's specific conditions. Such brain research help increase the understanding of how the brain grows and works and the effects of genes and environment on mental health. This ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... the results can affect many aspects of life. Scientists are continually learning more about how the brain ... the normal brain's structure develops and matures helps scientists understand what goes wrong in mental illnesses. Scientists ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... works in healthy people, and how normal brain development and function can go awry, leading to mental ... and are working to compare that with brain development in people mental disorders. Genes and environmental cues ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... the anatomy, physiology, and chemistry of the nervous system. When the brain cannot effectively coordinate the billions ... basic working unit of the brain and nervous system. These cells are highly specialized for the function ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... normal brain development and function can go awry, leading to mental illnesses. Brain Basics will introduce you ... of DNA. Sometimes this copying process is imperfect, leading to a gene mutation that causes the gene ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses ... depression experience when starting treatment. Gene Studies Advanced technologies are also making it faster, easier, and more ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... The brain continues maturing well into a person's early 20s. Knowing how the brain is wired and ... for mental disorders. This could greatly help in early detection, more tailored treatments, and possibly prevention of ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... related to changes in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot ... her feelings. Brain Research Modern research tools and techniques are giving scientists a more detailed understanding of ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a ... blues" from time to time. In contrast, major depression is a serious disorder that lasts for weeks. ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... the brain cannot effectively coordinate the billions of cells in the body, the results can affect many ... unit of the brain and nervous system. These cells are highly specialized for the function of conducting ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses ... the body's response to stress. impulse —An electrical communication signal sent between neurons by which neurons communicate ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a ... in her life. She began to think of suicide because she felt like things weren't going ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot effectively coordinate the billions ... the basic working unit of the brain and nervous system. These cells are highly specialized for the function ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... Neurons & Neural Circuits Neurons are the basic working unit of the brain and nervous system. These cells ... A nerve cell that is the basic, working unit of the brain and nervous system, which processes ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... the brain, which is linked to thought and emotion. It is also linked to reward systems in ... or problems using dopamine in the thinking and feeling regions of the brain may play a role ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... brain may play a role in disorders like schizophrenia or attention deficit hyperactivity disorder (ADHD) . Glutamate —the ... mental disorders, including autism , obsessive compulsive disorder (OCD) , schizophrenia , and depression . Brain Regions Just as many neurons ...

  11. Brain Basics

    Medline Plus

    Full Text Available ... can be related to changes in the anatomy, physiology, and chemistry of the nervous system. When the ... healthy people, and how normal brain development and function can go awry, leading to mental illnesses. Brain ...

  12. Brain Basics

    Medline Plus

    Full Text Available ... as: How the brain develops How genes and the environment affect the brain The basic structure of the ... leaves the cell, and responds to signals from the environment; this all helps the cell maintain its balance ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... and her husband questions about Sarah's symptoms and family medical history. Epigenetic changes from stress or early- ... brain's structure, studies show that brain growth in children with autism appears to peak early. And as ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a middle- ... and her husband questions about Sarah's symptoms and family medical history. Epigenetic changes from stress or early- ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... and aiding the flow of information to the front of the brain, which is linked to thought ... and aiding the flow of information to the front of the brain. DNA —The "recipe of life," ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... or problems using dopamine in the thinking and feeling regions of the brain may play a role ... depression helps Sarah to better cope with her feelings. Brain Research Modern research tools and techniques are ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... mainly involved in controlling movement and aiding the flow of information to the front of the brain, ... the neuron will fire. This enhances the electrical flow among brain cells required for normal function and ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... control specific body functions such as sleep and speech. The brain continues maturing well into a person's ... as in areas of the brain that control movement. When electrical signals are abnormal, they can cause ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... it increases the chance that the neuron will fire. This enhances the electrical flow among brain cells ... This area of the brain also helps to control the amygdala during stressful events. Some research shows ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... to slow or stop them from progressing. Functional magnetic resonance imaging (fMRI) is another important research tool in understanding how the brain functions. Another type of brain scan called magnetoencephalography, ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit of ... but sometimes give rise to disabilities or diseases. neural circuit —A network of neurons and their interconnections. ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... the brain How different parts of the brain communicate and work with each other How changes in ... occur when this process does not work correctly. Communication between neurons can also be electrical, such as ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... affect many aspects of life. Scientists are continually learning more about how the brain grows and works ... early brain development. It may also assist in learning and memory. Problems in making or using glutamate ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... treatment for a person's specific conditions. Such brain research help increase the understanding of how the brain grows and works and the effects of genes and environment on mental health. This knowledge is allowing scientists ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... How the brain develops How genes and the environment affect the brain The basic structure of the ... inside contents of the cell from its surrounding environment and controls what enters and leaves the cell, ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit of ... of contact for receiving impulses on a neuron, branching off from the cell body. dopamine —A neurotransmitter ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... Publications Help for Mental Illnesses Clinical Trials Outreach Research Priorities Funding Labs at NIMH News & Events About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The ...

  8. The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5.

    Science.gov (United States)

    Canova-Davis, E; Waskell, L

    1984-02-25

    Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.

  9. Different effects of dihydropyridine calcium channel antagonists on CYP3A4 enzyme of human liver microsomes.

    Science.gov (United States)

    Xia, Zongling; Wang, Mingli; Zou, Sulan; Chen, Rong

    2012-09-01

    The present study investigated inhibitory effects of 1,4-dihydropyridines (1,4-DHPs) calcium channel antagonists (1,4-DHP-CCAs) on cytochromeP450 3A4 (CYP3A4) of human liver microsomes and further explored importance of 1,4-DHPs molecular structural descriptors. Partial Least Squares method was applied to probe the quantitative relationships between the 1,4-DHPs molecular structural descriptors and its inhibitory actions, which demonstrated that different 1,4-DHP-CCAs could inhibit CYP3A4 enzyme's activity differently. The K (i) values of nicardipine, lercandipine, cilnidipine, nitrendipine, lacidipine, nifedipine, felodipine were 10.13, 10.17, 11.44, 23.90, 29.34, 29.06 and 32.64 μmol L⁻¹, respectively. It is suggested that the 1,4-DHPs molecular structural descriptors are the most important for its inhibitory effects based on the quantitative structure-activity relationship (QSAR) formula. The LogP was positively correlated to the K (i), whereas molecular weight and molecule volume were negatively correlated. It is concluded that analysis of K (i) of 1,4-DHPs derivatives on the CYP3A4 activity may apply for the QSAR formula at the initial stage of clinical application of new drugs.

  10. Effect of Honokiol on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Yong Yeon Cho

    2013-09-01

    Full Text Available Honokiol is a bioactive component isolated from the medicinal herbs Magnolia officinalis and Magnolia grandiflora that has antioxidative, anti-inflammatory, antithrombotic, and antitumor activities. The inhibitory potentials of honokiol on eight major human cytochrome P450 (CYP enzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, and four UDP-glucuronosyltransferases (UGTs 1A1, 1A4, 1A9, and 2B7 in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Honokiol strongly inhibited CYP1A2-mediated phenacetin O-deethylation, CYP2C8-mediated amodiaquine N-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4-hydroxylation, and UGT1A9-mediated propofol glucuronidation with Ki values of 1.2, 4.9, 0.54, 0.57, and 0.3 μM, respectively. Honokiol also moderately inhibited CYP2B6-mediated bupropion hydroxylation and CYP2D6-mediated bufuralol 1'-hydroxylation with Ki values of 17.5 and 12.0 μM, respectively. These in vitro results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9.

  11. Mouse microsomal triglyceride transfer protein large subunit: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Nakamuta, Makoto; Chang, Benny Hung-Junn; Hoogeveen, R. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1996-04-15

    Microsomal triglyceride transfer protein (MTP) catalyzes the transfer of triglyceride, cholesteryl ester, and phospholipid between membranes. It is essential for the secretion of apolipoprotein B from the cell. Mutations in MTP are a major cause of abetalipoproteinemia. The mouse is a popular animal model for lipoprotein metabolism. We have cloned and sequenced mouse MTP cDNA. The DNA-deduced amino acid sequence indicates that mouse protein shows 93, 86, and 83% sequence indicates that mouse MTP contains 894 amino acids; the mouse protein shows 93, 86, and 83% sequence identity to the hamster, human, and bovine sequences, respectively. Northern blot analysis indicates that mouse MTP mRNA is expressed at high levels in the small intestine and at substantially lower levels in the liver and that it is not detectable in six other tissues examined. The mouse MTP gene has been localized to the distal region of chromosome 3 by Southern blots of interspecific backcross panels using progeny derived from matings of (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei. Comparison of MTP sequences from human, bovine, hamster, and mouse indicates that the C-terminal region of MTP is better conserved than its N-terminal region. 21 refs., 2 figs.

  12. Reversible formation of fatty acid esters of budesonide, an antiasthma glucocorticoid, in human lung and liver microsomes.

    Science.gov (United States)

    Tunek, A; Sjödin, K; Hallström, G

    1997-11-01

    Microsomes from human lung and liver catalyze the formation of fatty acid esters of budesonide, a glucocorticoid used for inhalation treatment of asthma. The conjugation was dependent on coenzyme A and ATP. Addition of free fatty acids to the incubations affected the pattern of metabolites, but ester formation was observed also without such addition. Budesonide oleate, palmitate, linoleate, palmitoleate, and arachidonate were identified as metabolites. The fatty acid conjugates of budesonide were shown to be substrates for lipase in vitro, thus budesonide is regainable from the conjugates. The data suggest that an equilibrium between budesonide and these pharmacologically inactive lipoidal conjugates will be established in tissues at repeated exposure to budesonide. Since the fatty acid conjugates most likely will be retained intracellularly for a longer time than unchanged budesonide, the duration of tissue exposure to budesonide will depend partly on the rate of lipase-catalyzed hydrolysis of the conjugates. The findings in this study provide a possible explanation for the efficacy of budesonide in mild asthmatics also when inhaled once daily.

  13. Mutagenicity testing (+/-)-camphor, 1,8-cineole, citral, citronellal, (-)-menthol and terpineol with the Salmonella/microsome assay.

    Science.gov (United States)

    Gomes-Carneiro, M R; Felzenszwalb, I; Paumgartten, F J

    1998-08-07

    The essential oils and their monoterpenoid constituents have been widely used as fragrances in cosmetics, as flavouring food additives, as scenting agents in a variety of household products, as active ingredients in some old drugs, and as intermediates in the synthesis of perfume chemicals. The present study was undertaken to investigate the mutagenic potential of six monoterpenoid compounds: two aldehydes (citral and citronellal), a ketone ((+/-)-camphor), an oxide (1,8-cineole, also known as eucalyptol), and two alcohols (terpineol and (-)-menthol). It is part of a more comprehensive toxicological screening of monoterpenes under way at our laboratory. Mutagenicity was evaluated by the Salmonella/microsome assay (TA97a, TA98, TA100 and TA102 tester strains), without and with addition of an extrinsic metabolic activation system (lyophilized rat liver S9 fraction induced by Aroclor 1254). In all cases, the upper limit of the dose interval tested was either the highest non-toxic dose or the lowest dose of the monoterpene toxic to TA100 strain in the preliminary toxicity test. No mutagenic effect was found with (+/-) camphor, citral, citronellal, 1,8-cineole, and (-) menthol. Terpineol caused a slight but dose-related increase in the number of his+ revertants with TA102 tester strain both without and with addition of S9 mixture. The results from this study therefore suggest that, with the exception of terpineol, the monoterpenoid compounds tested are not mutagenic in the Ames test.

  14. Aromatization of 16alpha-hydroxyandrostenedione by human placental microsomes: effect of preincubation with suicide substrates of androstenedione aromatization.

    Science.gov (United States)

    Numazawa, Mitsuteru; Tachibana, Mii; Mutsumi, Ayako; Yoshimura, Akiko; Osawa, Yoshio

    2002-06-01

    Estrogen synthase (aromatase) catalyzes the aromatization of androstenedione (AD) as well as 16alpha-hydroxyandrostenedione (16alpha-OHAD) leading to estrone and estriol, respectively. We found that several steroid analogs including 4-hydroxyandrostenedione (1), 6-oxoandrostenedione (6-oxoAD, 2) and its 19-hydroxy analog (3), 10beta-acetoxyestr-5-ene-7,17-dione (4), androst-5-ene-4,7,17-trione (5), and 17alpha-ethynyl-19-norteststerone (6), which are known suicide inactivators of AD aromatization, are not effective in inactivating 16alpha-OHAD aromatization in a time-dependent manner. The compounds were tested with the use of human placental microsomes and 1beta-tritiated-16alpha-OHAD as the substrate. The results of the tritium water method of 16alpha-OHAD aromatization was confirmed by the gas chromatography-mass spectrometry (GC-MS) method of estriol formation. The 1beta-tritiated-AD was used to measure AD aromatization as a positive control for these experiments. The compounds were tested at concentrations up to 40-fold higher than the K(i)'s determined for inhibition of AD aromatization. These studies suggest that differences exist in the binding site structures responsible for aromatization of 16alpha-OHAD and AD.

  15. Potency of Semecarpus anacardium Linn. nut milk extract against aflatoxin B(1)-induced hepatocarcinogenesis: reflection on microsomal biotransformation enzymes.

