Sample records for solubilized microsomal preparations

  1. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans.

    Robinson, M L; Carman, G M


    CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 m), and manganese ions (0.5 mm). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mm) or magnesium (30 mm) ions, and Triton X-100 (3.6 mm) at pH 8.0 with Tris-HCl buffer. The apparent K(m) values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mm and 46 mum, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30 degrees C.

  2. Preparation of rough microsomes from rat liver.

    Sabatini, David D


    This protocol describes how to prepare rat liver rough microsomes that contain undegraded membrane-bound polysomes and can function very well in an in vitro translation system. It uses endogenous ribonuclease inhibitor in all steps, avoiding pelleting rough microsomes in all steps and sacrificing good recovery.

  3. Evaluation of surfactants as solubilizing agents in microsomal metabolism reactions with lipophilic substrates.

    Randall, Kathleen; Cheng, Shun Wen; Kotchevar, Anne Therese


    Solubilizing agents are routinely added when investigating the biotransformation of lipophilic substrates using hepatic microsomes. For highly lipophilic compounds, the concentration of solvent or surfactant necessary for dissolution can be detrimental to enzyme activity. This study evaluates the effect of 12 surfactants on microsomal metabolism and the ability of the same surfactants to improve the aqueous solubility of the pentabrominated diphenyl ether BDE-100, a lipophilic environmental contaminant previously found to be recalcitrant to in vitro metabolism. Of the surfactants investigated, Cremophor EL and Tween 80 displayed the best combination of increased BDE-100 solubility and minimal inhibition of microsomal metabolism. However, a comparison of the in vitro metabolism products of BDE-100 in the presence of the two surfactants revealed varying amounts of metabolites depending on the surfactant used.

  4. [Kinetic characteristics of microsomal NAD-glycohydrolase natural and solubilized with a non-ionic surface-active substance].

    Sestini, S; Cinci, G; Ricci, C


    Microsomal rat spleen NAD-glycohydrolase was solubilized by Nonidet P40. The solubilized enzyme shows Nicotinamide inhibition and pH dependence at the same extent as unsolubilized microsomal one. It differs from the latter in having a higher affinity for NAD and NADP, and in showing two peaks, instead of one, on electrofocusing: the former with a pH 5 pI without any activity, the latter with a pH 4, 1 pI with a high NAD-ase activity.

  5. Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC.

    Mandal, Amritlal; Das, Sudip; Chakraborti, Tapati; Kar, Pulak; Ghosh, Biswarup; Chakraborti, Sajal


    The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured

  6. Preparation of microsomes to study Ca2+ channels.

    Bezprozvanny, Ilya


    Native cerebellar microsomes are used to study the properties of native cerebellar inositol(1,4,5)-trisphosphate receptor 1 (InsP3R1) and ryanodine receptor 1 (RyanR1) Ca(2+) release channels. Additionally, microsomes prepared from Sf9 cells infected with InsP3R- or RyanR-expressing recombinant baculoviruses can be used to compare properties of different InsP3R and RyanR isoforms and to perform structure-function studies of both types of receptors. This protocol describes how to prepare ER microsomes from native cerebellar tissue or Sf9 cells infected with InsP3R- or RyanR-expressing recombinant baculoviruses for planar lipid bilayer (also called black lipid membranes or BLM) experiments. Prepared material is aliquoted and can be stored at -80°C freezer for many months before BLM experiments.

  7. Preparation and characterization of rodent intestinal microsomes: Comparative assessment of two methods

    Damre Anagha


    Full Text Available 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 (100000xg 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.

  8. Isolation and catalytic competence of different animal liver microsomal fractions prepared by calcium-aggregation method

    Walawalkar Pradnya


    Full Text Available Rat, mouse, rabbit, and guinea pig liver microsomes were prepared, using the calcium-aggregation method. The mean specific spectral cytochrome P450 content obtained for rat, mouse, rabbit, and guinea pig liver microsomes were 0.547, 0.394, 0.677, and 0.378 nmol cytochrome P450/mg protein, respectively. All the microsomal samples showed the ability to biotransform p-nitrophenol to p-nitrocatechol. The mean rate of formation of p-nitrocatechol by rat, mouse, rabbit, and guinea pig liver microsomes was 0.272, 0.247, 0.497 and 0.424 nmole/min/nmole cytochrome 450, respectively. This method circumvents the need of an ultracentrifuge for isolation of microsomes, by the conventional differential centrifugation method.

  9. Preparation of crude rough microsomes from tissue culture cells.

    Sabatini, David D


    There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose.

  10. Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation.

    Chang, Cheng-Hsiung; Yang, Shang-Shyng


    In order to prepare the multi-functional biofertilizer, thermo-tolerant phosphate-solubilizing microbes including bacteria, actinomycetes, and fungi were isolated from different compost plants and biofertilizers. Except Streptomycesthermophilus J57 which lacked pectinase, all isolates possessed amylase, CMCase, chitinase, pectinase, protease, lipase, and nitrogenase activities. All isolates could solubilize calcium phosphate and Israel rock phosphate; various isolates could solubilize aluminum phosphate, iron phosphate, and hydroxyapatite. During composting, biofertilizers inoculated with the tested microbes had a significantly higher temperature, ash content, pH, total nitrogen, soluble phosphorus content, and germination rate than non-inoculated biofertilizer; total organic carbon and carbon-to-nitrogen ratio showed the opposite pattern. Adding these microbes can shorten the period of maturity, improve the quality, increase the soluble phosphorus content, and enhance the populations of phosphate-solubilizing and proteolytic microbes in biofertilizers. Therefore, inoculating thermo-tolerant phosphate-solubilizing microbes into agricultural and animal wastes represents a practical strategy for preparing multi-functional biofertilizer.

  11. Chromatographic separation of piracetam and its metabolite in a mixture of microsomal preparations, followed by an MS/MS analysis.

    Sahu, Kapendra; Siddiqui, Anees A; Shaharyar, Mohammad; Ahmad, Niyaz; Anwar, Mohammad; Ahmad, Farhan J


    A rapid bioanalytical method was evaluated for the simultaneous determination of piracetam and its metabolite (M1) in human microsomal preparations by fast ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). In addition, a validated method of M1 in rat plasma was developed and successfully applied on pharmacokinetic studies. The present study was carried out to determine the metabolic pathways of piracetam for phase I metabolism and used cytochrome P450 isoforms responsible for the piracetam metabolism in human liver microsomes (HLMs). While additional potential metabolites of piracetam were suggested by computer-modeling. The resulting 2-(2-oxopyrrolidin-1-yl) acetic acid was the sole metabolite detected after the microsomal treatment. The amide hydrolysis mainly underwent to form a metabolite i.e., 2-(2-oxopyrrolidin-1-yl) acetic acid (M1). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  12. Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride

    Scott-Burden, T.; Hawtrey, A. O.


    1. Treatment of washed rat liver microsomes in a medium containing 0·12m-sucrose, 12·5mm-potassium chloride, 2·5mm-magnesium chloride and 25mm-tris–hydrochloric acid buffer, pH7·6, with 2m-lithium chloride at 5° for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting 14C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH–2,6-dichlorophenol-indophenol reductase, NADH–neo-tetrazolium reductase, NADH–cytochrome c reductase and ribonuclease activities. 5. 3H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of 3H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap. ImagesPLATE 1 PMID:4311814

  13. Separation of 1-acylglycerolphosphate acyltransferase and 1-acylglycerolphosphorylcholine acyltransferase of rat liver microsomes.

    Yamashita, S; Nakaya, N; Miki, Y; Numa, S


    1-Acylglycerolphosphate acyltransferase (Ec 2.3.1-) and 1-acylglycerolphosphorylcholine acyltransferase (EC of rat liver microsomes were separated from each other. The separation was achieved by sucrose density gradient centrifugation of the enzyme preparation that was obtained by solubilizing microsomes with a nonionic detergent, Triton X-100, and subjecting the solubilized microsomes to molecular-sieve chromatography. The two acyltransferases are distinguishable from each other also with respect to their stabilities to heat and to Triton X-100. Hence, it is concluded that these acyltransferases are distinct enzymes. These results, together with our previous finding that glycerolphosphate acyltransferase is also a separate enzyme, demonstrate the presence of distinct acyltransferases responsible for the acylation of the different acyl acceptors. Furthermore, the acyl-donor specificities of these acyltransferases provide the enzymatic basis for the nonrandom distribution of fatty acids in naturally occurring glycerolipids. PMID:1054842

  14. Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.).

    Smith, M A; Jonsson, L; Stymne, S; Stobart, K


    The major b-type cytochrome in microsomal membrane preparations from developing endosperm of castor bean (Ricinus communis) was cytochrome b5. Cytochrome P-450 was also present. The microsomal membranes had delta 12-hydroxylase activity and catalysed the NAD(P)H-dependent hydroxylation of oleate to yield ricinoleic acid. CO had no effect on the hydroxylase activity. Rabbit polyclonal antibodies were raised against the hydrophilic cytochrome b5 fragment purified from cauliflower (Brassica oleracea) floret microsomes. The anti-(cytochrome b5) IgG inhibited delta 12-hydroxylase, delta 12-desaturase and cytochrome c reductase activity in the microsomes. The results indicate that electrons from NAD(P)H were transferred to the site of hydroxylation via cytochrome b5 and that cytochrome P-450 was not involved. Images Fig. 1. PMID:1417766

  15. The Disposition of Oxymatrine in the Vascularly Perfused Rat Intestine-Liver Preparation and Its Metabolism in Rat Liver Microsomes.

    Huang, Li Hua; Zhong, Yun Ming; Xiong, Xiao Hong; Cen, Mei Feng; Cheng, Xuan Ge; Wang, Gui Xiang; Chen, Ji Sheng; Wang, Su Jun


    The study was aimed to investigate the absorption and metabolism of oxymatrine (OMT) which contributed to its poor bioavailability. Determinations of OMT absorption and metabolism in rats were evaluated using techniques of the in situ perfused rat intestine-liver preparation and recirculated intestine preparation. Furthermore, chemical inhibition experiments in rat liver microsomes were used to determine the principal cytochrome P450 (CYP) isoforms involved in OMT metabolism. In the intestine-liver preparation, the steady state liver extraction ratio (0.753 ± 0.054) of OMT was 33 times higher than that for the intestine (0.023 ± 0.002). The portal vein mainly consisted of OMT, and was devoid of the metabolite matrine, whereas both OMT and matrine were detected in hepatic vein. With the intestine preparation, the extent of OMT absorption at the end of 120 min of perfusion was 4.79 ± 0.352%. The first-order rate constant for OMT absorption was 0.05 ± 0.003 min(-1). The inhibitor of CYP3A2 had strong inhibitory effect on OMT metabolism in a concentration-dependent manner, and value was reduced to 29.73% of control. The 2 perfusion techniques indicated that poor bioavailability of OMT in rats is due mostly to poor absorption and higher hepatic elimination and CYP3A2 appears to contribute to OMT metabolism in rat liver.

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

    Moonen, H.; Bosch, H. van den


    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 co

  17. Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures.

    Hanley, K; Chappell, J


    Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-beta-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K(m) for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.

  18. Insulin phosphorylates calmodulin in preparations of solubilized rat hepatocyte insulin receptors

    Sacks, D.B.; McDonald, J.M.


    It has previously been shown that insulin stimulates the phosphorylation of calmodulin in adipocyte insulin receptor preparations. Here they demonstrate that insulin also stimulates the phosphorylation of calmodulin in wheat germ lectin-enriched insulin receptor preparations obtained from rat hepatocytes. Standard phosphorylation assays were performed at 30C in the presence of 50mM Tris-HCl (pH 7.5), 0.1% (v/v) Triton X-100, 1mM EGTA, 50 M (el-TSP)ATP, 5mM MgCl2, 0.25 M polylysine, 1.2 M calmodulin and various CaS and insulin concentrations. The phosphorylation of calmodulin was determined by SDS-PAGE and autoradiography. Phosphorylation of calmodulin had an absolute requirement for insulin receptors, insulin and certain basic proteins. Phosphorylation was maximal above 13 nM insulin and at submicromolar CaS concentrations, whereas supramicromolar CaS concentrations were inhibitory. As was observed in the adipocyte insulin receptor system, calmodulin phosphorylation was dependent upon the presence of co-factors, such as polylysine, histone H/sub f/2b and protamine sulfate. The role played by these co-factors has not yet been established. These data suggest that both CaS and calmodulin participate in post receptor insulin events in hepatocytes.

  19. Hydrolysis of solubilized hemicellulose derived from wet-oxidized wheat straw by a mixture of commercial fungal enzyme preparations

    Skammelsen Schmidt, Anette; Thomsen, Alle Belinda; Woidemann, Anders [Risoe National Lab. (Denmark); Tenkanen, Maija [VTT Biotechnology and Food Research (Finland)


    The enzymatic hydrolysis of the solubilized hemicellulose fraction from wet-oxidized wheat straw was investigated for quantification purposes. An optimal hydrolysis depends on factors such as composition of the applied enzyme mixture and the hydrolysis conditions (enzyme loading, hydrolysis time, pH-value, and temperature). A concentrated enzyme mixture was used in this study prepared at VTT Biotechnology and Food Research, Finland, by mixing four commercial enzyme preparations. No distinctive pH-value and temperature optima were identified after a prolonged incubation of 24 hours. By reducing the hydrolysis time to 2 hours a temperature optimum was found at 50 deg. C, where a pH-value higher than 5.2 resulted in reduced activity. An enzyme-substrate-volume-ratio of 0.042, a pH-value of 5.0, and a temperature of 50 deg. C were chosen as the best hydrolysis conditions due to an improved monosaccharide yield. The hydrolysis time was chosen to be 24 hours to ensure equilibrium and total quantification. Even under the best hydrolysis conditions, the overall sugar yield from the enzymatic hydrolysis was only 85% of that of the optimal acid hydrolysis. The glucose yield were approximately the same for the two types of hydrolyses, probably due to the high cellulase activity in the VTT-enzyme mixture. For xylose and arabinose the enzymatic hydrolysis yielded only 80% of that of the acid hydrolysis. As the pentoses existed mainly as complex polymers their degradation required many different enzymes, some of which might be missing from the VTT-enzyme mixture. Furthermore, the removal of side-choins from the xylan backbone during the wet-oxidation pretreatment process might enable the hemicellulosic polymers to interact and precipitate, hence, reducing the enzymatic digestibility of the hemicellulose. (au) 8 tabs., 10 ills., 65 refs.

  20. Phosphatidylcholine mobility in bile salt depleted rat liver microsomes

    Oliveira Filgueiras, O.M. de; Defize, B.; Echteld, C.J.A. van; Bosch, H. van den


    Rat liver microsomes prepared by differential centrifugation are known to contain measurable levels of bile salts. More than 90% of these can be removed by passing the microsomal preparation through a Bio-Gel A-150m column. Bile salt depleted microsomes show a high level (> 95%) of mannose-6-phospha

  1. Phagocyte NADPH-oxidase. Studies with flavin analogues as active site probes in triton X-100-solubilized preparations.

    Parkinson, J F; Gabig, T G


    NADPH-oxidase of stimulated human neutrophil membranes was solubilized in Triton X-100 and activity reconstituted with FAD, 8-F-FAD, 8-phenyl-S-FAD, and 8-S-FAD. The enzyme had similar affinities for all the flavins with Km values in the 60-80 nM range. Vmax was found to increase 4-fold with increasing redox midpoint potential of the flavin. 8-F-FAD reconstituted with the enzyme was reactive toward thiophenol, suggesting exposure of the 8-position to solvent, a finding supported by unsuccessful attempts to label the enzyme with the photoaffinity probe 8-N3-[32P]FAD. Solubilized oxidase stabilized the red thiolate form of 8-S-FAD, a characteristic of flavoproteins of the dehydrogenase/electron transferase classes which stabilize the blue neutral form of the flavin semiquinone radical.

  2. The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower.

    Stobart, A K; Stymne, S


    The utilisation of [(14)C]glycerol 3-phosphate and [(14)C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C18-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.

  3. Aspirin-induced asthma. Hypersensitivity to fenoprofen and ibuprofen in relation to their inhibitory action on prostaglandin generation by different microsomal enzymic preparations.

    Szczeklik, A; Gryglewski, R J; Czerniawska-Mysik, G; Zmuda, A


    Eighteen patients with asthma and aspirin hypersensitivity have been challenged with increasing doses of aspirin, fenoprofen, ibuprofen, and dextropropoxyphene. Low doses of the first three drugs induced bronchoconstriction in all the patients as evidenced by fall in peak expiratory flow and appearance of clinical symptoms. There were no reactions to therapeutic doses of dextropropoxyphene. Aspirin, fenoprofen, and ibuprofen, but not dextropropoxyphene, inhibited prostaglandin synthetase activity in three different microsomal preparations, i.e., in bovine seminal vesicles, in rabbit brain, and in rabbit kidney medulla. Expected in vivo antienzymic potency of a drug, calculated from experiments using rabbit brain microsomes, corresponded roughly with its potency to induce bronchoconstriction in the challenge tests. An individual pattern of sensitivity to threshold doses of prostaglandin synthetase inhibitors was demonstrated for each patient. The results obtained suggest that precipitation of asthmatic attacks by nonsteroidal anti-inflammatory drugs is mediated through inhibition of prostaglandin biosynthesis. The degree of enzymic inhibition, which is sufficient to precipitate bronchoconstriction, is an individual hallmark. Knowing the threshold dose for any of prostaglandin synthetase inhibitors in a patient, one can predict the threshold doses for the rest of aspirin-like drugs in this particular patient.

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

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


    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).

  5. Molecular morphology of the tetrodotoxin-binding sodium channel protein from Electrophorus electricus in solubilized and reconstituted preparations


    The appearance of detergent-solubilized voltage-regulated sodium channel protein was recently characterized by this laboratory. Negative- staining revealed rod-shaped particles measuring 40 X 170 A. Further studies have suggested that the actual configuration of this protein may be quite different from the rod-shaped structures. Freeze-fracture and freeze-etch images of the protein in reconstituted membranes indicated that the channel is cylindrical with a diameter of 100 A and a minimum length of 80 A. Experiments with two detergent systems (Lubrol-PX and sodium cholate) enabled us to explain the discrepancy between this structure and the rod-shaped particles visualized earlier. Negative staining in either detergent at low pH (4.5) produced rod- shaped structures. As the pH was increased, doughnut-shaped particles, consistent with the structure of the protein in freeze-etch, appeared in negative stain. The tendency of the protein to change shape under different pH conditions appears to be a peculiar property of this protein. PMID:6315745

  6. OP-7/氯仿反胶束体系的制备及其增溶水量的研究%Study on preparation of OP-7/chloroform reverse micelle systems and their water solubilization performance

    赵群; 陈怡秀; 朱青; 沈炎冰; 邓龙根; 马超; 阎克路


    The novel OP-7/chloroform reverse micelle system have been prepared by substituting chloroform for the alkanes of traditional reverse micelle systems,and the water solubilization behaviors of the systems have been investigated.The impact of OP-7' s concentration and the type of cosurfactant on the water solubilization amount of reverse micelle systems and the relationship between the water solubilization amount and the size and the conductivity of the systems were studied.The results showed that both the dosage of OP-7,the type and the concentration of the alcohol as cosufactant affect the water solubilization amount ; at the same time,the size and conductivity of the systems increased with increasing water solubilization amount,the OP-7/n-octanol/chloroform has the maximum water solubilization amount.%以氯仿代替传统的烃类溶剂,制备新型OP-7(非离子表面活性剂)/氯仿反胶束体系,并对该体系的增溶行为进行研究.考察了OP-7的浓度和不同的助表面活性剂对该体系增溶水量的影响及增溶水量与体系的粒径、电导率的关系.结果表明,OP-7的浓度、助表面活性剂醇的种类和用量都影响着体系的增溶水量;同时增溶水量增加,体系的粒径和电导率随之增加,OP-7/正辛醇/氯仿反胶束体系增溶水量最大.

  7. Partial purification of the microsomal rat liver iodothyronine deiodinase. II. Affinity chromatography.

    Mol, J A; van den Berg, T P; Visser, T J


    Iodothyronine deiodinase has been solubilized and purified approximately 2400 times from liver microsomal fractions of male Wistar rats pretreated with thyroxine. The deiodinase was solubilized with 1% cholate, and stripped of adhering phospholipids by ammonium sulfate precipitation followed by solubilization with the non-ionic detergent Emulgen 911. The enzyme was further purified by successive ion-exchange chromatography on DEAE-Sephacel and Cellex-P and affinity chromatography on 3,3',5-triiodothyronine-Sepharose. Finally, the deiodinase was reacted with 6-propionyl-2-thiouracil-Sepharose, a derivative of the mechanism-based inhibitor 6-propyl-2-thiouracil. Covalent binding was observed only in the presence of substrate in agreement with the proposed mechanism of deiodination. The deiodinase was eluted from the affinity column by reduction of the enzyme-propylthiouracil mixed disulfide with 50 mM dithiothreitol. The enzyme was approximately 50% pure as judged by SDS-PAGE, exhibiting a subunit molecular weight of 25,000. This preparation was equally enriched in outer ring and inner ring deiodinase activities in keeping with the view that both are intrinsic to a single, type I deiodinase.

  8. The composition of rat liver microsomes. The structural proteins of rat liver microsomes

    Ward, K. A.; Pollak, J. K.


    1. The structural-protein component of microsomal membranes was isolated by three separate methods. Analysis by polyacrylamide-gel electrophoresis indicated that the microsomal structural component is made up of a heterogeneous group of proteins. These proteins were further characterized by their phospholipid-binding capacity. The electrophoretic patterns of microsomal structural proteins were found to differ significantly from those of mitochondrial structural proteins. 2. The reticulosomal fraction was also characterized by electrophoresis with reference to total microsomal proteins, microsomal structural proteins and ribosomal proteins. The reticulosomes gave an electrophoretic pattern significantly different from those of the other three preparations examined. It is suggested that reticulosomes consist largely of enzymic proteins of the endoplasmic reticulum. ImagesPLATE 1PLATE 2PLATE 3 PMID:5810063

  9. Purification of RNA by SDS solubilization and phenol extraction.

    Rio, Donald C; Ares, Manuel; Hannon, Gregory J; Nilsen, Timothy W


    This protocol describes a method for RNA purification by sodium dodecyl sulfate (SDS) solubilization and phenol extraction. It is of wide utility and is used routinely to deproteinize RNAs in biological material that has been solubilized in SDS, an ionic detergent that dissolves membranes, disrupts protein-nucleic acid interactions, and inactivates ribonucleases. Once solubilized, addition of phenol or phenol:chloroform:isoamyl alcohol (PCA) completely denatures the protein, and it becomes insoluble in aqueous solution. PCA extraction is the method of choice for preparing cytoplasmic RNA from tissue culture cells or in any other situation (e.g., enzyme reactions) where solubilization in SDS is easily achievable.

  10. Study on preparation of D5 reverse micelle systems and their water solubilization performance%D5反胶束体系的制备及其增溶特性的研究

    万伟; 刘今强; 李莎; 鲁凤鸣; 王际平; 张玉高


    以对生态环境友好的D5(十甲基环五硅氧烷)替代传统反胶束体系中的烷烃作为连续相介质,制备新型的D5反胶束体系,并对该体系的增溶行为进行研究,分别比较了阴离子表面活性剂、阳离子表面活性剂、非离子表面活性剂的D5反胶束体系的增溶水量.结果表明,用非极性的D5替代传统反胶束体系中的烷烃来制备对生态环境友好的反胶束体系的方法可行;在某些需要助表面活性剂才能形成的D5反胶束体系里,助表面活性剂醇的用量和表面活性剂的类型及其浓度都影响着体系的增溶水量;非离子表面活性剂形成的TX-10/正辛醇/D5反胶束体系增溶水量最大.%The novel D5 reverse micelle systems have been prepared by substituting the eco-friendly D5 (decamethyl cyclopentasiloxane) for the alkanes of traditional reverse micelle systems as continuous phase medium, and the water solubilization behaviors of the systems have been investigated by comparing the water solubilization amount of D5 reverse micelle systems prepared with anionic surfactant, cau'onic surfactant and non-ionic surfactant respectively. The results show that it is a feasible way to substitute the non-polar D5 for the traditional alkanes for preparing reverse micelle systems, and it is much more eco-friendly ; in the cases of that cosurfactants are necessary for preparing reverse micelle systems, both the dosage of the alcohol as cosurfactant and the type and the concentration of the surfactant affect the water solubilization amount;the TX-10/n-octanol/D5 reverse micelle system formed from non-ionic surfactant has the maximum water solubilization amount.

  11. Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties.

    Rodrigo, Lourdes; Gil, Fernando; Hernandez, Antonio F; Lopez, Olga; Pla, Antonio


    Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435-33442] and liver [Ozols (1999) Biochem. J. 338, 265-275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A-Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, K(m) and heat and pH stability. PMID:12946270

  12. Solubilization of coal in organic media

    Lahaye, P.; Decroocq, D.


    The use of solvent extraction to solubilize coal is discussed. Simple extractions are described which are conducted at moderate temperatures to exclude extraneous chemical reactions which would lead to uncontrolled changes in the components of the treated coal. Sample preparation, extraction apparatus, and the determination of extract yield for different experiments are also described.


    Orrenius, Sten


    The TPNH- and O2-dependent drug hydroxylation system of liver microsomes has been studied using normal rats and rats in which the drug-hydroxylating activity has been enhanced by repeated injections of phenobarbital. The oxidative demethylation of aminopyrine is employed as an assay. Optimal conditions for the assay with regard to the concentrations of TPNH and aminopyrine are established. TPN inhibits the reaction in a competitive manner, similarly to its effect on the microsomal TPNH-cytochrome c reductase. Drug hydroxylation, but not the "TPNH oxidase," TPNH-cytochrome c, -2,6-dichlorophenolindophenol, or -neotetrazolium reductase reaction, or the TPNH-dependent lipid peroxidation, is blocked by carbon monoxide. Microsomes from phenobarbital-treated rats exhibit increased activities of the various TPNH-linked reductase reactions, parallel to the increased drug hydroxylation activity, whereas the "TPNH oxidase" activity does not change appreciably. Measurements with microsomes from drug-treated animals reveal a 1:1:1 stoichiometry of aminopyrine-dependent oxygen uptake, TPNH oxidation, and formaldehyde formation. Attempts to solubilize the drug-hydroxylating enzyme system are also presented. It is concluded that the drug-hydroxylating enzyme system involves the microsomal TPNH-cytochrome c reductase and CO-binding pigment, and a hypothetic reaction scheme accounting for the data presented is proposed. PMID:19866674

  14. Nonionic Microemulsions as Solubilizers of Hydrophobic Drugs: Solubilization of Paclitaxel

    Jen-Ting Lo


    Full Text Available The strategy using nonionic microemulsion as a solubilizer for hydrophobic drugs was studied and is demonstrated in this work. The aqueous phase behaviors of mixed nonionic surfactants with various oils at 37 °C are firstly constructed to give the optimal formulations of nonionic microemulsions with applications in the enhanced solubilization of the model hydrophobic drug, paclitaxel, at 37 °C. Briefly, the suitable oil phase with paclitaxel significantly dissolved is microemulsified with appropriate surfactants. Surfactants utilized include Tween 80, Cremophor EL, and polyethylene glycol (4.3 cocoyl ether, while various kinds of edible oils and fatty esters are used as the oil phase. On average, the apparent solubility of paclitaxel is increased to ca. 70–100 ppm in the prepared microemulsions at 37 °C using tributyrin or ethyl caproate as the oil phases. The sizes of the microemulsions attained are mostly from ca. 60 nm to ca. 200 nm. The cytotoxicity of the microemulsion formulations is assessed with the cellular viability of 3T3 cells. In general, the cell viability is above 55% after 24 h of cultivation in media containing these microemulsion formulations diluted to a concentration of total surfactants equal to 50 ppm and 200 ppm.

  15. Subcellular fractionation of rough microsomes.

    Sabatini, David D


    When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.

  16. Association of endogenous substrate with solubilized bovine brain sialidase.

    Schengrund, C L; Repman, M A


    Nonidet P40 solubilized up to 90% of the sialidase, active towards added ganglioside substrate, that was associated with the total membrane fraction prepared from gray matter of bovine brains. Solubilized sialidase acted upon endogenous substrate (sialic acid containing compounds solubilized with the enzyme), hydrolyzing approximately 50% of the readily available sialosyl residues within 20 min. During a 2-hr reaction time 80% of the polysialylated gangliosides solubilized with the enzyme were acted upon. A 20-min lag was observed before sialidase acted upon added ganglioside substrate. The lag could be reduced to less than 2 min when the enzyme was allowed to act on endogenous substrate prior to exposure to exogenous substrate, suggesting that the solubilized enzyme acted preferentially on endogenous substrate. A protease inhibitor prevented much of the 86% loss of activity towards added substrate that was seen when the enzyme was stored at 4 degrees C for 6 days; activity towards endogenous substrate decreased only 34%.

  17. Purification of NADPH-cytochrome c reductase from swine testis microsomes by chromatofocusing and characterization of the purified reductase.

    Kuwada, M; Ohsawa, Y; Horie, S


    A purified NADPH-cytochrome c reductase (NADPH: ferricytochrome oxidoreductase, EC was prepared from swine testis microsomes by detergent solubilization followed by a procedure including chromatofocusing. The reductase was eluted at an isoelectric point of 4.8 from the chromatofocusing column. 730-fold purification was achieved with an overall yield of 1.2%. The preparation was found to be homogeneous upon polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). Upon SDS-polyacrylamide gel electrophoresis, however, the purified preparation resolved into one major band (Mr 78 000) and two minor bands (Mr 60 000 and 15 000). The enzyme contained about 1 mol each of FMN and FAD, which were both extractable with trichloroacetic acid and also boiling water. The oxidized form of the enzyme showed the absorption spectrum of a typical flavoprotein. Aerobic reduction with NADPH resulted in conversion of the spectrum into one of an air-stable semiquinone form. The activity of the purified preparation was 26 mumol cytochrome c reduced/min per mg protein under the standard assay conditions at 22 degrees C. The enzyme catalyzed the reaction through a ping-pong mechanism.

  18. Precipitation and ultimate pH effect on chemical and gelation properties of protein prepared by isoelectric solubilization/precipitation process from pale, soft, exudative (PSE)-like chicken breast meat.

    Zhao, X; Xing, T; Chen, X; Han, M-Y; Li, X; Xu, X-L; Zhou, G-H


    Pale, soft, exudative (PSE)-like chicken breast is considered deteriorated raw material in the poultry meat industry that has inferior processing ability. The chemical and gelation properties of PSE-like chicken breast meat paste were studied. These pastes were prepared by the pH adjustment method and protein isolation using the isoelectric solubilization/precipitation (ISP) process from PSE-like chicken meat. The ISP-isolated samples were solubilized at pH 11.0 and recovered at pH 5.5 and 6.2. The ultimate pH of the ISP-isolated protein and meat paste was adjusted to 6.2 and 7.0. The ultimate pH in this article referred to the final pH of the extracted protein and meat paste. Higher reactive sulfhydryl content and surface hydrophobicity were found in the precipitation at pH 6.2 than at pH 5.5. However, various ultimate pH values showed no significant influence on the surface hydrophobicity. The hardness of gel, as measured by textural profile analysis, was improved using 6.2 as the precipitation pH compared with pH 5.5. The viscoelastic modulus (G(')) of gel pastes prior to the thermal gelation was higher with ISP treatment. However, lower G(') was seen after thermal gelation compared with the control. Dynamic rheological measurement demonstrated a different gel-forming mechanism for protein precipitated at pH values of 5.5 and 6.2 compared with the meat paste. The cooking loss showed that the recovered protein failed to form a gel with good water-retention capacity unless the ultimate pH was adjusted to 7.0. Gels made from protein extracted by the ISP method had higher yellowness and lower redness values, probably due to protein denaturation. Precipitation at pH 6.2 formed a harder gel with lower water-retention ability than that at pH 5.5, and this result was possibly due to higher surface hydrophobicity and S-S bridge formation. Overall, network characteristics of ISP-treated protein gels were strongly dependent on precipitation pH and ultimate pH.

  19. In vitro identification of metabolitesof verapamil in rat liver microsomes

    LuSUN; Shu-qiuZHANG; Da-fangZHONG


    AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MSn), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eightmetabolites, including two novel metabolites (M4 and MS), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (MS-MT), and N, O-didemethyl-verapamil (MS). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.


    The in vitro metabolism of phenol at 11 degrees C has been studied using juvenile and immature adult rainbow (Orcorhynchus mykiss), immature adult brook (Salvelinus fontinalis), and immature adult lake trout (Salvelinus namacush) hepatic microsomal preparations.


    In vitro metabolism of phenol at 11 degrees C has been studied using immature adult rainbow (Oncorhynchus mykiss), brook (Salvelinus fontinalis), and lake trout (Salvelinus namaycush) hepatic microsomal preparations. Incubations were optimized for time, cofactor concentration, pH...

  2. Effects of diffusible products of peroxidation of rat liver microsomal lipids

    Benedetti, Angelo; Casini, Alessandro F.; Ferrali, Marco; Comporti, Mario


    The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the

  3. Solubilization of bovine gelatin using power ultrasound: gelation without heating.

    Farahnaky, Asgar; Zendeboodi, Fatemeh; Azizi, Rezvan; Mesbahi, Gholamreza; Majzoobi, Mahsa


    The aim of this study was to investigate the efficacy of power ultrasound without using any heating stage in solubilizeing gelatin dispersions, and to characterize the mechanical and microstructural properties of the resulting gels using texture analysis and scanning electron microscopy, respectively. Usually to prepare a gel from gelatin, a primary heating stage of at about 40C or above is required to solubilize gelatin macromolecules. In this study solubilizing gelatin dispersions using power ultrasound without any heating was successfully performed. For solubilising gelatin, an ultrasound equipment with a frequency of 20 kHz, amplitude of 100% and power range of 50-150 W was used. Aqueous gelatin dispersions (4% w/v) were subjected to ultrasound for different times (40-240 s) at a constant temperature of 13C. Applying ultrasound to gelatin dispersions caused increases in water absorption and water solubility of the hydrocolloid. The textural parameters of the resulting gelatin gels, increased with increasing time and power of ultrasound. Moreover, a generalized Maxwell model with three elements was used for calculating relaxation times of the gels. The microstructural observations by SEM showed that the structural cohesiveness of the gels increased by increasing ultrasonication time. Ultrasound-assisted solubilization of gelatin can have emerging implications for industrial uses in pharmaceuticals, food and non-food systems. Usually to prepare a gel from gelatin, a primary heating stage of at about 40C or above is required to solubilize gelatin macromolecules. Therefore, the use of gelatin as a hydrocolloid in food processings or pharmaceutical formulations which lack a heating step has been a technological and practical challenge. In this study solubilizing gelatin dispersions using power ultrasound without any heating was successfully performed. Ultrasound-assisted solubilisation of gelatin can have emerging implications for industrial uses in pharmaceuticals

  4. Preparation of liquid bacterial fertilizer of phosphate-solubilizing bacteria from food waste-recycling wastewater%餐厨垃圾废水制备液态解磷菌剂研究

    郭新愿; 祁光霞; 王永京; 李雨桥; 张希; 李冬; 任连海


    The feasibility of using food waste-recycling wastewater as fermentation substrate for producing liquid phosphate-solubilizing bacillus megaterium fertilizer was investigated. The results showed that bacillus megaterium cultured in the food waste-recycling wastewater reached logarithmic growth stage in as short as 3~4 days of adaptation period, and the number of strains reached the maxima on the 6~7th day. The wastewater II generated from hydrothermal pre-treatment was more suitable to be the fermentation substrate than wastewater I. The strain numbers cultured in wastewater II was 5times of what was cultured in wastewater I (4.8×1015CFU/mL). Salt concentration in the wastewater had great influence on the growth and metabolism of Bacillus megaterium: the strain numbers first increased and then steeply decreased with the increase of NaCl concentration, indicating that the optimal NaCl concentration for bacterial culture was 10g/L. The variation of pH and temperature affected the growth of bacillus megaterium significantly, while shaking speed and strain inoculation volume were not key influencing factors. The optimal culture conditions were pH 8, T=35℃, shaking speed 80r/min, and inoculums of 2%(V/V) as determined by orthogonal experiment. Phosphate-solubilizing fertilizer prepared by food waste-recycling wastewater was able to achieve effective phosphating of immobilized phosphorus: the dry weight of soy beans grown in soils amended with 0.025‰~2.5‰ of phosphate-solubilizing fertilizer was 70.7%~84.5%of that grown in the soil amended with 5%of inorganic fertilizer on weight basis. Moreover, the optimal amended ratio of the microbial fertilizer was 0.25‰.%本研究探讨了餐厨垃圾废水用作发酵基质生产液态解磷巨大芽孢杆菌菌肥的可行性.结果表明,餐厨垃圾废水培养的巨大芽孢杆菌经过3~4d的调整期即进入对数生长期,第6~7d活菌数达到最大,而经过湿热预处理得到的II类废水较I类废水

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

    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:


    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.

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

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


    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.

  7. In vitro characterization of borneol metabolites by GC-MS upon incubation with rat liver microsomes.

    Zhang, Rong; Liu, Chang-hui; Huang, Tian-lai; Wang, Ning-sheng; Mi, Sui-qing


    The metabolism of borneol is studied by the analysis of incubations of in vitro-prepared rat liver microsomes. A sensitive gas chromatography (GC)-mass spectrometry (MS) method is developed for the identification of borneol and its metabolites. Four novel metabolites, which have not previously been reported, are isolated and confirmed by comparison of the GC-MS method. The biotransformation pathway of borneol in rat liver microsomes is proposed based on the in vitro results.

  8. Glycerophosphate acylation by microsomes and mitochondria of normal and dystrophic human muscle.

    Kunze, D; Rüstow, B; Olthoff, D


    The incorporation of [14C]glycerophosphate into phosphatidic acid, diacylglycerol, triacylglycerol and phosphatidylcholine by microsomes and mitochondria prepared from normal and dystrophic human muscle incubated in vitro in the presence of 0.3 mmol/l CDP-choline was measured. In mitochondria only phosphatidic acid and diacylglycerol are labelled; the rate of incorporation into these two compounds showed no difference between dystrophic and normal mitochondria. In dystrophic microsomes the incorporation into phosphatidic acid was delayed and decreased. No incorporation of glycerol into diacylglycerol, phosphatidylcholine and triacylglycerol could be measured. Thus in dystrophic muscle microsomes only PA was labelled during an incubation of up to 45 min. In both types of microsomes the concentration of endogenous free fatty acids and diacylglycerol was nearly identical. The level of phosphatidylcholine was 186 and 79 nmol/mg microsomal protein in normal and dystrophic muscle microsomes, respectively. Whether the results could be explained as secondary changes was discussed. Despite some unsolved problems the conclusion was drawn that a better explanation of the results is to assume a primary defect involving microsomal-bound phosphatidic acid phosphohydrolase and possibly glycerol-P-acyltransferases.

  9. Physical characterization and antioxidant activity of thymol solubilized Tween 80 micelles

    Deng, Ling-Li; Taxipalati, Maierhaba; Que, Fei; Zhang, Hui


    Attempts were made to solubilize thymol in Tween 80 micelle to study the solubilization mechanism of thymol and the effect of solubilization on its antioxidant activity. The maximum solubilized concentration of thymol in a 2.0% (w/v) Tween 80 micelle solution is 0.2 wt%. There was no significant difference in Z-average diameter between the empty micelles and thymol solubilized micelles. 1H NMR spectra indicated that 3-H and 4-H on the benzene ring of thymol interacted with the ester group between the hydrophilic head group and the hydrophobic tail group of Tween 80 by Van der Waals’ force. Ferric reducing antioxidant potential (FRAP) and cupric ion reducing antioxidant capacity (CUPRAC) assays showed that the reducing antioxidant activity of free thymol did not change after solubilized in Tween 80 micelles. Compared to free thymol, the solubilized thymol showed higher activities to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) and hydroxyl radicals. The present study suggested a possible preparation of thymol-carrying micelles with enhanced antioxidant activities that could be applied in food beverages.

  10. Recovery Act - Large Scale SWNT Purification and Solubilization

    Michael Gemano; Dr. Linda B. McGown


    The goal of this Phase I project was to establish a quantitative foundation for development of binary G-gels for large-scale, commercial processing of SWNTs and to develop scientific insight into the underlying mechanisms of solubilization, selectivity and alignment. In order to accomplish this, we performed systematic studies to determine the effects of G-gel composition and experimental conditions that will enable us to achieve our goals that include (1) preparation of ultra-high purity SWNTs from low-quality, commercial SWNT starting materials, (2) separation of MWNTs from SWNTs, (3) bulk, non-destructive solubilization of individual SWNTs in aqueous solution at high concentrations (10-100 mg/mL) without sonication or centrifugation, (4) tunable enrichment of subpopulations of the SWNTs based on metallic vs. semiconductor properties, diameter, or chirality and (5) alignment of individual SWNTs.

  11. Maize endophytic bacteria as mineral phosphate solubilizers.

    de Abreu, C S; Figueiredo, J E F; Oliveira, C A; Dos Santos, V L; Gomes, E A; Ribeiro, V P; Barros, B A; Lana, U G P; Marriel, I E


    In the present study, we demonstrated the in vitro activity of endophytic phosphate-solubilizing bacteria (PSB). Fifty-five endophytic PSB that were isolated from sap, leaves, and roots of maize were tested for their ability to solubilize tricalcium phosphate and produce organic acid. Partial sequencing of the 16S rRNA-encoding gene showed that the isolates were from the genus Bacillus and different species of Enterobacteriaceae. The phosphate solubilization index on solid medium and phosphate solubilization in liquid medium varied significantly among the isolates. There was a statistically significant difference (P ≤ 0.05) for both, the values of phosphate-solubilizing activity and pH of the growth medium, among the isolates. Pearson correlation was statistically significant (P ≤ 0.05) between P-solubilization and pH (R = -0.38), and between the gluconic acid production and the lowering of the pH of the liquid medium at 6 (R = 0.28) and 9 days (R = 0.39). Gluconic acid production was prevalent in all the PSB studied, and Bacillus species were most efficient in solubilizing phosphate. This is the first report on the characterization of bacterial endophytes from maize and their use as potential biofertilizers. In addition, this may provide an alternative strategy for improving the phosphorus acquisition efficiency of crop plants in tropical soils.

  12. Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity

    Bennett, W.M.; Mela-Riker, L.M.; Houghton, D.C.; Gilbert, D.N.; Buss, W.C.


    Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. (3H)leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis.

  13. Lipid peroxidation in adrenal and testicular microsomes.


    Studies were carried out to determine the actions of and interactions between ascorbate, NADPH, Fe2+, and Fe3+ on lipid peroxidation in adrenal and testicular microsomes. Ascorbate-induced malonaldehyde production was maximal in adrenal and testicular microsomes at an ascorbate concentration of 1 X 10(-4)M. Fe2+, at levels between 10(-6) and 10(-3)M, produced concentration-dependent increases in lipid peroxidation in adrenal and testicular microsomes; Fe2+ had a far greater effect than Fe3+ i...

  14. Fungal rock phosphate solubilization using sugarcane bagasse.

    Mendes, Gilberto O; Dias, Carla S; Silva, Ivo R; Júnior, José Ivo Ribeiro; Pereira, Olinto L; Costa, Maurício D


    The effects of different doses of rock phosphate (RP), sucrose, and (NH(4))(2)SO(4) on the solubilization of RP from Araxá and Catalão (Brazil) by Aspergillus niger, Penicillium canescens, Eupenicillium ludwigii, and Penicillium islandicum were evaluated in a solid-state fermentation (SSF) system with sugarcane bagasse. The factors evaluated were combined following a 2(3) + 1 factorial design to determine their optimum concentrations. The fitted response surfaces showed that higher doses of RP promoted higher phosphorus (P) solubilization. The addition of sucrose did not have effects on P solubilization in most treatments due to the presence of soluble sugars in the bagasse. Except for A. niger, all the fungi required high (NH(4))(2)SO(4) doses to achieve the highest level of P solubilization. Inversely, addition of (NH(4))(2)SO(4) was inhibitory to P solubilization by A. niger. Among the fungi tested, A. niger stood out, showing the highest solubilization capacity and for not requiring sucrose or (NH(4))(2)SO(4) supplementation. An additional experiment with A. niger showed that the content of soluble P can be increased by adding higher RP doses in the medium. However, P yield decreases with increasing RP doses. In this experiment, the maximal P yield (approximately 60 %) was achieved with the lower RP dose (3 g L(-1)). Our results show that SSF can be used to obtain a low cost biofertilizer rich in P combining RP, sugarcane bagasse, and A. niger. Moreover, sugarcane bagasse is a suitable substrate for SSF aiming at RP solubilization, since this residue can supply the C and N necessary for the metabolism of A. niger within a range that favors RP solubilization.

  15. Effect of acid and alkaline solubilization on the properties of surimi based film

    Thummanoon Prodpran


    Full Text Available The effect of acid and alkaline solubilizing processes on the properties of the protein based film from threadfin bream surimi was investigated. Surimi films prepared from both processes had the similar light transmission, tensile strength (TS and elongation at break (EAB (P<0.05. However, film with alkaline process had slightly lower water vapor permeability (WVP, compared to that prepared by acid solubilizing process. The protein concentration in the film-forming solution directly affected the properties of the film. Increase in protein concentration resulted in an increase in TS, EAB as well as WVP. The film prepared by acid solubilizing process had an increase in yellowish color as evidenced by the continuous increase in b* and E* values during the storage at r oom temperature. The acid and alkali solubilizing processes caused the degradation of muscle protein in surimi, especially with increasing exposure time. Therefore, solubilizing process had the influence on the properties of the protein film from threadfin bream surimi.

  16. Lung protection by inhalation of exogenous solubilized extracellular matrix

    Wu, Jinglei; Ravikumar, Priya; Nguyen, Kytai T.; Hsia, Connie C. W.


    Decellularized extracellular matrix (ECM) contains complex tissue-specific components that work in concert to promote tissue repair and constructive remodeling and has been used experimentally and clinically to accelerate epithelial wound repair, leading us to hypothesize that lung-derived ECM could mitigate acute lung injury. To explore the therapeutic potential of ECM for noninvasive delivery to the lung, we decellularized and solubilized porcine lung ECM, then characterized the composition, concentration, particle size and stability of the preparation. The ECM preparation at 3.2 mg/mL with average particle size <3 μm was tested in vitro on human A549 lung epithelial cells exposed to 95% O2 for 24 hours, and in vivo by tracheal instillation or nebulization into the lungs of rats exposed intermittently or continuously to 90% O2 for a cumulative 72 hours. Our results showed that the preparation was enriched in collagen, reduced in glycosaminoglycans, and contained various bioactive molecules. Particle size was concentration-dependent. Compared to the respective controls treated with cell culture medium in vitro or saline in vivo, ECM inhalation normalized cell survival and alveolar morphology, and reduced hyperoxia-induced apoptosis and oxidative damage. This proof-of-concept study established the methodology, feasibility and therapeutic potential of exogenous solubilized ECM for pulmonary cytoprotection, possibly as an adjunct or potentiator of conventional therapy. PMID:28151947

  17. Complement-mediated solubilization of immune complexes. Solubilization inhibition and complement factor levels in SLE patients

    Baatrup, Gunnar; Petersen, Ivan; Kappelgaard, E;


    Thirty-two of 36 serum samples from 19 SLE patients showed reduced capacity to mediate complement-dependent solubilization of immune complexes (IC). SLE patients with nephritis exerted the lowest complement-mediated solubilization capacity (CMSC) whereas sera from patients with inactive disease g...

  18. Solubilization of Hydrophobic Dyes in Surfactant Solutions

    Ali Reza Tehrani-Bagha


    Full Text Available In this paper, the use of surfactants for solubilization of hydrophobic organic dyes (mainly solvent and disperse dyes has been reviewed. The effect of parameters such as the chemical structures of the surfactant and the dye, addition of salt and of polyelectrolytes, pH, and temperature on dye solubilization has been discussed. Surfactant self-assemble into micelles in aqueous solution and below the concentration where this occurs—the critical micelle concentration (CMC—there is no solubilization. Above the CMC, the amount of solubilized dye increases linearly with the increase in surfactant concentration. It is demonstrated that different surfactants work best for different dyes. In general, nonionic surfactants have higher solubilization power than anionic and cationic surfactants. It is likely that the reason for the good performance of nonionic surfactants is that they allow dyes to be accommodated not only in the inner, hydrocarbon part of the micelle but also in the headgroup shell. It is demonstrated that the location of a dye in a surfactant micelle can be assessed from the absorption spectrum of the dye-containing micellar solution.

  19. Degradation of methyl and ethyl mercury into inorganic mercury by hydroxyl radical produced from rat liver microsomes

    Suda, Ikuo (Kumamoto Univ. (Japan). Dept. of Pharmacology); Hirayama, Kimiko (Kumamoto Univ. (Japan). Coll. of Medical Science)


    Liver microsomes were prepared from Wistar rat by the Ca{sup 2+} aggregation method. Under various conditions, ethyl mercury chloride (EtHgCl) or methyl mercury chloride (MeHgCl) was incubated with the microsomal preparations. After the incubation, the amounts of inorganic Hg and hydroxyl radical (.OH) in the preparations were determined. Although the preparations alone produced a small amount of inorganic Hg and .OH, the addition of NADPH to the preparations increased both inorganic Hg and .OH production, which were further accelerated by the addition of KCN. The addition of Fe(III)EDTA, a .OH formation promoter, to the microsome-NADPH-KCN system increased inorganic Hg production, whereas the addition of diethylenetriamine pentaacetic acid, a .OH formation inhibitor, decreased inorganic Hg production. When .OH scavengers such as mannitol and dimethyl sulfoxide were added to this system, the inorganic Hg production decreased. These results suggested that the .OH produced from liver microsomes was responsible for the degradation of MeHg and EtHg. Since both .OH and inorganic Hg production decreased with a concomitant decrease in NADPH-cytochrome P-450 reductase activities, it is suggested that this enzyme may be involved in the microsomal degradation of MeHg and EtHg. (orig.).

  20. The potential of phosphate solubilizing bacteria isolated from sugarcane wastes for solubilizing phosphate



    Full Text Available Most of P in agricultural soils is in unavailable forms for plant growth. Phosphate solubilizing bacteria can increase soil P availability. This study was aimed to isolate phosphate solubilizing bacteria from sugarcane waste compost and to test ability of the isolated bacterial to dissolve phosphate. The bacteria were isolated from three types of sugarcane waste, i.e. filter cake compost, bagasse compost, and a mixture of filter cake + bagasse + trash biomass compost. The potential colony was further purified by the Pikovskaya method on selective media. Eight isolates of phosphate solubilizing bacteria were obtained from all wasted studied. Amongst them, T-K5 and T-K6 isolates were superior in dissolving P from Ca3(PO42 in the media studied. The two isolates were able to solubilize P with solubilizing index of 1.75 and 1.67 for T-K5 and T-K6, respectively. Quantitatively, T-K6 isolate showed the highest P solubilization (0.74 mg / L, followed by T-K5 isolate (0.56 mg / L, while the lowest P solubilization (0.41 mg / L was observed for T-K4 isolate. The increase of soluble P was not always followed by the decrease in pH.

  1. Effects of Traditional Chinese Medicine Preparations on Rat CYP3A4 of Liver Microsomes in Vitro%大鼠肝微粒体法测定3种中药对CYP3A4亚型的作用

    刘艳; 王海霞; 黄丽军; 张一飞; 徐静; 田玮


    目的 建立肝微粒体测定法,观察中药制剂对CYP3A4 亚型的作用.方法 取大鼠肝脏,制备肝微粒体,分别优化肝微粒体体外温孵系统的反应时间、蛋白浓度及探针药物咪达唑仑的浓度;高效液相色谱-质谱联用(HPLC-MS-MS)法测定大鼠肝微粒体中咪达唑仑的浓度,计算咪达唑仑的活性;在肝微粒体体外温孵系统中分别加入不同浓度的血脂康胶囊、通心络胶囊、枣仁安神胶囊内容物及对照药物酮康唑,测定其半数抑制浓度(IC50)及抑制常数(Ki).结果 肝微粒体体外温孵系统的反应条件为0.4 g·L-1大鼠肝微粒体,4 μmol·L-1咪达唑仑溶液,37 ℃温育5 min.酮康唑、血脂康胶囊、通心络胶囊的IC50分别为(3.4±0.2),(25.1±0.3),(56.2±0.3) mg·L-1,枣仁安神胶囊的IC50﹥150 mg·L-1;酮康唑、血脂康胶囊、通心络胶囊的Ki分别为(1.7±0.1),(25.0±0.2),(50.0±0.3) mg·L-1.结论 建立了中药制剂对CYP3A4亚型作用的大鼠肝微粒体研究模型.血脂康胶囊和通心络胶囊对大鼠CYP3A4亚型有较弱的抑制作用.%Objective To investigate the influence of traditional Chinese medicine preparations on rat liver CYP 3A4 activity in vitro. Methods The rat liver microsomes were prepared from rat livers. The reaction time, protein content and midazolam concentration in the microsomal incubation was optimalized respectively ; the metabolism of midazolam and activity were measured by HPLC-MS-MS;IC50 and Ki of ketoconazole , xuezhikang ,tongxinluo and zaoren ansheng capsule in microsomal incubated system were determined after incubation with the midazolam ( CYP3A4 ). Results The optimum protein content of rat liver microsomes for kinetic analysis was 0.4 g·L-1 with 4 [junol· L midazolam at 37 ℃ for incubation up to 5 min. The IC50 of ketoconazole, xuezhikang and tongxinluo was (3.4±0.2) ,(25.l±0.3) , (56.2±0.3) mg» L ,respectively,but the IC50 of zaoren ansheng capsule was greater than 150 mg · L

  2. Benzo(a)pyrene metabolism by murine spleen microsomes

    Kawabata, T.T.; White, K.L. Jr. (Virginia Commonwealth Univ., Richmond (USA))


    The immunosuppressive actions of benzo(a)pyrene have been proposed to be mediated by reactive metabolites rather than the parent compound. Reactive metabolites which suppress splenic humoral immune responses are thought to be generated within the spleen rather than in distant tissues. Although the spleen has been shown to be capable of metabolizing benzo(a)pyrene, the relative amounts and types of metabolites generated have not been determined. In this study, high-pressure liquid chromatography was used to separate benzo(a)pyrene metabolites generated by splenic microsomes. The major metabolites generated by the splenic microsomal preparations of untreated female B6C3F1 mice were found to be the 9,10- and 7,8-dihydrodiols and 9-, 7-, and 3-hydroxy benzo(a)pyrene. The 1,3-, 3,6-, and 6,12-diones and 4,5-dihydrodiol constituted only a small fraction of the metabolites generated. The generation of all metabolites were inhibited by alpha-naphthoflavone and antiserum to NADPH-cytochrome P-450 reductase, whereas SKF 525-A had only a minimal effect. Dihydrodiol production was completely inhibited by the epoxide hydrolase inhibitor, trichloropropylene oxide. Benzo(a)pyrene pretreatment of mice produced a dramatic increase in the amount of metabolites formed; however, the pattern of metabolites remained similar to that generated by splenic microsomes of untreated mice. The role of prostaglandin synthetase in generating these metabolites was also examined. The addition of arachidonic acid in place of NADPH resulted in the formation of only quinones. Dihydrodiols and phenols were undetectable. The results of this study indicate that splenocytes may be capable of generating the 7,8-dihydrodiol, the precursor to the highly reactive 7,8-dihydrodiol-9,10-epoxide.

  3. Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6 alpha-oxide and certain xenobiotic alkene and arene oxides.

    Levin, W; Michaud, D P; Thomas, P E; Jerina, D M


    Metabolism of cholesterol 5,6 alpha-oxide to the 5,6-glycol is catalyzed by a rat liver microsomal epoxide hydrolase that is distinct from the microsomal epoxide hydrolase that metabolizes a wide range of xenobiotic alkene and arene oxides. The two enzymes are antigenically distinct, and the purified microsomal epoxide hydrolase that metabolizes xenobiotic oxides does not catalyze the hydration of cholesterol 5,6 alpha-oxide. In vivo treatment of rats with inducers of microsomal epoxide hydrolase does not enhance the activity of cholesterol 5,6 alpha-oxide hydrolase and, in some cases, actually depresses enzyme activity in the resultant microsomal preparations. Octene 1,2-oxide and benz[a]anthracene 5,6-oxide, both good substrates for xenobiotic epoxide hydrolase, are not competitive inhibitors of cholesterol oxide hydration by rat liver microsomes. The above results establish the existence of a liver microsomal epoxide hydrolase that is under different regulatory control and that appears to have a different substrate specificity than the well-characterized microsomal epoxide hydrolase involved in the metabolism of a widely diverse group of alkene and arene oxides.

  4. Solubilization of paraffinic deposits for microemulsions

    Gomes, Erika A.S.; Soares, Ranieri G.F.; Nascimento, Roseane E.S.; Dantas Neto, Afonso A.; Barros Neto, Eduardo L. [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil)


    The oil company has been intensifying its efforts to find more efficient solutions for the problems related to the paraffin in wells and transport lines. When applied in the flow lines, the solvents dissolve the paraffin and they must be used hot, since the temperature increases the solubility of the wax and, consequently, its removal rate. The microemulsions appear as an alternative capable of acting in the solubilization and in the inhibition of the formation of deposits due to its great interfacial area, low superficial tension and high capacity of solubilization. They present some advantages in relation to the methods of use of chemical products due to its flexibility of composition in which they can be used, presenting low toxicity and inflammability, without any loss of its capacity of solubilization. The use of oil-in-water microemulsion aims to solubilized paraffin in the disperse phase, where one can find the apolar part of the molecule of the surfactant and the also apolar chain of paraffin, occurring, therefore the 'encapsulation' of the crystal, prohibiting the growth of the chain due to the affinity of paraffin and oil. In this in case, it is possible to transport the inserted paraffin in direct micelles, reducing the precipitation and optimizing the transport. (author)

  5. Computational prediction of solubilizers' effect on partitioning.

    Hoest, Jan; Christensen, Inge T; Jørgensen, Flemming S; Hovgaard, Lars; Frokjaer, Sven


    A computational model for the prediction of solubilizers' effect on drug partitioning has been developed. Membrane/water partitioning was evaluated by means of immobilized artificial membrane (IAM) chromatography. Four solubilizers were used to alter the partitioning in the IAM column. Two types of molecular descriptors were calculated: 2D descriptors using the MOE software and 3D descriptors using the Volsurf software. Structure-property relationships between each of the two types of descriptors and partitioning were established using partial least squares, projection to latent structures (PLS) statistics. Statistically significant relationships between the molecular descriptors and the IAM data were identified. Based on the 2D descriptors structure-property relationships R(2)Y=0. 99 and Q(2)=0.82-0.83 were obtained for some of the solubilizers. The most important descriptor was related to logP. For the Volsurf 3D descriptors models with R(2)Y=0.53-0.64 and Q(2)=0.40-0.54 were obtained using five descriptors. The present study showed that it is possible to predict partitioning of substances in an artificial phospholipid membrane, with or without the use of solubilizers.

  6. Multicomponent mixtures for cryoprotection and ligand solubilization

    Lidia Ciccone


    Full Text Available Mixed cryoprotectants have been developed for the solubilization of ligands for crystallization of protein–ligand complexes and for crystal soaking. Low affinity lead compounds with poor solubility are problematic for structural studies. Complete ligand solubilization is required for co-crystallization and crystal soaking experiments to obtain interpretable electron density maps for the ligand. Mixed cryo-preserving compounds are needed prior to X-ray data collection to reduce radiation damage at synchrotron sources. Here we present dual-use mixes that act as cryoprotectants and also promote the aqueous solubility of hydrophobic ligands. Unlike glycerol that increases protein solubility and can cause crystal melting the mixed solutions of cryo-preserving compounds that include precipitants and solubilizers, allow for worry-free crystal preservation while simultaneously solubilizing relatively hydrophobic ligands, typical of ligands obtained in high-throughput screening. The effectiveness of these mixture has been confirmed on a human transthyretin crystals both during crystallization and in flash freezing of crystals.

  7. Solubilization of an Arabinan Arabinosyltransferase Activity from Mung Bean Hypocotyls1

    Nunan, Kylie Joy; Scheller, Henrik Vibe


    The biosynthesis of polysaccharides destined for the plant cell wall and the subsequent assembly of the cell wall are poorly understood processes that are currently the focus of much research. Arabinan, a component of the pectic polysaccharide rhamnogalacturonan I, is composed of arabinosyl residues connected via various glycosidic linkages, and therefore, the biosynthesis of arabinan is likely to involve more than one arabinosyltransferase. We have studied the transfer of [14C]arabinose (Ara) from UDP-l-arabinopyranose onto polysaccharides using microsomal membranes isolated from mung bean (Vigna radiata) hypocotyls. [14C]arabinosyl and [14C]xylosyl residues were incorporated into endogenous products due to the presence of UDP-Xyl-4-epimerase activity. Enzymatic digestion of endogenous products with endo-arabinanase released very little radiolabeled sugars, whereas digestion with arabinofuranosidase released some [14C]Ara. Microsomal membranes solubilized with the detergent octyl glucoside were able to add a single [14C]Ara residue onto (1→5)-linked α-l-arabino-oligosaccharide acceptors. The reaction had a pH optimum of 6.5 and a requirement for manganese ions. However, enzymatic digestion of the radiolabeled oligosaccharides with endo-arabinanase and arabinofuranosidases could not fully release the radiolabeled Ara residue, indicating that the [14C]Ara residue was not a (1→2)-, (1→3)-, or (1→5)-linked α-l-arabinofuranosyl residue. Rather, mild acid treatment of the product suggested that the radiolabeled Ara residue was in a pyranose conformation, and this result was confirmed by thin-layer chromatography of radiolabeled partially methylated sugars. Using microsomal membranes separated on a discontinuous sucrose gradient, the arabinosyltransferase activity appears to be mainly localized to Golgi membranes. PMID:12746538

  8. Effect of Water Miscible Organic Solvents on p-Nitrophenol Hydroxylase (CYP2E1) Activity in Rat Liver Microsomes.

    Patil, Pranali G; Kamble, S H; Shah, T S; Iyer, K R


    Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400) on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration). Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated) reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.