    Science.gov (United States)

    Premalatha, B; Sachdanandam, P

    2000-08-01

    The effect of Semecarpus anacardium Linn. nut milk extract on host detoxification system in aflatoxin B(1) induced hepatocellular carcinoma, which is a vital mechanism in cancer treatment, was studied in male albino rats. Oral administration of nut extract (200 mg kg(-1)body weight per day for 14 days) is found to be highly effective in inducing phase I and phase II biotransformation enzymes. The obtained results have shown an overall decrease of liver microsomal cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, and aniline hydroxylase with a subsequent decrease of phase II enzymes, glutathione-S-transferase and UDP-glucuronyl transferase in cancer-bearing animals. The Semecarpus anacardium nut extract affords anticancer activity by enhancing both phase I and phase II enzymes to near normal levels. We propose that, much of the anticarcinogenic potency of Semecarpus anacardium nut extract on aflatoxin B(1)-induced hepatocarcinogenesis is mediated through the induction of hepatic biotransformation enzymes. Copyright 2000 Academic Press.

  16. Brain Basics

    Medline Plus

    Full Text Available ... a major mood circuit called the hypothalamic-pituitary-adrenal (HPA) axis. Brain Basics in Real Life Brain ... in creating and filing new memories. hypothalmic-pituitary-adrenal (HPA) axis —A brain-body circuit which plays ...

  17. Brain Aneurysm

    Science.gov (United States)

    A brain aneurysm is an abnormal bulge or "ballooning" in the wall of an artery in the brain. They are sometimes called berry aneurysms because they ... often the size of a small berry. Most brain aneurysms produce no symptoms until they become large, ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... early brain development. It may also assist in learning and memory. Problems in making or using glutamate have been ... early brain development, and may also assist in learning and memory. hippocampus —A portion of the brain involved in ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... affect many aspects of life. Scientists are continually learning more about how the brain grows and works in healthy people, and how ... early brain development, and may also assist in learning and memory. hippocampus —A portion of the brain involved in creating and filing new memories. hypothalmic- ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... including autism , obsessive compulsive disorder (OCD) , schizophrenia , and depression . Brain Regions Just as many neurons working together form ... Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a middle-aged woman ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... pituitary-adrenal (HPA) axis. Brain Basics in Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah ... having trouble coping with the stresses in her life. She began to think of suicide because she ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... These circuits control specific body functions such as sleep and speech. The brain continues maturing well into a person's early 20s. ... that regulates many functions, including mood, appetite, and sleep. synapse —The tiny gap between neurons, where nerve impulses are sent from one neuron to ... of Deep Brain Stimulation Brain’s Alertness Circuitry Revealed New BRAIN Grants ...

  3. Brain Aneurysm

    Science.gov (United States)

    ... inside of the brain (ventricles) or surrounding your brain and spinal cord to drain the excess fluid into an external bag. Sometimes it may then be necessary to introduce a shunt system — which consists of a ... brain and ending in your abdominal cavity. Rehabilitative therapy. ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of the brain's structure, studies show that brain growth in children with autism appears to peak early. And as they grow there are differences in ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... another important research tool in understanding how the brain functions. Another type of brain scan called magnetoencephalography, or ... highly developed area at the front of the brain that, in humans, plays a role in executive functions such as judgment, decision making and problem solving, ...

  6. Acyl-CoA synthetase activity links wild-type but not mutant a-Synuclein to brain arachidonate metabolism

    DEFF Research Database (Denmark)

    Golovko, Mikhail; Rosenberger, Thad; Færgeman, Nils J.

    2006-01-01

    Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of alpha-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-(14)C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using...... an established steady-state kinetic model. Liver was used as a negative control, and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored...... after the addition of exogenous wild-type mouse or human alpha-synuclein, but not by A30P, E46K, and A53T forms of alpha-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools were markedly reduced...

  7. Left Brain. Right Brain. Whole Brain

    Science.gov (United States)

    Farmer, Lesley S. J.

    2004-01-01

    As the United States student population is becoming more diverse, library media specialists need to find ways to address these distinctive needs. However, some of these differences transcend culture, touching on variations in the brain itself. Most people have a dominant side of the brain, which can affect their personality and learning style.…

  8. Oxidation of R- and S-omeprazole stereoselectively mediated by liver microsomal cytochrome P450 2C19 enzymes from cynomolgus monkeys and common marmosets.

    Science.gov (United States)

    Uehara, Shotaro; Kawano, Mirai; Murayama, Norie; Uno, Yasuhiro; Utoh, Masahiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2016-11-15

    Racemic omeprazole has been used for clinically treating gastric acid-related diseases and also as a typical human cytochrome P450 (P450) 2C19 probe substrate in preclinical studies. S-Omeprazole has been developed as a single enantiomer medicine, which has been reported not to be associated with polymorphic human P450 2C19 phenotypes. In this study, 5-hydroxylation and sulfoxidation activities, with respect to stereoselective R- and S-omeprazole oxidations by liver microsomes from experimental animals including non-human primates and humans, were investigated in vitro. Liver microsomes from humans, cynomolgus monkeys, and mice preferentially mediated R-omeprazole 5-hydroxylations, however those from marmosets, minipigs, dogs, and rats preferentially mediated S-omeprazole 5-hydroxylations. High catalytic activities were observed for recombinant human P450 2C19 in R-omeprazole 5-hydroxlations, cynomolgus monkey P450 2C19 in both R- and S-omeprazole 5-hydroxlations, and marmoset P450 2C19 in S-omeprazole 5-hydroxlations. On the other hand, human, cynomolgus monkey, and marmoset P450 3A enzymes preferentially mediated S-omeprazole sulfoxidations. Correlation and kinetic analyses revealed a high affinity of polymorphic cynomolgus monkey and marmoset liver microsomal P450 2C19 enzymes with respect to R- and S-omeprazole 5-hydroxylations, respectively, and a high capacity of cynomolgus monkey and marmoset liver microsomal P450 3A4 for omeprazole 5-hydroxylations and sulfoxidations. R-and S-omeprazole 5-hydroxylation activities in cynomolgus monkey and marmoset liver microsomes were significantly different among wild-type, heterozygous, and homozygous animals genotyped for cynomolgus monkey P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] and for marmoset P450 2C19 p.[(Phe7Leu; Ser254Leu; Ile469Thr)], respectively. The results of this study demonstrate polymorphic cynomolgus monkey and marmoset P450 2C19-dependent omeprazole oxidation activities with individual variations

  9. Identification of cytochrome P450 2E1 as the predominant enzyme catalyzing human liver microsomal defluorination of sevoflurane, isoflurane, and methoxyflurane.

    Science.gov (United States)

    Kharasch, E D; Thummel, K E

    1993-10-01

    Renal and hepatic toxicity of the fluorinated ether volatile anesthetics is caused by biotransformation to toxic metabolites. Metabolism also contributes significantly to the elimination pharmacokinetics of some volatile agents. Although innumerable studies have explored anesthetic metabolism in animals, there is little information on human volatile anesthetic metabolism with respect to comparative rates or the identity of the enzymes responsible for defluorination. The first purpose of this investigation was to compare the metabolism of the fluorinated ether anesthetics by human liver microsomes. The second purpose was to test the hypothesis that cytochrome P450 2E1 is the specific P450 isoform responsible for volatile anesthetic defluorination in humans. Microsomes were prepared from human livers. Anesthetic metabolism in microsomal incubations was measured by fluoride production. The strategy for evaluating the role of P450 2E1 in anesthetic defluorination involved three approaches: for a series of 12 human livers, correlation of microsomal defluorination rate with microsomal P450 2E1 content (measured by Western blot analysis), correlation of defluorination rate with microsomal P450 2E1 catalytic activity using marker substrates (para-nitrophenol hydroxylation and chlorzoxazone 6-hydroxylation), and chemical inhibition by P450 isoform-selective inhibitors. The rank order of anesthetic metabolism, assessed by fluoride production at saturating substrate concentrations, was methoxyflurane > sevoflurane > enflurane > isoflurane > desflurane > 0. There was a significant linear correlation of sevoflurane and methoxyflurane defluorination with antigenic P450 2E1 content (r = 0.98 and r = 0.72, respectively), but not with either P450 1A2 or P450 3A3/4. Comparison of anesthetic defluorination with either para-nitrophenol or chlorzoxazone hydroxylation showed a significant correlation for sevoflurane (r = 0.93, r = 0.95) and methoxyflurane (r = 0.78, r = 0

  10. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying; Takagi, Akira [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kayano, Hidekazu [Department of Pathology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Koyama, Isamu [Department of Digestive and General Surgery, Saitama International Medical Center, Faculty of Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  11. Microsomal Glutathione Transferase 1 Protects Against Toxicity Induced by Silica Nanoparticles but Not by Zinc Oxide Nanoparticles

    Science.gov (United States)

    2012-01-01

    Microsomal glutathione transferase 1 (MGST1) is an antioxidant enzyme located predominantly in the mitochondrial outer membrane and endoplasmic reticulum and has been shown to protect cells from lipid peroxidation induced by a variety of cytostatic drugs and pro-oxidant stimuli. We hypothesized that MGST1 may also protect against nanomaterial-induced cytotoxicity through a specific effect on lipid peroxidation. We evaluated the induction of cytotoxicity and oxidative stress by TiO2, CeO2, SiO2, and ZnO in the human MCF-7 cell line with or without overexpression of MGST1. SiO2 and ZnO nanoparticles caused dose- and time-dependent toxicity, whereas no obvious cytotoxic effects were induced by nanoparticles of TiO2 and CeO2. We also noted pronounced cytotoxicity for three out of four additional SiO2 nanoparticles tested. Overexpression of MGST1 reversed the cytotoxicity of the main SiO2 nanoparticles tested and for one of the supplementary SiO2 nanoparticles but did not protect cells against ZnO-induced cytotoxic effects. The data point toward a role of lipid peroxidation in SiO2 nanoparticle-induced cell death. For ZnO nanoparticles, rapid dissolution was observed, and the subsequent interaction of Zn2+ with cellular targets is likely to contribute to the cytotoxic effects. A direct inhibition of MGST1 by Zn2+ could provide a possible explanation for the lack of protection against ZnO nanoparticles in this model. Our data also showed that SiO2 nanoparticle-induced cytotoxicity is mitigated in the presence of serum, potentially through masking of reactive surface groups by serum proteins, whereas ZnO nanoparticles were cytotoxic both in the presence and in the absence of serum. PMID:22303956

  12. Cardiac expression of microsomal triglyceride transfer protein is increased in obesity and serves to attenuate cardiac triglyceride accumulation.

    Directory of Open Access Journals (Sweden)

    Emil D Bartels

    Full Text Available Obesity causes lipid accumulation in the heart and may lead to lipotoxic heart disease. Traditionally, the size of the cardiac triglyceride pool is thought to reflect the balance between uptake and beta-oxidation of fatty acids. However, triglycerides can also be exported from cardiomyocytes via secretion of apolipoproteinB-containing (apoB lipoproteins. Lipoprotein formation depends on expression of microsomal triglyceride transfer protein (MTP; the mouse expresses two isoforms of MTP, A and B. Since many aspects of the link between obesity-induced cardiac disease and cardiac lipid metabolism remain unknown, we investigated how cardiac lipoprotein synthesis affects cardiac expression of triglyceride metabolism-controlling genes, insulin sensitivity, and function in obese mice. Heart-specific ablation of MTP-A in mice using Cre-loxP technology impaired upregulation of MTP expression in response to increased fatty acid availability during fasting and fat feeding. This resulted in cardiac triglyceride accumulation but unaffected cardiac insulin-stimulated glucose uptake. Long-term fat-feeding of male C57Bl/6 mice increased cardiac triglycerides, induced cardiac expression of triglyceride metabolism-controlling genes and attenuated heart function. Abolishing cardiac triglyceride accumulation in fat-fed mice by overexpression of an apoB transgene in the heart prevented the induction of triglyceride metabolism-controlling genes and improved heart function. The results suggest that in obesity, the physiological increase of cardiac MTP expression serves to attenuate cardiac triglyceride accumulation albeit without major effects on cardiac insulin sensitivity. Nevertheless, the data suggest that genetically increased lipoprotein secretion prevents development of obesity-induced lipotoxic heart disease.

  13. Heterocyclic compounds: toxic effects using algae, daphnids, and the Salmonella/microsome test taking methodical quantitative aspects into account.

    Science.gov (United States)

    Eisentraeger, Adolf; Brinkmann, Corinna; Hollert, Henner; Sagner, Anne; Tiehm, Andreas; Neuwoehner, Judith

    2008-07-01

    Heterocyclic aromatic hydrocarbons containing nitrogen, sulfur, or oxygen (NSO-HET), have been detected in air, soil, sewage sludge, marine environments, and freshwater sediments. Since toxicity data on this class of substances are scarce, the present study focuses on possible implications NSO-HET have for ecotoxicity (algae and daphnids) and mutagenicity (Salmonella/microsome test). A combination of bioassays and chemical-analytical quantification of the test compounds during toxicity assays should aid in determination of the hazard potential. Samples of the test concentrations of 14 NSO-HET were taken at the beginning and end of the bioassays; these samples were then quantified by high-performance liquid chromatography. The toxicity potential of the substances was evaluated and compared with the toxicity calculated with the nominal concentrations. Significantly different results were obtained primarily for volatile or highly hydrophobic NSO-HET. The concentration of heterocyclic hydrocarbons can change significantly during the algae and Daphnia test. The EC50 values (effective concentration value: the concentration of a chemical that is required to produce a 50% effect) calculated with the nominal concentrations underestimate the toxicity by a factor of up to 50. Prioritizing the tested compounds according to toxicity, the mutagenic and toxic compounds quinoline, 6-methylquinoline, and xanthene have to be listed first. The greatest ecotoxic potential on algae and daphnids was determined for dibenzothiophene followed by acridine. In the Daphnia magna immobilization test, benzofuran, dibenzofuran, 2-methylbenzofuran, and 2,3-dimethylbenzofuran and also carbazole are ecotoxicologically relevant with EC50 values below 10 mg/L. These substances are followed by indole with a high ecotoxic effect to daphnids and less effect to algae. Only minor toxic effects were observed for 2-methylpyridine and 2,4,6-trimethylpyridine.

  14. Identification of metabolites of meisoindigo in rat, pig and human liver microsomes by UFLC-MS/MS.

    Science.gov (United States)

    Huang, Meng; Ho, Paul C

    2009-04-15

    3-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-1,3-dihydro-1-methyl-2H-indol-2-one, abbreviated as meisoindigo, has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. To gain an understanding of the interspecies differences in the metabolism of meisoindigo, the relevant metabolism studies were carried out for the first time in rat, pig and human liver microsomes of different genders by ultra fast liquid chromatography/tandem mass spectrometry (UFLC-MS/MS). The qualitative metabolite identification was accomplished by multiple reaction monitoring (MRM) in combination with Enhanced Product Ion (EPI). The semi-quantitative metabolic stability and metabolite formation were simultaneously measured by MRM. The in vitro metabolic pathways of meisoindigo in three species were proposed as 3,3' double bond reduction, followed by N-demethylation, and reduction followed by phenyl mono-oxidation. Two novel metabolic pathways involving direct phenyl mono-oxidation without reduction in the three species, and direct N-demethylation without reduction in only pig and human, were also proposed. It may be noted that the two metabolites formed after reduction followed by phenyl mono-oxidation at positions 4, 5, 6 or 7, as well as one metabolite formed from direct phenyl mono-oxidation at either of the two phenyl rings without reduction were found to be uniquely present in human. The in vitro t(1/2) and in vitro CL(int) values of meisoindigo were calculated. Statistical analysis showed there were no significant differences in the metabolic stability profiles of meisoindigo among three species, and gender effect on the metabolic stability of meisoindigo was negligible. Formation profiles of the most significant reductive metabolites were obtained in the three species.

  15. Prediction of human drug clearance by multiple metabolic pathways: integration of hepatic and intestinal microsomal and cytosolic data.

    Science.gov (United States)

    Cubitt, Helen E; Houston, J Brian; Galetin, Aleksandra

    2011-05-01

    The current study assesses hepatic and intestinal glucuronidation, sulfation, and cytochrome P450 (P450) metabolism of raloxifene, quercetin, salbutamol, and troglitazone using different in vitro systems. The fraction metabolized by conjugation and P450 metabolism was estimated in liver and intestine, and the importance of multiple metabolic pathways on accuracy of clearance prediction was assessed. In vitro intrinsic sulfation clearance (CL(int, SULT)) was determined in human intestinal and hepatic cytosol and compared with hepatic and intestinal microsomal glucuronidation (CL(int, UGT)) and P450 clearance (CL(int, CYP)) expressed per gram of tissue. Hepatic and intestinal cytosolic scaling factors of 80.7 mg/g liver and 18 mg/g intestine were estimated from published data. Scaled CL(int, SULT) ranged between 0.7 and 11.4 ml · min(-1) · g(-1) liver and 0.1 and 3.3 ml · min(-1) · g(-1) intestine (salbutamol and quercetin were the extremes). Salbutamol was the only compound with a high extent of sulfation (51 and 28% of total CL(int) for liver and intestine, respectively) and also significant renal clearance (26-57% of observed plasma clearance). In contrast, the clearance of quercetin was largely accounted for by glucuronidation. Drugs metabolized by multiple pathways (raloxifene and troglitazone) demonstrated improved prediction of intravenous clearance using data from all hepatic pathways (44-86% of observed clearance) compared with predictions based only on the primary pathway (22-36%). The assumption of no intestinal first pass resulted in underprediction of oral clearance for raloxifene, troglitazone, and quercetin (3-22% of observed, respectively). Accounting for the intestinal contribution to oral clearance via estimated intestinal availability improved prediction accuracy for raloxifene and troglitazone (within 2.5-fold of observed). Current findings emphasize the importance of both hepatic and intestinal conjugation for in vitro-in vivo extrapolation

  16. Comparative kinetics of fatty acid-amino acid conjugate elicitor biosynthesis by midgut tissue microsomes of Lepidopterous caterpillar larvae.

    Science.gov (United States)

    Lait, Cameron G; Lobaido, Melanie J; Wiester, Amy J; Kossak, Sarah; Tumlinson, James H

    2010-12-01

    N-Linolenoyl-L-glutamine is one of several structurally similar fatty acid-amino acid conjugate (FAC) elicitors found in the oral secretions of Lepidopterous caterpillars and its biosynthesis is catalyzed by membrane-associated alimentary tissue enzyme(s). FAC elicitors comprise 17-hydroxylated or non-hydroxylated linolenic acid coupled with L-glutamine or L-glutamate by an amide bond. We demonstrate in vitro biosynthesis of N-linolenoyl-L-glutamine by Manduca sexta, Heliothis virescens, and Helicoverpa zea tissue microsomes. Comparison of N-linolenoyl-L-glutamine biosynthesis kinetics for these species suggests that concurrent biosynthesis and hydrolysis contribute to proportions of FAC elicitors found in their oral secretions. The apparent K(m) values for coupling of sodium linolenate were 8.75±0.79, 14.3±3.7 and 20.7±3.4 mM and V(max) values were 2.92±0.14, 6.81±1.2 and 4.95±0.55 nmol/min/mg protein for H. zea, H. virescens and M. sexta, respectively. The K(m) values for coupling of L-glutamine were 10.5±0.26, 22.3±2.0 and 18.9±2.4 mM and V(max) values were 1.78±0.21, 3.71±0.50 and 2.49±0.41 nmol/min/mg of protein for H. zea, H. virescens and M. sexta, respectively. © 2010 Wiley Periodicals, Inc.

  17. Brain glycogen

    DEFF Research Database (Denmark)

    Obel, Linea Lykke Frimodt; Müller, Margit S; Walls, Anne B

    2012-01-01

    Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g., liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia....... In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies-it is a highly dynamic molecule with versatile implications in brain function, i.e., synaptic...... activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms...

  18. Brain death.

    Science.gov (United States)

    Beresford, H R

    1999-05-01

    Current law in the United States authorizes physicians to diagnose brain death by applying generally accepted neurologic criteria for determining loss of function of the entire brain. This article offers a medical-legal perspective on problems that may arise with respect to the determination of brain death. These include the possibility of diagnostic error, conceptual disagreements that may constrain the use of neurologic criteria to diagnose death, and the conflation of brain death and loss of consciousness. This article also addresses legal aspects of the debate over whether to expand the definition of brain death to include permanent unconsciousness. Although existing laws draw a clear distinction between brain death and the persistent vegetative state, many courts have authorized removal of life support from individuals whose unconsciousness is believed to be permanent on proof that removal accords with preferences expressed before sentience was lost.

  19. Brain Basics

    Medline Plus

    Full Text Available ... have been linked to many mental disorders, including autism , obsessive compulsive disorder (OCD) , schizophrenia , and depression . Brain Regions Just as many neurons working together form ...

  20. In vitro characterization of the oxidative cleavage of the octyl side chain of olanexidine, a novel antimicrobial agent, in dog liver microsomes.

    Science.gov (United States)

    Umehara, K; Kudo, S; Hirao, Y; Morita, S; Ohtani, T; Uchida, M; Miyamoto, G

    2000-12-01

    The metabolism of olanexidine [1-(3,4-dichlorobenzyl)-5-octylbiguanide], a new potent biguanide antiseptic, was investigated in dog liver microsomes to characterize the enzyme(s) catalyzing the biotransformation of olanexidine to C-C bond cleavage metabolites. Olanexidine was initially biotransformed to monohydroxylated metabolite 2-octanol (DM-215), and DM-215 was subsequently oxidized to diol derivatives threo-2,3-octandiol (DM-221) and erythro-2,3-octandiol (DM-222). Diols were further biotransformed to a ketol derivative and C-C bond cleavage metabolite (DM-210, hexanoic acid derivative), an in vivo end product, in the incubation with dog liver microsomes. The formations of DM-215, DM-221, DM-222, and DM-210 followed Michaelis-Menten kinetics, and Eadie-Hofstee analysis of the metabolite formation activity confirmed single-enzyme Michaelis-Menten kinetics. The K(m) and V(max) values for the formation of DM-210 appeared to be 2.42 microM and 26.6 pmol/min/mg in the oxidation of DM-221 and 2.48 microM and 30.2 pmol/min/mg in the oxidation of DM-222. The intrinsic clearance (V(max)/K(m)) of the C-C bond cleavage reactions was essentially the same with either DM-221 or DM-222 as substrate. These oxidative reactions were significantly inhibited by quinidine, a selective inhibitor of CYP2D subfamilies, indicating the metabolic C-C bond cleavage of the octyl side chain of olanexidine to likely be mediated via the CYP2D subfamily in dog liver microsomes. This aliphatic C-C bond cleavage by cytochrome P450s may play an important role in the metabolism of other drugs or endogenous compounds possessing aliphatic chains.

  1. Metabolism of novel opioid agonists U-47700 and U-49900 using human liver microsomes with confirmation in authentic urine specimens from drug users.

    Science.gov (United States)

    Krotulski, Alex J; Mohr, Amanda L A; Papsun, Donna M; Logan, Barry K

    2017-06-13

    Recently, the number of adverse events, including death, involving novel opioids has continued to increase, providing additional and sustained challenges for forensic and medical communities. Identification of emerging novel opioids can be challenging, compounded by detection windows and unknown metabolic profiles. In this study, human liver microsomes were used for the generation of in vitro metabolic profiles of U-47700 and U-49900. Generated metabolites were analyzed via a SCIEX TripleTOF® 5600+ quadrupole time-of-flight mass spectrometer and resulting data files were processing using MetabolitePilot™. Characterized metabolites were verified in vivo by analysis of authentic human urine specimens collected after analytically confirmed cases of overdose involving U-47700 or U-49900. In total, four metabolites were identified and present in urine specimens for U-47700, and five metabolites for U-49900. N-Desmethyl-U-47700 was determined to be the primary metabolite of U-47700. Parent U-47700 was identified in all urine specimens. N-Desmethyl-U-47700 and N,N-didesmethyl-U-47700 were structurally confirmed for the first time during this study following acquisition of standard reference material. N-Desethyl-U-49900 was determined to be the primary metabolite of U-49900 following microsomal incubations, while N,N-didesethyl-N-desmethyl-U-49900 was the most abundant in a urine specimen. Similarities in metabolic transformation were identified between U-47700 and U-49900, resulting in a common metabolite and isomeric species. These phenomena should be considered in cases involving U-47700 or U-49900. This study is the first to map the metabolic profiles of U-47700 and U-49900 using human liver microsomes, as well as the first to report any literature involving U-49900 and analysis of case specimens. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Effectiveness of human cytochrome P450 3A4 present in liposomal and microsomal nanoparticles in formation of covalent DNA adducts by ellipticine.

    Science.gov (United States)

    Sulc, Miroslav; Mrizova, Iveta; Cerna, Tereza; Frei, Eva; Eckschlager, Tomas; Adam, Vojtech; Kopeckova, Katerina; Stiborova, Marie

    2016-12-18

    Ellipticine is an anticancer agent that functions through multiple mechanisms participating in cell cycle arrest and initiation of apoptosis. This drug forms covalent DNA adducts after its enzymatic activation with cytochrome P450 (CYP), which is one of the most important ellipticine DNA-damaging mechanisms of its cytotoxic effects. The improvements of cancer treatment are the major challenge in oncology research. Nanotransporters (nanoparticles) are promising approaches to target tumor cells, frequently leading to improve drug therapeutic index. Ellipticine has already been prepared in nanoparticle forms. However, since its anticancer efficiency depends on the CYP3A4-mediated metabolism in cancer cells, the aim of our research is to develop nanoparticles containing this enzyme that can be transported to tumor cells, thereby potentiating ellipticine cytotoxicity. The CYP3A4 enzyme encapsulated into two nanoparticle forms, liposomes and microsomes, was tested to activate ellipticine to its reactive species forming covalent DNA adducts. Ellipticine-derived DNA adducts were determined by the 32P-postlabeling method. The CYP3A4 enzyme both in the liposome and microsome nanoparticle forms was efficient to activate ellipticine to species forming DNA adducts. Two DNA adducts, which are formed from ellipticine metabolites 12-hydroxy- and 13-hydroxyellipticine generated by its oxidation by CYP3A4, were formed by both CYP3A4 nanoparticle systems. A higher effectiveness of CYP3A4 in microsomal than in liposomal nanoparticles to form ellipticine-DNA adducts was found. Further testing in a suitable cancer cell model is encouraged to investigate whether the DNA-damaging effects of ellipticine after its activation by CYP3A4 nanoparticle forms are appropriate for active targeting of this enzyme to specific cancer cells.