  9. Effect of water miscible organic solvents on p-nitrophenol hydroxylase (CYP2E1 activity in rat liver microsomes

    Pranali G Patil


    Full Text Available Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400 on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration. Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.

  10. Metabolism of 4'-(9-acridinylamino)methanesulfon-m-anisidide by rat liver microsomes

    Shoemaker, D.D.; Cysyk, R.L.; Gormley, P.E.; DeSouza, J.J.; Malspeis, L.


    4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) is metabolized by a hepatic microsomal enzyme system composed of rat liver microsomes, a reduced nicotinamide adenine dinucleotide phosphate-generating system, cytosolic protein (or glutathione), and oxygen. Omission of any one of the components, or incubation under an atmosphere of CO or N/sub 2/, results in inhibition of the reaction. Also, the addition of inhibitors of microsomal metabolism (alpha-naphthoflavone, metyrapone, or SKF 525-A) decreases m-AMSA metabolism. Metabolism of m-AMSA is more rapid with microsomes prepared from rats pretreated with phenobarbital or 3-methylcholanthrene. Two microsomal oxidation products of m-AMSA were isolated and identified as N1'-methanesulfonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohex adiene-1', 4'-dimine (m-AQDI) and 3'-methoxy-4'-(9-acridinylamino-2',5'-cyclohexadien-1'-one (m-AQI). m-AQDI reacts with glutathione to form a product previously identified in in vivo studies as the principal rat biliary metabolite and which is not cytotoxic to cultured L1210 cells. Thus, the end result of the microsomal metabolism of m-AMSA is detoxification. However, the two primary oxidation products (m-AQDI and m-AQI) are considerably more cytotoxic to L1210 cells in vitro than is m-AMSA. The concentration of m-AMSA required to produce a 5-log kill is 1.0 microgram/ml compared to 0.01 microgram/ml for m-AQDI and m-AQI. These results indicate that m-AMSA might undergo bioactivation to form the active cytotoxic species of the drug.

  11. Interaction of complement-solubilized immune complexes with CR1 receptors on human erythrocytes. The binding reaction

    Jepsen, H H; Svehag, S E; Jarlbaek, L


    showed no binding. IC solubilized in 50% human serum in the presence of autologous RBC bound rapidly to RBC-CR1, with maximal binding within less than 1 min at 37 degrees C. Release of CR1-bound IC under these conditions occurred slowly, requiring more than 30 min. Only binding of 'partially' solubilized...... of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solubilized IC could be absorbed to solid-phase conglutinin or antibody to C3c and C4c, and these ligands were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect...

  12. Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes.

    Pham, Minh Hien; Auzeil, Nicolas; Regazzetti, Anne; Dauzonne, Daniel; Dugay, Annabelle; Menet, Marie-Claude; Scherman, Daniel; Chabot, Guy G


    Flavone-8-acetic acid (FAA) is a potent anticancer agent in mouse but has not shown activity in humans. Because FAA metabolism could play a role in this interspecies difference, our aim was to identify the metabolites formed in vitro using mouse microsomes compared with those in human microsomes. Mouse microsomes produced six metabolites as detected by reversed-phase high-performance liquid chromatography-mass spectrometry (MS). Three metabolites were identified as the 3'-, 4'-, or 6-hydroxy-FAA, by comparison with retention times and UV and MS spectra of standards. Two metabolites presented a molecular weight of 296 (FAA = 280) indicating the presence of one oxygen but did not correspond to any monohydroxylated FAA derivative. These two metabolites were identified as epoxides because they were sensitive to epoxide hydrolase. The position of the oxygen was determined by the formation of the corresponding phenols under soft acidic conditions: one epoxide yielded the 3'- and 4'-hydroxy-FAA, thus corresponding to the 3',4'-epoxy-FAA, whereas the other epoxide yielded 5- and 6-hydroxy-FAA, thus identifying the 5,6-epoxy-FAA. The last metabolite was assigned to the 3',4'-dihydrodiol-FAA because of its molecular weight (314) and sulfuric acid dehydration that indicated that the 3'- and 4'-positions were involved. Compared with mouse microsomes, human microsomes (2 pools and 15 individual microsomes) were unable to metabolize FAA to a significant extent. In conclusion, we have identified six new FAA metabolites formed by mouse microsomes, whereas human microsomes could not metabolize this flavonoid to a significant extent. The biological importance of the new metabolites identified herein remains to be evaluated.

  13. Microsomal metabolism of trenbolone acetate metabolites ...

    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

  14. Hydroxylation of pentamidine by rat liver microsomes.

    Berger, B J; Reddy, V V; Le, S T; Lombardy, R J; Hall, J E; Tidwell, R R


    The antiprotozoal/antifungal drug pentamidine [1,5-bis(4-amidinophenoxy)pentane] has been recently shown to be metabolized by rat liver fractions to at least six putative metabolites as detected by high-performance liquid chromatography. Two minor metabolites have been previously identified as N-hydroxypentamidine and N,N'-dihydroxypentamidine. In this study, the two major microsomal metabolites have been identified as the 2-pentanol and 3-pentanol analogs of pentamidine [1,5-di(4-amidinophenoxy)-2-pentanol; and 1,5-bis(4-amidinophenoxy)-3-pentanol]. As well, a seventh putative metabolite has been discovered and identified as para-hydroxybenzamidine, a fragment of the original drug. Whereas the cytochromes P-450 have been demonstrated as the enzyme system responsible for pentamidine metabolism, hydroxylation of the drug was not inducible by phenobarbital, beta-naphthoflavone, clofibrate, isosafrole, pregnenolone-16 alpha-carbonitrile, ethanol or pentamidine pretreatment of rats. The kinetics of the production of the two major microsomal metabolites has been determined as Km = 56 +/- 19 microM and Vmax = 126 +/- 21 pmol/min/mg microsomal protein for the 2-pentanol analog, and Km = 28 +/- 0.28 microM and Vmax = 195 +/- 2.4 pmol/min/mg microsomal protein for the 3-pentanol analog. Therefore, the mixed-function oxidases readily convert pentamidine to hydroxylated metabolites, but exactly which isozyme(s) of cytochrome P-450 is responsible is not clear.

  15. Solubilization isotherms of aromatic solutes in surfactant aggregates

    Gadelle, F.; Koros, W.J.; Schechter, R.S. (Univ. of Texas, Austin, TX (United States). Dept. of Chemical Engineering)


    Several factors affecting solubilization of aromatic solutes in surfactant micelles have been investigated. Solubilization isotherms of benzene, toluene, and chlorobenzene in various aqueous micellar solutions were determined using head space gas chromatography. Cationic surfactants such as cetylpyridinium chloride or cetyltrimethylammonium bromide present high solubilization capacities. Comparable anionic surfactants exhibit lower solubilization and a greater tendency to precipitate. It was observed that nonionic surfactants show high solubilization on a molar basis. Solubilization in mixed cationic-anionic micelles was also investigated. It also appears that the molecular size of the solute determines the extent of the solubilization. Finally, the shape of the different isotherms indicates that knowing the amount solubilized at saturation of the micellar solution is not sufficient to estimate solubilization at solute concentrations lower than the solute aqueous solubility. Solubilization of organics in surfactant micelles is of major importance in many applications. One new application is micellar-enhanced ultrafiltration. Another application of interest is the surfactant-enhanced aquifer remediation, a process in which a micellar aqueous solution is flushed into contaminated groundwaters to enhance recovery of pollutants by micellar solubilization.

  16. Phosphate solubilizing ability of two Arctic Aspergillus niger strains

    Shiv Mohan Singh,


    Full Text Available Many filamentous fungi were isolated from the soils of Ny-Ålesund, Spitsbergen, Svalbard, and were screened in vitro for their phosphate solubilizing ability. Two strains of Aspergillus niger showed good tricalcium phosphate (TCP solubilizing ability in Pikovskaya's medium. The TCP solubilization index was calculated at varying levels of pH and temperatures. The ability of Aspergillus niger strain-1 to solubilize and release inorganic-P was 285 µg ml–1, while Aspergillus niger strain-2 solubilized 262 µg ml–1 from 0.5% TCP after seven days. This is the first report of TCP solubilization by Arctic strains that may serve as very good phosphate solubilizers in the form of biofertilizer.

  17. Antitumor activity of two gelsemine metabolites in rat liver microsomes.

    Zhao, Qing-Chun; Hua, Wei; Zhang, Lin; Guo, Tao; Zhao, Ming-Hong; Yan, Ming; Shi, Guo-Bing; Wu, Li-Jun


    Gelsemine is one of the major alkaloids from Gelsemium elegans Benth., which has been used as an antitumor remedy in clinic. In this paper, metabolism of gelsemine has been investigated in vitro in phenobarbital-treated rat liver microsomes. The metabolites of gelsemine were separated and evaluated using the flash silica gel column, preparative HPLC, using NMR and MS methods. According to the spectral data, two metabolites, M1 and M2, were identified as 4-N-demethylgelsemine and 21-oxogelsemine, respectively. By the MTT method in vitro, the antitumor activities between gelsemine and its metabolites were compared in the HepG2 and HeLa cell lines. Moreover, the main metabolic pathway was further proposed.

  18. Stress-tolerant P-solubilizing microorganisms.

    Vassilev, N; Eichler-Löbermann, B; Vassileva, M


    Drought, high/low temperature, and salinity are abiotic stress factors accepted as the main reason for crop yield losses in a world with growing population and food price increases. Additional problems create nutrient limitations and particularly low P soil status. The problem of phosphate fertilizers, P plant nutrition, and existing phosphate bearing resources can also be related to the scarcity of rock phosphate. The modern agricultural systems are highly dependent on the existing fertilizer industry based exclusively of this natural, finite, non-renewable resource. Biotechnology offers a number of sustainable solutions that can mitigate these problems by using plant beneficial, including P-solubilizing, microorganisms. This short review paper summarizes the current and future trends in isolation, development, and application of P-solubilizing microorganisms in stress environmental conditions bearing also in mind the imbalanced cycling and unsustainable management of P. Special attention is devoted to the efforts on development of biotechnological strategies for formulation of P-solubilizing microorganisms in order to increase their protection against adverse abiotic factors.

  19. Enhanced solubilization of curcumin in mixed surfactant vesicles.

    Kumar, Arun; Kaur, Gurpreet; Kansal, S K; Chaudhary, Ganga Ram; Mehta, S K


    Self-assemblies of equimolar double and single chain mixed ionic surfactants, with increasing numbers of carbon atoms of double chain surfactant, were analyzed on the basis of fluorescence and conductivity results. Attempts were also made to enhance the solubilization of curcumin in aqueous equimolar mixed surfactant systems. Mixed surfactant assembly was successful in retarding the degradation of curcumin in alkaline media (only 25-28 40% degraded in 10h at pH 13). Fluorescence spectroscopy and fluorescence quenching methods were employed to predict the binding position and mechanism of curcumin with self-assemblies. Results indicate that the interactions take place according to both dynamic and static quenching mechanisms and curcumin was distributed in a palisade layer of mixed aggregates. Antioxidant activity (using DPPH radical) and biocompatibility (using calf-thymus DNA) of curcumin-loaded mixed surfactant formulations were also evaluated. The prepared systems improved the stability, solubility and antioxidant activity of curcumin and additionally are biocompatible.

  20. Isolation of phosphate-solubilizing fungus and its application in solubilization of rock phosphates.

    Wu, Yingben; He, Yuelin; Yin, Hongmei; Chen, Wei; Wang, Zhen; Xu, Lijuan; Zhang, Aiqun


    Microorganisms have been obtained to improve the agronomic value of rock phosphates (RPs), but the phosphorus solubilizing rate by these approaches is very slow. It is important to explore a high-efficient phosphate-solubilizing approach with a kind of microorganisms. This study aimed to isolate a high-efficient level of phosphate-solubilizing fungus from rhizosphere soil samples phosphate mines (Liuyang County, Hunan province, China) and apply it in solubilization of RPs. The experiments were carried out by the conventional methodology for morphological and biochemical fungus characterization and the analysis of 18s rRNA sequence. Then the effects of time, temperature, initial pH, phosphorus (P) sources, RPs concentration, shaking speed and silver ion on the content of soluble P released by this isolate were investigated. The results showed this isolate was identified as Galactomyces geotrichum P14 (P14) in GeneBank and the maximum amount of soluble P was 1252.13 mg L(-1) within 40 h in a modified phosphate growth agar's medium (without agar) where contained tricalcium phosphate (TCP) as sole phosphate source. At the same time, it could release phosphate and solubilize various rock phosphates. The isolated fungus can convert RPs from insoluble form into plant available form and therefore it hold great potential for biofertilizers to enhance soil fertility and promote plant growth.

  1. Solubilization and characterization of haloperidol-sensitive (+)-( sup 3 H)SKF-10,047 binding sites (sigma sites) from rat liver membranes

    McCann, D.J.; Su, T.P. (National Institute on Drug Abuse, Baltimore, MD (USA))


    The zwitterionic detergent 3-((3-cholamidopropyl)dimethylamino)-1-propanesulfonate (CHAPS) produced optimal solubilization of (+)-({sup 3}H)SKF-10,047 binding sites from rat liver membranes at a concentration of 0.2%, well below the critical micellular concentration of the detergent. The pharmacological selectivity of the liver (+)-({sup 3}H)SKF-10,047 binding sites corresponds to that of sigma sites from rat and guinea pig brain. When the affinities of 18 different drugs at (+)-({sup 3}H)SKF-10,047 binding sites in membranes and solubilized preparations were compared, a correlation coefficient of 0.99 and a slope of 1.03 were obtained, indicating that the pharmacological selectivity of rat liver sigma sites is retained after solubilization. In addition, the binding of 20 nM ({sup 3}H)progesterone to solubilized rat liver preparations was found to exhibit a pharmacological selectivity appropriate for sigma sites. A stimulatory effect of phenytoin on (+)-({sup 3}H)SKF-10,047 binding to sigma sites persisted after solubilization. When the solubilized preparation was subjected to molecular sizing chromatography, a single peak exhibiting specific (+)-({sup 3}H)SKF-10,047 binding was obtained. The binding activity of this peak was stimulated symmetrically when assays were performed in the presence of 300 microM phenytoin. The molecular weight of the CHAPS-solubilized sigma site complex was estimated to be 450,000 daltons. After solubilization with CHAPS, rat liver sigma sites were enriched to 12 pmol/mg of protein. The present results demonstrate a successful solubilization of sigma sites from rat liver membranes and provide direct evidence that the gonadal steroid progesterone binds to sigma sites. The results also suggest that the anticonvulsant phenytoin binds to an associated allosteric site on the sigma site complex.

  2. Sulfation of p-nitrophenyl-N-acetyl-beta-D-galactosaminide with a microsomal fraction from cultured chondrocytes

    Habuchi, O.; Conrad, H.E.


    Chick embryo chondrocyte microsomes containing intact Golgi vesicles took up 3'-phosphoadenosine-5'-phospho(TVS)sulfate ((TVS)PAPS) in a time- and temperature-dependent, substrate-saturable manner. When (TVS)PAPS and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-GalNAc) were added to the incubation in the absence of detergent, the microsomes catalyzed the transfer of sulfate from (TVS)PAPS to pNP-GalNAc to form pNP-GalNAc-6-TVSO4. The apparent Km values for PAPS in the uptake and the pNP-GalNAc sulfation reactions were 2 X 10(-7) and 2 X 10(-6) M, respectively. The sulfation of pNP-GalNAc by the microsomal preparation was inhibited by detergent. The microsomal fraction also catalyzed the transfer of sulfate from (TVS)PAPS to oligosaccharides prepared from chondroitin. However, in contrast to the sulfation of pNP-GalNAc, the rate of sulfation of these oligosaccharides was low in the absence of detergent and was markedly stimulated when detergent was added. Sulfation of pNP-GalNAc by the freeze-thawed microsomes was inhibited when the octasaccharide prepared from chondroitin was present in the reaction mixture. As the PAPS that had been internalized in the microsomal vesicles was consumed in the sulfation of pNP-GalNAc, more (TVS)PAPS was taken up and the sulfated pNP-GalNAc was released from the vesicles. These observations suggest that pNP-GalNAc may serve as a model membrane-permeable substrate for study of the 6-sulfo-transferase reaction involved in sulfation of chondroitin sulfate in intact Golgi vesicles.

  3. Interrogating Circulating Microsomes and Exosomes Using Metal Nanoparticles.

    Zhou, Yi-Ge; Mohamadi, Reza M; Poudineh, Mahla; Kermanshah, Leyla; Ahmed, Sharif; Safaei, Tina Saberi; Stojcic, Jessica; Nam, Robert K; Sargent, Edward H; Kelley, Shana O


    A chip-based approach for electrochemical characterization and detection of microsomes and exosomes based on direct electro-oxidation of metal nanoparticles (MNPs) that specifically recognize surface markers of these vesicles is reported. It is found that exosomes and microsomes derived from prostate cancer cells can be identified by their surface proteins EpCAM and PSMA, suggesting the potential of exosomes and microsomes for use as diagnostic biomarkers.

  4. Characterization of (/sup 3/H)pirenzepine binding to muscarinic cholinergic receptors solubilized from rat brain

    Luthin, G.R.; Wolfe, B.B.


    Membranes prepared from rat cerebral cortex were solubilized in buffer containing 1% digitonin. Material present in the supernatant after centrifugation at 147,000 X g was shown to contain binding sites for both (/sup 3/H)quinuclidinyl benzilate ((/sup 3/H)QNB) and (/sup 3/H)pirenzepine ((/sup 3/H)PZ). Recovery of binding sites was approximately 25% of the initial membrane-bound (/sup 3/H)QNB binding sites. The Kd values for (/sup 3/H)QNB and (/sup 3/H)PZ binding to solubilized receptors were 0.3 nM and 0.1 microM, respectively. As has been observed previously in membrane preparations, (/sup 3/H)PZ appeared to label fewer solubilized binding sites than did (/sup 3/H)QNB. Maximum binding values for (/sup 3/H)PZ and (/sup 3/H)QNB binding to solubilized receptors were approximately 400 and 950 fmol/mg of protein, respectively. Competition curves for PZ inhibiting the binding of (/sup 3/H)QNB, however, had Hill slopes of 1, with a Ki value of 0.24 microM. The k1 and k-1 for (/sup 3/H)PZ binding were 3.5 X 10(6) M-1 min-1 and 0.13 min-1, respectively. The muscarinic receptor antagonists atropine, scopolamine and PZ inhibited the binding of (/sup 3/H)QNB and (/sup 3/H)PZ to solubilized receptors with Hill slopes of 1, as did the muscarinic receptor agonist oxotremorine. The muscarinic receptor agonist carbachol competed for (/sup 3/H)QNB and (/sup 3/H)PZ binding with a Hill slope of less than 1 in cerebral cortex, but not in cerebellum. GTP did not alter the interactions of carbachol or oxotremorine with the solubilized receptor. Together, these data suggest that muscarinic receptor sites solubilized from rat brain retain their abilities to interact selectively with muscarinic receptor agonists and antagonists.

  5. Solubilization of poorly water-soluble compounds using amphiphilic phospholipid polymers with different molecular architectures.

    Mu, Mingwei; Konno, Tomohiro; Inoue, Yuuki; Ishihara, Kazuhiko


    To achieve stable and effective solubilization of poorly water-soluble bioactive compounds, water-soluble and amphiphilic polymers composed of hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) units and hydrophobic n-butyl methacrylate (BMA) units were prepared. MPC polymers having different molecular architectures, such as random-type monomer unit sequences and block-type sequences, formed polymer aggregates when they were dissolved in aqueous media. The structure of the random-type polymer aggregate was loose and flexible. On the other hand, the block-type polymer formed polymeric micelles, which were composed of very stable hydrophobic poly(BMA) cores and hydrophilic poly(MPC) shells. The solubilization of a poorly water-soluble bioactive compound, paclitaxel (PTX), in the polymer aggregates was observed, however, solubilizing efficiency and stability were strongly depended on the polymer architecture; in other words, PTX stayed in the poly(BMA) core of the polymer micelle formed by the block-type polymer even when plasma protein was present in the aqueous medium. On the other hand, when the random-type polymer was used, PTX was transferred from the polymer aggregate to the protein. We conclude that water-soluble and amphiphilic MPC polymers are good candidates as solubilizers for poorly water-soluble bioactive compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Phosphate solubilizing bacteria and their role in plant growth promotion.

    Rodríguez, H; Fraga, R


    The use of phosphate solubilizing bacteria as inoculants simultaneously increases P uptake by the plant and crop yield. Strains from the genera Pseudomonas, Bacillus and Rhizobium are among the most powerful phosphate solubilizers. The principal mechanism for mineral phosphate solubilization is the production of organic acids, and acid phosphatases play a major role in the mineralization of organic phosphorous in soil. Several phosphatase-encoding genes have been cloned and characterized and a few genes involved in mineral phosphate solubilization have been isolated. Therefore, genetic manipulation of phosphate-solubilizing bacteria to improve their ability to improve plant growth may include cloning genes involved in both mineral and organic phosphate solubilization, followed by their expression in selected rhizobacterial strains. Chromosomal insertion of these genes under appropriate promoters is an interesting approach.

  7. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes

    Eagling, Victoria A; Tjia, John F; Back, David J


    Aims Chemical inhibitors of cytochrome P450 (CYP) are a useful tool in defining the role of individual CYPs involved in drug metabolism. The aim of the present study was to evaluate the selectivity and rank the order of potency of a range of isoform-selective CYP inhibitors and to compare directly the effects of these inhibitors in human and rat hepatic microsomes. Methods Four chemical inhibitors of human cytochrome P450 isoforms, furafylline (CYP1A2), sulphaphenazole (CYP2C9), diethyldithiocarbamate (CYP2E1), and ketoconazole (CYP3A4) were screened for their inhibitory specificity towards CYP-mediated reactions in both human and rat liver microsomal preparations. Phenacetin O-deethylation, tolbutamide 4-hydroxylation, chlorzoxazone 6-hydroxylation and testosterone 6β-hydroxylation were monitored for enzyme activity. Results Furafylline was a potent, selective inhibitor of phenacetin O-deethylation (CYP1A2-mediated) in human liver microsomes (IC50 = 0.48 μm), but inhibited both phenacetin O-deethylation and tolbutamide 4-hydroxylation (CYP2C9-mediated) at equimolar concentrations in rat liver microsomes (IC50 = 20.8 and 24.0 μm respectively). Sulphaphenazole demonstrated selective inhibition of tolbutamide hydroxylation in human liver microsomes but failed to inhibit this reaction in rat liver microsomes. DDC demonstrated a low level of selectivity as an inhibitory probe for chlorzoxazone 6-hydroxylation (CYP2E1-mediated). DDC also inhibited testosterone 6β-hydroxylation (CYP3A-mediated) in man and rat, and tolbutamide 4-hydroxylase activity in rat. Ketoconazole was a very potent, selective inhibitor of CYP3A4 activity in human liver (IC50 = 0.04 μm). Although inhibiting CYP3A in rat liver it also inhibited all other reactions at concentrations ≤5 μm. Conclusions It is evident that CYP inhibitors do not exhibit the same selectivity in human and rat liver microsomes. This is due to differential selectivity of the inhibitors and/or differences in the CYP

  8. Large scale solubilization of coal and bioconversion to utilizable energy. Eleventh quarterly technical progress report, April 1, 1996--June 30, 1996

    Mishra, N.C.


    Neurospora has the capability to solubilize coal and the protein fraction accounting for this ability has been isolated. During this period the cola solubilizing activity (CSA) was fractionated and partially sequenced. The activity has been determined to be a tyrosinase and/or a phenol oxidase. The amino acid sequence of the protein was used to prepare oligonucleotides to identify the clone carrying Neurospora CSA. It is intended to clone the Neurospora gene into yeast, since yeast cannot solubilize coal, to further characterize the CSA.

  9. Characterization of the mechanisms for coal solubilization by filamentous fungi

    Fernandez, M.; Luna, N.; Monistrol, I.F.; Laborda, F. [Madrid Univ. (Spain). Dept. de Microbiologia y Parasitologia


    The mechanisms implicated on coal solubilization have been studied in this work. Two fungal strains (Trichoderma and Penicillium) has been isolated which were able to solubilize different kinds of Spanish coals (hard coal, subbituminous coal and lignite) when growing in liquid media in the presence of coal. Extracellular peroxidase, esterase and some times phenol oxidase enzymes were present in the culture supernatant when a high degree of coal solubilization was attained. Those enzymes could be involved in the coal attack. (orig.)

  10. Phosphate solubilizing bacteria around Indian peninsula

    DeSouza, M.J.B.D; Nair, S.; Chandramohan, D

    nitrophenylphosphate by the culture. One mole of nitrophenol is equivalent to 1 atom of phosphorus. The absorbance was read at 418 nm using DU-6 Beckmann spectrophotometer. The activity was expressed as ?mol.Pml-1d-1. The cultures which showed good phosphate... strengthening the fact that the isolates are of marine origin. Maximum activity of 5 ? was observed for strain P6 in 4.5% NaCl Location Beaches Islands Coasts Offshore Table 1?Phosphate solubilizing bacteria in various niches. No. of cultures...

  11. Fluoranthene metabolism: Human and rat liver microsomes display different stereoselective formation of the trans-2,3-dihydrodiol

    Day, B.W.; Sahali, Y.; Hutchins, D.A.; Wildschuette, M.P.; Pastorelli, R.; Nguyen, T.T.; Naylor, S.; Skipper, P.L.; Wishnok, J.S.; Tannenbaum, S.R. (Univ. of Pittsburgh, PA (United States))


    The metabolism of the environmental carcinogen fluoroanthene by human liver microsomes was compared to that by liver microsomes from rats treated with Aroclor 1254. Although the human-derived system gave primarily one product, similar metabolites were noted from each system. Enantiomers of the major metabolic product, in both cases the trans-2,3-dihydrodiol, were separated by chiral stationary-phase chromatography. Absolute configurations were assigned by application of the benzoate exciton chirality rules to the CD spectra of the 4-(dimethylamino)benzoyl esters. Liver microsomes from Aroclor 1254-treated rats produced the R,R enantiomer of the diol in 75-78% enantiomeric excess, while human liver microsomes produced this enantiomer in only 6-12% excess. The activities of these enantiomers were compared in Salmonella typhimurium strain TM677 mutagenicity assays employing the 9000g supernatant of Aroclor 1254-induced rat liver homogenates. Both the syn- and anti-2,3-dihydrodiol 1,10b-epoxides, which had only been inferred to be metabolites in previous studies, were isolated from the microsomal incubations by preparative reverse-phase HPLC. The evident exceptional aqueous stabilities of these diol epoxides were further examined by half-life determination experiments. Their tetrahydrotetrol hydrolysis products were also noted in the metabolite HPLC profiles. The structures of the tetrahydrotetrols were confirmed by total synthesis.

  12. Isolation and biochemical characterization of the microsomal fraction from the digestive gland of mussel Mytilus galloprovincialis Lam.

    Viarengo, A; Pertica, M; Mancinelli, G; Palmero, S; Orunesu, M


    A procedure to prepare microsomes from the mussel digestive gland is proposed. The data concerning the biochemical characterization of this subcellular fraction shows a typical RNA:protein ratio, but the presence of hydrolytic enzymes was also found; therefore a mixture of hydrolase inhibitors to study the different biochemical characteristics was used. The biochemical data demonstrate that glucose-6-phosphatase activity (G6Pase), a typical microsomal marker in mammalian cells, is not present in mussel digestive gland microsomes but a high non-specific phosphatase activity was detected. Benzo[a]pyrene hydroxylase activity was found to be present although in a minimal amount. The evaluation of the molecular weight of the rRNA demonstrates that the larger ribosomal subunit contains RNA of Mr 1.40 X 10(-6) (approximately 26S) and the smaller subunit is composed of RNA of Mr 0.65 X 10(-6) (18S). The data from mussel digestive gland microsomes was compared with that experimentally obtained from rat liver microsomes and discussed from a functional or an evolutionary point of view.

  13. Liquefaction/solubilization processes of Spanish coals by microorganisms

    Laborda, F.; Monistrol, I.F.; Luna, N.; Fernandez, M. [Madrid Univ. (Spain). Dept. de Microbiologia y Parasitologia


    Several fundamental aspects of microbial coal liquefaction/solubilization have been studied in this work. The first one is the mechanisms implicated on coal transformation. During coal solubilization, fungal cells produced extracellular peroxidase, esterase and some times phenol oxidase enzymes which appear to be involved in solubilization. Moreover, the analysis of liquefaction/solubilization products was done. In this regard, a reduction on the average size of humic acids derived from lignite was observed, probably due to depolymerization caused by microorganisms. Finally, microorganisms showed a specific adherence to the coal surface, that seems to promote the microbial attack to coal. (orig.)

  14. Solubilizing excipients in oral and injectable formulations.

    Strickley, Robert G


    A review of commercially available oral and injectable solution formulations reveals that the solubilizing excipients include water-soluble organic solvents (polyethylene glycol 300, polyethylene glycol 400, ethanol, propylene glycol, glycerin, N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide), non-ionic surfactants (Cremophor EL, Cremophor RH 40, Cremophor RH 60, d-alpha-tocopherol polyethylene glycol 1000 succinate, polysorbate 20, polysorbate 80, Solutol HS 15, sorbitan monooleate, poloxamer 407, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire 44/14, Softigen 767, and mono- and di-fatty acid esters of PEG 300, 400, or 1750), water-insoluble lipids (castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil and palm seed oil), organic liquids/semi-solids (beeswax, d-alpha-tocopherol, oleic acid, medium-chain mono- and diglycerides), various cyclodextrins (alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, and sulfobutylether-beta-cyclodextrin), and phospholipids (hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, L-alpha-dimyristoylphosphatidylcholine, L-alpha-dimyristoylphosphatidylglycerol). The chemical techniques to solubilize water-insoluble drugs for oral and injection administration include pH adjustment, cosolvents, complexation, microemulsions, self-emulsifying drug delivery systems, micelles, liposomes, and emulsions.

  15. Effect of sedimentation through sucrose solutions on the protein-synthesizing ability of rat liver microsomes

    Leslie, Ruth A.; Mansbridge, J. N.


    1. Centrifugation of the postmitochondrial supernatant of rat liver through 1.0m-sucrose produces particles that have 85–95% less incorporating ability in a cell-free protein-synthesizing system than either ribosomes or microsomes. 2. The incorporation of [14C]phenylalanine into protein by particles prepared by sedimentation through 1.0m-sucrose is stimulated about 20-fold by addition of poly U. 3. The content of rapidly labelled RNA of microsomes is decreased during centrifugation through 1.0m-sucrose. 4. It is suggested that degradation of mRNA occurs during the formation of the pellet in the centrifuge tube as a result of a membrane-bound alkaline ribonuclease, after removal of the ribonuclease inhibitor of the soluble fraction. PMID:5451910

  16. Amiloride and diltiazem inhibition of microsomal and mitochondrial Na+ and Ca2+ transport.

    Sordahl, L A; LaBelle, E F; Rex, K A


    Amiloride, a K+-sparing diuretic, and diltiazem, a Ca2+ channel antagonist, have both been reported to inhibit Na+ transport-associated processes in different subcellular membrane systems. In this report, similar inhibitory effects of both agents are demonstrated on Na+-induced Ca2+ release from rabbit heart mitochondria and on Na+ uptake in a kidney medulla microsomal preparation. Both amiloride and diltiazem produce 50% inhibition of Na+ uptake in kidney microsomes at the same concentrations. Heart mitochondrial Na+-induced Ca2+ release was 50% inhibited by 6 microM diltiazem and 200 microM amiloride. No effects of either agent on mitochondrial respiratory activity were observed. The results suggest a specific effect of both drugs on a Na+-binding site associated with an antiport exchange process. These data also extend previous observations suggesting the use of these agents as tools to define further ion transport mechanisms in biological membranes.

  17. Metabolism of bupropion by baboon hepatic and placental microsomes.

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


    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.

  18. In vitro biotransformation of flavonoids by rat liver microsomes

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


    microsomes but only to a minor extent by uninduced microsomes. No metabolites were detected from eriodictyol, taxifolin, luteolin, quercetin, myricetin, fisetin, morin or isorhamnetin. 2. The identity of the metabolites was elucidated using lc-ms and H-1-nmr, and was consistent with a general metabolic...

  19. In vitro metabolic studies using homogenized horse liver in place of horse liver microsomes.

    Wong, Jenny K Y; Tang, Francis P W; Wan, Terence S M


    The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of using homogenized horse liver instead of liver microsomes with the aim of saving preparation time and improving the correlation between in vitro and in vivo results. Indeed, the preparation of the homogenized horse liver was very simple, needing only to homogenize the required amount of liver. Even though no further purification steps were performed before the homogenized liver was used, the cleanliness of the extracts obtained, based on gas chromatography-mass spectrometry (GC-MS) analysis, was similar to that for liver microsomes. Herein, the results of the in vitro experiments carried out using homogenized horse liver for five anabolic steroids-turinabol, methenolone acetate, androst-4-ene-3,6,17-trione, testosterone, and epitestosterone-are discussed. In addition to the previously reported in vitro metabolites, some additional known in vivo metabolites in the equine could also be detected. As far as we know, this is the first report of the successful use of homogenized liver in the horse for carrying out in vitro metabolism experiments. Copyright © 2011 John Wiley & Sons, Ltd. Copyright © 2011 John Wiley & Sons, Ltd.

  20. Haloarchaea Endowed with Phosphorus Solubilization Attribute Implicated in Phosphorus Cycle.

    Yadav, Ajar Nath; Sharma, Divya; Gulati, Sneha; Singh, Surender; Dey, Rinku; Pal, Kamal Krishna; Kaushik, Rajeev; Saxena, Anil Kumar


    Archaea are unique microorganisms that are present in ecological niches of high temperature, pH and salinity. A total of 157 archaea were obtained from thirteen sediment, water and rhizospheric soil samples collected from Rann of Kutch, Gujarat, India. With an aim to screen phosphate solubilizing archaea, a new medium was designed as Haloarchaea P Solubilization (HPS) medium. The medium supported the growth and P solubilization activity of archaea. Employing the HPS medium, twenty isolates showed the P-solubilization. Phosphate solubilizing archaea were identified as seventeen distinct species of eleven genera namely Haloarcula, Halobacterium, Halococcus, Haloferax, Halolamina, Halosarcina, Halostagnicola, Haloterrigena, Natrialba, Natrinema and Natronoarchaeum. Natrinema sp. strain IARI-WRAB2 was identified as the most efficient P-solubilizer (134.61 mg/L) followed by Halococcus hamelinensis strain IARI-SNS2 (112.56 mg/L). HPLC analysis detected seven different kinds of organic acids, namely: gluconic acid, citric acid, formic acid, fumaric acid succinic acid, propionic acid and tartaric acid from the cultures of these isolates. These phosphate solubilizing halophilic archaea may play a role in P nutrition to vegetation growing in these hypersaline soils. This is the first report for these haloarchaea to solubilize considerable amount of P by production of organic acids and lowering of pH.

  1. [The solubilization of four insoluble phosphates by some microorganisms].

    Zhao, Xiaorong; Lin, Qimei; Li, Baoguo


    Four insoluble phosphates of ferric phosphate (Fe-P), aluminum phosphate (Al-P), fluorapatite (FAP) and rock phosphate (RP) were used as a sole phosphorus resource for some phosphate-solubilizing microorganisms. It was found that there was significant difference in solubilizing these phosphates by the tested isolates. The fungi normally were more powerful than the bacteria in dissolving the phsophates. The microorganisms generally solubilized more phosphate when supplied with NO3- than with NH4+. However, the isolates of 2TCiF2 and 4TCiF6 had much higher capacity to solubilize FAP and Al-P respectively in NH4+ medium. Most of the isolates solubilized readily FAP and RP, and then Al-P. Ferric phosphate was the least soluble to these isolates. Only isolate 2TCiF2 showed strong ability to solubilize Fe-P. In particular, two Aspergillus sp. had much higher capacity of dissolving Fe-P when suppled with NO3-. The isolates of Evwinia sp. 4TCRi22 and Enterobacter sp. 1TCRi15 had higher capacity of solubilizing FAP. But two Arthrobacter sp. showed the highest activity in RP medium. It is supposed that complexion of organic acids with metals may be the main reason for these isolates to solubilize the phosphates. However, other chelant substances may be much more important for Enterobacter sp. and Erwinia sp. to release phosphorus from the phsphates.

  2. Development of cholinephosphotransferase in guinea pig lung mitochondria and microsomes.

    Stith, I E; Das, S K


    Development of mitochondrial and microsomal choline phosphotransferase in the fetal guinea pig lung was investigated. The activity in fetal mitochondria was more than twice of that in fetal microsomes. However, in adult lung, the enzyme was distributed mostly in microsomes. In fetal lung, both the mitochondrial and microsomal enzyme activity was greatest at approx. 81% of the total gestation period (55 days). The specific activity in the microsomal fraction than declined until term, but increased again in the 24-h newborn from 1.0 to 2.3 nmol/min per mg protein. The activity in the mitochondrial fraction declined after 61 days (2.8 nmol/min per mg) to a minimal level at term (0.6 nmol/min per mg). Although the enzyme activity decreased from day 55 (1.2 nmol/min per mg), the amount of phosphatidylcholine gradually increased between day 55 and term.

  3. Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

    Socaciu, Carmen; Jessel, Robert; Diehl, Horst A.


    The carotenoids β-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.

  4. The binding of pirenzepine to digitonin-solubilized muscarinic acetylcholine receptors from the rat myocardium.

    Birdsall, N. J.; Hulme, E. C.; Keen, M.


    The binding of pirenzepine to digitonin-solubilized rat myocardial muscarinic acetylcholine receptors has been examined at 4 degrees C. Solubilization produced only small changes in the binding of N-methylscopolamine and atropine. In contrast to the low affinity binding of pirenzepine found to be present in in the membranes, high affinity binding was detected in the soluble preparation. In both preparations, pirenzepine binding was complex. High affinity pirenzepine binding (KD approximately 3 X 10(-8)M) to the soluble myocardial receptors could be monitored directly using [3H]-pirenzepine. [3H]-pirenzepine-labelled soluble myocardial receptors have a sedimentation coefficient of 11.1 s. This indicates that [3H]-pirenzepine binds predominantly to the uncoupled form of the receptor. However, [3H]-pirenzepine-agonist competition experiments indicated that the high affinity pirenzepine binding sites are capable of coupling with a guanosine 5'-triphosphate (GTP)-binding protein. Pirenzepine affinities for the soluble myocardial receptors were unaffected by their state of association with the GTP-binding proteins found in the heart. The equilibrium binding properties of the soluble cortical and myocardial receptors were very similar. However, the binding kinetics of the myocardial receptor were much slower. It appears that the membrane environment can affect the affinity of pirenzepine for the rat myocardial muscarinic receptor. Removal of the constraint by solubilization allows the expression of high affinity pirenzepine binding. PMID:3754173

  5. Phosphate Solubilizing Bacteria Adaptive to Vinasse

    Kahar Muzakhar


    Full Text Available Microorganisms identified as phosphate solubilizing bacteria (PSB adaptive to vinasse were successfully screened from sugarcane soil from an agriculatural estate in Jatiroto. By conducting a screening on Pikovskaya’s agar medium (PAM, we found that five different isolates were detected as PSB (pvk-5a, pvk-5b, pvk-6b, pvk-7a, and pvk-8a. Of the five isolates only three could be grown and were found to be adaptive to vinasse based medium without any nutrients added (pvk-5a, pvk-5b and pvk-7a. The three isolates were characterized as coccus and Gram negative with no endospores detected. We suggest that these three isolates can be used as biofertilizer agent to support organic farming.

  6. Reduced complement-mediated immune complex solubilizing capacity and the presence of incompletely solubilized immune complexes in SLE sera

    Baatrup, Gunnar; Petersen, I; Jensenius, J C


    Reduced complement-mediated solubilization (CMS) of pre-formed immune complexes (IC) was demonstrated in sera from 11 out of 12 SLE patients. The presence of incompletely solubilized endogeneous IC in SLE sera was indicated by the following findings: (1) When IC positive SLE sera with reduced CMS...

  7. Metabolism of (-)-cis- and (-)-trans-rose oxide by cytochrome P450 enzymes in human liver microsomes.

    Nakahashi, Hiroshi; Yamamura, Yuuki; Usami, Atsushi; Rangsunvigit, Pramoch; Malakul, Pomthong; Miyazawa, Mitsuo


    The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes.

  8. Preparation

    M.M. Dardir


    Full Text Available Some hexanamide-mono and di-linoleniate esters were prepared by the reaction of linolenic acid and hexanamide (derived from the reaction of hexanoic acid and diethanolamine. The chemical structure for the newly prepared hexanamide-mono and di-linoleniate esters were elucidated using elemental analysis, (FTIR, H 1NMR and chemical ionization mass spectra (CI/Ms spectroscopic techniques. The results of the spectroscopic analysis indicated that they were prepared through the right method and they have high purity. The new prepared esters have high biodegradability and lower toxicity (environmentally friendly so they were evaluated as a synthetic-based mud (ester-based mud for oil-well drilling fluids. The evaluation included study of the rheological properties, filtration and thermal properties of the ester based-muds formulated with the newly prepared esters compared to the reference commercial synthetic-based mud.

  9. Enantioselective Metabolism of Flufiprole in Rat and Human Liver Microsomes.

    Lin, Chunmian; Miao, Yelong; Qian, Mingrong; Wang, Qiang; Zhang, Hu


    The enantioselective metabolism of flufiprole in rat and human liver microsomes in vitro was investigated in this study. The separation and determination were performed using a liquid chromatography system equipped with a triple-quadrupole mass spectrometer and a Lux Cellulose-2 chiral column. The enantioselective metabolism of rac-flufiprole was dramatically different in rat and human liver microsomes in the presence of the β-nicotinamide adenine dinucleotide phosphate regenerating system. The half-lives (t1/2) of flufiprole in rat and human liver microsomes were 7.22 and 21.00 min, respectively, for R-(+)-flufiprole, whereas the values were 11.75 and 17.75 min, respectively, for S-(-)-flufiprole. In addition, the Vmax of R-(+)-flufiprole was about 3-fold that of S-(-)-flufiprole in rat liver microsomes, whereas its value in the case of S-(-)-flufiprole was about 2-fold that of R-(+)-flufiprole in human liver microsomes. The CLint of rac-flufiprole also showed opposite enantioselectivy in rat and human liver microsomes. The different compositions and contents of metabolizing enzyme in the two liver microsomes might be the reasons for the difference in the metabolic behavior of the two enantiomers.

  10. A Biochemical and Morphological Study of Rat Liver Microsomes

    Moulé, Y.; Rouiller, C.; Chauveau, J.


    Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point of view. 1. Under these experimental conditions, the "total microsome" fraction was obtained by centrifuging the cytoplasmic extract free of nuclei and mitochondria, for 3 hours at 145,000 g. Morphologically, the total microsomes consist mainly of "rough-surfaced membranes" and "smooth" ones. 2. The total microsomes have been divided into 2 subfractions so that the 1st microsomal fraction contains the "rough" vesicles (2 hours centrifugation at 40,000 g) while the 2nd microsomal fraction consists essentially of smooth vesicles, free particles, and ferritin (centrifugation of the supernatant at 145,000 g for 3 hours). 3. By the action of 0.4 per cent sodium deoxycholate in 0.88 M sucrose, it was possible to obtain a pellet for each of the 2 fractions which consisted of dense particles, rich in RNA, poor in lipids, and which represented about 50 to 60 percent of the RNA and 10 to 15 per cent of the proteins. The results have been discussed taking into consideration the hypothesis of the presence of RNA in the membranes of microsomal vesicles. PMID:14424705

  11. Morphine metabolism in human skin microsomes.

    Heilmann, S; Küchler, S; Schäfer-Korting, M


    For patients with severe skin wounds, topically applied morphine is an option to induce efficient analgesia due to the presence of opioid receptors in the skin. However, for topical administration it is important to know whether the substance is biotransformed in the skin as this can eventually reduce the concentration of the active agent considerably. We use skin microsomes to elucidate the impact of skin metabolism on the activity of topically applied morphine. We are able to demonstrate that morphine is only glucuronidated in traces, indicating that the biotransformation in the skin can be neglected when morphine is applied topically. Hence, there is no need to take biotransformation into account when setting up the treatment regimen.

  12. Phosphate Solubilization Potentials of Rhizosphere Isolates from Central Anatolia (Turkey)

    Ogut, M.; Er, F.


    Plant available-phosphorus (P) is usually low in Anatolian soils due mainly to the precipitation as calcium (Ca) and magnesium (Mg) phosphates in alkaline conditions. Phosphate solubilizing microorganisms (PSM) can enhance plant P-availability by dissolving the hardly soluble-P within the rhizosphere, which is the zone that surrounds the plant roots. PSM's can be used as seed- or soil-inocula to increase plant P-uptake and the overall growth. A total of 162 PSM's were isolated from the rhizosphere of wheat plants excavated from different fields located along a 75 km part of a highway in Turkey. The mean, the standart deviation, and the median for solubilized-P (ppm) in a 24 h culture in a tricalcium phosphate broth were 681, 427, and 400 for glucose; 358, 266, and 236 for sucrose; and 102, 117, and 50 for starch, respectively. There was not a linear relationship between the phosphate solubilized in the liquid cultures and the solubilization index obtained in the Pikovskaya's agar. Nine isolates representing both weak and strong solubilizers [Bacillus megaterium (5), Bacillus pumilis (1), Pseudomonas syringae pv. phaseolica (1), Pseudomonas fluorescens (1), Arthrobacter aurescens (1) as determined by the 16S rRNA gene sequence analysis] were further studied in a five day incubation. Pseudomonas syringae pv. phaseolica solubilized statistically (P<0.05) higher phosphate (409 ppm) than all the other strains did. There was not a statistically significant (P<0.05) difference in solubilized-P among the Bacillus strains. The pH of the medium fell to the levels between 4 and 5 from the initial neutrality. The phosphate solubilizing strains variably produced gluconic, 2-keto-D-gluconic, glycolic, acetic and butyric acids. The organic acids produced by these microorganisms seem to be the major source of phosphate solubilization in vitro.

  13. Cell-free synthesis of membrane proteins: tailored cell models out of microsomes.

    Fenz, Susanne F; Sachse, Rita; Schmidt, Thomas; Kubick, Stefan


    Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.

  14. Metabolism of bupropion by baboon hepatic and placental microsomes


    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-...

  15. Selective bioreduction of nitroxides by rat liver microsomes

    Rosen, G.M.; Rauckman, E.J.; Hanck, K.W.


    Presented are possible explanations for the inability of rat liver microsomes to reduce 2-ethyl-2,4,4-trimethyl-3-oxazolidinyloxy (OXAN) even though the reduction potential for OXAN is identical to that for 2,2,6,6-tetramethylpiperidinoxyl (TEMPO) and di-tert-butyl nitroxide (DTBN), both of which are reduced by these microsomes. It is suggested that the conformation of OXAN prevents these enzymes from reducing the compound. Administration of phenobarbital was found to induce the synthesis of a form of cytochrome P-450, which enabled the rat liver microsomes to reduce the OXAN. (2 diagrams, 9 references)

  16. Processes of liquefaction/solubilization of Spanish coals by microorganisms

    Laborda, F.; Monistrol, I.F.; Luna, N.; Fernandez, M. [Universidad de Alcala de Henares, Madrid (Spain). Dept. de Microbiologia y Parasitologia


    Several fundamental aspects of microbial coal liquefaction/solubilization were studied. The liquefied/solubilized products from coal by microorganisms were analysed. The liquid products analysed by IR titration and UV/visible spectrometry showed some alterations with regard to the original coal. Humic acids extracted from the liquefied lignite showed a reduction in the average molecular weight and a increase in the condensation index, probably due to depolymerization caused by microorganisms. The mechanisms implicated in coal biosolubilization by two fungal strains, M2 (Trichoderma sp.) and M4 (Penicillium sp.) were also studied. Extracellular peroxidase, esterase and phenoloxidase enzymes appear to be involved in coal solubilization. (orig.)

  17. Solubilization of proteins: the importance of lysis buffer choice.

    Peach, Mandy; Marsh, Noelle; Miskiewicz, Ewa I; MacPhee, Daniel J


    The efficient extraction of proteins of interest from cells and tissues is not always straightforward. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 from choriocarcinoma cells using NP-40 and RIPA lysis buffer. Furthermore, we demonstrate the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness for solubilization of small heat-shock proteins from smooth muscle with the often utilized RIPA lysis buffer. Overall, the results demonstrate the importance of establishing the optimal lysis buffer for specific protein solubilization within the experimental workflow.

  18. Solubilization of benzene and cyclohexane by sodium deoxycholate micelles

    Christian, S.D.; Smith, L.S.; Bushong, D.S.; Tucker, E.E.


    Vapor pressure-solubility data were obtained for the aqueous systems benzene-sodium deoxycholate and cyclohexane- sodium deoxycholate at 25/sup 0/C. The results are consistent with a mass action model similar to the BET equation. Equilibrium constants are inferred to characterize interactions of hydrocarbons with solubilization sites assumed to consist of units of four deoxycholate anions. Although addition of sodium chloride increases the middle aggregation number, solubilization results are affected very little by variation in salt concentration. When pure liquid hydrocarbon standard states are employed, solubilization results for benzene and cyclohexane (at varying salt concentrations) are quite similar. 26 references.

  19. New competition assay for the solubilized hepatic galactosyl receptor

    Ray, D.A.; Weigel, P.H.


    The present method of quantitating soluble asialoglycoprotein (galactosyl) receptor activity relies on the selective precipitation of receptor-ligand complexes to allow separation from free ligand. To provide an alternative to selective precipitation procedures, a simple and rapid method to assay for detergent-solubilized galactosyl receptor activity has been developed which uses permeabilized, fixed cells as a source of immobilized solid-phase receptors. Isolated rat hepatocytes were treated with digitonin to make available the internal as well as the external receptors. The permeable cells were also treated with glutaraldehyde to prevent further protein loss during subsequent exposure to detergents such as Triton X-100. The permeable/fixed cells, which retained about 70% of their total /sup 125/I-asialo-orosomucoid (/sup 125/I-ASOR)-binding activity, with 89% specific binding, were insoluble even in 0.5% Triton X-100 and were easily pelleted. The permeable/fixed cells can be prepared in advance and stored frozen for months. A detergent extract of receptor is mixed with a constant amount of both /sup 125/I-ASOR and permeable/fixed cells. Soluble active receptors compete with immobilized receptors on the treated cell for binding of the /sup 125/I-ASOR. The assay is reproducible, linear over a broad range of soluble receptor concentration, and can quantitate receptor activity from as few as 10(5) hepatocytes. A modified purification procedure for the rat hepatic galactosyl receptor using this competition assay is also described.

  20. In vitro effects of various heterocyclic thiol compounds and beta-lactam antibiotics on vitamin K-dependent gamma-glutamylcarboxylation activity in liver microsomes.

    Oka, T; Touchi, A; Ezumi, K; Yamakawa, M; Matsubara, T


    The in vitro effect of N-methyltetrazolethiol (NMTT), one of the common substituents at the 3'-position of the cephem in various beta-lactam antibiotics, on liver microsomal gamma-glutamylcarboxylation (gamma-carboxylation) activity was examined using solubilized rat liver enzyme. The enzyme activity was inhibited by coexisting with NMTT and NADH, and this inhibitory activity could be suppressed by the addition of a sulfhydryl compound such as dithiothreitol (DTT), glutathione or cysteine. Various five-membered heterocyclic thiol compounds exhibited concentration-dependent inhibition of microsomal gamma-carboxylation activity. These inhibitory actions diminished markedly in the presence of 1 mM DTT. In vitro gamma-carboxylation activity also decreases upon addition of various beta-lactam antibiotics at 1 or 10 mM, depending upon the concentration of the drug. Among the heterocyclic thiol compounds, there is a correlation between their inhibitory activities and hydrophobicities. Thus, the in vitro inhibitory activity of heterocyclic thiol compounds and beta-lactam antibiotics on microsomal gamma-carboxylation activity is not correlated with their molecular structures, but rather depends on their hydrophobicities and with the concentrations in the reaction mixture.

  1. Application of Potential Phosphate-Solubilizing Bacteria and Organic Acids on Phosphate Solubilization from Phosphate Rock in Aerobic Rice

    Qurban Ali Panhwar; Shamshuddin Jusop; Umme Aminun Naher; Radziah Othman; Mohd Ismail Razi


    A study was conducted at Universiti Putra Malaysia to determine the effect of phosphate-solubilizing bacteria (PSB) and organic acids (oxalic & malic) on phosphate (P) solubilization from phosphate rock (PR) and growth of aerobic rice. Four rates of each organic acid (0, 10, 20, and 30 mM), and PSB strain (Bacillus sp.) were applied to aerobic rice. Total bacterial populations, amount of P solubilization, P uptake, soil pH, and root morphology were determined. The results of the study showed ...

  2. Solubilization of silk protein by radiation

    Sudatis, Boonya; Pongpat, Suchada [Office of Atomic Energy of Peace, Bangkok (Thailand)


    Gamma irradiated silk fibroin at doses of 0, 5, 10, 20, 40, 60, 80, 100, 125, 250, 500, 750 and 1000 kGy were soaked in water for 1 hr. Silk fibroin solubilized percentage was investigated from lost weight of sample (dried at 105{sup 0}C), they were 0, 0, 0.7, 0, 0.11, 0.11, 0, 0.73, 0.77, 4.38, 8.32, 10.22 and 18.52 respectively. It showed that at the higher dose up to 250 kGy had direct effect to solubility, and increased with increasing dose. In addition, silk sericin dissolved 77.76, 82.22, 83.55, 84.31, 86.04, 86.67 and 87.37% after gamma irradiation at the doses of 0, 50, 100, 200, 500, 750 and 1000 kGy respectively. It presents that radiation can cause silk protein, fibroin and sericin dissolve because of their degradation. (author)

  3. Solubilization and Humanization of Paraoxonase-1

    Mohosin Sarkar


    Full Text Available Paraoxonase-1 (PON1 is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we “humanized” an engineered variant of PON1 with high activity against cyclosarin (GF and found that it was still very active against GF with much greater similarity to the human sequence.

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

    Kornbrust, D.J.


    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)

  5. Crude oil degradation by phosphate-solubilizing bacteria

    DeSouza, M.J.B.D.; Nair, S.; David, J.J.; Chandramohan, D.

    Phosphate-solubilizing bacteria were isolated from tropical areas around the Indian peninsula. Two of the isolates showed high phosphatase activity. The isolates were identified as Klebsiella pneumoniae and Bacillus pumilus, and they showed high...

  6. Moroccan rock phosphate solubilization during a thermo-anaerobic ...



    Dec 4, 2013 ... thermo-anaerobic grassland waste biodegradation process. Moussa S. .... plating the nutrient agar isolate on the solid NBRIP medium. (Nautiyal, 1999) ..... solubilizing fungi isolated from phosphate mines. Ecol. Eng. 33:187-.

  7. Rock Phosphate Solubilization Mechanisms of One Fungus and One Bacterium

    LIN Qi-mei; ZHAO Xiao-rong; ZHAO Zi-juan; LI Bao-guo


    Many microorganisms can dissolve the insoluble phosphates like apatite. However, the mechanisms are still not clear. This study was an attempt to investigate the mechanisms of rock phosphate solubilization by an Aspergillus 2TCiF2 and an Arthrobacter1TCRi7. The results indicated that the fungus produced a large amount of organic acids, mainly oxalic acid. The total quantity of the organic acids produced by the fungus was 550 times higher than that by the bacterium. Different organic acids had completely different capacities to solubilize the rock. Oxalic acid and citric acid had stronger capacity to dissolve the rock than malic acid, tartaric acid, lactic acid, acetic acid, malonic acid and succinic acid. The fungus solubilized the rock through excreting both proton and organic acids. The rock solubilization of the bacterium depended on only proton.

  8. Glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes.

    Yamanaka, Hiroyuki; Nakajima, Miki; Katoh, Miki; Yokoi, Tsuyoshi


    Glucuronidation of thyroxine is a major metabolic pathway facilitating its excretion. In this study, we characterized the glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes, and identified human UDP-glucuronosyltransferase (UGT) isoforms involved in the activity. Human jejunum microsomes showed a lower K(m) value (24.2 microM) than human liver (85.9 microM) and kidney (53.3 microM) microsomes did. Human kidney microsomes showed a lower V(max) value (22.6 pmol/min/mg) than human liver (133.4 pmol/min/mg) and jejunum (184.6 pmol/min/mg) microsomes did. By scaling-up, the in vivo clearances in liver, intestine, and kidney were estimated to be 1440, 702, and 79 microl/min/kg body weight, respectively. Recombinant human UGT1A8 (108.7 pmol/min/unit), UGT1A3 (91.6 pmol/min/unit), and UGT1A10 (47.3 pmol/min/unit) showed high, and UGT1A1 (26.0 pmol/min/unit) showed moderate thyroxine glucuronosyltransferase activity. The thyroxine glucuronosyltransferase activity in microsomes from 12 human livers was significantly correlated with bilirubin O-glucuronosyltransferase (r = 0.855, p microsomes was mainly catalyzed by UGT1A8 and UGT1A10 and to a lesser extent by UGT1A1, and the activity in human kidney microsomes was mainly catalyzed by UGT1A7, UGT1A9, and UGT1A10. The changes of activities of these UGT1A isoforms via inhibition and induction by administered drugs as well as genetic polymorphisms may be a causal factor of interindividual differences in the plasma thyroxine concentration.

  9. Enriching vermicompost by nitrogen fixing and phosphate solubilizing bacteria.

    Kumar, V; Singh, K P


    The effect of inoculation of vermicompost with nitrogen-fixing Azotobacter chroococcum strains, Azospirillum lipoferum and the phosphate solubilizing Pseudomonas striata on N and P contents of the vermicompost was assessed. Inoculation of N2 fixing bacteria into vermicompost increased contents of N and P. Enriching vermicompost with rock phosphate improved significantly the available P when inoculated with P. striata. During the incubation period, the inoculated bacterial strains proliferated rapidly, fixed N and solubilized added and native phosphate.

  10. Research and engineering assessment of biological solubilization of phosphate

    Rogers, R.D.; McIlwain, M.E.; Losinski, S.J.; Taylor, D.D.


    This research and engineering assessment examined a microbial phosphate solubilization process as a method of recovering phosphate from phosphorus containing ore compared to the existing wet acid and electric arc methods. A total of 860 microbial isolates, collected from a range of natural environments were tested for their ability to solubilize phosphate from rock phosphate. A bacterium (Pseudomonas cepacia) was selected for extensive characterization and evaluation of the mechanism of phosphate solubilization and of process engineering parameters necessary to recover phosphate from rock phosphate. These studies found that concentration of hydrogen ion and production of organic acids arising from oxidation of the carbon source facilitated microbial solubilization of both pure chemical insoluble phosphate compounds and phosphate rock. Genetic studies found that phosphate solubilization was linked to an enzyme system (glucose dehydrogenase). Process-related studies found that a critical solids density of 1% by weight (ore to liquid) was necessary for optimal solubilization. An engineering analysis evaluated the cost and energy requirements for a 2 million ton per year sized plant, whose size was selected to be comparable to existing wet acid plants.

  11. Phosphate-solubilizing bacteria associated with runner bean rhizosphere

    Mihalache Gabriela


    Full Text Available Soil microorganisms, especially rhizobacteria, play a key role in soil phosphorus (P dynamics and the subsequent availability of phosphate to plants. Utilization of phosphate-solubilizing bacteria as biofertilizers instead of synthetic chemicals is known to improve plant growth through the supply of plant nutrients, and may help to sustain environmental health and soil productivity. The main purpose of this study was to identify new phosphate-solubilizing bacteria isolated from runner bean rhizosphere. Ten out of 25 isolated bacterial strains solubilized Ca3(PO42 in qualitative and quantitative P-solubilization. The strain that exhibited the highest potential to solubilize Ca3(PO42, was selected for further determination of the mechanisms involved in the process. The medium pH was measured, organic acids released in the culture medium were identified by HPLC analysis, and the acid and alkaline phosphatase activities were determined. Our results showed that strain R7 solubilized phosphorous through the production of various organic acids such as lactic, isocitric, tartaric and pyruvic acids, and that it can be used as a potential biofertilizer.