  3. In vitro metabolism of cis- and trans-permethrin by rat liver microsomes, and its effect on estrogenic and anti-androgenic activities.

    Science.gov (United States)

    Tange, Satoko; Fujimoto, Nariaki; Uramaru, Naoto; Sugihara, Kazumi; Ohta, Shigeru; Kitamura, Shigeyuki

    2014-05-01

    Permethrin is a widely applied broad-spectrum pyrethroid insecticide that consists of a mixture of cis- and trans-isomers. We examined the changes of estrogenic and anti-androgenic activities resulting from metabolism of the isomers. Both cis- and trans-permethrin were hydrolyzed to 3-phenoxybenzyl alcohol (PBAlc) by rat liver microsomes, but the extent of hydrolysis of trans-permethrin was much greater than that of the cis-isomer. In the presence of NADPH, PBAlc was further transformed to 4'-hydroxylated PBAlc (4'-OH PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid). cis-Permethrin, but not trans-permethrin, also afforded its 4'-hydroxylated derivative (4'-OH cis-permethrin). trans-Permethrin was an anti-androgen, but also showed weak estrogenic activity, while cis-permethrin was a weak estrogen and a weak anti-androgen. After incubation with rat liver microsomes in the presence of NADPH, cis-permethrin but not trans-permethrin was metabolically activated for estrogenic activity. On the other hand, estrogenic activity of trans-permethrin was not changed, but its anti-androgenic activity was enhanced after incubation. 4'-OH PBAlc and PBAlc showed estrogenic activity, while PBAld and PBAlc showed anti-androgenic activity. PBAcid showed neither activity. 4'-OH cis-permethrin showed both estrogenic and anti-androgenic activities. Overall, our results indicate that permethrin is metabolically activated for estrogenic and anti-androgen activities, and the microsomal transformation of permethrin to 4'-OH cis-permethrin, 4'-OH PBAlc and PBAlc contributes to the both metabolic activations. Copyright © 2014. Published by Elsevier B.V.

  4. Characterization of in vitro glucuronidation clearance of a range of drugs in human kidney microsomes: comparison with liver and intestinal glucuronidation and impact of albumin.

    Science.gov (United States)

    Gill, Katherine L; Houston, J Brian; Galetin, Aleksandra

    2012-04-01

    Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL(int, UGT)) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL(int, UGT) on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL(int, UGT) in different tissues. Although BSA increased CL(int, UGT) in all tissues, the extent was tissue- and drug-dependent. Scaled CL(int, UGT) in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min(-1) · g tissue(-1) in liver, kidney, and intestinal microsomes. Renal CL(int, UGT) (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL(int, UGT) for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CL(int, UGT) (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL(int, UGT) is particularly important for UGT1A9 substrates.

  5. Fluorometric assay using high-pressure liquid chromatography for the microsomal metabolism of certain substituted aliphatics to 1,N6-ethenoadenine-forming metabolites.

    Science.gov (United States)

    Rinkus, S J; Legator, M S

    1985-11-01

    Monohaloacetaldehydes and monohalooxiranes are early oxidative metabolites of several carcinogenic haloaliphatics. Since monohaloacetaldehydes and supposedly monohalooxiranes react with adenines to form fluorescent 1,N6-ethenoadenines, it was hypothesized that in vitro metabolic systems that produce an ethenoadenine-forming metabolite could be assayed quantitatively by trapping the metabolite in situ with an adenine and identifying it by its characteristic retention and fluorescence during HPLC. Bromoacetaldehyde was chosen as a model haloacetaldehyde to develop an assay based on this concept for measurements in a microsomal system. The optimal trapping reaction requires a postmetabolic step involving acidification and heating. Cyclic AMP was found to be a suitable adenine for the trapping reaction under these conditions. The chromatographic analysis utilizes tetrabutylammonium phosphate and a nonsilica reversed-phase stationary phase (Hamilton PRP-1). The chromatography is isocratic and allows an analysis time of less than 5 min per sample. The titration of bromoacetaldehyde in a microsomal system is affected by typically studied metabolic conditions: incubation time, pH, and protein concentration. Using this assay, the following were found to be metabolized by rat liver microsomes to etheno-adenine-forming products: 1,2-dibromoethane, 1,2-dichloroethane, cyclophosphamide, vinyl chloride, and acrylonitrile. Chloroacetone and 1,3-dichloroacetone also are fluorochromogenic without metabolism but the latter apparently forms a positively charged, nonetheno adduct. The proposed assay should be useful for in vitro metabolic studies of 1,2-dihaloethanes and mustards and has potential application for similar studies of monohalogenated ethanes, ethanols, and ethenes. The positive results with acrylonitrile suggest also that many types of substituted aliphatics may be studied with this proposed assay.

  6. Chemical behaviour of seven aromatic diisocyanates (toluenediisocyanates and diphenylmethanediisocyanates) under in vitro conditions in relationship to their results in the Salmonella/microsome test.

    Science.gov (United States)

    Seel, K; Walber, U; Herbold, B; Kopp, R

    1999-01-13

    There are conflicting results on the mutagenicity of toluenediisocyanate (TDI) and diphenylmethanediisocyanate (MDI). It was found that the organic solvent chosen to dissolve the compounds dictates the outcome of the bacterial tests. The Salmonella/microsome tests showed uniformly mutagenic effects for all the compounds that were predissolved in DMSO. Due to the instability of aromatic diisocyanates in DMSO this solvent was replaced by ethyleneglycoldimethylether (EGDE). TDI and MDI endured the dissolving and were therefore still available for the subsequent bacterial tests. Furthermore, no aromatic diamines (TDA or MDA) could be detected in EGDE prior to the start of the assays. The Salmonella/microsome tests, however, revealed unexpected differences between TDI and MDI. As previously published the four types of MDI showed negative results, whereas the data presented in this paper demonstrated mutagenic effects of all three types of TDI if EGDE is the solvent. To gain deeper insight into the chemical changes that occurred during the Salmonella/microsome test, the possible reactions were modelled in the laboratory by mixing predissolved diisocyanates with a defined surplus of water and monitoring the progress of the chemical reactions by analytical methods. Additionally, the quality of the model was checked by exposing solutions of 2,6-TDI and 4,4'-MDI to the real biological test environment. In both cases, the reaction patterns of TDI were different to those of MDI. Within 1 min, which is the maximum time needed to mix the predissolved compounds with water before they are poured onto the agar plate, the TDI content was reduced in favour of different ureas and TDA. In addition water was replaced by the complete set of test ingredients. While the TDA content remained more or less constant, the amount of residual TDI was reduced considerably. Reactions of MDI were markedly slower than those of TDI. More than 90% of the predissolved MDI remained intact when it was

  7. Affinity of drugs for cytochrome P-450 determined by inhibition of p-nitrophenetole O-deethylation by rat liver microsomes

    DEFF Research Database (Denmark)

    Jørgensen, L; Johansen, Torben

    1983-01-01

    The rate of conversion of p-nitrophenetole to p-nitrophenol by rat liver microsomes was studied. Inhibition of the reaction by CO and by SKF 525A and the absolute dependence on NADPH and oxygen indicate that cytochrome P-450 catalyzes the reaction. The apparent Km for oxygen was 0.07 microM. Furt...... microM) and chloramphenicol (Ki, 20 microM), whereas a mixed-type inhibition by isoniazid was observed (Ki, 1,3 mM and Kii, 10,6 mM)....

  8. Brain Basics

    Medline Plus

    Full Text Available ... the brain, which is linked to thought and emotion. It is also linked to reward systems in the brain. Problems in producing dopamine can result in Parkinson's disease, a disorder that affects a person's ability to move as they want to, resulting in ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... The brain's "fear hub," which activates our natural "fight-or-flight" response to confront or escape from a dangerous ... The brain's "fear hub," which helps activate the fight-or-flight response and is also involved in emotions and ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... will fire. This enhances the electrical flow among brain cells required for normal function and plays an important ... of neurons and their interconnections. neuron —A nerve cell that is the basic, working unit of the brain and nervous system, which processes and transmits information. ...

  11. Brain Basics

    Medline Plus

    Full Text Available ... Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of ... to slow or stop them from progressing. Functional magnetic resonance imaging (fMRI) is another important research tool in understanding ...

  12. Brain Basics

    Medline Plus

    Full Text Available ... Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures ... to slow or stop them from progressing. Functional magnetic resonance imaging (fMRI) is another important research tool in understanding ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a ... MSC 9663 Bethesda, MD 20892-9663 Follow Us Facebook Twitter YouTube Google Plus NIMH Newsletter NIMH RSS ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a middle-aged woman who seemed to have it all. She was happily married and successful in business. Then, after a serious setback at work, she ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... to the front of the brain, which is linked to thought and emotion. It is also linked to reward systems in the brain. Problems in ... Problems in making or using glutamate have been linked to many mental disorders, including autism , obsessive compulsive ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... little dopamine or problems using dopamine in the thinking and feeling regions of the brain may play ... axis —A brain-body circuit which plays a critical role in the body's response to stress. impulse — ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... And as they grow there are differences in brain development in children who develop bipolar disorder than children who do not. Studies comparing such children to those with normal brain development may help scientists to pinpoint when and where ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... These circuits control specific body functions such as sleep and speech. The brain continues maturing well into a person's early 20s. ... Basics in Real Life—How Depression affects the Brain Meet Sarah ... had problems getting to sleep and generally felt tired, listless, and had no ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... in brain development in children who develop bipolar disorder than children who do not. Studies comparing such children to those with normal brain development may help scientists to pinpoint when and where mental disorders begin and perhaps how to slow or stop ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... as they grow there are differences in brain development in children who develop bipolar disorder than children who do not. Studies comparing such children to those with normal brain development may help scientists to pinpoint when and where ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... thinking and feeling regions of the brain may play a role in disorders like schizophrenia or attention deficit hyperactivity ... the front of the brain that, in humans, plays a role in executive functions such as judgment, decision making ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... PTSD) . Prefrontal cortex (PFC) —Seat of the brain's executive functions, such as judgment, decision making, and problem solving. ... brain that, in humans, plays a role in executive functions such as judgment, decision making and problem solving, ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... our physical surroundings but also factors that can affect our bodies, such as sleep, diet, or stress. These factors may act alone ... Life Brain Basics in Real Life—How Depression affects the Brain ... had problems getting to sleep and generally felt tired, listless, and had no ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... her symptoms were not caused by a stroke, brain tumor, or similar conditions, Sarah's doctor referred her to a psychiatrist, a type of medical doctor who is an expert on mental ... of serotonin in the brain and help reduce symptoms of depression. Sarah also ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... begun to chart how the brain develops over time in healthy people and are working to compare that with brain development in ... Other medical professionals who can diagnose mental disorders are psychologists or ... gets "the blues" from time to time. In contrast, major depression is a ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of the brain's structure, studies ... imaging (MRI) mdash;An imaging technique that uses magnetic fields to take pictures of the brain's structure. mutation — ...

  7. 3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2.

    Science.gov (United States)

    Arlt, Volker M; Hewer, Alan; Sorg, Bernd L; Schmeiser, Heinz H; Phillips, David H; Stiborova, Marie

    2004-08-01

    3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat

  8. Effects of antidepressant drugs on the activity of cytochrome P-450 measured by caffeine oxidation in rat liver microsomes.

    Science.gov (United States)

    Danie, W A; Syrek, M; Ryłko, Z; Wójcikowski, J

    2001-01-01

    Caffeine is a marker drug for testing the activity of CYP1A2 (3-N-demethylation) in humans and rats. Moreover, it is also a relatively specific substrate of CYP3A (8-hydroxylation). In the case of 1-N- and in particular 7-N-demethylation of caffeine, apart from CYP1A2, other cytochrome P-450 isoenzymes play a considerable role. The aim of the present study was to investigate the influence of imipramine, amitriptyline and fluoxetine on cytochrome P-450 activity measured by caffeine oxidation in rat liver microsomes. The obtained results showed that imipramine exerted a most potent inhibitory effect on caffeine metabolism. Imipramine decreased the rate of 3-N-, 1-N- and 7-N-demethylations, and 8-hydroxylation of caffeine, the effect on 3-N-demethylation being most pronounced (Ki = 33 microM). Amitriptyline showed distinct inhibition of 3-N- and 1-N-demethylation of caffeine, though its effect was less potent than in the case of imipramine (Ki = 57 and 61 pM, respectively). The influence of amitriptyline on 8-hydroxylation and especially on 7-N-demethylation of caffeine was weaker (Ki = 108 and 190 pM, respectively) than on 3-N- or 1-N-demethylation, suggesting a narrower spectrum of cytochrome P-450 inhibition by amitriptyline than by imipramine, involving mainly the subfamily CYP1A2, and--to a lesser degree--CYP3A. In contrast to the tested tricyclic antidepressants, fluoxetine did not exert any considerable effect on the 3-N- or 1-N-demethylation of caffeine (Ki = 152 and 196 microM, respectively), which indicates its low affinity for CYP1A2. However, fluoxetine displayed a clear inhibitory effect on caffeine 7-N-demethylation (Ki = 72 microM), the reaction which is catalyzed mainly by other than CYP1A2 isoenzymes. Fluoxetine diminished markedly the 8-hydroxylation of the marker drug; as reflected by Ki values, the potency of inhibition of rat CYP3A by fluoxetine was similar to that of imipramine (Ki = 40 and 45 microM, respectively). In summary, CYP1A2 was

  9. Radiocesium bioaccumulation in freshwater plankton: Influences of cation concentrations (K{sup +} and Na{sup +}) on direct uptake of {sup 137}Cs in Chlamydomonas, Scenedesmus and Daphnia. Food-chain transfer of {sup 137}Cs from Chlamydomonas to Daphnia at different K{sup +} concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Hagstroem, J. [Uppsala Univ., Dept. of Limnology, Uppsala (Sweden)

    2002-04-01

    The influences of cation concentrations (K{sup +} and Na{sup +}) on radiocesium ({sup 137}Cs) bioaccumulation in two freshwater phytoplankton species (Scenedesmus quadricauda and Chlamydomonas noctigama) were systematically investigated in batch-cultures monitored during two weeks. Both species were cultured at 9 {mu}E M{sup -2} s{sup -1} constant illumination at 20 deg. C. The exponential growth phase lasted for more than 100 hours ({mu} {approx_equal} 0.02 h{sup -1} for C. noctigama and 0.03 h{sup -1} for S, quadricauda). Over cation concentration ranges encountered in natural fresh waters ([K{sup +}] from 0.1 {mu}M to 3 mM, [Na{sup +}] from 20 {mu}M to 3 mM), a more than three order of magnitude variation was found for both intake rate and observed bioconcentration factors (BCF) at apparent steady-state (from less than 10{sup 3} to 10{sup 6} L (kg C){sup -1}). For both species, the major effector on BCF and uptake rate was external [K{sup +}], which was inversely proportional to these parameters over wide ranges (1-1000 {mu}M for S. quadricauda and 0.1 to 300 {mu}M for C. noctigama). At concentrations above these ranges K{sup +} still reduced {sup 137} Cs bio-uptake, but less effectively. A minor influence of external [Na{sup +}] on {sup 137}Cs bioaccumulation was indicated for S. quadricauda, whereas no such influence was significant for C. noctigama. A biphasic pattern for {sup 137}Cs bioaccumulation was discovered in C. noctigama. A rapid 'quasi-steady state' with an effective equilibration time of less than 100 hours was approached during the exponential growth phase. A surge in the uptake occurred when exponential growth ceased, and this pattern was consistent over the range 30 {mu}M to 1.4 mM external [K{sup +}]. Since depletion of external [K{sup +}] was not detected for these treatments, this pattern can only be explained if there are at least two different cellular compartments involved. Although less certain, a second steady-state BCF

  10. [Brain concussion].

    Science.gov (United States)

    Pälvimäki, Esa-Pekka; Siironen, Jari; Pohjola, Juha; Hernesniemi, Juha

    2011-01-01

    Brain concussion is a common disturbance caused by external forces or acceleration affecting the head. It may be accompanied by transient loss of consciousness and amnesia. Typical symptoms include headache, nausea and dizziness; these may remain for a week or two. Some patients may experience transient loss of inability to create new memories or other brief impairment of mental functioning. Treatment is symptomatic. Some patients may suffer from prolonged symptoms, the connection of which with brain concession is difficult to show. Almost invariably the prognosis of brain concussion is good.

  11. Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide.

    Science.gov (United States)

    Silva, Elias C C; Masui, Douglas C; Furriel, Rosa P; McNamara, John C; Barrabin, Hector; Scofano, Helena M; Perales, Jonas; Teixeira-Ferreira, André; Leone, Francisco A; Fontes, Carlos Frederico L

    2012-11-01

    This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Grapefruit juice, lyophilized grapefruit juice, and powdered whole grapefruit inhibit cytochrome P450-mediated triazolam hydroxylation by beagle dog liver microsomes.

    Science.gov (United States)

    Hanley, M J; Cerundolo, R; Radwanski, N; Court, M H

    2010-04-01

    Coadministration of grapefruit juice (GFJ) has been proposed to enhance the systemic availability and decrease the required dose of drugs such as cyclosporine that are extensively metabolized in the intestine and liver. Although GFJ inhibits human cytochrome P450 (CYP) 3A, effects on dog CYP have not yet been reported. Consequently, we determined whether GFJ inhibits triazolam hydroxylation by Beagle dog liver microsomes (DLM) using human liver microsomes (HLM) as positive control. Results were compared with the effects of lyophilized GFJ and commercially-available powdered grapefruit capsules, which may be more convenient dosage forms. GFJ inhibited alpha-hydroxytriazolam formation in both DLM and HLM with similar IC(50) (inhibitor concentration producing a 50% decrease in reaction velocity) values of 0.56% and 0.52% (v/v), respectively. Lyophilized GFJ and powdered grapefruit also inhibited DLM alpha-hydroxytriazolam formation with IC(50) values of 0.76 and 1.2 mg/mL, respectively. Consistent with mechanism-based enzyme inhibition, preincubation of DLM with any of the grapefruit products for 20 min resulted in significant enhancement of inhibition of triazolam alpha-hydroxylation by 8-20%. The results indicate that 16 g of lyophilized GFJ or 23 g of powdered grapefruit would be equivalent to dosing 100 mL of GFJ. In vivo pharmacokinetic interaction studies are needed to confirm these in vitro findings.

  13. Effect of Curcuma longa on CYP2D6- and CYP3A4-mediated metabolism of dextromethorphan in human liver microsomes and healthy human subjects.

    Science.gov (United States)

    Al-Jenoobi, Fahad Ibrahim; Al-Thukair, Areej A; Alam, Mohd Aftab; Abbas, Fawkeya A; Al-Mohizea, Abdullah M; Alkharfy, Khalid M; Al-Suwayeh, Saleh A

    2015-03-01

    Effect of Curcuma longa rhizome powder and its ethanolic extract on CYP2D6 and CYP3A4 metabolic activity was investigated in vitro using human liver microsomes and clinically in healthy human subjects. Dextromethorphan (DEX) was used as common probe for CYP2D6 and CYP3A4 enzymes. Metabolic activity of CYP2D6 and CYP3A4 was evaluated through in vitro study; where microsomes were incubated with NADPH in presence and absence of Curcuma extract. In clinical study phase-I, six healthy human subjects received a single dose (30 mg) of DEX syrup, and in phase-II DEX syrup was administered with Curcuma powder. The enzyme CYP2D6 and CYP3A4 mediated O- and N-demethylation of dextromethorphan into dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. Curcuma extract significantly inhibited the formation of DOR and 3-MM, in a dose-dependent and linear fashion. The 100 μg/ml dose of curcuma extract produced highest inhibition, which was about 70 % for DOR and 80 % for 3-MM. Curcuma significantly increases the urine metabolic ratio of DEX/DOR but the change in DEX/3-MM ratio was statistically insignificant. Present findings suggested that curcuma significantly inhibits the activity of CYP2D6 in in vitro as well as in vivo; which indicates that curcuma has potential to interact with CYP2D6 substrates.

  14. Dietary saturated and monounsaturated fats protect against acute acetaminophen hepatotoxicity by altering fatty acid composition of liver microsomal membrane in rats.

    Science.gov (United States)

    Hwang, Jinah; Chang, Yun-Hee; Park, Jung Hwa; Kim, Soo Yeon; Chung, Haeyon; Shim, Eugene; Hwang, Hye Jin

    2011-10-20

    Dietary polyunsaturated fats increase liver injury in response to ethanol feeding. We evaluated the effect of dietary corn oil (CO), olive oil (OO), and beef tallow (BT) on fatty acid composition of liver microsomal membrane and acute acetaminophen hepatotoxicity. Male Sprague-Dawley rats were fed 15% (wt/wt) CO, OO or BT for 6 weeks. After treatment with acetaminophen (600 mg/kg), samples of plasma and liver were taken for analyses of the fatty acid composition and toxicity. Treatment with acetaminophen significantly elevated levels of plasma GOT and GPT as well as hepatic TBARS but reduced hepatic GSH levels in CO compared to OO and BT groups. Acetaminophen significantly induced protein expression of cytochrome P450 2E1 in the CO group. In comparison with the CO diet, lower levels of linoleic acid, higher levels of oleic acids and therefore much lower ratios of linoleic to oleic acid were detected in rats fed OO and BT diets. Dietary OO and BT produces similar liver microsomal fatty acid composition and may account for less severe liver injury after acetaminophen treatment compared to animals fed diets with CO rich in linoleic acid. These findings imply that types of dietary fat may be important in the nutritional management of drug-induced hepatotoxicity.

  15. Utilization of estimated physicochemical properties as an integrated part of predicting hepatic clearance in the early drug-discovery stage: Impact of plasma and microsomal binding.

    Science.gov (United States)

    Emoto, C; Murayama, N; Rostami-Hodjegan, A; Yamazaki, H

    2009-03-01

    Rapid prediction of hepatic clearance for drug candidates plays an important role for decision-making in the early drug-discovery stage. Although knowledge of protein binding in both plasma and microsomal components is needed in the prediction of metabolic clearance from metabolic stability studies, the capacity of protein binding assays are generally lower than those of metabolic stability assays. However, many in silico prediction methods for protein binding are now available and software packages such as ACDLabs, ADMET Predictor and SimCYP incorporate various aspects of in silico predictions relevant to estimating binding and clearance. This has facilitated the use of various estimated or measured physicochemical parameters, relevant to binding, to predict clearance. In this study, prediction of protein binding for 33 drugs was evaluated using various combinations of estimated physicochemical properties. Subsequently, the most accurate estimated protein binding values were used to predict hepatic clearance using the SimCYP software. For the drugs used herein, SimCYP provided the most accurate prediction for protein binding in both plasma and microsomes using physiochemical properties estimated with the ACDLabs software. In conclusion, the use of in silico methods as an integrated part of predicting hepatic clearance in early drug-discovery stage is recommended.

  16. Evaluation of the stereoselective biotransformation of permethrin in human liver microsomes: contributions of cytochrome P450 monooxygenases to the formation of estrogenic metabolites.

    Science.gov (United States)

    Lavado, Ramon; Li, Jiwen; Rimoldi, John M; Schlenk, Daniel

    2014-04-21

    Permethrin (PM) is a pyrethroid insecticide that exists as 4 enantiomers. Biotransformation of PM to estrogen receptor agonists (3-phenoxybenzyl alcohol (PBOH) and 3-(4'-hydroxyphenoxy)-benzyl alcohol (3,4 PBOH)) has been shown to be stereoselective in other vertebrate species. This study evaluated the biotransformation of PM enantiomers in human liver microsomes and with recombinant CYP3A4 and CYP2C19. PBOH and 3,4 PBOH were the only metabolites detected from in vitro incubations including each of the 4 enantiomers of PM with 1R-trans PM having the most efficient NADPH-catalyzed biotransformation to both metabolites. Coincubation with the CYP inhibitor ketoconazole and time course experiments with liver microsomes and recombinant CYP2C19 and CYP3A4 indicated CYP-catalyzed stereoselective cleavage of the ester followed by 4-hydoxylation to 3,4' PBOH. These data indicate potential dispositional differences may occur with PM enantiomers and a shift in putative molecular targets. While cleavage of pyrethroid esters lead to detoxification of the acute neurological effects, formation of the benzyl alcohol and hydroxylated metabolite may lead to estrogenic responses, since each of these metabolites are estrogen receptor ligands. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. The identification of lobeglitazone metabolites in rat liver microsomes and the kinetics of the in vivo formation of the major metabolite M1 in rats.

    Science.gov (United States)

    Lee, Jong-Hwa; Ahn, Sung Hoon; Maeng, Han-Joo; Lee, Wooin; Kim, Dae-Duk; Chung, Suk-Jae

    2015-11-10

    The objective of this study was to elucidate the chemical structure of the metabolites derived from lobeglitazone (LB) during its incubation with rat liver microsomes and to characterize the kinetics of formation of the major metabolite M1 in vivo. Using high performance liquid chromatography coupled with a hybrid quadrupole linear ion trap, the metabolites were derived from LB during its incubation with rat liver microsomes. From various fragmentation patterns obtained from the metabolites, LB was biotransformed into 5 metabolites in the incubation, in which demethylation and hydroxylation appeared to be the principle metabolic pathways in vitro; Amongst the five primary metabolites, M1, a demethylated derivative of LB, appeared to be the major metabolite of LB, based on a comparison on the peak intensities in the ion chromatogram. In a study of the in vivo kinetics of formation of M1 in rats, the rate of formation of M1 from LB was determined to be 0.252 and 0.216mL/min/kg at doses of 0.5mg/kg and 2mg/kg of LB, respectively, suggesting that the kinetics of M1 formation were linear in the dose range tested. Considering the fact that LB is primarily eliminated by hepatic metabolism in rats, the formation of M1 accounts for approximately 7.50-9.76% of the overall elimination of LB in rats. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Properties of a microsomal enzyme system from Linum usitatissimum (linen flax) which oxidizes valine to acetone cyanohydrin and isoleucine to 2-methylbutanone cyanohydrin.