  12. Solubilization and fractionation of paired helical filaments.

    González, P J; Correas, I; Avila, J


    Paired helical filaments isolated from brains of two different patients with Alzheimer's disease were extensively treated with the ionic detergent, sodium dodecyl sulphate. Filaments were solubilized at different extents, depending on the brain examined, thus suggesting the existence of two types of paired helical filaments: sodium dodecyl sulphate-soluble and insoluble filaments. In the first case, the number of structures resembling paired helical filaments greatly decreased after the detergent treatment, as observed by electron microscopy. Simultaneously, a decrease in the amount of sedimentable protein was also observed upon centrifugation of the sodium dodecyl sulfate-treated paired helical filaments. A sodium dodecyl sulphate-soluble fraction was isolated as a supernatant after low-speed centrifugation of the sodium dodecyl sulphate-treated paired helical filaments. The addition of the non-ionic detergent Nonidet-P40 to this fraction resulted in the formation of paired helical filament-like structures. When the sodium dodecyl sulphate-soluble fraction was further fractionated by high-speed centrifugation, three subfractions were observed: a supernatant, a pellet and a thin layer between these two subfractions. No paired helical filaments were observed in any of these subfractions, even after addition of Nonidet P-40. However, when they were mixed back together, the treatment with Nonidet P-40 resulted in the visualization of paired helical filament-like structures. These results suggest that at least two different components are needed for the reconstitution of paired helical filaments as determined by electron microscopy. The method described here may allow the study of the components involved in the formation of paired helical filaments and the identification of possible factors capable of blocking this process.

  13. Human liver microsomal metabolism of (+)-discodermolide.

    Fan, Yun; Schreiber, Emanuel M; Day, Billy W


    The polyketide natural product (+)-discodermolide is a potent microtubule stabilizer that has generated considerable interest in its synthetic, medicinal, and biological chemistry. It progressed to early clinical oncology trials, where it showed some efficacy in terms of disease stabilization but also some indications of causing pneumotoxicity. Remarkably, there are no reports of its metabolism. Here, we examined its fate in mixed human liver microsomes. Due to limited availability of the agent, we chose a nanoflow liquid chromatography-electrospray ionization-mass spectrometry analytical approach employing quadrupolar ion trap and quadrupole-quadrupole-time-of-flight instruments for these studies. (+)-Discodermolide was rapidly converted to eight metabolites, with the left-side lactone (net oxidation) and the right-side diene (epoxidation followed by hydrolysis, along with an oxygen insertion product) being the most metabolically labile sites. Other sites of metabolism were the allylic and pendant methyl moieties in the C12-C14 region of the molecule. The results provide information on the metabolic soft spots of the molecule and can be used in further medicinal chemistry efforts to optimize discodermolide analogues.

  14. Kavalactone metabolism in rat liver microsomes.

    Fu, Shuang; Rowe, Anthony; Ramzan, Iqbal


    The specific CYP enzymes involved in kavalactone (KLT) metabolism and their kinetics have not been fully examined. This study used rat liver microsomes (RLM) to determine kavain (KA), methysticin (MTS) and desmethoxyyangonin (DMY) enzyme kinetic parameters, to elucidate the major CYP450 isoforms involved in KLT metabolism and to examine gender differences in KLT metabolism. Formation of the major KLT metabolites was first-order, consistent with classic enzyme kinetics. In both male and female RLM, clotrimazole (CLO) was the most potent inhibitor of KA and MTS metabolism. This suggests CYP3A1/3A23 (females) and CYP3A2 (males) are the main isoenzymes involved in the metabolism of these KLTs in rats, while the roles of CYP1A2, -2 C6, -2 C9, -2E1 and -3A4 are limited. Desmethoxyyangonin metabolism was equally inhibited by cimetidine (CIM) and CLO in females, and CIM and nortriptyline in males. This implies that DMY metabolism involves CYP2C6 and CYP2C11 in males, and CPY2C12 in females. CYP3A1/3A23 may also be involved in females.

  15. The microsomal dicarboxylyl-CoA synthetase.

    Vamecq, J; de Hoffmann, E; Van Hoof, F


    Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo.

  16. Effect of the beta-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases.

    Oleson, Lauren; Court, Michael H


    Glucuronidation studies using microsomes and recombinant uridine diphosphoglucuronosyltransferases (UGTs) can be complicated by the presence of endogenous beta-glucuronidases, leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used beta-glucuronidase inhibitor, although it is not clear whether this reagent should be added routinely to glucuronidation incubations. Here we have determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and recombinant UGTs (rUGTs). Despite the use of buffered incubation solutions, it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM did not enhance any of the glucuronidation activities evaluated that could be considered consistent with inhibition of beta-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for glucuronidation of 5-hydroxytryptamine and estradiol by pHLMs, with a 35% decrease at 20 mM saccharolactone concentration. Endogenous beta-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes and insect-cell expressed rUGTs, but not for kidney, intestinal or human embryonic kidney HEK293 microsomes. However, the extent of hydrolysis was relatively small, representing only 9-19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations. If saccharolactone is used, concentrations should be titrated to achieve activity enhancement without inhibition.

  17. Influence of induction and flavones on lung microsomal oxygen metabolism

    Gregory, E.M.; Barlow, R.; Ebel, R.E.


    NADPH oxidase activity, H/sub 2/O/sub 2/ production and O/sub 2/ production were studied using microsomes isolated from lungs of control rats and rats treated with phenobarbital (PB), 3-methylcholanthrene (3-MC), 5,6-benzoflavone (..beta..-NF), or chlordecone (CD). 3-MC, ..beta..-NF or Cd treatment resulted in induction of lung P-450 and P-450 reductase. NADPH oxidase activity was slightly higher in 3-MC or ..beta..-NF lung microsomes than in control, PB or CD microsomes. 5 mM metyrapone inhibited oxidase activity of these microsomes by 20-50% depending upon their source. The rates of H/sub 2/O/sub 2/ and O/sub 2/ production did not appear to vary significantly as a function of induction and metyrapone had only a marginal effect on these rates. The rates of H/sub 2/O/sub 2/ and O/sub 2/ production at 37/sup 0/ were 1.8 +/- 0.2 and 1.8 +/- 0.3 nmol/min/mg, respectively. 50 ..beta..-NF stimulated H/sub 2/O/sub 2/ production by about 2-fold regardless of microsomal source while the stimulation of O/sub 2/ production was marginal. In each case, the effect of ..beta..-NF was greater than a comparable amount of 7,8-benzoflavone (..cap alpha..-NF). NADPH oxidase activity was dramatically stimulated by ..beta..-NF. For example, at 37/sup 0/ 100 ..beta..-NF increased the oxidase activity of control lung microsomes by about 9-fold from 3.4 to 31.6 nmol/min/mg. Lung microsomes from induced rats were comparably stimulated by ..beta..-NF. This stimulation was concentration dependent although not hyperbolic. Metyrapone did not inhibit the ..beta..-NF stimulated NADPH oxidase activity.

  18. Evidence for the activation of organophosphate pesticides by cytochromes P450 3A4 and 2D6 in human liver microsomes.

    Sams, C; Mason, H J; Rawbone, R


    The role of specific cytochrome P450 isoforms in catalysing the oxidative biotransformation of the organophosphorothioate pesticides parathion, chlorpyrifos and diazinon into structures that inhibit cholinesterase has been investigated in human liver microsomes using chemical inhibitors. Pesticides were incubated with human liver microsomes and production of the anticholinergic oxon metabolite was investigated by the inhibition of human serum cholinesterase. Quinidine and ketoconazole at 10 micromol/l inhibited oxidative biotransformation. Compared to control incubations (no inhibitor) where cholinesterase activity was inhibited to between 1 and 4% of control levels, incorporation of the CYP2D6 inhibitor quinidine into the microsomal incubation resulted in cholinesterase activity of 50% for parathion, 38% for diazinon and 30% for chlorpyrifos. Addition of the CYP3A4 inhibitor ketoconazole to microsomal incubations resulted in 66% cholinesterase activity with diazinon, 20% with parathion and 5% with chlorpyrifos. The unexpected finding that CYP2D6, as well as CYP3A4, catalysed oxidative biotransformation was confirmed for chlorpyrifos and parathion using microsomes prepared from a human lymphoblastoid cell line expressing CYP2D6. While parathion has been investigated only as a model compound, chlorpyrifos and diazinon are both very important, widely used pesticides and CYP2D6 appears to be an important enzyme in their bioactivation pathway. CYP2D6 is polymorphic and hence may influence individual susceptibility to exposure to chlorpyrifos and diazinon as well as other structurally similar pesticides.

  19. Solubilization of trichloroethylene by polyelectrolyte/surfactant complexes

    Uchiyama, Hirotaka; Christian, S.D.; Tucker, E.E.; Scamehorn, J.F. (Univ. of Oklahoma, Norman, OK (United States))


    An automated vapor pressure method is used to obtain solubilization isotherms for trichloroethylene (TCE) in polyelectrolyte/surfactant complexes throughout a wide range of solute activities at 20 and 25 C. The polyelectrolyte chosen is sodium poly(styrenesulfonate), PSS< and the surfactant is cetylpyridinium chloride or N-hexadecylpyridinium chloride, CPC. Data are fitted to the quadratic equation K = K[sub 0](1[minus][alpha]X + [beta]X[sup 2]), which correlates the solubilization equilibrium constant (K) with the mole fraction of TCE (X) in the micelles or complexes at each temperature. Activity coefficients are also obtained for TCE in the PSS/CPC complexes as a function of X. The general solubilization of TCE in PSS/CPC complexes resembles that of TCE in CPC micelles, as well as that of benzene or toluene in CPC micelles, suggesting that TCE solubilizes in ionic micelles both within the hydrocarbon micellar interior and near the micellar surface. The presence of the polyelectrolyte causes a small decrease in the ability of the cationic surfactant to solubilize TCE, while greatly reducing the concentration of the surfactant present in monomeric form. PSS/CPC complexes may be useful in colloid-enhanced ultrafiltration processes to purify organic-contaminated water.

  20. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    Jeffery, Constance J


    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  1. Factors determining rock phosphate solubilization by microorganisms isolated from soil.

    Nahas, E


    Forty two soil isolates (31 bacteria and 11 fungi) were studied for their ability to solubilize rock phosphate and calcium phosphate in culture medium. Eight bacteria and 8 fungi possessed solubilizing ability. Pseudomonas cepacia and Penicillium purpurogenum showed the highest activity. There was a correlation between final pH value and titratable acidity (r=-0.29 to -0.87) and between titratable acidity and soluble phosphate (r=0.22 to 0.99). Correlation values were functions of insoluble phosphate and of the group of microorganisms considered. A high correlation was observed between final pH and soluble phosphate only for the rock phosphates inoculated with the highest concentration of solubilizing bacteria (r=-0.73 to -0.98).

  2. Bilirubin diglucuronide synthesis by a UDP-glucuronic acid-dependent enzyme system in rat liver microsomes.

    Blanckaert, N; Gollan, J; Schmid, R


    Incubation of rat liver homogenate or microsomal preparations with bilirubin or bilirubin monoglucuronide with (BMG) resulted in formation of bilirubin diglucuronide (BDG). Both synthesis of BMG and its conversion to BDG were critically dependent on the presence of UDP-glucuronic acid. Pretreatment of the animals with phenobarbital stimulated both reactions. When 33 microM bilirubin was incubated with microsomal preparations from phenobarbital-treated rats, 80-90% of the substrate was converted to bilirubin glucuronides; the reaction products consisted of almost equal amounts of BMG and BDG. When phenobarbital pretreatment was omitted or when the substrate concentration was increased to 164 microM bilirubin, proportionally more BMG and less BDG were formed. Homogenate and microsomes from homozygous Gunn rats neither synthesized BMG nor converted BMG to BDG. These findings in vitro suggest an explanation for the observations in vivo that, in conditions of excess bilirubin load or of genetically decreased bilirubin UDP glucuronosyltransferase (EC activity, proportionally more BMG and less BDG are excreted in bile. PMID:109837

  3. The stability studies and in vitro hepatic microsomal metabolism of some alpha-phenyl-N-substituted nitrones in rats.

    Bulut, Gülen; Oktav, Mehmet; Ulgen, Mert


    Nitrones are a very important class of synthetic chemicals as synthetic intermediates, antioxidant agents, and metabolic oxidation products of secondary amines and imines used drug, food, cosmetic and printing industry. In the present study, the stability experiments and in vitro metabolism studies using rat microsomal preparations fortified with NADPH were carried out using three different alpha-phenyl-N-substituted nitrones ie alpha-phenyl-N-tert-butylnitrone (PTBN), alpha-(2,6-dichlorophenyl)-N-phenylnitrone (DCPPN) and alpha-phenyl-N-adamantanylnitrone (PADN). The separation of these compounds from the potential degradation, isomerization and metabolic products were performed using a reverse phase HPLC system with a diodearray uv detection. Following stability experiments at 37 degrees C using methanolic nitrone solutions, it was observed that PTBN produced trace amounts of benzaldehyde and the corresponding amide. DCPPN also produced trace amounts of amide. After 12 hours, the amount of the amide significantly increased. PADN produced trace amount of benzaldehyde but not any amide. The proposed compounds were incubated with rat microsomal preparations fortified with NADPH, extracted into dichloromethane (DCM) and finally evaporated under nitrogen in the dark conditions. PTBN was metabolized into corresponding amide whereas DCPPN and PADN did not. With all of the substrates, the corresponding aldehydes are observed with both test and control tubes using denaturated microsomes and without co-factors.

  4. Study of Viability of Phosphate Solubilizing Bacteria in Phosphate granules

    hajar rajabi


    Full Text Available Introduction: sustainable development and the environment are interconnected. Sustainable agriculture is continuous utilization of a farm with respect to various aspects of environmental conditions by using fewer inputs (other than Bio-fertilizers. Phosphorus is one of the essential elements for the plants. Management of soil is possible by using biological fertilizers pillar of sustainable agriculture and providing some of the phosphorus needed by plants via bio-fertilizers. Phosphorus deficiency is extremely effective on the plant growth and productivity. The application of phosphorus fertilizers is expensive and dangerous. In addition, phosphorus in the soilmay become insoluble and will be unavailable to the plants. Studies showed that phosphate solubilizing bacteria in the soil rhizosphere are active and by root exudates solve insoluble phosphates such as tricalcium phosphate, and form absorbable P for plant. Consequently, the use of microbial fertilizers could reduce excessive use of chemical fertilizers and lead to decrease their harmful effects and protect the environment and conservation of available resources. The biological phosphate fertilizer industry uses sugar beet molasses as a binder and drying granules at high temperatures. Therefore, it is important to evaluate the durability of the bacteria in molasses at high temperature. Materials and Methods: This study was designed as completely randomized design in a factorial arrangement.10 isolates were selected and the ratios of 50%, 25%, 15% and 10% of the apatite, organic matter, sulfur and soluble granule (ratio 1: 1 and 2: 1 bacteria and molasses, respectively, for each isolate was prepared. The final product was dried at 28 and 40 °C and remained for 4 months and population counted at first day and 10, 20, 30, 60, 90 and 120 days after the preparing. The population was counted by the serial dilution technique and cultured at Sperber media. Results and Discussion:Comparing the

  5. Stereoselective propranolol metabolism in two drug induced rat hepatic microsomes

    Xin Li; Su Zeng


    AIM To study the influence of inducers BNF and PB on the stereoselective metabolism of propranolol in rat hepatic microsomes.METHODS Phase Ⅰ metabolism of propranolol was studied by using the microsomes induced by BNF and PB and the non-induced microsome as the control. The enzymatic kinetic parameters of propranolol enantiomers were calculated by regression analysis of Lineweaver-Burk plots.Propranolol concentrations were assayed by HPLC.RESULTS A RP-HPLC method was developed to determine propranolol concentration in rat hepatic microsomes. The linearity equations for R( + )-propranolol and S ( - )-propranolol were A=705.7C+ 311.2C (R =0.9987) and A=697.2C +311.4C (R = 0.9970) respectively. Recoveries of each enantiomer were 98.9%, 99.5%, 101.0% at 60 μmol/L, 120 μmol/L, 240 μmol/L respectively. At the concentration level of 120 μmol/L, propranolol enantiomers were metabolized at different rates in different microsomes. The concentration ratio R (+)/S (-) of control and PB induced microsomes increased with time, whereas that of microsome induced by BNF decreased. The assayed enzyme parameters were: 1. Km. Control group: R( + )30±8, S( - )18 ± 5; BNF group: R( + )34 ± 3, S (-)39±7; PB group: R(+)38±17, S(-)36± 10.2. Vmax. Control group: R(+ )1.5 ±0.2, S( - )2.9±0.3; BNF group: R(+)3.8±0.3, S(-)3.3±0.5; PB group: R( + )0.07±0.03, S( - )1.94±0.07.3.Clint. Control group: R( + )60±3, S(- )170±30; BNF group: R( + )111.0 ±1, S(- ) 84±5; PB group: R(+)2.0 ± 2, S(- )56.0 ± 1. The enzyme parameters compared with unpaired t tests showed that no stereoselectivity was observed in enzymatic affinity of three microsomes to enantiomers and their catalytic abilitieswere quite different and had stereoselectivities. Compared with the control,microsome induced by BNF enhanced enzyme activity to propranolol R ( + )-enantiomer, and microsome induced by PB showed less enzyme activity to propranolol S(- )-enantiomer which remains the same stereoselectivities as

  6. [Solubilization Specificities Interferon beta-1b from Inclusion Bodies].

    Zhuravko, A S; Kononova, N V; Bobruskin, A I


    A new solubilization method of recombinant interferon beta-1b (IFNβ-1b) from the inclusion bodies was developed. This method allows to extract the target protein selectively in the solutions of different alcohols, such as ethanol, propanol and isopropanol. It was shown that the more effective IFNβ-1b solubilization was achieved in the 55% propanol solution. This method allowed to extract the target protein from inclusion bodies around 85-90%, and significantly reduced Escherichia coli content in the solubilizate, in comparison with standard methods.

  7. Water-solubilization and functionalization of semiconductor quantum dots.

    Tyrakowski, Christina M; Isovic, Adela; Snee, Preston T


    Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals that have abundant potential for uses in biological imaging and sensing. However, the best materials are synthesized in hydrophobic surfactants that prevent direct aqueous solubilization. While several methods have been developed to impart water-solubility, an aqueous QD dispersion has no inherent useful purpose and must be functionalized further. Due to the colloidal nature of QD dispersions, traditional methods of chemical conjugation in water either have low yields or cause irreversible precipitation of the sample. Here, we describe several methods to water-solubilize QDs and further functionalize the materials with chemical and/or biological vectors.

  8. Environmentally Persistent Free Radicals Inhibit Cytochromes P450 Activity in Rat Liver Microsomes

    Reed, James R.; Cawley, George F.; Ardoin, Taylor G.; Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W.; Backes, Wayne L.


    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) are 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 affect 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. PMID:24713513

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

    Reed, James R; Cawley, George F; Ardoin, Taylor G; Dellinger, Barry; Lomnicki, Slawomir M; Hasan, Farhana; Kiruri, Lucy W; Backes, Wayne L


    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.

  10. Biotransformation of myrislignan by rat liver microsomes in vitro.

    Li, Fei; Yang, Xiu-Wei


    Myrislignan (1), erythro-(1R,2S)-2-(4-allyl-2,6-dimethoxyphenoxyl)-1-(4-hydroxy-3-methoxyphenyl) propan-1-ol, is a major acyclic neolignan in seeds of Myristica fragrans. Studies have suggested that myrislignan may deter feeding activity, but little is known about its metabolism. We investigated the biotransformation of myrislignan by rat liver microsomes in vitro. Seven metabolites were produced by liver microsomes from rats pre-treated with sodium phenobarbital. These were identified, using spectroscopic methods, as myrislignanometins A-G (2-8), respectively.

  11. Biotransformation of chlorpyrifos and diazinon by human liver microsomes and recombinant human cytochrome P450s (CYP).

    Sams, C; Cocker, J; Lennard, M S


    The cytochrome P450 (CYP)-mediated biotransformation of the organophosphorothioate insecticides chlorpyrifos and diazinon was investigated. Rates of desulphuration to the active oxon metabolite (chlorpyrifos-oxon and diazinon-oxon) and dearylation to non-toxic hydrolysis products were determined in human liver microsome preparations from five individual donors and in recombinant CYP enzymes. Chlorpyrifos and diazinon underwent desulphuration in human liver microsome with mean Km = 30 and 45 microM and V(max) = 353 and 766 pmol min(-1) mg(-1), respectively. Dearylation of these compounds by human liver microsome proceeded with Km = 12 and 28 microM and V(max) = 653 and 1186 pmol min(-1) mg(-1), respectively. The apparent intrinsic clearance (V(max)/Km) of dearylation was 4.5- and 2.5-fold greater than desulphuration for chlorpyrifos and diazinon, respectively. Recombinant human CYP2B6 possessed the highest desulphuration activity for chlorpyrifos, whereas CYP2C19 had the highest dearylation activity. In contrast, both desulphuration and dearylation of diazinon were catalysed at similar rates, in the rank order CYP2C19 > CYP1A2 > CYP2B6 > CYP3A4. Both organophosphorothioates were more readily detoxified (dearylation) than bioactivated (desulphuration) in all human liver microsome preparations. However, the role of individual CYP enzymes in these two biotransformation pathways varied according to the structure of the organophosphorothioate, which was reflected in different activation/detoxification ratios for chlorpyrifos and diazinon. Variability in activity of individual CYP enzymes may influence interindividual sensitivity to the toxic effects of chlorpyrifos and diazinon.

  12. Solubilization of the chromatin-bound estrogen receptor from chicken liver and fractionation on hydroxylapatite.

    Gschwendt, M


    1. High-affinity estrogen-binding sites can be solubilized from the liver chromatin of estrogenized chickens by treatment of the chromatin with 2 M KCL/5 M urea and fractionation on hydroxylapatite. Two estrogen-binding proteins are eluted from hydroxylapatite columns by 20mM phosphate (binding protein I) and 200mMphosphate (binding protein II), respectively. 2. The binding protein I is part of a non-histone protein fraction containing acid-soluble and insoluble proteins, whereas the binding protein II elutes together with high molecular weight nonhistone proteins containing acid insoluble proteins only. Both binding proteins exhibit the smae affinity for estradiol (Kd approximately 10(-9) M). 3. From chromatin of untreated chickens very small amounts of binding protein I (0.1 pmol/mg protein compared to 1.9 pmol/mg protein from estrogenized chickens) with the smae affinity for estradiol as that from estrogenized animals can be solubilized. Binding protein II is not detectable. 4. The "soluble nuclear estrogen receptor" extracted from crude liver nucleir of estrogenized chickens by 0.5 M KCL behaves on hydroxylapatite very similarly to salt/urea-dissociated chromatin with respect to the binding protein I. No binding protein II, however, can be demonstrated. 5. Chromatography of various preparations on Bio-Gel A-1.5 m indicates that the binding protein II is a residual chromatin fragment containing an unseparated binding protein-DNA complex, whereas the binding protein I represents the solubilized nucleic-acid-free chromosomal estrogen receptor. The "soluble nuclear receptor" and the binding protein I, however, are not identical with respect to their chromatographic behaviour on Bio-Gel A-1.5m, even though their estrogen binding entity remaining after trypsin treatment seems to be very similar.

  13. Immunoreactive proteins of Acanthocheilonema viteae (Nematoda: Filarioidea) adults: solubilization in various detergents.

    Misra, S; Singh, D P; Fatma, N; Chatterjee, R K


    For optimal solubilization of immunoreactive proteins of filarial parasites, adult worms of Acanthocheilonema viteae were extracted in different detergents including anionic, cationic, nonionic and zwitterionic agents under varying incubation periods. Each preparation was then analysed by SDS-PAGE and immunoblotting using pooled sera from Mastomys natalensis infected with A. viteae. Amongst the detergents used, maximum immunoreactive proteins were exposed by sodium dodecylsulphate (SDS), closely followed by sodium deoxycholate (DOC). Nevertheless, a few additional protein bands were recognized by infected sera in DOC preparations, but not in SDS, and vice versa. Most of the proteins were completely or partially dissolved within 2 hrs extraction time. It is felt that DOC may be used in place of SDS because of the strong denaturing character of the latter.

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

    Reed, James R., E-mail: [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)


    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

  15. Solubilization of silica: Synthesis, characterization and study of penta-coordinated pyridine N-oxide silicon complexes

    Subramania Ranganathan; Ch Chandrashekhar Rao; Suvarchala Devi Vudayagiri; Y B R D Rajesh; B Jagadeesh


    In an effort to design agents that could solubilize silica in water, under ambient conditions and pH, as takes place in nature, novel zwitterionic, penta-oxo-coordinated silicon compounds with siliconate cores have been prepared from 4-substituted pyridine N-oxides (H, OMe, morpholino, NO2) as donor ligands, their structures established by 1H, 13C and MS, and the coordination number of silicon, by 29Si NMR. The formation of complexes from pyridine N-oxides is noteworthy since they arise from interaction with a weakly nucleophilic oxygen centre. The ability of the pyridine N-oxides to enhance the solubilization of silica in water has been experimentally demonstrated. Possible rationalization of this observation on the basis of O→Si coordination via the oxygen atom of pyridine N-oxide is suggested.

  16. Amphiphilic biopolymers (amphibiopols) as new surfactants for membrane protein solubilization

    Duval-Terrié, Caroline; Cosette, Pascal; Molle, Gérard; Muller, Guy; Dé, Emmanuelle


    The aim of this study was to develop new surfactants for membrane protein solubilization, from a natural, biodegradable polymer: the polysaccharide pullulan. A set of amphiphilic pullulans (HMCMPs), differing in hydrophobic modification ratio, charge ratio, and the nature of the hydrophobic chains introduced, were synthesized and tested in solubilization experiments with outer membranes of Pseudomonas fluorescens. The membrane proteins were precipitated, and then resolubilized with various HMCMPs. The decyl alkyl chain (C10) was the hydrophobic graft that gave the highest level of solubilization. Decyl alkyl chain-bearing HMCMPs were also able to extract integral membrane proteins from their lipid environment. The best results were obtained with an amphiphilic pullulan bearing 18% decyl groups (18C10). Circular dichroism spectroscopy and membrane reconstitution experiments were used to test the structural and functional integrity of 18C10-solubilized proteins (OmpF from Escherichia coli and bacteriorhodopsin from Halobacterium halobium). Whatever their structure type (α or β), 18C10 did not alter either the structure or the function of the proteins analyzed. Thus, HMCMPs appear to constitute a promising new class of polymeric surfactants for membrane protein studies. PMID:12649425

  17. [Effect of titanium dioxide nanoparticles on protein expression profiles in rat liver microsomes].

    Tananova, O N; Arianova, E A; Gmoshinskiĭ, I V; Aksenov, I V; Zgoda, V G; Khotimchenko, S A


    There was studied an influence of intragastric administration of titanium dioxide (anatase form) nanoparticles (NP) on protein expression profiles in rat's liver microsomes by methods of proteomics. Animals received water suspension of NP in doses from 0,1 to 10 mg per kg body weight intragastrically daily during 28 days. Microsomes were isolated from liver by means of preparative ultracentrifugation. Proteins composition was studied by 2D-electrophoresis in acrylamide gel. Protein spots were identified by MALDI-TOF analysis. The results demonstrated appearance of 53 new protein spots and disappearance of 19 spots in animals subjected to NP irrespective of their dose. In addition 25 new spots appeared and 3 disappeared in higher doses of NP when compared to low dose group and control animals. Mass-spectrum analysis showed presence of few polypeptides registered in international database among proteins expressed under influence of NP. One of this dominant expressed proteins corresponded to enzyme glutathione transpherase Mu 2 isoform (M=41,55 kD, pI=8,0). The conclusion was made of well advances of proteomic analysis in artificial NP influences on biosynthetic processes estimation.

  18. The cytochromes in microsomal fractions of germinating mung beans.

    Hendry, G A; Houghton, J D; Jones, O T


    Detailed studies of microsomal cytochromes from mung-bean radicles showed the presence of cytochrome P-420, particularly in dark-grown seedlings, accompanied by smaller quantities of cytochrome P-450. Similar proportions of cytochrome P-420 to cytochrome P-450 were found spectrophotometrically in vivo with whole radicles and hypocotyls. Assayed in vitro, maximum concentrations of both cytochromes were attained after 4 days of growth, before undergoing rapid degradation. Illumination of seedlings stabilized cytochrome P-450 and decreased the amount of cytochrome P-420. Three b cytochromes were present in the microsomal fraction, namely cytochromes b-562.5 (Em + 105 +/- 23 mV), b-560.5 (Em + 49 +/- 13 mV) and b5 (Em - 45 +/- 14 mV), all at pH 7.0. Of the b cytochromes, cytochrome b5 alone undergoes a rapid degradation after day 4, Changes in cytochrome b concentrations were confined to the microsomal fraction: mitochondrial b cytochrome concentrations were unaltered with age. Protohaem degradation (of exogenous methaemalbumin) was detected in microsomal fractions of mung beans. The rates of degradation were highest in extracts of young tissue and declined after day 4. The degradation mechanism and products did not resemble those of mammalian haem oxygenase. PMID:7306021

  19. Two New Lactones Metabolized from Isoline by Rat Liver Microsomes

    Jun TANG; Zheng Tao WANG; Teruaki AKAO; Norio NAKAMURA; Masao HATTORI


    Two new metabolites, namely bisline lactone and isolinecic acid lactone, were isolated from the resultant incubates after a scale-up incubation of isoline with rat liver microsomes. Their structures were determined by spectroscopic data, especially those from 1D and 2D NMR experiments.

  20. Zinc solubilizing Bacillus spp. potential candidates for biofortification in maize.

    Mumtaz, Muhammad Zahid; Ahmad, Maqshoof; Jamil, Moazzam; Hussain, Tanveer


    Bioaugmentation of Zn solubilizing rhizobacteria could be a sustainable intervention to increase bioavailability of Zn in soil which can be helpful in mitigation of yield loss and malnutrition of zinc. In present study, a number of pure rhizobacterial colonies were isolated from maize rhizosphere and screened for their ability to solubilize zinc oxide. These isolates were screened on the basis of zinc and phosphate solubilization, IAA production, protease production, catalase activity and starch hydrolysis. All the selected isolates were also positive for oxidase activity (except ZM22), HCN production (except ZM27) and utilization of citrate. More than 70% of isolates produces ammonia, hydrogen cyanide, siderophores, exopolysaccharides and cellulase. More than half of isolates also showed potential for urease activity and production of lipase. The ZM31 and S10 were the only isolates which showed the chitinase activity. All these isolates were evaluated in a jar trial for their ability to promote growth of maize under axenic conditions. Results revealed that inoculation of selected zinc solubilizing rhizobacterial isolates improved the growth of maize. In comparison, isolates ZM20, ZM31, ZM63 and S10 were best compared to other tested isolates in stimulating the growth attributes of maize like shoot length, root length, plant fresh and dry biomass. These strains were identified as Bacillus sp. (ZM20), Bacillus aryabhattai (ZM31 and S10) and Bacillus subtilis (ZM63) through 16S rRNA sequencing. This study indicated that inoculation of Zn solubilizing strains have potential to promote growth and can be the potential bio-inoculants for biofortification of maize to overcome the problems of malnutrition. Copyright © 2017 Elsevier GmbH. All rights reserved.