    Science.gov (United States)

    Cutler, A J; Sternberg, M; Conn, E E

    1985-04-01

    Microsomal preparations from flax seedlings have recently been shown to convert L-valine to acetone cyanohydrin, the precursor of the cyanogenic glucoside linamarin [A. J. Cutler and E. E. Conn (1981) Arch. Biochem. Biophys. 212, 468-474]. Further details of this four-step biosynthetic sequence and also details of the analogous reactions in lotaustralin biosynthesis have been obtained. The lotaustralin precursor, 2-methylbutyraldoxime, is the best substrate for cyanide production (Vmax = 413 nmol h-1 g fresh wt-1) and inhibits the conversion of valine and isoleucine into products. Similarly, the linamarin precursor isobutyraldoxime is an excellent substrate (Vmax = 400 nmol h-1 g fresh wt-1) and also inhibits oxidation of the amino acids. The substrate specificity of the oxime-metabolizing step is low and a variety of aliphatic oximes are converted to cyanide. On the other hand, the activity of the microsomal extract is highly selective with regard to the amino acid substrate since, of the aliphatic amino acids tested, only valine and isoleucine are metabolized. We were unable to demonstrate product formation from isobutyronitrile (a linamarin precursor) but did observe detectable cyanide formation from 2-methylcyanobutane, the corresponding precursor of lotaustralin. Competition experiments showed that the biosynthesis of linamarin and lotaustralin is not likely to be catalyzed by separate enzyme systems.

  19. Antithyroid microsomal antibody

    Science.gov (United States)

    ... to confirm the cause of thyroid problems, including Hashimoto thyroiditis . The test is also used to find ... positive test may be due to: Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also ...

  20. Getting the MAX out of Computational Models: The Prediction of Unbound-Brain and Unbound-Plasma Maximum Concentrations.

    Science.gov (United States)

    Mente, Scot; Doran, Angela; Wager, Travis T

    2012-06-14

    The objective of this work was to establish that unbound maximum concentrations may be reasonably predicted from a combination of computed molecular properties assuming subcutaneous (SQ) dosing. Additionally, we show that the maximum unbound plasma and brain concentrations may be projected from a mixture of in vitro absorption, distribution, metabolism, excretion experimental parameters in combination with computed properties (volume of distribution, fraction unbound in microsomes). Finally, we demonstrate the utility of the underlying equations by showing that the maximum total plasma concentrations can be projected from the experimental parameters for a set of compounds with data collected from clinical research.

  1. Brain tumor - primary - adults

    Science.gov (United States)

    ... Vestibular schwannoma (acoustic neuroma) - adults; Meningioma - adults; Cancer - brain tumor (adults) ... Primary brain tumors include any tumor that starts in the brain. Primary brain tumors can start from brain cells, ...

  2. Brain radiation - discharge

    Science.gov (United States)

    Radiation - brain - discharge; Cancer-brain radiation; Lymphoma - brain radiation; Leukemia - brain radiation ... Decadron) while you are getting radiation to the brain. It may make you hungrier, cause leg swelling ...

  3. Brain Stimulation Therapies

    Science.gov (United States)

    ... Magnetic Seizure Therapy Deep Brain Stimulation Additional Resources Brain Stimulation Therapies Overview Brain stimulation therapies can play ... for a shorter recovery time than ECT Deep Brain Stimulation Deep brain stimulation (DBS) was first developed ...

  4. Anatomy of the Brain

    Science.gov (United States)

    ... Young Adult Guidelines For brain tumor information and support Call: 800-886-ABTA (2282) or Complete our contact form Brain Tumor Information Brain Anatomy Brain Structure Neuron Anatomy Brain Tumor Symptoms Diagnosis ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... the brain, which is linked to thought and emotion. It is also linked to reward systems in ... stay focused on a task, and managing proper emotional reactions. Reduced ACC activity or damage to this ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... at some point. Such disorders include depression , anxiety disorders , bipolar disorder , attention deficit hyperactivity disorder (ADHD) , and many others. ... differences in brain development in children who develop bipolar disorder than children who do not. Studies comparing such ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... Chemical signals from other cells guide neurons in forming various brain structures. Neighboring neurons make connections with ... our original set of DNA. Sometimes this copying process is imperfect, leading to a gene mutation that ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... the anatomy, physiology, and chemistry of the nervous system. When the brain cannot effectively coordinate the billions of cells in the body, the results can affect many aspects of life. ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... little dopamine or problems using dopamine in the thinking and feeling regions of the brain may play ... but can still remember past events and learned skills, and carry on a conversation, all which rely ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... medicine, the term environment includes not only our physical surroundings but also factors that can affect our ... the brain, which is linked to thought and emotion. It is also linked to reward systems in ...

  11. Brain Basics

    Medline Plus

    Full Text Available ... are sent from one neuron to another. Share Science News NIMH Director Gordon Elected AAAS Fellow NIMH Launches Director Twitter Account Ethics of Deep Brain Stimulation More General Health Information ...

  12. Brain Basics

    Medline Plus

    Full Text Available ... Us Home About the Director Advisory Boards and Groups Strategic Plan Offices and Divisions Budget Careers at NIMH ... of the brain's executive functions, such as judgment, decision making, and problem solving. Different parts of the PFC ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... early brain development. It may also assist in learning and memory. Problems in making or using glutamate ... The amygdala also appears to be involved in learning to fear an event, such as touching a ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... and certain abilities, such as a good singing voice. A gene is a segment of DNA that ... as in areas of the brain that control movement. When electrical signals are abnormal, they can cause ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... with symptoms of mental illness every day. They can be moderate, or serious and cause severe disability. ... disorders are brain disorders. Evidence shows that they can be related to changes in the anatomy, physiology, ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... Criteria (RDoC) Funding Funding Home Opportunities & Announcements Funding Strategy for Grants Application Process Managing Grants Clinical Research ... affect many aspects of life. Scientists are continually learning more about how the brain grows and works ...

  17. Brain abscess

    Science.gov (United States)

    ... including those with certain heart disorders, may receive antibiotics before dental or other procedures to help reduce the risk of infection. Alternative Names Abscess - brain; Cerebral abscess; CNS abscess Patient ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... early brain development. It may also assist in learning and memory. Problems in making or using glutamate have been linked to many mental disorders, including autism , obsessive compulsive disorder (OCD) , schizophrenia , and ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... focused on a task, and managing proper emotional reactions. Reduced ACC activity or damage to this brain ... 9663 Bethesda, MD 20892-9663 Follow Us Facebook Twitter YouTube Google Plus NIMH Newsletter NIMH RSS Feed ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... highly developed area at the front of the brain that, in humans, plays a role in executive functions such as ... component of the U.S. Department of Health and Human Services. Top

  1. Brain Basics

    Medline Plus

    Full Text Available ... related to changes in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot ... medicine, the term environment includes not only our physical surroundings but also factors that can affect our ...

  2. Brain Health

    Science.gov (United States)

    ... that combining good nutrition with mental, social and physical activities may have a greater benefit in maintaining or improving brain health than any single activity. At the Alzheimer’s Association ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... focused on a task, and managing proper emotional reactions. Reduced ACC activity or damage to this brain ... of how environmental factors like diet, stress and post-natal care can change gene expression (when genes ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... specific protein. Scientists believe epigenetics play a major role in mental disorders and the effects of medications. ... feeling regions of the brain may play a role in disorders like schizophrenia or attention deficit hyperactivity ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... to reward systems in the brain. Problems in producing dopamine can result in Parkinson's disease, a disorder ... and her husband questions about Sarah's symptoms and family medical history. Epigenetic changes from stress or early- ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... are sent from one neuron to another. Share Science News NIMH Launches Director Twitter Account Ethics of Deep Brain Stimulation Brain’s Alertness Circuitry Revealed More General Health Information from NIH MEDLINEPlus : Authoritative information from government ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... control specific body functions such as sleep and speech. The brain continues maturing well into a person's ... as sleep, diet, or stress. These factors may act alone or together in complex ways, to change ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... they can cause tremors or symptoms found in Parkinson's disease. Serotonin —helps control many functions, such as ... brain. Problems in producing dopamine can result in Parkinson's disease, a disorder that affects a person's ability ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... These circuits control specific body functions such as sleep and speech. The brain continues maturing well into ... factors that can affect our bodies, such as sleep, diet, or stress. These factors may act alone ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... illnesses, such as depression, can occur when this process does not work correctly. Communication between neurons can also be electrical, such as in areas of the brain that control movement. When electrical signals are abnormal, they can ...

  11. Brain Basics

    Medline Plus

    Full Text Available ... that contains codes to make proteins and other important body chemicals. DNA also includes information to control ... cells required for normal function and plays an important role during early brain development. It may also ...

  12. Brain Basics

    Medline Plus

    Full Text Available ... focused on a task, and managing proper emotional reactions. Reduced ACC activity or damage to this brain ... listless, and had no appetite most of the time. Weeks later, Sarah realized she was having trouble ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take ... slow or stop them from progressing. Functional magnetic resonance imaging (fMRI) is another important research tool in ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... to produce a specific protein. Scientists believe epigenetics play a major role in mental disorders and the ... thinking and feeling regions of the brain may play a role in disorders like schizophrenia or attention ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... another as chemical or electrical signals. The brain begins as a small group of cells in the ... how she's responding to the treatment. She also begins regular talk therapy sessions with her psychiatrist. In ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... studied in mental health research are listed below. Amygdala —The brain's "fear hub," which activates our natural " ... confront or escape from a dangerous situation. The amygdala also appears to be involved in learning to ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... related to changes in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot ... and information that the cell needs for growth, metabolism, and repair. Cytoplasm is the substance that fills ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... This area of the brain also helps to control the amygdala during stressful events. Some research shows that people who have PTSD or ADHD have reduced activity in their PFCs. Anterior cingulate cortex (ACC) — the ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... Us Home About the Director Advisory Boards and Groups Strategic Plan Offices and Divisions Budget Careers at ... electrical signals. The brain begins as a small group of cells in the outer layer of a ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... some point. Such disorders include depression , anxiety disorders , bipolar disorder , attention deficit hyperactivity disorder (ADHD) , and many others. ... differences in brain development in children who develop bipolar disorder than children who do not. Studies comparing such ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... begun to chart how the brain develops over time in healthy people and are working to compare ... listless, and had no appetite most of the time. Weeks later, Sarah realized she was having trouble ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... related to changes in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot ... who can diagnose mental disorders are psychologists or clinical social workers. The psychiatrist asked Sarah and her ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... also linked to reward systems in the brain. Problems in producing dopamine can result in Parkinson's disease, ... studies suggest that having too little dopamine or problems using dopamine in the thinking and feeling regions ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... related to changes in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot ... and epigenetic changes can be passed on to future generations. Further understanding of genes and epigenetics may ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... possibly prevention of such illnesses. The Working Brain Neurotransmitters Everything we do relies on neurons communicating with ... axon, most neurons release a chemical message (a neurotransmitter) which crosses the synapse and binds to receptors ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... Using MEG, some scientists have found a specific pattern of brain activity that may help predict who ... people with depression experience when starting treatment. Gene Studies Advanced technologies are also making it faster, easier, ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... Some people who develop a mental illness may recover completely; others may have repeated episodes of illness ... in early detection, more tailored treatments, and possibly prevention of such illnesses. The Working Brain Neurotransmitters Everything ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... all. She was happily married and successful in business. Then, after a serious setback at work, she ... from one neuron to another. Share Science News Ethics of Deep Brain Stimulation Brain’s Alertness Circuitry Revealed ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... affects the Brain Meet Sarah Sarah is a middle-aged woman who seemed to have it all. ... and works and the effects of genes and environment on mental health. This knowledge is allowing scientists ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... each other How changes in the brain can lead to mental disorders, such as depression. The Growing ... understanding of genes and epigenetics may one day lead to genetic testing for people at risk for ...

  11. Brain Basics

    Medline Plus

    Full Text Available ... glutamate —The most common neurotransmitter in a person's body, which increases neuronal activity, is involved in early brain development, and may also assist in learning and memory. ...