  1. Characterization of Phosphate Solubilizing and Potassium Decomposing Strains and Study on their Effects on Tomato Cultivation

    Tin Mar Lynn; Hlaing Swe Win; Ei Phyu Kyaw; Zaw Ko Latt; San San Yu


    Seven strains were collected for phosphate solubilizing and potassium decomposing activities from Microbiology Laboratory, Department of Biotechnology, Shweziwa Biofertilizer Plant. When phosphate solubilizing activity of selected strains was qualitatively determined, all strains except from B1 strain, gave clear zone formation on NBRIP media. But when quantitatively determined by spectrophotometric method, all strains solubilized insoluble tricalcium phosphate. Among seven strains, Ps strain...

  2. [Metabolism of mitomycin C by human liver microsomes in vitro].

    Hao, Fu-rong; Yan, Min-fen; Hu, Zhuo-han; Jin, Yi-zun


    To provide the profiles of metabolism of mitomycin C (MMC) by human liver microsomes in vitro, MMC was incubated with human liver microsomes, then the supernatant component was isolated and detected by HPLC. Types of metabolic enzymes were estimated by the effect of NADPH or dicumarol (DIC) on metabolism of MMC. Standard, reaction, background control (microsomes was inactivated), negative control (no NADPH), and inhibitor group (adding DIC) were assigned, the results were analyzed by Graphpad Prism 4. 0 software. Reaction group compared with background control and negative control groups, 3 NADPH-dependent absorption peaks were additionally isolated by HPLC after MMC were incubated with human liver microsomes. Their retention times were 10. 0, 14. 0, 14. 8 min ( named as Ml, M2, M3) , respectively. Their formation was kept as Sigmoidal dose-response and their Km were 0. 52 (95% CI, 0. 40 - 0.67) mmol x L(-1), 0. 81 (95% CI, 0. 59 - 1. 10) mmol x L(-1), 0. 54 (95% CI, 0. 41 -0. 71) mmol x L(-1) , respectively. The data indicated that the three absorption peaks isolated by HPLC were metabolites of MMC. DIC can inhibit formation of M2, it' s dose-effect fitted to Sigmoidal curve and it' s IC50 was 59. 68 (95% CI, 40. 66 - 87. 61) micromol x L(-1) , which indicated DT-diaphorase could take part in the formation of M2. MMC can be metabolized by human liver microsomes in vitro, and at least three metabolites of MMC could be isolated by HPLC in the experiment, further study showed DT-diaphorase participated in the formation of M2.

  3. Application of potential phosphate-solubilizing bacteria and organic acids on phosphate solubilization from phosphate rock in aerobic rice.

    Panhwar, Qurban Ali; Jusop, Shamshuddin; Naher, Umme Aminun; Othman, Radziah; Razi, Mohd Ismail


    A study was conducted at Universiti Putra Malaysia to determine the effect of phosphate-solubilizing bacteria (PSB) and organic acids (oxalic & malic) on phosphate (P) solubilization from phosphate rock (PR) and growth of aerobic rice. Four rates of each organic acid (0, 10, 20, and 30 mM), and PSB strain (Bacillus sp.) were applied to aerobic rice. Total bacterial populations, amount of P solubilization, P uptake, soil pH, and root morphology were determined. The results of the study showed significantly high P solubilization in PSB with organic acid treatments. Among the two organic acids, oxalic acid was found more effective compared to malic acid. Application of oxalic acid at 20 mM along with PSB16 significantly increased soluble soil P (28.39 mg kg(-1)), plant P uptake (0.78 P pot(-1)), and plant biomass (33.26 mg). Addition of organic acids with PSB and PR had no influence on soil pH during the planting period. A higher bacterial population was found in rhizosphere (8.78 log10 cfu g(-1)) compared to the nonrhizosphere and endosphere regions. The application of organic acids along with PSB enhanced soluble P in the soil solution, improved root growth, and increased plant biomass of aerobic rice seedlings without affecting soil pH.

  4. Application of Potential Phosphate-Solubilizing Bacteria and Organic Acids on Phosphate Solubilization from Phosphate Rock in Aerobic Rice

    Qurban Ali Panhwar


    Full Text Available A study was conducted at Universiti Putra Malaysia to determine the effect of phosphate-solubilizing bacteria (PSB and organic acids (oxalic & malic on phosphate (P solubilization from phosphate rock (PR and growth of aerobic rice. Four rates of each organic acid (0, 10, 20, and 30 mM, and PSB strain (Bacillus sp. were applied to aerobic rice. Total bacterial populations, amount of P solubilization, P uptake, soil pH, and root morphology were determined. The results of the study showed significantly high P solubilization in PSB with organic acid treatments. Among the two organic acids, oxalic acid was found more effective compared to malic acid. Application of oxalic acid at 20 mM along with PSB16 significantly increased soluble soil P (28.39 mg kg−1, plant P uptake (0.78 P pot−1, and plant biomass (33.26 mg. Addition of organic acids with PSB and PR had no influence on soil pH during the planting period. A higher bacterial population was found in rhizosphere (8.78 log10 cfu g−1 compared to the nonrhizosphere and endosphere regions. The application of organic acids along with PSB enhanced soluble P in the soil solution, improved root growth, and increased plant biomass of aerobic rice seedlings without affecting soil pH.

  5. A Comparative Study of the Metabolism of 6-Methylbenzo [A] Pyrene and Benzo [A] Pyrene by Rat Liver Microsomes


    Rat Liver Microsomes Name...pyrena by Rat Liver Microsomes Karen Lee Hamernik, Doctor of Philosophy, 1984 Dissertation directed by: Shan K. Yang, Ph.D., Professor, Department of...Microsomal Enzymes 85 Metabolism of 6-OHMBaP by Rat Liver Microsomes 94 Identification of 6-OHMBaP Metabolites 94 UV absorption spectral analysis


    Dunaitsev I. A.


    Full Text Available Two granular formulations of phosphorus biofertilizers combining rock phosphate and two highly active phosphate solubilizing strains: Acinetobacter species 305 and Pseudomonas species 181а have been investigated. Granules of about 3 mm in size were obtained by contact-convective drying of a mixture of ground ore, concentrated biomass of two different strains, starch and glucose. Micro granules with size of 0.1- 0.5 mm were obtained by spray drying the biomass of two different strains and application of dried cells on the particles of the ground ore. Starch was used as a binder. In the model liquid medium it was shown that the microorganisms have retained the ability to solubilize mineral phosphates in granular formulations prepared. In laboratory pot trial on marigold (Tagetes patula it was demonstrated that both formulations of biofertilizer increased the dry weight of the plants to the same level as that of chemical fertilizer - double superphosphate, but were inferior in the concentration of phosphorus in plants. Both formulations exceeded the effectiveness of rock phosphate and biomass used as biofertilizers both separately and jointly. No significant differences were noted between the two strains and the two granular formulations both for plant dry weight, and the content of phosphorus therein. Both granular formulations of biofertilizer retained their structure and avoided aggregating over a year of storage at 4 oC. The average persistence of living cells in the microbeads was about 1.5%, in granules - 32 %

  7. Solubilization properties of polycyclic aromatic hydrocarbons by saponin, a plant-derived biosurfactant

    Zhou Wenjun, E-mail: [Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang 310028 (China); Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Hangzhou, Zhejiang 310028 (China); Yang Juanjuan; Lou Linjie [Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang 310028 (China); Zhu Lizhong [Department of Environmental Science, Zhejiang University, Hangzhou, Zhejiang 310028 (China); Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Hangzhou, Zhejiang 310028 (China)


    The enhanced solubilization of polycyclic aromatic hydrocarbons (PAHs) by saponin, a plant-derived non-ionic biosurfactant, was investigated. The results indicated that the solubilization capabilities of saponin for PAHs were greater than some representative synthetic non-ionic surfactants and showed strong dependence on solution pH and ionic strength. The molar solubilization ratio (MSR) of saponin for phenanthrene was about 3-6 times of those of the synthetic non-ionic surfactants, and decreased by about 70% with the increase of solution pH from 4.0 to 8.0, but increased by approximately 1 times with NaCl concentration increased from 0.01 to 1.0 M. Heavy metal ions can enhance saponin solubilization for phenanthrene and the corresponding MSR values increased by about 25% with the presence of 0.01 M of Cd{sup 2+} or Zn{sup 2+}. Saponin is more effective in enhancing PAHs solubilization than synthetic non-ionic surfactants and has potential application in removing organic pollutants from contaminated soils. - Highlights: > The enhanced solubilization of PAHs by saponin was investigated in this study. > Saponin showed great solubilization capability for PAHs. > Saponin is more effective in enhancing HOCs solubilization at lower solution pH. > Increasing ionic strength can enhance HOCs solubilization in saponin solution. > Heavy metal ions can also enhance phenanthrene solubilization in saponin solution. - Saponin showed different solubilization properties for PAHs from the synthetic non-ionic surfactants and anionic rhamnolipid biosurfactants.

  8. Solubilization of Australian lignites by fungi and other microorganisms

    Catcheside, D.E.A.; Mallett, K.J. (Flinders University, Bedford Park, SA (Australia). School of Biological Sciences)

    Lignites (brown coals) from the Latrobe Valley in Victoria are solubilized by {ital Coriolus versicolor}, {ital Phanerochaete chrysosporium}, and five other species known to be active on Leonardite and various acid-treated North America lignites. Run-of-mine coal from Morwell and Loy Yang is refractory but is soluble after pretreatment with acid. A weathered deposit at Loy Yang, like Leonardite, is susceptible to biosolubilization without pretreatment. The white rot fungi {ital Ganoderma applanatum}, {ital Perenniporia tephropora} ({ital Fomes lividus}), {ital Pleurotus ostreatus}, {ital Pycnoporus cinnabarinus}, {ital Rigidoporus ulmarius}, and {ital Xylaria hypoxylon} were found to be capable of solubilizing lignite. In contrast, brown rot fungi were weakly active or inactive under the same test conditions. Lignite-degrading fungi, actinomycetes, and other bacteria, including some active on untreated run-of-mine coal, were isolated from natural lignite exposures and mining sites. 15 refs., 5 tabs.

  9. Mobilization and micellar solubilization of NAPL contaminants in aquifer rocks

    Javanbakht, Gina; Goual, Lamia


    Surfactant-enhanced aquifer remediation is often performed to overcome the capillary forces that keep residual NAPL phases trapped within contaminated aquifers. The surfactant selection and displacement mechanism usually depend on the nature of NAPL constituents. For example, micellar solubilization is often used to cleanup DNAPLs from aquifers whereas mobilization is desirable in aquifers contaminated by LNAPLs. Although the majority of crude oils are LNAPLs, they often contain heavy organic macromolecules such as asphaltenes that are classified as DNAPLs. Asphaltenes contain surface-active components that tend to adsorb on rocks, altering their wettability. Previous studies revealed that surfactants that formed Winsor type III microemulsions could promote both mobilization and solubilization. However the extent by which these two mechanisms occur is still unclear, particularly in oil-contaminated aquifers. In this study we investigated the remediation of oil-contaminated aquifers using an environmentally friendly surfactant such as n-Dodecyl β-D-maltoside. Focus was given on asphaltenes to better understand the mechanisms of surfactant cleanup. Through phase behavior, spontaneous imbibition, dynamic interfacial tension and contact angle measurements, we showed that microemulsions formed by this surfactant are able to mobilize bulk NAPL (containing 9 wt.% asphaltenes) in the porous rock and solubilize DNAPL (i.e., 4-6 wt.% adsorbed asphaltenes) from the rock surface. Spontaneous imbibition tests, in particular, indicated that the ratio of mobilized to solubilized NAPL is about 6:1. Furthermore, aging the cores in NAPL beyond 3 days allowed for more NAPL to be trapped in the large pores of the rock but did not alter the amount of asphaltenes adsorbed on the mineral surface.

  10. Hydrotropic Solubilization by Urea Derivatives: A Molecular Dynamics Simulation Study

    Yong Cui


    Full Text Available Hydrotropy is a phenomenon where the presence of a large quantity of one solute enhances the solubility of another solute. The mechanism of this phenomenon remains a topic of debate. This study employed molecular dynamics simulation to investigate the hydrotropic mechanism of a series of urea derivatives, that is, urea (UR, methylurea (MU, ethylurea (EU, and butylurea (BU. A poorly water-soluble compound, nifedipine (NF, was used as the model solute that was solubilized. Structural, dynamic, and energetic changes upon equilibration were analyzed to supply insights to the solubilization mechanism. The study demonstrated that NF and urea derivatives underwent significant nonstoichiometric molecular aggregation in the aqueous solution, a result consistent with the self-aggregation of urea derivatives under the same conditions. The analysis of hydrogen bonding and energy changes revealed that the aggregation was driven by the partial restoration of normal water structure. The energetic data also suggested that the promoted solubilization of NF is favored in the presence of urea derivatives. While the solutes aggregated to a varying degree, the systems were still in single-phase liquid state as attested by their active dynamics.

  11. Extractant efficiency in the solubilization of alternative sources of potassium

    Daniele Nogueira dos Reis


    Full Text Available Knowledge of a rock’s composition allows for inferences regarding several properties, ranging from its physical characteristics to its solubility. This study aimed to evaluate the use of different extractants to solubilize the K present in rocks as a potential source of nutrients and the effects of extractant contact time and temperature on rock solubilization. Samples of two rocks and a mineral concentrated from a granitic rock were treated with ammonium dihydrogen phosphate (NH4(H2PO4, calcium dihydrogen phosphate (Ca(H2PO42, sodium hydroxide (NaOH and water (control. Sample-extractant treatments were performed using a water bath shaker at temperatures of 25 and 50°C for periods of 3, 7, 10, 20, and 30 days. The amounts of K extracted from rocks using the extractants were in the following order: NH4H2PO4>Ca(HPO42>NaOH>water. The sequence of K release (ppm based on the rocks studied was as follows: nepheline syenite>green banded argillite>concentrated biotite. Increasing the extractant contact time and temperature enhanced the solubilized K content.

  12. Solubilization of diabase and phonolite dust by filamentous fungus

    Juliana Andréia Vrba Brandão


    Full Text Available The objective of this study was to evaluate the effect of the fungus Aspergillus niger strain CCT4355 in the release of nutrients contained in two types of rock powder (diabase and phonolite by means of in vitro solubilization trials. The experimental design was completely randomized in a 5 x 4 factorial design with three replications. It was evaluated five treatments (phonolite dust + culture medium; phonolite dust + fungus + culture medium; diabase powder + culture medium; diabase powder + fungus + culture medium and fungus + culture medium and four sampling dates (0, 10, 20 and 30 days. Rock dust (0.4% w/v was added to 125 mL Erlenmeyer flasks containing 50 mL of liquid culture medium adapted to A. niger. The flasks were incubated at 30°C for 30 days, and analysis of pH (in water, titratable acidity, and concentrations of soluble potassium, calcium, magnesium, zinc, iron and manganese were made. The fungus A. niger was able to produce organic acids that solubilized ions. This result indicates its potential to alter minerals contained in rock dust, with the ability to interact in different ways with the nutrients. A significant increase in the amount of K was found in the treatment with phonolite dust in the presence of the fungus. The strain CCT4355 of A. niger can solubilize minerals contained in these rocks dust.

  13. 4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase.

    Lambooy, J P; Koffman, B M


    Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.

  14. [Enzyme kinetics of ligustilide metabolism in rat liver microsomes].

    Qian, Min; Shi, Li-fu; Hu, Jin-hong


    To study the enzyme kinetics of ligustilide metabolism and the effects of selective CYP450 inhibitors on the metabolism of ligustilide in liver microsomes of rat, a LC-MS method was established for quantitative analysis of ligustilide in liver microsomes incubation system with nitrendipine as internal standard. The determination m/z for ligustilide was 173, and for nitrendipine, 315. An optimum incubation system was found and various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of ligustilide. The results showed that enzyme kinetics of ligustilide could be significantly inhibited by ketoconazole, trimethoprim and a-naphthoflavon but scarcely inhibited by omeprazole, 4-methylpyrazole and quinidine. Therefore, CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme participated in in vitro metabolism of ligustilide.

  15. Hepatic microsomal. omega. -oxidation of leukotriene B/sub 4/

    Mukhtar, H.; Bik, D.P.; Das, M.; Bickers, D.R.


    In this study the authors investigated the metabolism of LTB/sub 4/ by rat hepatic microsomes. /sup 3/H-LTB/sub 4/ (9 was incubated for 20 min in the presence3 of oxygen, NADPH and liver microsomes (1.5 mg). Incubation was followed by extraction and analysis by HPLC. Metabolite identification was based on cochromatography with reference standards. At least three radioactive peaks were observed; two of which were products 20-OH-LTB/sub 4/ and 20-COOH-LTB/sub 4/. The rate of formation of 20-OH-LTB/sub 4/ (14.6 nmoles/min/mg protein) was higher than that of 20-COOH-LTB/sub 4/ (2.5 nmoles/min/mg protein). The third radioactive peak remains unidentified. Product formation was negligible with boiled microsomes. LTB/sub 4/ activity required NADPH and oxygen, was linear with respect to incubation time and protein, and was maximal at pH 7.4. Enzyme activity was inhibited (>90%) by carbon monoxide, SKF 525A (1 mM), but was not affected by ..cap alpha..-naphthoflavone. Phenobarbital (PB, 80 mg/kg for 3 days), AROCLOR 1254 (100 mg/kg for 3 days) or 3-methylcholanthrene (40 mg/kg for 3 days) administration to rats resulted in only slight (<20%) or no increase in LTB/sub 4/ activity. However, the formation of the unidentified peak was increased (>100%) by PB treatment. These results suggest that is the major pathway for biotransformation of LTB/sub 4/ in liver microsomes and that this reaction is mediated by cytochrome P-450.

  16. Solubilization of a guanyl nucleotide-sensitive alpha/sub 1/ adrenergic receptor from liver membranes

    Harris, S.I.; Moss, J.


    Rat liver membranes incubated with norepinephrine before solubilization with digitonin yielded a soluble hormone-receptor complex from which the release of tightly bound norepinephrine was facilitated by guanyl nucleotides. Binding of the alpha/sub 1/-adrenergic receptor antagonist, (/sup 3/H)-prazosin, to the soluble preparation was utilized as a gauge of guanyl nucleotide-induced release of receptor-bound agonist. The following potency series was obtained with regard to the ability of guanyl nucleotides to facilitate (/sup 3/H)-prazosin binding to the solubilized preparation: guanosine 5'-0-(3-thiotriphosphate)(K/sub 1/2/ = 2.5 nM), guanylyl-imidodiphosphate (K/sub 1/2/ = 10 nM), guanosine triphosphate (K/sub 1/2/ = 34 nM) and adenylyl-imidodiphosphate (K/sub 1/2/ > 1 mM). In the presence of guanylyl-imidodiphosphate (0.4 mM), the receptor population displayed monotonic binding parameters with a K/sub d/ for (/sup 3/H)-prazosin of 1.16 nM by Scatchard analysis. Competition curves against (/sup 3/H)-prazosin with the antagonists phentolamine and yohimbine revealed respective K/sub i/'s of .089 and 1.8; curves with the agonists norepinephrine and isoproterenol yielded respective K/sub i/'s of and 360 Competition curves performed in the absence of guanyl nucleotide were complex demonstrating an apparent increase in affinity for agonists and an apparent decrease in affinity for antagonists. These curve shifts are consistent with the conversion of receptor to and from the guanyl nucleotide-sensitive state as a function of competing ligand concentration.

  17. Stereoselective glucuronidation of carvedilol by Chinese liver microsomes


    Objective: To study the stereoselective glucuronidation of carvedilol (CARV) by three Chinese liver microsomes.Methods: The metabolites of CARV were identified by a hydrolysis reaction with β-glucuronidase and HPLC-MS/MS. The enzyme kinetics for CARV enantiomers glucuronidation was determined by a reversed phase-high pressure liquid chromatography (RP-HPLC) assay using (S)-propafenone as internal standard after precolumn derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylisothiocyanate. Results: Two CARV glucuronides were found in three Chinese liver microsomes incubated with CARV. The non-linear regression analysis showed that the values of Km and Vmax for (S)-CARV and (R)-CARV enantiomers were (118±44) μmol/L, (2 500±833) pmol/( protein) and (24±7) μmol/L, (953+399) pmol/( protein),respectively. Conclusion: These results suggested that there was a significant (P<0.05) stereoselective glucuronidation of CARV enantiomers in three Chinese liver microsomes, which might partly explain the enantioselective pharmacokinetics of CARV.

  18. Oxidation of esterified arachidonate by rat liver microsomes

    Davis, H.W.; Suzuki, T.; Schenkman, J.B.


    The authors have previously demonstrated a relationship between phospholipid arachidonate in liver microsomes and malondialdehyde (MDA) formation during lipid peroxidation. In this study arachidonic acid (U-/sup 14/C) was incorporated into rat liver microsomes and NADPH-supported peroxidation was carried out at 37/sup 0/C for 15 minutes. The microsomes were pelleted by centrifugation and the labeled products in the supernatant were isolated by a solid phase method. Pellets were hydrolyzed with phospholipase A/sub 2/ and extracted with diethyl ether and the products from both fractions were separated by reverse phase HPLC. The results show that (1) oxidation occurs in all of the major phospholipids but that phosphatidylethanolamine is the most susceptible; (2) a linear correlation exists between MDA formation and supernatant radioactivity; (3) several different polar products are found in both the supernatant and the hydrolyzed pellet but that the ratios of product peaks in HPLC do not change during the peroxidation, indicating no secondary metabolism or propagation; and (4) cytochrome P-450 is not involved in the peroxidative reactions since no oxidation occurs in the absence of Fe/sup 3 +/ and since product formation is unaffected in the presence of carbon monoxide.

  19. Thin film voltammetry of metabolic enzymes in rat liver microsomes

    Krishnan, Sadagopan; Rusling, James F.


    We report herein thin film voltammetry and kinetics of electron transfer for redox proteins in rat liver microsomes for the first time. Films were made layer-by-layer from liver microsomes and polycations on pyrolytic graphite electrodes. Cyclic voltammograms were chemically reversible with a midpoint potential of −0.48 V vs SCE at 0.1 V s−1 in pH 7.0 phosphate buffer. Reduction peak potentials shifted negative at higher scan rates, and oxidation-reduction peak current ratios were ∼1 consistent with non-ideal quasireversible thin film voltammetry. Analysis of oxidation-reduction peak separations gave an average apparent surface electron transfer rate constant of 30 s−1. Absence of significant electrocatalytic reduction of O2 or H2O2 and lack of shift in midpoint potential when CO is added that indicates lack of an iron heme cofactor suggest that peaks can be attributed to oxidoreductases present in the microsomes rather than cytochrome P450 enzymes. PMID:18037986

  20. Stereoselective metabolism of tetrahydropalmatine enantiomers in rat liver microsomes.

    Zhao, Ming; Li, Li-Ping; Sun, Dong-Li; Sun, Si-Yuan; Huang, Shan-Ding; Zeng, Su; Jiang, Hui-Di


    Tetrahydropalmatine (THP), with one chiral center, is an active alkaloid ingredient in Rhizoma Corydalis. The aim of the present paper is to study whether THP enantiomers are metabolized stereoselectively in rat, mouse, dog, and monkey liver microsomes, and then, to elucidate which Cytochrome P450 (CYP) isoforms are predominately responsible for the stereoselective metabolism of THP enantiomers in rat liver microsomes (RLM). The results demonstrated that (+)-THP was preferentially metabolized by liver microsomes from rats, mice, dogs, and monkeys, and the intrinsic clearance (Cl(int)) ratios of (+)-THP to (-)-THP were 2.66, 2.85, 4.24, and 1.67, respectively. Compared with the metabolism in untreated RLM, the metabolism of (-)-THP and (+)-THP was significantly increased in dexamethasone (Dex)-induced and β-naphthoflavone (β-NF)-induced RLM; meanwhile, the Cl(int) ratios of (+)-THP to (-)-THP in Dex-induced and β-NF-induced RLM were 5.74 and 0.81, respectively. Ketoconazole had stronger inhibitory effect on (+)-THP than (-)-THP, whereas fluvoxamine had stronger effect on (-)-THP in untreated and Dex-induced or β-NF-induced RLM. The results suggested that THP enantiomers were predominately metabolized by CYP3A1/2 and CYP1A2 in RLM, and CYP3A1/2 preferred to metabolize (+)-THP, whereas CYP1A2 preferred (-)-THP.

  1. Stereoselective degradation of chiral fungicide myclobutanil in rat liver microsomes.

    Yan, Jin; Zhang, Ping; Wang, Xinru; Wang, Yao; Zhou, Zhiqiang; Zhu, Wentao


    Myclobutanil, (RS)-2-(4-chlorophenyl)-2-(1H-1, 2, 4-triazol-1-ylmethyl)hexanenitrile is a broad-spectrum systemic triazole fungicide which consists of a pair of enantiomers. The stereoselective degradation of myclobutanil was investigated in rat liver microsomes. The concentrations of myclobutanil enantiomers were determined by high-performance liquid chromatography (HPLC) with a cellulose-tris-(3,5-dimethyl-phenylcarbamate)-based chiral stationary phase (CDMPC-CSP) under reversed phase condition. The t(1/2) of (+)-myclobutanil is 8.49 min, while the t(1/2) of (-)-myclobutanil is 96.27 min. Such consequences clearly indicated that the degradation of myclobutanil in rat liver microsomes was stereoselective and the degradation rate of (+)-myclobutanil was much faster than (-)-myclobutanil. In addition, significant differences between two enantiomers were also observed in enzyme kinetic parameters. The V(max) of (+)-myclobutanil was about 4-fold of (-)-myclobutanil and the CL(int) of (+)-myclobutanil was three times as much as (-)-myclobutanil after incubation in rat liver microsomes. Corresponding consequences may shed light on the environmental and ecological risk assessment for myclobutanil and may improve human health.

  2. Antigen-loaded ER microsomes from APC induce potent immune responses against viral infection.

    Sofra, Vassiliki; Mansour, Salah; Liu, Mengya; Gao, Bin; Primpidou, Elisavet; Wang, Ping; Li, Suling


    Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER-enriched microsomal membranes (microsomes) isolated from DC contain more peptide-receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide-MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide-loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide-pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide-MHC complexes armed microsomes from DC can be an important alternative to DC-based vaccines for protection from viral infection.

  3. Evaluation of Five Jet Fuels in the Salmonella-Escherichia coli / Microsome Plate Incorporation Assay


    AFRL-RH-WP-TR-2010-0138 Evaluation of Five Jet Fuels in the Salmonella-Escherichia coli / Microsome Plate Incorporation Assay Edward S...31 Jul 2010 4. TITLE AND SUBTITLE Evaluation of Five Jet Fuels in the Salmonella-Escherichia coli / Microsome Plate Incorporation Assay 5a...the Salmonella typhimurium-Escherichia coli/ microsome plate incorporation assay. The assay was performed using the plate incorporation procedure

  4. Increase in phorbol ester binding in liver microsomes after chronic administration of phenobarbital

    Menez, J.F.; Deitrich, R.A. (Univ. of Colorado, Denver (United States))


    The effect of chronic administration of phenobarbital on the binding of phorbol-12,13-dibutyrate (({sup 3}H)PDBu), an activator of protein kinase C (PKC), was examined in rat liver microsomes. A significant increase in the number of binding sites was observed in microsomes of Fisher 344 rats. However, no change appeared in liver cytosol binding of PDBu. Consequently, a translocation process of PKC is unlikely. The increase in ({sup 3}H)PDBu binding in liver microsomes is significant 24 h. after one injection of phenobarbital and reaches its maximum in 2 days. In other strains of rats (ACI and lean Zucker), significant differences were found in the increase of ({sup 3}H)PDBu binding in microsomes. Fisher 344 were the most sensitive, lean Zucker rats, the least sensitive. Those results parallel the pentoxy-resorufin O demethylase activity in the microsomes of the same animals. EC{sub 50} values for inhibition of ({sup 3}H)PDBu binding by pentobarbital were determined in control microsomes from Fisher and Zucker rats. In Fisher rats, ({sup 3}H)PDBu binding in microsomes was found to be more sensitive to the inhibitory effect of pentobarbital than in lean Zucker rats, which suggests that the more microsomes are inhibited in vitro the greater the increase in PKC in microsomes following chronic barbiturate treatment.

  5. Specific Genomic Fingerprints of Phosphate Solubilizing Pseudomonas Strains Generated by Box Elements

    Javadi Nobandegani, Mohammad Bagher; Saud, Halimi Mohd; Yun, Wong Mui


    Primers corresponding to conserved bacterial repetitive of BOX elements were used to show that BOX-DNA sequences are widely distributed in phosphate solubilizing Pseudomonas strains. Phosphate solubilizing Pseudomonas was isolated from oil palm fields (tropical soil) in Malaysia. BOX elements were used to generate genomic fingerprints of a variety of Pseudomonas isolates to identify strains that were not distinguishable by other classification methods. BOX-PCR, that derived genomic fingerprints, was generated from whole purified genomic DNA by liquid culture of phosphate solubilizing Pseudomonas. BOX-PCR generated the phosphate solubilizing Pseudomonas specific fingerprints to identify the relationship between these strains. This suggests that distribution of BOX elements' sequences in phosphate solubilizing Pseudomonas strains is the mirror image of their genomic structure. Therefore, this method appears to be a rapid, simple, and reproducible method to identify and classify phosphate solubilizing Pseudomonas strains and it may be useful tool for fast identification of potential biofertilizer strains. PMID:25580434

  6. In vitro identification of metabolites of verapamil in rat liver microsomes%维拉帕米在大鼠肝微粒体中代谢产物的体外鉴定

    孙璐; 张淑秋; 钟大放


    AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MSn), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eight metabolites, including two novel metabolites (M4 and M8), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (M5-M7), and N, O-didemethyl-verapamil (M8). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.

  7. Fluidized-bed bioreactor system for the microbial solubilization of coal

    Scott, C.D.; Strandberg, G.W.


    A fluidized-bed bioreactor system for the conversion of coal into microbially solubilized coal products. The fluidized-bed bioreactor continuously or periodically receives coal and bio-reactants and provides for the production of microbially solubilized coal products in an economical and efficient manner. An oxidation pretreatment process for rendering coal uniformly and more readily susceptible to microbial solubilization may be employed with the fluidized-bed bioreactor. 2 figs.

  8. Fixed-bed bioreactor system for the microbial solubilization of coal

    Scott, C.D.; Strandberg, G.W.


    A fixed-bed bioreactor system for the conversion of coal into microbially solubilized coal products. The fixed-bed bioreactor continuously or periodically receives coal and bio-reactants and provides for the large scale production of microbially solubilized coal products in an economical and efficient manner. An oxidation pretreatment process for rendering coal uniformly and more readily susceptible to microbial solubilization may be employed with the fixed-bed bioreactor. 1 fig., 1 tab.

  9. Biochemical solubilization of toxic salts from residual geothermal brines and waste waters

    Premuzic, Eugene T.; Lin, Mow S.


    A method of solubilizing metal salts such as metal sulfides in a geothermal sludge using mutant Thiobacilli selected for their ability to metabolize metal salts at high temperature is disclosed, The method includes the introduction of mutated Thiobacillus ferrooxidans and Thiobacillus thiooxidans to a geothermal sludge or brine. The microorganisms catalyze the solubilization of metal salts, For instance, in the case of metal sulfides, the microorganisms catalyze the solubilization to form soluble metal sulfates.

  10. Fatty acid solubilizer from the oral disk of the blowfly.

    Yuko Ishida

    Full Text Available BACKGROUND: Blowflies are economic pests of the wool industry and potential vectors for epidemics. The establishment of a pesticide-free, environmentally friendly blowfly control strategy is necessary. Blowflies must feed on meat in order to initiate the cascade of events that are involved in reproduction including juvenile hormone synthesis, vitellogenesis, and mating. During feeding blowflies regurgitate salivary lipase, which may play a role in releasing fatty acids from triglycerides that are found in food. However, long-chain fatty acids show low solubility in aqueous solutions. In order to solubilize and ingest the released hydrophobic fatty acids, the blowflies must use a solubilizer. METHODOLOGY: We applied native PAGE, Edman degradation, cDNA cloning, and RT-PCR to characterize a protein that accumulated in the oral disk of the black blowfly, Phormia regina. In situ hybridization was carried out to localize the expression at the cellular level. A fluorescence competitive binding assay was used to identify potential ligands of this protein. CONCLUSION: A protein newly identified from P. regina (PregOBP56a belonged to the classic odorant-binding protein (OBP family. This gene was expressed in a cluster of cells that was localized between pseudotracheae on the oral disk, which are not accessory cells of the taste peg chemosensory sensilla that normally synthesize OBPs. At pH 7 and pH 6, PregOBP56a bound palmitic, stearic, oleic, and linoleic acids, that are mainly found in chicken meat. The binding affinity of PregOBP56a decreased at pH 5. We propose that PregOBP56a is a protein that solubilizes fatty acids during feeding and subsequently helps to deliver the fatty acids to the midgut where it may help in the process of reproduction. As such, PregOBP56a is a potential molecular target for controlling the blowfly.


    O. S. Talalaeva


    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

  12. Solubilization of nerve growth factor receptors of rabbit superior cervical ganglia.

    Banerjee, S P; Cuatrecasas, P; Snyder, S H


    Nerve growth factor (NGF) receptors of rabbit superior cervical ganglia can be solubilized by treatment with detergents and readily assayed in the soluble state. Triton X-100 and deoxycholate reduce specific binding of NGF to ganglia membranes. In membranes treated with Triton X-100 (0.5 to 2.0%) the reduction in NGF binding by membranes is accompanied by a corresponding increase in binding in the supernatant fluid. NGF binding in soluble preparations can be rapidly assayed by precipitating NGF bound to receptors with polyethylene glycol under conditions in which unbound NGF is not precipitated. NGF binding to soluble preparations is saturable whether evaluated by the binding of 125I-NGF or by diluting 125I-NGF with native NGF. Using both techniques, the dissociation constant for NGF binding to soluble receptors is about 0.2 nM, the same as its dissociation constant from receptor sites in intact membranes. NGF binding to soluble receptors displays a high degree of peptide specificity, similar to receptor sites in intact membranes of superior cervical ganglia. A method of labeling NGF with 125I-3(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester is described which leads to binding properties that are superior to those obtained with previously described 125I-NGF preparations.

  13. Bio-solubilization of Chinese lignite Ⅱ: protein adsorption onto the lignite surface

    YIN Su-dong; TAO Xiu-xiang; SHI Kai-yi


    Lignite bio-solubilization is a promising technology for converting solid lignite into oil. This study concerns the adsorption of lignite-solubilizing enzymes onto the lignite surface. Adsorption capacity, infrared spectral analysis and driving forces analysis are studied as a way to help understand the bio-solubilization mechanism. The results show that the amount of lignite bio-solubilization is proportional to the amount of adsorbed lignite-solubilizing enzymes. An increase in lignite-solubilizing enzyme adsorption of 10% leads to a 7% increase in lignite bio-solubilization. However, limited amounts of enzymes can be adsorbed by the lignite, thus resulting in low percentages of bio-solubilization. Infrared spectral analysis shows that side chains, such as hydroxyl and carbonyl, of the lignite structure are the main, and necessary, structures where lignite-solubilizing enzymes attachto the lignite. Furthermore, driving force analysis indicates that the electrostatic force between lignite and enzymes is the main adsorption mechanism. The forces are influenced by solution pH levels, the zeta potential of the lignite and the isoelectric points of the enzymes.

  14. A standardized method for quantitating the complement-mediated immune complex solubilizing capacity of human serum

    Baatrup, G; Peterson, I; Svehag, S E;


    A standardized radioassay for measuring the complement-mediated immune complex solubilizing capacity (CMSC) and the initial kinetics of the solubilization (IKS) reaction is described. The total complement (C)-mediated solubilizing capacity was determined after incubation of diluted serum and 125I......-BSA-anti-BSA. Percentage C-mediated solubilization (CMS) was measured after centrifugation by determining the distribution of radioactivity. The dependency of CMSC upon factors such as serum dilution and buffer system used, amount of IC added to serum, serum storage conditions and centrifugation conditions...

  15. Complement-mediated solubilization of immune complexes and their interaction with complement C3 receptors

    Petersen, Ivan; Baatrup, Gunnar; Jepsen, H H;


    Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components of the me......Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components...

  16. Comparative metabolism of mycophenolic acid by glucuronic acid and glucose conjugation in human, dog, and cat liver microsomes.

    Slovak, J E; Mealey, K; Court, M H


    Use of the immunosuppressant mycophenolic acid (MPA) in cats is limited because MPA elimination depends on glucuronidation, which is deficient in cats. We evaluated formation of major (phenol glucuronide) and minor (acyl glucuronide, phenol glucoside, and acyl glucoside) MPA metabolites using liver microsomes from 16 cats, 26 dogs, and 48 humans. All MPA metabolites were formed by human liver microsomes, while dog and cat liver microsomes formed both MPA glucuronides, but only one MPA glucoside (phenol glucoside). Intrinsic clearance (CLint) of MPA for phenol glucuronidation by cat liver microsomes was only 15-17% that of dog and human liver microsomes. However, CLint for acyl glucuronide and phenol glucoside formation in cat liver microsomes was similar to or greater than that for dog and human liver microsomes. While total MPA conjugation CLint was generally similar for cat liver microsomes compared with dog and human liver microsomes, relative contributions of each pathway varied between species with phenol glucuronidation predominating in dog and human liver microsomes and phenol glucosidation predominating in cat liver microsomes. MPA conjugation variation between cat liver microsomes was threefold for total conjugation and for phenol glucosidation, sixfold for phenol glucuronidation, and 11-fold for acyl glucuronidation. Our results indicate that total MPA conjugation is quantitatively similar between liver microsomes from cats, dogs, and humans despite large differences in the conjugation pathways that are utilized by these species.

  17. Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

    Baumgold, J.


    Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and the remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.

  18. Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut.

    Pasti, M B; Pometto, A L; Nuti, M P; Crawford, D L


    The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2167628

  19. Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

    David J Leibly

    Full Text Available Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2, proline, xylitol, NDSB 201, CTAB and K(2PO(4 solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40% were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

  20. Inhibitory effects of beryllium chloride on rat liver microsomal enzymes.

    Teixeira, C F; Yasaka, W J; Silva, L F; Oshiro, T T; Oga, S


    A single i.v. dose (0.1 mmol Be2+/kg) of beryllium chloride prolonged the duration of pentobarbital-induced sleep and zoxazolamine-induced paralysis, in rats. The effects are correlated with changes of the pharmacokinetic parameters and with the in vitro inhibition of both aliphatic and aromatic hydroxylation of pentobarbital and zoxazolamine. In vitro N-demethylation of meperidine and aminopyrine was partially inhibited while O-demethylation of quinidine was unaffected by liver microsomes of rats pretreated with beryllium salt. The findings give clues that beryllium chloride inhibits some forms of cytochrome P-450, especially those responsible for hydroxylation of substrates, like pentobarbital and zoxazolamine.

  1. Corticosteroids increase superoxide anion production by rat liver microsomes.

    Nelson, D H; Ruhmann-Wennhold, A


    Superoxide anion production by liver microsomes from intact, adrenalectomized, and cortisoltreated adrenalectomized rats has been determined. The amount formed was roughly proportionate to the amount of cortisol given, and a similar response was seen in the activity of NADPH-cytochrome c reductase. The amount of measurable superoxide anion was markedly reduced by the addition of superoxide dismutase. The increased production of this potent free radical with cortisol therapy suggests that its formation may contribute to some of the harmful effects of corticosteroids given in more than physiologic amounts. PMID:239969

  2. Metabolism of 3-methylcholanthrene by rat liver microsomes: a reinvestigation

    Stoming, T.A. (Medical Coll., Augusta, GA); Bornstein, W.; Bresnick, E.


    Metabolites of 3-methylcholanthrene (3-MC) formed by rat liver microsomes were analyzed by high pressure liquid chromatography. The metabolic profile is significantly different from previous studies using thin layer chromatography. The major metabolites include 1- and 2-hydroxy-3-MC. Use of the high pressure liquid chromatographic system allows for the separation of at least seven new metabolites. The amounts of three of these new metabolites are substantially decreased when the potent epoxide hydrase inhibitor 3,3,3-trichloropropene oxide is added to the incubation system. These results then suggest the formation of epoxides of 3-methylcholanthrene other than the K-region oxide.

  3. Microsomal prostaglandin E synthase-1 in rheumatic diseases

    Marina eKorotkova


    Full Text Available Microsomal prostaglandin E synthase-1 (mPGES-1 is a well recognized target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases and other inflammatory conditions. In this review, we focus on mPGES-1 in rheumatic diseases with the aim to cover the most recent advances in the understanding of mPGES-1 in rheumatoid arthritis, osteoarthritis and inflammatory myopathies. Novel findings regarding regulation of mPGES1 cell expression as well as enzyme inhibitors are also summarized.

  4. Studies on the solubilization of German coal by fungi

    Reiss, J. (Grahamhaus Stadt kg, Bad Kreuznach (Germany))


    The capability of seven basidiomycetes (Trametes versicolor, Poria placenta, Pleurotus florida, P. ostreatus, P. sajor-caju, P. eryngii, Stropharia sp.), one ascomycete (Chaetomium globosum) and five hyphomycetes and moulds (Humicola grisea, Trichoderma viride, Aspergillus terreus, Paecilomyces varioti, Papulaspora immersa) to solubilize medium and high volatile bituminous coals (types A and B) as well as four types of lignite B from Germany was tested in surface cultures. The intensity of bioliquefaction was determined by estimating the rate of droplet formation and by measuring the loss of weight of the coal granules gravimetrically. The bituminous coals with a relative high degree of coalification were only moderately converted by Trametes versicolor, Pleurotus florida, P. ostreatus and P. sajor-caju. The three species of Pleurotus caused the greatest rate of biosolubilization of lignite, yielding a loss of weight of the coal granules of more than 5.8% with a maximum of 7.6% with P. florida. The non-basidiomycetes proved to be less active with a liquefaction rate of up to 3.5% with Trichoderma viride. In general, the geologically younger lignite coals were more effectively solubilized than the older hard coals. The volatile matter and the oxygen content proved to be the principal factors influencing the intensity of bioconversion.

  5. Solubilization and electrophoretic characterization of select edible nut seed proteins.

    Sathe, Shridhar K; Venkatachalam, Mahesh; Sharma, Girdhari M; Kshirsagar, Harshal H; Teuber, Suzanne S; Roux, Kenneth H


    The solubility of almond, Brazil nut, cashew nut, hazelnut, macadamia, pecan, pine nut, pistachio, walnut, and peanut proteins in several aqueous solvents was qualitatively and quantitatively assessed. In addition, the effects of extraction time and ionic strength on protein solubility were also investigated. Electrophoresis and protein determination (Lowry, Bradford, and micro-Kjeldahl) methods were used for qualitative and quantitative assessment of proteins, respectively. Depending on the seed, buffer type and ionic strength significantly affected protein solubility. The results suggest that buffered sodium borate (BSB; 0.1 M H(3)BO(3), 0.025 M Na(2)B(4)O(7), 0.075 M NaCl, pH 8.45) optimally solubilizes nut seed proteins. Qualitative differences in seed protein electrophoretic profiles were revealed. For a specific seed type, these differences were dependent on the solvent(s) used to solubilize the seed proteins. SDS-PAGE results suggest the polypeptide molecular mass range for the tree nut seed proteins to be 3-100 kDa. The results of native IEF suggested that the proteins were mainly acidic, with a pI range from >4.5 to <7.0. Western immunoblotting experiments indicated that rabbit polyclonal antibodies recognized substantially the same polypeptides as those recognized by the corresponding pooled patient sera IgE.

  6. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian


    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

  7. A comparative study of precision cut liver slices, hepatocytes, and liver microsomes from the Wistar rat using metronidazole as a model substance

    Sidelmann, U. G.; Cornett, Claus; Tjornelund, J.


    , 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...... higher in microsomes than in the other liver preparations. The metabolic rates in hepatocytes in primary culture and in suspension with respect to the oxidative metabolites were higher than in liver slices. The metabolic turnover observed in liver slices was predicted to correlate with in vivo data...... 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...

  8. Nutritional influences on the solubilization of metal phosphate by ericoid mycorrhizal fungi.

    Gibson, Brian R; Mitchell, Derek T


    Four ericoid mycobionts (two isolates of Hymenoscyphus ericae, and two dark, sterile ericoid mycobionts isolated from metal-contaminated mine sites) were grown on solid agar plates supplemented with zinc phosphate (0.25 %) containing different forms of nitrogen (nitrate, ammonium or alanine) and different concentrations of carbon (glucose) and phosphorus (K2HPO4). The influence of nutrient variation on solubilizing ability of the fungi was assessed by measuring the zones of solubilization appearing beneath the growing colonies. All four mycobionts were capable of zinc phosphate solubilization in the presence of all three nitrogen sources and in media containing no nitrogen. No solubilization was observed at 0 mM glucose-C but was observed with increasing glucose concentration from 300 to 600 mM C. Increasing phosphorus concentration (0-5 mM P) had no effect on the solubilizing ability of the isolates. All but one of the mycobionts were capable of solubilizing calcium phosphate (CaHPO4), while no solubilization was observed in media containing aluminium phosphate (AlPO4), iron phosphate (FePO4 x 4H2O) or copper phosphate (Cu3O8P2 x 2H2O) under conditions which were found to be optimal for zinc phosphate solubilization. Under conditions of glucose at 300 mM C and alanine as the N source in the zinc phosphate-amended agar medium, one of the mycobionts produced new crystals, which were morphologically distinct from the original zinc phosphate crystals. It is concluded that medium composition influences the metal-phosphate solubilizing ability of ericoid mycobionts. The results are discussed in relation to the possible mechanisms involved in solubilization and the potential benefits of metal-phosphate solubilization to ericoid mycobionts and their host plants.

  9. Identification of saturated and unsaturated fatty acids released during microsomal incubations.

    Bushee, Jennifer L; Liang, Guiqing; Dunne, Christine E; Harriman, Shawn P; Argikar, Upendra A


    1. In vitro clearance in liver microsomes is routinely measured in drug discovery and development for new chemical entities. Literature reports indicate that long chain fatty acids such as arachidonic, linoleic and oleic acids may be released over a period of time during microsomal incubations. Some fatty acids have been shown to interfere with oxidative and conjugative reactions in microsomes, thus potentially inhibiting microsomal clearance of compounds. 2. The present study was aimed at deciphering the fatty acids present or released from microsomes. Analytical methods were developed to characterize and quantitatively assess the fatty acids without chemical derivatization in rat, monkey and human liver microsomes. Additionally, incubations with uridine-5'-diphosphoglucuronic acid (UDPGA) were utilized to trap the released fatty acids as their glucuronate esters, which were characterized and confirmed by high-resolution LC-MS/MS. 3. Our results indicate for the first time that timnodonic, trans-eicosenoic, gondoic, behenic, and nervonic acid were released during microsomal incubations. Additionally, α- and γ-linolenic, timnodonic, palmitoleic, linoleic, arachidonic, palmitic, oleic, and stearic acid were identified as their corresponding acyl-glucuronides in rat, monkey and human liver microsomes, providing the first direct evidence that the released fatty acids are capable of forming glucuronides under incubation conditions.

  10. Permeability of Rat Liver Microsomes to Sucrose and Carboxypoly-glucose in vitro

    Share, Leonard; Hansrote, Ronald W.


    A study was made of the permeability of the microsomes to C14-sucrose and to C14-carboxypolyglucose, a branch-chained glucose polymer with a molecular weight of approximately 50,000. It was concluded that the microsomal membranes are permeable to sucrose on the basis of the following evidence: the volume of distribution of C14-sucrose was 84 per cent of the total microsomal pellet water; the sucrose unavailable volume, the per cent dry weights of the microsomal pellets, and the optical density of microsomal suspensions were independent of the concentration of sucrose in the suspending medium. It is suggested that the microsomal water which is unavailable to sucrose may be bound to protein and/or ribonucleic acid of the microsomes. The volume of distribution of C14-carboxypolyglucose was 44 per cent of the total pellet water, and it is considered that the microsomal membranes may be impermeable to this compound. Pretreatment with ribonuclease resulted in small increases in the volumes of distribution of both C14-sucrose and C14-carboxypolyglucose. PMID:14445567

  11. Effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism.

    Klaassen, C D; Hood, A M


    The effects of microsomal enzyme inducers on thyroid hormone homeostasis and the thyroid gland are of concern. We have investigated the effects of microsomal enzyme inducers on thyroid follicular cell proliferation and thyroid hormone metabolism in rats. We have shown that small increases in serum TSH can result in large increases in thyroid follicular cell proliferation. Furthermore, only those microsomal enzyme inducers that increase serum TSH--that is, phenobarbital (PB) and pregnenolone-16alpha-carbonitrile (PCN)-increase thyroid follicular cell proliferation, whereas those microsomal enzyme inducers that do not increase serum TSH--that is, 3-methylcholanthrene (3MC) and Aroclor 1254 (PCB)-do not increase thyroid follicular cell proliferation. Deiodination does not appear to be the reason why serum T3 concentrations are maintained in microsomal enzyme inducer-treated rats. We have also shown that those microsomal enzyme inducers that increase serum TSH increase T3 UDP-glucuronosyltransferase (UGT) activity, whereas those microsomal enzyme inducers that do not increase serum TSH do not increase T3 UGT activity. This finding suggests that induction of T3 glucuronidation, rather than T4 glucuronidation, mediates increases in serum TSH of microsomal enzyme inducer treated rats.

  12. Metabolism of 20(S-Ginsenoside Rg2 by Rat Liver Microsomes: Bioactivation to SIRT1-Activating Metabolites

    Li-Yuan Ma


    Full Text Available 20(S-Ginsenoside Rg2 (1 has recently become a hot research topic due to its potent bioactivities and abundance in natural sources such as the roots, rhizomes and stems-leaves of Panax ginseng. However, due to the lack of studies on systematic metabolic profiles, the prospects for new drug development of 1 are still difficult to predict, which has become a huge obstacle for its safe clinical use. To solve this problem, investigation of the metabolic profiles of 1 in rat liver microsomes was first carried out. To identify metabolites, a strategy of combined analyses based on prepared metabolites by column chromatography and ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS was performed. As a result, four metabolites M1–M4, including a rare new compound named ginsenotransmetin A (M1, were isolated and the structures were confirmed by spectroscopic analyses. A series of metabolites of 1, MA–MG, were also tentatively identified by UPLC-Q-TOF/MS in rat liver microsomal incubate of 1. Partial metabolic pathways were proposed. Among them, 1 and its metabolites M1, M3 and M4 were discovered for the first time to be activators of SIRT1. The SIRT1 activating effects of the metabolite M1 was comparable to those of 1, while the most interesting SIRT1 activatory effects of M3 and M4 were higher than that of 1 and comparable with that of resveratrol, a positive SIRT1 activator. These results indicate that microsome-dependent metabolism may represent a bioactivation pathway for 1. This study is the first to report the metabolic profiles of 1 in vitro, and the results provide an experimental foundation to better understand the in vivo metabolic fate of 1.

  13. Metabolism of 20(S)-Ginsenoside Rg₂ by Rat Liver Microsomes: Bioactivation to SIRT1-Activating Metabolites.

    Ma, Li-Yuan; Zhou, Qi-Le; Yang, Xin-Bao; Wang, Hong-Ping; Yang, Xiu-Wei


    20(S)-Ginsenoside Rg₂ (1) has recently become a hot research topic due to its potent bioactivities and abundance in natural sources such as the roots, rhizomes and stems-leaves of Panax ginseng. However, due to the lack of studies on systematic metabolic profiles, the prospects for new drug development of 1 are still difficult to predict, which has become a huge obstacle for its safe clinical use. To solve this problem, investigation of the metabolic profiles of 1 in rat liver microsomes was first carried out. To identify metabolites, a strategy of combined analyses based on prepared metabolites by column chromatography and ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) was performed. As a result, four metabolites M1-M4, including a rare new compound named ginsenotransmetin A (M1), were isolated and the structures were confirmed by spectroscopic analyses. A series of metabolites of 1, MA-MG, were also tentatively identified by UPLC-Q-TOF/MS in rat liver microsomal incubate of 1. Partial metabolic pathways were proposed. Among them, 1 and its metabolites M1, M3 and M4 were discovered for the first time to be activators of SIRT1. The SIRT1 activating effects of the metabolite M1 was comparable to those of 1, while the most interesting SIRT1 activatory effects of M3 and M4 were higher than that of 1 and comparable with that of resveratrol, a positive SIRT1 activator. These results indicate that microsome-dependent metabolism may represent a bioactivation pathway for 1. This study is the first to report the metabolic profiles of 1 in vitro, and the results provide an experimental foundation to better understand the in vivo metabolic fate of 1.

  14. Dynamic headspace analysis of volatile metabolites from the reductive dehalogenation of trichloro- and tetrachloroethanes by hepatic microsomes.

    Thompson, J A; Ho, B; Mastovich, S L


    A dynamic headspace technique was developed to facilitate the identification and quantitation of low levels of volatile metabolites produced in vitro by subcellular preparations. The method is complementary to commonly used static headspace and solvent-extraction techniques, and involves purging the compounds from microsomal suspensions with an inert gas, trapping them on a short column of adsorbant resin, and transferring the metabolites to a gas chromatograph. An apparatus was designed to facilitate the incubations and isolations of volatile compounds. Recoveries of several chlorinated hydrocarbons with boiling points in the range 12 to 186 degrees C were 85% or higher, and the recovery of vinyl chloride (boiling point -13 degrees C) was 25%. The quantitative precision of the method was determined and calibration curves were established for each metabolite, demonstrating that no discrimination occurred over a wide range of concentrations. This technique was employed to investigate the reductive metabolism of 1,1,1-trichloroethane, 1,1,2-trichloroethane, and 1,1,2,2-tetrachloroethane by rat liver microsomes. The metabolites from these substrates were 1,1-dichloroethane, vinyl chloride, and 1,2-dichloroethylene, respectively. These conversions were NADPH-dependent, occurred only under anaerobic conditions, and indicate that chloroethanes with relatively low electron affinities can be reduced slowly by microsomal cytochrome P-450. The rates of formation of vinyl chloride, 1,1-dichloroethane, and 1,2-dichloroethylene with 1.0 mM substrate were 12.5 +/- 2.0, 122 +/- 14, and 147 +/- 12 pmol/min/mg of protein, respectively. The results show that there are distinct advantages of the purge/trap method over the static headspace method for studying volatile metabolites when high sensitivity is required.

  15. Sulfation of chondroitin. Specificity, degree of sulfation, and detergent effects with 4-sulfating and 6-sulfating microsomal systems

    Sugumaran, G.; Silbert, J.E.


    Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage, and from chondroitin 4-sulfate-producing mouse mastocytoma cells, were incubated with UDP-(14C)glucuronic acid and UDP-N-acetylgalactosamine to form non-sulfated proteo(14C)chondroitin. Aliquots of the incubations were then incubated with 3'-phosphoadenylylphosphosulfate (PAPS) in the presence or absence of various detergents. In the absence of detergents, there was good sulfation of this endogenous proteo(14C)chondroitin by the original microsomes from both sources. Detergents, with the exception of Triton X-100, markedly inhibited sulfation in the mast cell system but not in the chick cartilage system. These results indicate that sulfation and polymerization are closely linked on cell membranes and that in some cases this organization can be disrupted by detergents. When aliquots of the original incubation were heat inactivated, and then reincubated with new microsomes from chick cartilage and/or mouse mastocytoma cells plus PAPS, there was no significant sulfation of this exogenous proteo(14C) chondroitin with either system unless Triton X-100 was added. Sulfation of exogenous chondroitin and chondroitin hexasaccharide was compared with sulfation of endogenous and exogenous proteo(14C)chondroitin. Sulfate incorporation into hexasaccharide and chondroitin decreased as their concentrations (based on uronic acid) approached that of the proteo(14C)chondroitin. At the same time, the degree of sulfation in percent of substituted hexosamine increased. However, the degree of sulfation did not reach that of the endogenous proteo(14C)chondroitin. Hexasaccharide and chondroitin sulfation were stimulated by the presence of Triton X-100. However, in contrast to the exogenous proteo(14C)chondroitin, there was some sulfation of hexasaccharide and chondroitin in the absence of this detergent.

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

    Svobodová, Kateřina; Mikesková, Hana; Petráčková, Denisa


    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.

  17. In vitro covalent binding of /sup 14/C-mibolerone to rat liver microsomes

    Jaglan, P.S.


    Mibolerone (17-Hydroxy-7,17-dimethylestr-4-en-3-one; 7 alpha-17 alpha dimethyl-19-nortestosterone) is being marketed by The Upjohn Company for the inhibition of estrus in bitches. The aim of this study was to determine the extent of covalent binding of mibolerone to rat liver microsomes. Liver microsomes were obtained from Control and phenobarbitol-treated female Fisher rats, and were incubated with /sup 14/C-mibolerone at 37/sup 0/C for 10 minutes. No covalent binding to macromolecules was observed when /sup 14/C-mibolerone was incubated with rat liver microsomes. Under identical conditions, /sup 14/C-estradiol was covalently bound to macromolecules. Slightly higher covalent binding of estradiol was observed with microsomes from phenobarbitol-treated rats. Ascorbic acid and glutathione inhibited covalent binding of estradiol to macromolecules in the in vitro microsomal system.


    Tedeschi, Henry; James, Joseph M.; Anthony, William


    Electron microscope observations are consistent with the interpretation that the elements of the endoplasmic reticulum are osmotically active in situ as well as after isolation. More recently, it has been reported that microsomal suspensions equilibrate almost completely with added C14-sucrose and that no osmotic behavior is evident from photometric data. These findings were considered at variance with the electron microscope data. However, equilibration with added label simply attests to a relatively high permeability, and, in addition, the photometric data need not be critical. Osmotic volume changes, measured photometrically, may be masked by concomitant events (e.g., changes in the refractive index of the test solutions at varying osmotic pressures, breakdown of the particles, and agglutination). For these reasons the photometric experiments were repeated. In this work, the reciprocal of optical density of microsomal suspensions was found to vary linearly with the reciprocal of concentration of the medium at constant refractive index. These changes probably correspond to osmotic volume changes, since the effect was found to be (a) independent of substance used and (b) osmotically reversible. The transmission of the suspension was found to vary with the refractive index of the medium, the concentration of particles, and the wavelength of incident light, according to relationships that are similar to or identical with those obtained for mitochondrial suspensions. PMID:14064105

  19. Microsomal transformation of organophosphorus pesticides by white rot fungi.

    Jauregui, Juan; Valderrama, Brenda; Albores, Arnulfo; Vazquez-Duhalt, Rafael


    The enzymatic mechanism for the transformation of organophosphorus pesticides (OPPs) by different white-rot fungi strains was studied. With the exception of Ganoderma applanatum 8168, all strains from a collection of 17 different fungi cultures were able to deplete parathion. Three strains showing the highest activities were selected for further studies: Bjerkandera adusta 8258, Pleurotus ostreatus 7989 and Phanerochaete chrysosporium 3641. These strains depleted 50 to 96% of terbufos, azinphos-methyl, phosmet and tribufos after four-days exposure to the pesticides. In order to identify the cellular localization of the transformation activity, the extracellular and microsomal fractions of Pleuronts ostreatus 7989 were evaluated in vitro. While the activities of ligninolytic enzymes (lignin peroxidase, manganese peroxidase and laccase) were detected in the extracellular fraction, no enzymatic modification of any of the five pesticides tested could be found, suggesting the intracellular origin of the transformation activity. In accordance with this observation the microsomal fraction was found able to transform three OPPs with the following rates: 10 micromol mg prot(-1) h(-1) for phosmet, 5.7 micromol mg prot(-1) h(-1) for terbufos, and 2.2 micromol mg prot(-1) h(-1) for azinphos-methyl. The products from these reactions and from the transformation of trichlorfon and malathion, were identified by mass-spectrometry. These results, supported by specific inhibition experiments and the stringent requirement for NADPH during the in vitro assays suggest the involvement of a cytochrome P450.

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

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


    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.


    Kalariya Nikunj


    Full Text Available Sulfasalazine, a sulfonamide derivative, is used as an antimicrobial agent by inhibiting bacterial growth and activity mainly in the treatment of ulcerative colitis and in the treatment of rheumatoid arthritis. It is practically insoluble in water. The aim of study was to prepare aqueous injection of sulphasalazine, using the mixed solvency solubilization technique. Several blends were prepared by co-solvents such as polyethylene glycol 200 (PEG200, PEG 400, PEG 600, PEG 4000, propylene glycol (PG, polyvinyl pyrrolidine (PVP K 30, ethanol, glycerin, tween20, tween80, niacenamide, lignocain hydrochloride and sodium benzoate. The enhancement in the solubility of sulphasalazine in a mixed solvency was more than 50 folds (compared to its solubility in distilled water. This proved a synergistic enhancement in solubility of a poorly water soluble drug due to mixed solvency. Synergistic combination of mixed-solvency can minimize the amount of co-solvents employed, minimizing the chances of their toxicities. The developed formulation was studied for physical and chemical stability.