  12. Brain Basics

    Medline Plus

    Full Text Available ... network of neurons and their interconnections. neuron —A nerve cell that is the basic, working unit of the brain and nervous system, which processes and transmits information. neurotransmitter —A chemical ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... of the brain's executive functions, such as judgment, decision making, and problem solving. Different parts of the PFC ... a role in executive functions such as judgment, decision making and problem solving, as well as emotional control ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... of cells in the body, the results can affect many aspects of life. Scientists are continually learning more about how the brain grows and works in healthy people, and how normal ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... in the thinking and feeling regions of the brain may play a role in disorders like schizophrenia or attention deficit hyperactivity disorder (ADHD) . Glutamate —the most common neurotransmitter, glutamate has ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... bind onto, leading to more normal mood functioning. Dopamine —mainly involved in controlling movement and aiding the ... reward systems in the brain. Problems in producing dopamine can result in Parkinson's disease, a disorder that ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... little dopamine or problems using dopamine in the thinking and feeling regions of the brain may play a role in disorders like schizophrenia or attention deficit hyperactivity disorder (ADHD) . Glutamate —the most common ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... Amygdala —The brain's "fear hub," which activates our natural "fight-or-flight" response to confront or escape ... genes that appear to increase risk or provide protection from various mental disorders. Other genes may change ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... and information that the cell needs for growth, metabolism, and repair. Cytoplasm is the substance that fills ... as in areas of the brain that control movement. When electrical signals are abnormal, they can cause ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... doctor that she had experienced long periods of deep sadness throughout her teenage years, but had never ... the understanding of how the brain grows and works and the effects of genes and environment on ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... of genes and environment on mental health. This knowledge is allowing scientists to make important discoveries that ... increases neuronal activity, is involved in early brain development, and may also assist in learning and memory. ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... taking SSRIs and has joined an online support group. Sharing her experiences with others also dealing with depression helps Sarah to better cope with her feelings. Brain Research Modern research tools and techniques are giving scientists ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... her feelings. Brain Research Modern research tools and techniques are giving scientists a more detailed understanding of ... other. magnetic resonance imaging (MRI) mdash;An imaging technique that uses magnetic fields to take pictures of ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... neuron to another. Share Science News NIMH Director Gordon Elected AAAS Fellow NIMH Launches Director Twitter Account Ethics of Deep Brain Stimulation More General Health Information from NIH MEDLINEPlus : Authoritative information from government ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... cells required for normal function and plays an important role during early brain development. It may also assist in learning and memory. Problems in making or using glutamate ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... can be related to changes in the anatomy, physiology, and chemistry of the nervous system. When the ... at the front of the brain that, in humans, plays a role in executive functions such as ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... sends impulses and extends from cell bodies to meet and deliver impulses to another nerve cell. Axons ... in Real Life—How Depression affects the Brain Meet Sarah Sarah is a middle-aged woman who ...

  8. Brain Basics

    Medline Plus

    Full Text Available ... as in areas of the brain that control movement. When electrical signals are abnormal, they can cause ... normal mood functioning. Dopamine —mainly involved in controlling movement and aiding the flow of information to the ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... health research are listed below. Amygdala —The brain's "fear hub," which activates our natural "fight-or-flight" ... also appears to be involved in learning to fear an event, such as touching a hot stove, ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... the brain, which is linked to thought and emotion. It is also linked to reward systems in ... or-flight response and is also involved in emotions and memory. anterior cingulate cortex —Is involved in ...

  11. Brain Basics

    Medline Plus

    Full Text Available ... genes and epigenetics may one day lead to genetic testing for people at risk for mental disorders. ... brain. DNA —The "recipe of life," containing inherited genetic information that helps to define physical and some ...

  12. Brain Basics

    Medline Plus

    Full Text Available ... neuron's point of contact for receiving chemical and electrical signals called impulses from neighboring neurons. Axon which ... from one neuron to another as chemical or electrical signals. The brain begins as a small group ...

  13. Brain Basics

    Medline Plus

    Full Text Available ... of the cell from its surrounding environment and controls what enters and leaves the cell, and responds ... via axons) to form brain circuits. These circuits control specific body functions such as sleep and speech. ...

  14. Brain Basics

    Medline Plus

    Full Text Available ... Amygdala —The brain's "fear hub," which activates our natural "fight-or-flight" response to confront or escape ... Research Fact Sheets NIH Office of Science Education : Resources for science educators Pillbox: How to identify unknown ...

  15. Brain Basics

    Medline Plus

    Full Text Available ... heart rate to responding when we sense a mistake, helping us feel motivated and stay focused on ... peak early. And as they grow there are differences in brain development in children who develop bipolar ...

  16. Brain Basics

    Medline Plus

    Full Text Available ... Brain Neurotransmitters Everything we do relies on neurons communicating with one another. Electrical impulses and chemical signals ... depression, can occur when this process does not work correctly. Communication between neurons can also be electrical, ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... News & Events News & Events Home Science News Events Multimedia Social Media Press Resources Newsletters NIMH News Feeds ... affect many aspects of life. Scientists are continually learning more about how the brain grows and works ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... in mental illnesses. Scientists have already begun to chart how the brain develops over time in healthy ... our original set of DNA. Sometimes this copying process is imperfect, leading to a gene mutation that ...

  19. Brain Basics

    Medline Plus

    Full Text Available ... Labs at NIMH Home Research Areas Principal Investigators Administrative Oversight & Support Collaborations & Partnerships Join A Study News & ... unit of the brain and nervous system, which processes and transmits information. neurotransmitter —A chemical produced by ...

  20. Brain Basics

    Medline Plus

    Full Text Available ... some point. Such disorders include depression , anxiety disorders , bipolar disorder , attention deficit hyperactivity disorder (ADHD) , and many ... differences in brain development in children who develop bipolar disorder than children who do not. Studies comparing ...

  1. Brain Basics

    Medline Plus

    Full Text Available ... related to changes in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot ... NIH Research Fact Sheets NIH Office of Science Education : Resources for science educators Pillbox: How to identify ...

  2. Brain Basics

    Medline Plus

    Full Text Available ... brain and nervous system. Glutamate is an excitatory transmitter: when it is released it increases the chance ... at www.nimh.nih.gov . Glossary action potential —Transmission of signal from the cell body to the ...

  3. Brain Basics

    Medline Plus

    Full Text Available ... all. She was happily married and successful in business. Then, after a serious setback at work, she ... A nerve cell that is the basic, working unit of the brain and nervous system, which processes ...

  4. Brain Basics

    Medline Plus

    Full Text Available ... little dopamine or problems using dopamine in the thinking and feeling regions of the brain may play ... can diagnose mental disorders are psychologists or clinical social workers. The psychiatrist asked Sarah and her husband ...

  5. Brain Basics

    Medline Plus

    Full Text Available ... of the brain's executive functions, such as judgment, decision making, and problem solving. Different parts of the ... a role in executive functions such as judgment, decision making and problem solving, as well as emotional ...

  6. Brain Basics

    Medline Plus

    Full Text Available ... related to changes in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot ... people with depression often have lower than normal levels of serotonin. The types of medications most commonly ...

  7. Brain Basics

    Medline Plus

    Full Text Available ... resonance imaging (MRI), which uses magnetic fields to take pictures of the brain's structure, studies show that ... fast-acting antidepressant medications. Currently available antidepressants usually take four to six weeks to reach their full ...

  8. Brain death.

    Science.gov (United States)

    Wijdicks, Eelco F M

    2013-01-01

    The diagnosis of brain death should be based on a simple premise. If every possible confounder has been excluded and all possible treatments have been tried or considered, irreversible loss of brain function is clinically recognized as the absence of brainstem reflexes, verified apnea, loss of vascular tone, invariant heart rate, and, eventually, cardiac standstill. This condition cannot be reversed - not even partly - by medical or surgical intervention, and thus is final. Many countries in the world have introduced laws that acknowledge that a patient can be declared brain-dead by neurologic standards. The U.S. law differs substantially from all other brain death legislation in the world because the U.S. law does not spell out details of the neurologic examination. Evidence-based practice guidelines serve as a standard. In this chapter, I discuss the history of development of the criteria, the current clinical examination, and some of the ethical and legal issues that have emerged. Generally, the concept of brain death has been accepted by all major religions. But patients' families may have different ideas and are mostly influenced by cultural attitudes, traditional customs, and personal beliefs. Suggestions are offered to support these families. © 2013 Elsevier B.V. All rights reserved.

  9. In vitro hepatic microsomal metabolism of meloxicam in koalas (Phascolarctos cinereus), brushtail possums (Trichosurus vulpecula), ringtail possums (Pseudocheirus peregrinus), rats (Rattus norvegicus) and dogs (Canis lupus familiaris).

    Science.gov (United States)

    Kimble, B; Li, K M; Valtchev, P; Higgins, D P; Krockenberger, M B; Govendir, M

    2014-04-01

    Quantitative and qualitative aspects of in vitro metabolism of the non-steroidal anti-inflammatory drug meloxicam, mediated via hepatic microsomes of specialized foliage (Eucalyptus) eating marsupials (koalas and ringtail possums), a generalized foliage eating marsupial (brushtail possum), rats, and dogs, are described. Using a substrate depletion method, intrinsic hepatic clearance (in vitro Clint) was determined. Significantly, rates of oxidative transformation of meloxicam, likely mediated via cytochromes P450 (CYP), were higher in marsupials compared to rats or dogs. The rank order of apparent in vitro Clint was brushtail possums (n=3) (mean: 394μL/min/mg protein), >koalas (n=6) (50), >ringtail possums (n=2) (36) (with no significant difference between koalas and ringtail possums), >pooled rats (3.2)>pooled dogs (in which the rate of depletion, as calculated by the ratio of the substrate remaining was meloxicam, at a first-order rate constant, 5-hydroxymethyl metabolite (M1) was identified in the brushtail possums and the rat as the major metabolite. However, multiple hydroxyl metabolites were observed in the koala (M1, M2, and M3) and the ringtail possum (M1 and M3) indicating that these specialized foliage-eating marsupials have diverse oxidation capacity to metabolize meloxicam. Using a well-stirred model, the apparent in vitro Clint of meloxicam for koalas and the rat was further scaled to compare with published in vivo Cl. The closest in vivo Cl prediction from in vitro data of koalas was demonstrated with scaled hepatic Cl(total) (average fold error=1.9) excluding unbound fractions in the blood and microsome values; whereas for rats, the in-vitro scaled hepatic Cl fu(blood, mic), corrected with unbound fractions in the blood and microsome values, provided the best prediction (fold error=1.86). This study indicates that eutherians such as rats or dogs serve as inadequate models for dosage extrapolation of this drug to marsupials due to differences in

  10. In vitro bioactivation of 3-(N-phenylamino)propane-1,2-diol by human and rat liver microsomes and recombinant P450 enzymes. Implications for toxic oil syndrome

    NARCIS (Netherlands)

    Martinez-Cabot, A.; Morato, A.; Commandeur, J.N.M.; Vermeulen, N.P.E.; Messeguer, A.

    2007-01-01

    Toxic oil syndrome (TOS) was a massive food-borne intoxication that occurred in Spain in 1981. Epidemiological studies imputed 3-(N-phenylamino) propane-1,2-diol (PAP) derivatives as the toxic agents. The in vitro bioactivation of PAP by rat and human liver microsomes was studied. In both cases,

  11. In vitro modulatory effects of Terminalia arjuna, arjunic acid, arjunetin and arjungenin on CYP3A4, CYP2D6 and CYP2C9 enzyme activity in human liver microsomes

    Directory of Open Access Journals (Sweden)

    Alice Varghese

    2015-01-01

    Full Text Available Terminalia arjuna is a tree having an extensive medicinal potential in cardiovascular disorders. Triterpenoids are mainly responsible for cardiovascular properties. Alcoholic and aqueous bark extracts of T. arjuna, arjunic acid, arjunetin and arjungenin were evaluated for their potential to inhibit CYP3A4, CYP2D6 and CYP2C9 enzymes in human liver microsomes. We have demonstrated that alcoholic and aqueous bark extract of T. arjuna showed potent inhibition of all three enzymes in human liver microsomes with IC50 values less than 50 μg/mL. Arjunic acid, arjunetin and arjungenin did not show significant inhibition of CYP enzymes in human liver microsomes. Enzyme kinetics studies suggested that the extracts of arjuna showed reversible non-competitive inhibition of all the three enzymes in human liver microsomes. Our findings suggest strongly that arjuna extracts significantly inhibit the activity of CYP3A4, CYP2D6 and CYP2C9 enzymes, which is likely to cause clinically significant drug–drug interactions mediated via inhibition of the major CYP isozymes.

  12. Brain extraction and distribution of {sup 99m}{Tc}-bicisate in humans and in rats

    Energy Technology Data Exchange (ETDEWEB)

    Knudsen, G.M.; Andersen, A.R.; Somnier, F.E.; Videbaek, C.; Hasselbalch, S.; Paulson, O.B. [Univ. Clinic Rigshospitalet, Copenhagen (Denmark)

    1994-01-01

    Blood-brain barrier (BBB) passage of the flow tracer ethylenediylbis-L-cysteine diethylester (bicisate, ECD) was measured repeatedly in five patients by means of the intravenous (i.v.) double-indicator technique using {sup 24}Na{sup +} as an intravascular cotracer. After i.v. injection, the arterial concentration curve of {sup 99m}{Tc}-bicisate was delayed and dispersed compared with that of the intravascular cotracer, presumably due to lung retention of the flow tracer. The corrected cerebral venous output curves were fitted using a three-compartment model with four parameters. At resting cerebral blood flow (CBF) values, the unidirectional brain extraction was 0.57 {+-} 0.05, the permeability-surface area product for passage from blood to brain (PS{sub 1}) was 0.48 {+-} 0.07 ml/g/min, and the distribution volume for bicisate was 0.74 {+-} 0.20 (mean {+-} SD). In a single patient, BBB transport after i.v. injection of bicisate was compared with that of a similar flow tracer, d,l-hexamethylpropyleneamine oxime (HM-PAO), and similar values were found for the two tracers. In 19 rats, the brain extraction of bicisate was measured by means of the intracarotid double-indicator technique. The brain extraction was measured at resting, decreased, and increased CBF values. Low CBF values were obtained by hyperventilation and high values by hypercapnia. The degree of backflux of tracer from brain to blood was evaluated by means of the three-compartment model and was found to be negligible in these experiments. The brain extraction was 0.70 {+-} 0.1 and PS{sub 1} was 0.94 {+-} 0.27 ml/g/min. During hypercapnia, CBF increased from 0.77 to 1.09 ml/g/min, leading to a significant decrease in brain extraction, from 0.70 to 0.56. During hyperventilation, CBF decreased from 0.77 to 0.54 ml/g/min, leading to a significant increase in brain extraction, from 0.70 to 0.74. PS{sub 1} remained constant despite changes in CBF or bolus size. 12 refs., 5 figs., 3 tabs.

  13. Antimutagenic activity of some naturally occurring compounds towards cigarette-smoke condensate and benzo(a)pyrene in the Salmonella/microsome assay

    Energy Technology Data Exchange (ETDEWEB)

    Terwel, L.; van der Hoeven, J.C.

    1985-10-01

    Several compounds, occurring in food, were tested for antimutagenic activity towards cigarette-smoke condensate (CSC) and benzo(a)pyrene (BaP). Antimutagenicity was determined in the Salmonella/microsome test, with tester strain TA98, in the presence of rat-liver homogenate. Dose-response curves did show reduction of CSC- and BaP-induced mutagenicity by ellagic acid, riboflavin and chlorophyllin. Chlorophyll a and chlorophyll b, although less distinct, also inhibited CSC- and BaP-induced mutagenicity. Ascorbic acid, beta-carotene, tocopherol acetate, chlorogenic acid and butyl hydroxyanisole did not have any influence on the mutagenicity of CSC and BaP. The similarity in results for cigarette-smoke condensate and for BaP indicates that a general mechanism may be involved in the inhibition of CSC- and BaP-induced mutagenicity.

  14. A comparative study of precision cut liver slices, hepatocytes, and liver microsomes from the Wistar rat using metronidazole as a model substance

    DEFF Research Database (Denmark)

    Sidelmann, U. G.; Cornett, Claus; Tjornelund, J.

    1996-01-01

    1. Metronidazole is metabolized by rat liver in vitro models to form a hydroxy metabolite, an acetic acid metabolite, a glucuronic acid conjugate, and a sulphate conjugate. 2. Four different in vitro systems for investigation of drug metabolism based on liver preparations from the male Wistar rat...... have been investigated. 3. An incubation system where liver slices are incubated in 12-well culture plates was evaluated with respect to metabolism of metronidazole. Optimal viability was observed for a time period of up to 24 h. The Michaelis-Menten parameters for the metabolism of metronidazole......, whereas the intrinsic clearance with respect to formation of the glucuronic acid conjugate was lower in slices compared with hepatocytes. 4. The metabolism of metronidazole in liver slices, in hepatocytes in primary monolayer culture, in hepatocytes incubated in suspension, and in liver microsomes...

  15. Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene.

    Science.gov (United States)

    Anai, T; Koga, M; Tanaka, H; Kinoshita, T; Rahman, S M; Takagi, Y

    2003-06-01

    Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid alpha-linolenic acid during the seed developing stage. We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation. Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting. Furthermore, alpha-linolenic acid content of the T(1) seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T(2) and T(3) progenies. These results demonstrate that the alpha-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene.

  16. The effects of general anesthetics on ESR spectra of spin labels in phosphatidylcholine vesicles containing purified Na,K-ATPase or microsomal protein

    Energy Technology Data Exchange (ETDEWEB)

    Shibuya, Makiko, E-mail: shibu@den.hokudai.ac.jp [Department of Dental Anesthesiology, Graduate School of Dental Medicine, Hokkaido University (Japan); Hiraoki, Toshifumi [Division of Applied Physics, Graduate School of Engineering, Hokkaido University (Japan); Kimura, Kunie; Fukushima, Kazuaki [Department of Dental Anesthesiology, Graduate School of Dental Medicine, Hokkaido University (Japan); Suzuki, Kuniaki [Department of Molecular Cell Pharmacology, Graduate School of Dental Medicine, Hokkaido University (Japan)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer We studied the effects of general anesthetics on liposome using ESR spectra. Black-Right-Pointing-Pointer Two spin labels, 5-DSA and 16-DSA, were located in different position in liposome. Black-Right-Pointing-Pointer Anesthetics did not change the environment around the spin labels in the liposome. Black-Right-Pointing-Pointer Anesthetics remained on the surface of the lipid bilayer of liposome. Black-Right-Pointing-Pointer Proteins in the liposome did not change the effects of anesthetics on liposome. - Abstract: We investigated the effects of general anesthetics on liposome containing spin labels, 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA), and purified Na,K-ATPase or membrane protein of microsome using an electron spin resonance (ESR) spectroscopy. The spectra of 16-DSA in liposomes with both proteins showed three sharp signals compared with 5-DSA. The difference in the order parameter S value of 5-DSA and 16-DSA suggested that the nitroxide radical location of 5-DSA and 16-DSA were different in the membrane bilayer. The results were almost the same as those obtained in liposomes without proteins. The addition of sevoflurane, isoflurane, halothane, ether, ethanol and propofol increased the intensity of the signals, but the clinical concentrations of anesthetics did not significantly alter the S and {tau} values, which are indices of the fluidity of the membrane. These results suggest that anesthetics remain on the surface of the lipid bilayer and do not act on both the inside hydrophobic area and the relatively hydrophilic area near the surface. These results and others also suggest that the existence of Na,K-ATPase and microsomal proteins did not affect the environment around the spin labels in the liposome and the effects of anesthetics on liposome as a model membrane.

  17. Strategy for Hepatotoxicity Prediction Induced by Drug Reactive Metabolites Using Human Liver Microsome and Online 2D-Nano-LC-MS Analysis.

    Science.gov (United States)

    Zhuo, Yue; Wu, Jian-Lin; Yan, Xiaojing; Guo, Ming-Quan; Liu, Ning; Zhou, Hua; Liu, Liang; Li, Na

    2017-12-19

    Hepatotoxicity is a leading cause of drug withdrawal from the market; thus, the assessment of potential drug induced liver injury (DILI) in preclinical trials is necessary. More and more research has shown that the covalent modification of drug reactive metabolites (RMs) for cellular proteins is a possible reason for DILI. Unfortunately, so far no appropriate method can be employed to evaluate this kind of DILI due to the low abundance of RM-protein adducts in complex biological samples. In this study, we proposed a mechanism-based strategy to solve this problem using human liver microsomes (HLMs) and online 2D nano-LC-MS analysis. First, RM modification patterns and potential modified AA residues are determined using HLM and model amino acids (AAs) by UHPLC-Q-TOF-MS. Then, a new online 2D-nano-LC-Q-TOF-MS method is established and applied to separate the digested modified microsomal peptides from high abundance peptides followed by identification of RM-modified proteins using Mascot, in which RM modification patterns on specific AA residues are added. Finally, the functions and relationship with hepatotoxicity of the RM-modified proteins are investigated using ingenuity pathway analysis (IPA) to predict the possible DILI. Using this strategy, 21 proteins were found to be modified by RMs of toosendanin, a hepatotoxic drug with complex structure, and some of them have been reported to be associated with hepatotoxicity. This strategy emphasizes the identification of drug RM-modified proteins in complex biological samples, and no pretreatment is required for the drugs. Consequently, it may serve as a valuable method to predict potential DILI, especially for complex compounds.

  18. Use of the human monocytic leukemia THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and prohapten sensitizers.

    Science.gov (United States)

    Chipinda, Itai; Ruwona, Tinashe B; Templeton, Steven P; Siegel, Paul D

    2011-02-27

    Consumer and medical products can contain leachable chemical allergens which can cause skin sensitization. Recent efforts have been directed at the development of non-animal based tests such as in vitro cell activation assays for the identification of skin sensitizers. Prohapten identification by in vitro assays is still problematic due to the lack of prohapten bioactivation. The present study evaluated the effect of hapten and prohapten exposure on cell surface markers expression (CD86, CD54 and CD40) in the human monocytic leukemia, THP-1, cell line. Upregulation of activation and costimulatory markers are key events in the allergic sensitization process and have been reported to serve as indicators of skin sensitization. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), carvone oxime (COx), cinnamic alcohol (CA) and isoeugenol (IEG) at concentrations ranging from 1 to 10 μM for 24 and 48 h. The direct-binding haptens dinitrochlorobenzene (DNCB), benzoquinone (BQ), hydroxylethyl acrylate (HEA) and benzylbromide (BB) were used as positive controls. Cells were also exposed to the irritants sodium dodecyl sulfate (SDS) and sulfanilamide (SFA). Bioactivation of prohaptens was achieved by adding aroclor-induced rat liver microsomes (S9) to the cell cultures. Consistent upregulation of surface expressions of CD86, CD54 (ICAM-1) and CD40 was observed in THP-1 cells treated with direct-acting haptens (±S9) or prohapten (+S9). Upregulation of these markers was not observed after exposure to skin irritants or prohaptens in the absence of exogenously added S9. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from direct-binding haptens. Published by Elsevier Ireland Ltd.

  19. Biotransformation of the Flame Retardant 1,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) in Vitro by Human Liver Microsomes.

    Science.gov (United States)

    Nguyen, Khanh-Hoang; Abou-Elwafa Abdallah, Mohamed; Moehring, Thomas; Harrad, Stuart

    2017-09-19

    The technical mixture of 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH or DBE-DBCH) and the pure β-TBECH isomer were subjected to in vitro biotransformation by human liver microsomes (HLM). After 60 min of incubation, 5 potential metabolites of TBECH were identified in microsomal assays of both the TBECH mixture and β-TBECH using ultraperformance liquid chromatography-Q-Exactive Orbitrap mass spectrometry. These include mono- and dihydroxylated TBECH and mono- and dihydroxylated TriBECH as well as an α-oxidation metabolite bromo-(1,2-dibromocyclohexyl)-acetic acid. Our results indicate potential hepatic biotransformation of TBECH via cyctochrome P450-catalyzed hydroxylation, debromination, and α-oxidation. Kinetic studies revealed that the formation of monohydroxy-TBECH, dihydroxy-TBECH, and monohydroxy-TriBECH were best fitted to a Michaelis-Menten enzyme kinetic model. Respective estimated Vmax values (maximum metabolic rate) for these metabolites were 11.8 ± 4, 0.6 ± 0.1, and 10.1 ± 0.8 pmol min(-1) mg protein(-1) in TBECH mixture and 4992 ± 1340, 14.1 ± 4.9, and 66.1 ± 7.3 pmol min(-1) mg protein(-1) in β-TBECH. This indicates monohydroxy-TBECH as the major metabolite of TBECH by in vitro HLM-based assay. The estimated in vitro intrinsic clearance (Clint) of TBECH mixture was slower (P < 0.05) than that of pure β-TBECH. While the formation of monohydroxy-TBECH may reduce the bioaccumulation potential and provide a useful biomarker for monitoring TBECH exposure, further studies are required to fully understand the levels and toxicological implications of the identified metabolites.

  20. Kinetics of naphthalene metabolism in target and non-target tissues of rodents and in nasal and airway microsomes from the Rhesus monkey

    Energy Technology Data Exchange (ETDEWEB)

    Buckpitt, Alan, E-mail: arbuckpitt@ucdavis.edu [Department of Molecular Biosciences, School of Veterinary Medicine, UC Davis, Davis, CA 95616 (United States); Morin, Dexter [Department of Molecular Biosciences, School of Veterinary Medicine, UC Davis, Davis, CA 95616 (United States); Murphy, Shannon; Edwards, Patricia; Van Winkle, Laura [Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, UC Davis, Davis, CA 95616 (United States); Center for Health and the Environment, UC Davis, Davis, CA 95616 United States (United States)

    2013-07-15

    Naphthalene produces species and cell selective injury to respiratory tract epithelial cells of rodents. In these studies we determined the apparent K{sub m}, V{sub max}, and catalytic efficiency (V{sub max}/K{sub m}) for naphthalene metabolism in microsomal preparations from subcompartments of the respiratory tract of rodents and non-human primates. In tissues with high substrate turnover, major metabolites were derived directly from naphthalene oxide with smaller amounts from conjugates of diol epoxide, diepoxide, and 1,2- and 1,4-naphthoquinones. In some tissues, different enzymes with dissimilar K{sub m} and V{sub max} appeared to metabolize naphthalene. The rank order of V{sub max} (rat olfactory epithelium > mouse olfactory epithelium > murine airways ≫ rat airways) correlated well with tissue susceptibility to naphthalene. The V{sub max} in monkey alveolar subcompartment was 2% that in rat nasal olfactory epithelium. Rates of metabolism in nasal compartments of the monkey were low. The catalytic efficiencies of microsomes from known susceptible tissues/subcompartments are 10 and 250 fold higher than in rat airway and monkey alveolar subcompartments, respectively. Although the strong correlations between catalytic efficiencies and tissue susceptibility suggest that non-human primate tissues are unlikely to generate metabolites at a rate sufficient to produce cellular injury, other studies showing high levels of formation of protein adducts support the need for additional studies. - Highlights: • Naphthalene is metabolized with high catalytic efficiency in susceptible tissue. • Naphthalene is metabolized at low catalytic efficiency in non-susceptible tissue. • Respiratory tissues of the non human primate metabolize naphthalene slowly.