  2. Behavior of inorganic elements during sludge ozonation and their effects on sludge solubilization.

    Sui, Pengzhe; Nishimura, Fumitake; Nagare, Hideaki; Hidaka, Taira; Nakagawa, Yuko; Tsuno, Hiroshi


    The behavior of inorganic elements (including phosphorus, nitrogen, and metals) during sludge ozonation was investigated using batch tests and the effects of metals on sludge solubilization were elucidated. A decrease of ∼ 50% in the ratio of sludge solubilization was found to relate to a high iron content 80-120 mgFe/gSS than that of 4.7-7.4 mgFe/gSS. During sludge ozonation, the pH decreased from 7 to 5, which resulted in the dissolution of chemically precipitated metals and phosphorus. Based on experimental results and thermodynamic calculation, phosphate precipitated by iron and aluminum was more difficult to release while that by calcium released with decrease in pH. The release of barium, manganese, and chrome did not exceed 10% and was much lower than COD solubilization; however, that of nickel, copper, and zinc was similar to COD solubilization. The ratio of nitrogen solubilization was 1.2 times higher than that of COD solubilization (R(2)=0.85). Of the total nitrogen solubilized, 80% was organic nitrogen. Because of their high accumulation potential and negative effect on sludge solubilization, high levels of iron and aluminum in both sewage and sludge should be considered carefully for the application of the advanced sewage treatment process with sludge ozonation and phosphorus crystallization. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Improving the aqueous solubility of triclosan by solubilization, complexation, and in situ salt formation.

    Grove, Christine; Liebenberg, Wilna; du Preez, Jan L; Yang, Wenzhan; de Villiers, Melgardt M


    Triclosan, an antimicrobial, although widely incorporated into many skin care products, toothpastes, and liquid soaps, presents formulation difficulties because it is practically insoluble in water. The objective of this study was to improve the aqueous solubility of triclosan through solubilization, complexation, and salt formation. The solubility of triclosan in distilled water and in phosphate buffers (pH 7.4) was determined at 30 degrees C. The order of solubilizing performance of the solubilizers was: N-methylglucamine> or =L-arginine>sodium lauryl sulfate>beta-cyclodextrin> or =hydroxypropyl-beta-cyclodextrin>ethanolamine>sodium benzoate>sodium methyl 4-hydroxybenzoate>triethanolamine> or =diethanolamine. These solubilizers increased the solubility of triclosan from 80- to 6000-fold. Micellar solubilization and the formation of either salts or complexes are postulated as possible mechanisms for the increase in the solubility of triclosan by the surfactant sodium lauryl sulphate, the cyclic sugar derivatives beta-cyclodextrin and 2-hydropropyl-beta-cyclodextrin, the amino acid L-arginine, and the amino sugar alcohol N-methylglucamine. Furthermore, although the bacteriostatic efficacy of triclosan was significantly increased when solubilized with N-methylglucamine, L-arginine, and ethanolamine, increased solubilization did not increase the effectiveness of triclosan for all solubilizers tested.

  4. Effect of an organophosphate pesticide, monocrotophos, on phosphate-solubilizing efficiency of soil fungal isolates.

    Jain, Rachna; Garg, Veena; Saxena, Jyoti


    Soil is a sink of pesticide residues as well as microorganisms. Fungi are well known for solubilization of inorganic phosphates, and this activity of fungal isolates may be affected by the presence of pesticide residues in the soil. In the present study, five generically different fungal isolates, viz. Aspergillus niger JQ660373, Aspergillus flavus, Penicillium aculeatum JQ660374, Fusarium pallidoroseum and Macrophomina sp., were tested and compared for their phosphate-solubilizing ability in the absence and presence of monocrotophos (500 mg L(-1)). After 168 h of incubation, four times high amount of tricalcium phosphate was solubilized by isolates in the growth medium containing monocrotophos in comparison to control (without monocrotophos). Concurrently, 78 % of the applied monocrotophos was degraded by these fungal isolates. Kinetics of phosphate solubilization shifted from logarithmic to power model in the presence of monocrotophos. Similarly, the phosphatase activity was also found significantly high in the presence of monocrotophos. The combined order of phosphate solubilization as well as monocrotophos degradation was found to be A. niger JQ660373 > P. aculeatum JQ660374 > A. flavus > F. pallidoroseum > Macrophomina sp. On the contrary, phosphate solubilization negatively correlated with the pH of the growth medium. Hence, it could be concluded that these fungal species efficiently solubilize inorganic phosphates and monocrotophos poses a positive effect on their ability and in turn degraded by them. To the best of our knowledge, this is the first report on P solubilization by Macrophomina sp. and F. pallidoroseum.

  5. Carbon and Nitrogen Sources Influence Tricalcium Phosphate Solubilization and Extracellular Phosphatase Activity by Talaromyces flavus.

    Stefanoni Rubio, P J; Godoy, M S; Della Mónica, I F; Pettinari, M J; Godeas, A M; Scervino, J M


    The aim of this work was to study phosphate (P) solubilization (and the processes involved in this event) by Talaromyces flavus (BAFC 3125) as a function of carbon and/or nitrogen sources. P solubilization was evaluated in NBRIP media supplemented with different carbon (glucose, sorbitol, sucrose, and fructose) and nitrogen (L-asparagine, urea, ammonium sulfate (AS), and ammonium nitrate (AN) combinations. The highest P solubilization was related to the highest organic acid production (especially gluconic acid) and pH drop for those treatments where glucose was present. Also P solubilization was higher when an inorganic nitrogen source was supplemented to the media when compared to an organic one. Although not being present an organic P source, phosphatase activity was observed. This shows that P mineralization and P solubilization can occur simultaneously, and that P mineralization is not induced by the enzyme substrate. The combination that showed highest P solubilization was for AN-glucose. The highest acid phosphatase activity was for AS-fructose, while for alkaline phosphatase were for AS-fructose and AN-fructose. Acid phosphatase activity was higher than alkaline. P solubilization and phosphatase activity (acid and alkaline) were influenced by the different carbon-nitrogen combinations. A better understanding of phosphate-solubilizing fungi could bring a better use of soil P.

  6. Biochar enhances Aspergillus niger rock phosphate solubilization by increasing organic acid production and alleviating fluoride toxicity.

    Mendes, Gilberto de Oliveira; Zafra, David Lopez; Vassilev, Nikolay Bojkov; Silva, Ivo Ribeiro; Ribeiro, José Ivo; Costa, Maurício Dutra


    During fungal rock phosphate (RP) solubilization, a significant quantity of fluoride (F(-)) is released together with phosphorus (P), strongly inhibiting the process. In the present study, the effect of two F(-) adsorbents [activated alumina (Al2O3) and biochar] on RP solubilization by Aspergillus niger was examined. Al2O3 adsorbed part of the F(-) released but also adsorbed soluble P, which makes it inappropriate for microbial RP solubilization systems. In contrast, biochar adsorbed only F(-) while enhancing phosphate solubilization 3-fold, leading to the accumulation of up to 160 mg of P per liter. By comparing the values of F(-) measured in solution at the end of incubation and those from a predictive model, it was estimated that up to 19 mg of F(-) per liter can be removed from solution by biochar when added at 3 g liter(-1) to the culture medium. Thus, biochar acted as an F(-) sink during RP solubilization and led to an F(-) concentration in solution that was less inhibitory to the process. In the presence of biochar, A. niger produced larger amounts of citric, gluconic, and oxalic acids, whether RP was present or not. Our results show that biochar enhances RP solubilization through two interrelated processes: partial removal of the released F(-) and increased organic acid production. Given the importance of organic acids for P solubilization and that most of the RPs contain high concentrations of F(-), the proposed solubilization system offers an important technological improvement for the microbial production of soluble P fertilizers from RP.

  7. Isolation of phosphate solubilizing bacteria and their potential for lead immobilization in soil.

    Park, Jin Hee; Bolan, Nanthi; Megharaj, Mallavarapu; Naidu, Ravi


    Lead (Pb), a highly toxic heavy metal forms stable compounds with phosphate (P). The potential of phosphate solubilizing bacteria (PSB) to immobilize Pb by enhancing solubilization of insoluble P compounds was tested in this research. Eighteen different PSB strains isolated from P amended and Pb contaminated soils were screened for their efficiency in P solubilization. The PSB isolated from P amended soils solubilized 217-479 mg/L of P while the PSB from Pb contaminated soil solubilized 31-293 mg/L of P. Stepwise multiple regression analysis and P solubility kinetics indicated that the major mechanism of P solubilization by PSB is the pH reduction through the release of organic acids. From the isolated bacteria, two PSB were chosen for Pb immobilization and these bacteria were identified as Pantoea sp. and Enterobacter sp., respectively. The PSB significantly increased P solubilization by 25.0% and 49.9% in the case of Pantoea sp., and 63.3% and 88.6% in the case of Enterobacter sp. for 200 and 800 mg/kg of rock phosphate (RP) addition, respectively, thereby enhancing the immobilization of Pb by 8.25-13.7% in the case of Pantoea sp. and 14.7-26.4% in the case of Enterobacter sp. The ability of PSB to solubilize P, promote plant growth, and immobilize Pb can be used for phytostabilization of Pb contaminated soils.

  8. Isolation and functional characterization of hydrocarbon emulsifying and solubilizing factors produced by a Pseudomonas species

    Reddy, P.G.; Banuah, J.N.; Bhagat, S.D.; Pathak, M.G.; Singh, H.D.


    Pseudomonas PG-1 cultivated on pristane produced in good amount a heat-stable polymeric substance which showed strong hydrocarbon emulsifying and solubilizing properties. The substance was isolated in crude form and was found to contain 34% protein, 16% carbohydrate, and 40% lipid. The hydrocarbon solubilizing activity of the isolate was strongly inhibited by EDTA but the chelating agent had no effect on the hydrocarbon emulsifying activity. Both activities of the isolate were strongly inhibited by chymotrypsin treatment indicating the importance of the protein moiety for its activity. Hydrocarbon solubilization by the isolate showed a certain degree of specificity to pristane in modest agitation generally used in microbial cultivation, but this specificity was lost by vigorous agitation in a Waring blender. It was proposed that in the first case, solubilization was effected by a solubilizing factor specific to pristane, whereas in the latter ease, nonspecific solubilization occurred due to the action of the emulsifying factor. The rate of pristane solubilization by heat-treated culture broth under the conditions of agitation used in cultivation (rotary shaker, 120 rpm) was found to be ca. 750 mg L/sup -1/h/sup -1/ which was much larger than the maximal pristane uptake rate of 170 mg L/sup -1/h/sup -1/ observed during microbial growth on the substrate. It was concluded that hydrocarbon solubilization could satisfactorily account for the substrate uptake and growth.

  9. The mechanism of detergent solubilization of liposomes and protein-containing membranes.

    Kragh-Hansen, U; le Maire, M; Møller, J V


    The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.

  10. Chelating agents improve enzymatic solubilization of pectinaceous co-processing streams

    Ravn, Helle Christine; Meyer, Anne S.


    /substrate] at 60 °C, pH 6.0 for 1 min. Characterization of the released fractions demonstrated a significantly improved effect of chelating agents for polysaccharide solubilization from FiberBind 400, PUF, and citrus peel, whereas only low amounts of polysaccharides were solubilized from the sugar beet pulp...

  11. Solubilization of zinc compounds by the diazotrophic, plant growth promoting bacterium Gluconacetobacter diazotrophicus.

    Saravanan, V S; Madhaiyan, M; Thangaraju, M


    Gluconacetobacter diazotrophicus an endophytic diazotroph also encountered as rhizosphere bacterium is reported to possess different plant growth promoting characteristics. In this study, we assessed the zinc solubilizing potential of G. diazotrophicus under in vitro conditions with different Zn compounds using glucose or sucrose as carbon sources. G. diazotrophicus showed variations in their solubilization potential with the strains used and the Zn compounds tested. G. diazotrophicus PAl5 efficiently solubilized the Zn compounds tested and ZnO was effectively solubilized than ZnCO(3) or Zn(3)(PO(4))(2). The soluble Zn concentration was determined in the culture supernatant through Atomic Absorption Spectrophotometer. Gas chromatography coupled Mass Spectrometry analysis revealed 5-ketogluconic acid, a derivative of gluconic acid as the major organic acid produced by G. diazotrophicus PAl5 cultured with glucose as carbon source. This organic anion may be an important agent that helped in the solubilization of insoluble Zn compounds.

  12. Zinc metal solubilization by Gluconacetobacter diazotrophicus and induction of pleomorphic cells.

    Saravanan, Venkatakrishnan Sivaraj; Osborne, Jabez; Madhaiyan, Munusamy; Mathew, Lazar; Chung, Jongbae; Ahn, Kisup; Sa, Tongmin


    Gluconacetobacter diazotrophicus strain PAl5 exhibited a minimum inhibitory concentration value of 11 mM in an LGI medium amended with ZnCl2. When an LGI medium was amended with Zn metal, solubilization halos were observed in a plate assay, and further solubilization was confirmed in a broth assay. The maximum solubilization was recorded after 120 h with a 0.1% Zn metal amendment. During solubilization, the culture growth and pH of the broth were indirectly correlated. Using a Fourier Transform Infrared Spectroscopy analysis, one of the agents solubilizing the Zn metal was identified as gluconic acid. When the Zn-amended broth was observed under a bright field microscope, long involution cells were observed, and further analysis with Atomic Force Microscopy revealed highly deformed, pleomorphic, aggregate-like cells.

  13. Visualization of surfactant enhanced NAPL mobilization and solubilization in a two-dimensional micromodel



    Surfactant-enhanced aquifer remediation is an emerging technology for aquifers contaminated with nonaqueous phase liquids (NAPLs). A two-dimensional micromodel and image capture system were applied to observe NAPL mobilization and solubilization phenomena. In each experiment, a common residual NAPL field was established, followed by a series of mobilization and solubilization experiments. Mobilization floods included pure water floods with variable flow rates and surfactant floods with variations in surfactant formulations. At relatively low capillary numbers (N{sub ca}<10{sup {minus}3}), the surfactant mobilization floods resulted in higher NAPL saturations than for the pure water flood, for similar N{sub ca}.These differences in macroscopic saturations are explained by differences in micro-scale mobilization processes. Solubilization of the residual NAPL remaining after the mobilization stage was dominated by the formation of dissolution fingers, which produced nonequilibrium NAPL solubilization. A macroemulsion phase also as observed to form spontaneously and persist during the solubilization stage of the experiments.

  14. Mechanistic differences in permeation behavior of supersaturated and solubilized solutions of carbamazepine revealed by nuclear magnetic resonance measurements.

    Ueda, Keisuke; Higashi, Kenjirou; Limwikrant, Waree; Sekine, Shuichi; Horie, Toshiharu; Yamamoto, Keiji; Moribe, Kunikazu


    A solid dispersion (SPD) of carbamazepine (CBZ) with hydroxypropyl methylcellulose acetate succinate (HPMC-AS) was prepared by the spray drying method. The apparent solubility (37 °C, pH 7.4) of CBZ observed with the SPD was over 3 times higher than the solubility of unprocessed CBZ. The supersaturated solution was stable for 7 days. A higher concentration of CBZ in aqueous medium was also achieved by mixing with Poloxamer 407 (P407), a solubilizing agent. From permeation studies of CBZ using Caco-2 monolayers and dialysis membranes, we observed improved CBZ permeation across the membrane in the supersaturated solution of CBZ/HPMC-AS SPD. On the contrary, the CBZ-solubilized P407 solution exhibited poor permeation by CBZ. The chemical shifts of CBZ on the (1)H NMR spectrum from CBZ/HPMC-AS SPD solution were not altered significantly by coexistence with HPMC-AS. In contrast, an upfield shift of CBZ was observed in the CBZ/P407 solution. The spin-lattice relaxation time (T(1)) over spin-spin relaxation time (T(2)) indicated that the mobility of CBZ in the HPMC-AS solution was much lower than that in water. Meanwhile, the mobility of CBZ in P407 solution was significantly higher than that in water. NMR data indicate that CBZ does not strongly interact with HPMC-AS. CBZ mobility was suppressed due to self-association and microviscosity around CBZ, which do not affect permeation behavior. Most of the CBZ molecules in the CBZ/P407 solution were solubilized in the hydrophobic core of P407, and a few were free to permeate the membrane. The molecular state of CBZ, as evaluated by NMR measurements, directly correlated with permeation behavior.

  15. Marine actinobacteria showing phosphate-solubilizing efficiency in Chorao Island, Goa, India.

    Dastager, Syed G; Damare, Samir


    The occurrence and distribution of an actinobacteria group of bacteria capable of dissolving insoluble phosphates were investigated in this study in marine environments, especially in sediments of Chorao Island, Goa Province, India. A total of 200 bacterial isolates of actinobacteria was isolated. All isolates were screened for phosphate-solubilizing activity on Pikovskaya's agar. Thirteen different isolates exhibiting maximum formation of halos (zone of solubilization) around the bacterial colonies were selected for quantitative estimations of P-solubilization. Quantitative estimations for P-solubilization were analyzed for up to 10 days at intervals of 24 h. Maximum solubilization from 89.3 ± 3.1 to 164.1 ± 4.1 μg ml(-1) was observed after 6 days of incubation in six of all isolates, while the isolate NII-1020 showed maximum P-solubilization. The increase in solubilization coincided with the drop in pH. Many of these species showed wide range of tolerance to temperature, pH, and salt concentrations. Further, 16S rRNA gene sequence analyses were carried to identify the bacterial groups which are actively solubilized phosphate in vitro. Gene sequencing results reveal that all isolates were clustered into six different actinobacterial genera: Streptomyces, Microbacterium, Angustibacter, Kocuria, Isoptericola, and Agromyces. The presence of phosphate-solubilizing microorganisms and their ability to solubilize phosphate were indicative of the important role played by bacteria in the biogeochemical cycle of phosphorus and the plant growth in coastal ecosystems.

  16. Solubilization of glycoproteins of envelope viruses by detergents

    Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.; Isaeva, E.S.; Zhdanov, V.M.


    The action of a number of known ionic and nonionic detergents, as well as the new nonionic detergent MESK, on envelope viruses was investigated. It was shown that the nonionic detergents MESK, Triton X-100, and octyl-..beta..-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis. Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines.

  17. Phosphate solubilizing bacteria: Comparison between coastal and deep sea sediments

    Biche, S.; Pandey, S.; Gonsalves, M.J.B.D.; Das, A.; Mascarenhas-Pereira, M.B.L.; LokaBharathi, P.A.

    Phosphorus (P) is an essential nutrient for sustaining marine life (Karl, 2000). It is an important macromolecule and it plays vital role in the primary production in marine environment. In some organisms it can also be present as intracellular... organic compounds (Hoppe and Ullrich, 1999). Fig:3a Scatter plot of phosphate solubilizing activity v/s Pore water phosphate in the coast R² = 0.1423 0 0.01 0.02 0.03 0.04 0.05 0.06 0.00 0.50 1.00 1.50 Ph os ph at e uM PSA µg P ml-1 h-1 PSA v/s P...

  18. Combination of surfactant solubilization with permanganate oxidation for DNAPL remediation.

    Li, Zhaohui; Hanlie, Hong


    A combination of surfactant solubilization with permanganate oxidation of trichloroethylene (TCE) was studied in batch, flow-through column, and three-dimensional (3-D) tank tests. Batch results showed that chloride production, an indication of TCE degradation, followed a pseudo-first-order reaction kinetics with respect to KMnO4 in the presence of free-phase TCE. A higher chloride production rate was achieved when anionic surfactants were present. The observed pseudo-first-order reaction rate constant increased as the concentrations of anionic surfactants Ninate 411 and Calfax increased from 0% to 0.1%, 0.3%, and 1.0%. Column experiments on TCE reduction by permanganate in the presence and absence of surfactants were carried out using well-sorted coarse Ottawa sand. The peak effluent TCE concentration reached 1700 mg/L due to enhanced solubilization when both sodium dodecyl sulfate (SDS) and permanganate were used, in contrast to less than 300 mg/L when only permanganate solution was used. In addition, the effluent TCE concentration decreased much faster when SDS was present in the permanganate solution, compared with the case when SDS was absent. With an initial 1 mL of TCE emplaced in the columns, the effluent TCE concentration dropped to breakthrough occurred after 21-25 h of injection when SDS was present compared with 45-70 h later when SDS was absent. A slightly higher chloride concentration was observed in the earlier stage of the column experiment and the chloride concentration decreased quickly once KMnO4 was seen in the effluent. The 3-D tank test showed that the MnO2 precipitation front formed more quickly when 1% SDS was present, which further confirmed the observation from the column study.

  19. Improved solubilization of curcumin with a microemulsification formulation


    Full Text Available Due to the large number of bioactive substances, with low and very low solubility in water, new and improved investigation methods were developed. Researches in this area have shown that lipid systems in lipophilic substances formulation increase their bioavailability and prevent or reduce the toxicological risk because most of the components involved in the formulation are of natural origin, with a structure compatible with biological membranes components. Among the lipid systems used in the leaching, transport and release of lipophilic substances there are: liposomes, solid lipid nanoparticles, double and single emulsions, autoemulsionante and auto-microemulsionante lipid systems. The last are the subject of the present research and meet specialists in concern for the harmonization of cost-benefit-risk in order to improve population health. Curcumin [(1E, 6E-1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione] is a yellow pigment derived from the rhizome of the plant Curcuma Longa with phenol groups and conjugated double bounds which is unstable at light and basic pH, degrading within 30 minutes. The aim of this study is curcumin solubilization used as alimentary dye in automicroemulsionante systems. Dye/oil/surfactant/cosurfactant mixing ratio was made, based on quaternary phase diagrams. Mesofazice structures were revealed by conductivity and viscosimetric analysis. A curcumin solubilization system in aqueous medium was obtained. On the other hand, this paper studies the colour evolution of these automicroemulsionante systems comparing with hexane dye solution. The use of the chromatic attributes L*, a* and b* and L*, C* and hab, suggested by the Commission Internationale de l’Eclairage (CIE (i.e., the CIELAB system, obtained from direct transmitance measurements, which made it possible to follow the evolution of colour.

  20. Geochemical hosts of solubilized radionuclides in uranium mill tailings

    Landa, E.R.; Bush, C.A.


    The solubilization and subsequent resorption of radionuclides by ore components or by reaction products during the milling of uranium ores may have both economic and environmental consequences. Particle-size redistribution of radium during milling has been demonstrated by previous investigators; however, the identification of sorbing components in the tailings has received little experimental attention. In this study, uranium-bearing sandstone ore was milled, on a laboratory scale, with sulfuric acid. At regular intervals, filtrate from this suspension was placed in contact with mixtures of quartz sand and various potential sorbents which occur as gangue in uranium ores; the potential sorbents included clay minerals, iron and aluminum oxides, feldspar, fluorspar, barite, jarosite, coal, and volcanic glass. After equilibration, the quartz sand-sorbent mixtures were separated from the filtrate and radioassayed by gamma-spectrometry to determine the quantities of 238U, 230Th, 226Ra, and 210Pb sorbed, and the radon emanation coefficients. Sorption of 238U was low in all cases, with maximal sorptions of 1-2% by the bentonite- and coal-bearing samples. 230Th sorption also was generally less than 1%; maximal sorption here was observed in the fluorspar-bearing sample and appears to be associated with the formation of gypsum during milling. 226Ra and 210 Pb generally showed higher sorption than the other nuclides - more than 60% of the 26Ra solubilized from the ore was sorbed on the barite-bearing sample. The mechanism (s) for this sorption by a wide variety of substrates is not yet understood. Radon emanation coefficients of the samples ranged from about 5 to 30%, with the coal-bearing samples clearly demonstrating an emanating power higher than any of the other materials. ?? 1990.

  1. Butylbenzyl phthalate hydrolysis in liver microsomes of humans, monkeys, dogs, rats and mice.

    Takahara, Yuka; Kinashi, Yu; Takahara, Yuusuke; Hichiya, Hiroyuki; Okada, Kenji; Murata, Mikio; Shigeyama, Masato; Hanioka, Nobumitsu


    Butylbenzyl phthalate (BBzP) is used as a plasticizer to import flexibility to polyvinylchloride plastics. In this study, hydrolysis of BBzP to monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP) in liver microsomes of humans, monkeys, dogs, rats and mice was examined. The kinetics for MBP formation by human, dog and mouse liver microsomes followed the Michaelis-Menten model, whereas the kinetics by monkey and rat liver microsomes fitted the Hill model. The kinetics for MBzP formation fitted the Hill model for all liver microsomes. The Vmax and in vitro clearance (CLint or CLmax) ratios of MBP/MBzP formation varied among animal species, although the Km for MBP and MBzP formation in each liver microsomes were generally comparable. The hydrolysis of BBzP to monoester phthalates in mammalian liver microsomes could be classified into two types: MBzP>MBP type for humans and dogs, and MBP>MBzP type for monkeys, rats and mice. These findings suggest that the formation profile of MBzP and MBP from BBzP by liver microsomes differs extensively among animal species.

  2. Encapsulation of liver microsomes into a thermosensitive hydrogel for characterization of drug metabolism and toxicity.

    Yang, Huiying; Zheng, Yuanting; Zhao, Bei; Shao, Tengfei; Shi, Qingling; Zhou, Ning; Cai, Weimin


    This study reported the encapsulation of liver microsomes into a thermosensitive hydrogel to characterize drug metabolism and predict drug effects. Pluronic(®)F-127 (F127) and acrylamide-bisacrylamide (Acr-Bis) were utilized as the two precursors. After chemical crosslinking catalyzed by ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED), the resulting Pluronic F127-acrylamide-bisacrylamide (FAB) hydrogel could encapsulate microsomes at 4 °C and facilitate metabolic reactions at 37 °C. The gel morphology at different Acr-Bis concentrations was characterized using field emission scanning electron microscopy (FE-SEM). Higher concentrations of Acr-Bis could lead to higher degrees of cross-linking of the gel. A fluorescent staining assay was subsequently used to demonstrate successful encapsulation of microsomes into the gel as well as the free diffusion process of micromolecular substrates. The thermosensitivity of the FAB gel was studied using swelling ratio and protein release assay to verify its ability to encapsulate microsomes. The metabolic activity of microsomes encapsulated in gels was investigated by detecting the metabolites of FDA-approved substrates, including dextromethorphan, chlorzoxazone and testosterone. Compared with the traditional method of microsomal incubation, the FAB gel maintained 60%-70% of microsome activity. Lastly, the classic anticancer prodrug cyclophosphamide (CTX) was chosen as a model drug for the study of drug metabolism and the prediction of drug effects. When the microsomes encapsulated in the FAB gel were used in the cell culture system, CTX induced a higher level of apoptosis in MCF-7 cells compared with traditional microsomes.


    Ernster, Lars; Jones, Lois C.


    Rat liver microsomes catalyze the hydrolysis of the triphosphates of adenosine, guanosine, uridine, cytidine, and inosine into the corresponding diphosphates and inorganic orthophosphate. The activities are stimulated by Na2S2O4, and inhibited by atebrin, chlorpromazine, sodium azide, and deaminothyroxine. Sodium deoxycholate inhibits the ATPase activity in a progressive manner; the release of orthophosphate from GTP and UTP is stimulated by low, and inhibited by high, concentrations of deoxycholate, and that from CTP and ITP is unaffected by low, and inhibited by high, concentrations of deoxycholate. Subfractionation of microsomes with deoxycholate into ribosomal, membrane, and soluble fractions reveals a concentration of the triphosphatase activity in the membrane fraction. Rat liver microsomes also catalyze the hydrolysis of the diphosphates of the above nucleosides into the corresponding monophosphates and inorganic orthophosphate. Deoxycholate strongly enhances the GDPase, UDPase, and IDPase activities while causing no activation or even inhibition of the ADPase and CDPase activities. The diphosphatase is unaffected by Na2S2O4 and is inhibited by azide and deaminothyroxine but not by atebrin or chlorpromazine. Upon fractionation of the microsomes with deoxycholate, a large part of the GDPase, UDPase, and IDPase activities is recovered in the soluble fraction. Mechanical disruption of the microsomes with an Ultra Turrax Blender both activates and releases the GDPase, UDPase, and IDPase activities, and the former effect occurs more readily than the latter. The GDPase, UDPase, and IDPase activities of the rat liver cell reside almost exclusively in the microsomal fraction, as revealed by comparative assays of the mitochondrial, microsomal, and final supernatant fractions of the homogenate. The microsomes exhibit relatively low nucleoside monophosphatase and inorganic pyrophosphatase activities, and these are unaffected by deoxycholate or mechanical treatment

  4. In vitro glucuronidation of ochratoxin a by rat liver microsomes.

    Han, Zheng; Tangni, Emmanuel K; Di Mavungu, José Diana; Vanhaecke, Lynn; De Saeger, Sarah; Wu, Aibo; Callebaut, Alfons


    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-MS(n)) 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.

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

    Zheng Han


    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.

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

    Christopher R Pudney


    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

  7. A study of liver microsomal enzymes in rats following propoxur (Baygon) administration.

    Nelson, D L; Lamb, D W; Mihail, F


    Groups of rats were given either propoxur, were left as untreated controls, or were given phenobarbital, DDT, chlordane or toxaphene which are known to induce liver microsomal detoxification enzymes. Microsomal enzyme activity was measured by testing the ability of liver homogenates to degrade EPN (O-ethyl O-(4-nitrophenyl) phenylphosphonothioate) to p-nitrophenol. The activity of aminopyrine-N-demethylase, cytochrome P-450 and p-nitroanisole-O-demethylase in liver homogenates of rats receiving propoxur was measured. Liver microsomal detoxification enzymes were not induced by propoxur exposure.

  8. Toxic alkaloids and their interaction with microsomal cytochrome P-450 in vitro.

    Peeples, A; Dalvi, R R


    Studies on the binding spectra of certain alkaloids with rat liver microsomes revealed that brucine, scopolamine and strychnine are type I compounds, whereas boldine, emetine, nicotine, reserpine and sanguinarine show type II binding. In contrast, colchicine and solanine failed to produce any measurable binding spectra. In vitro incubation of colchicine, nicotine or scopolamine with microsomal suspensions and NADPH resulted in demethylation of these alkaloids, while the incubation of boldine, brucine, emetine, reserpine, sanguinarine or solanine showed little or no dealkylation reaction. Furthermore, the effect of these alkaloids on the in vitro microsomal metabolism of a drug, benzphetamine, has also been studied.

  9. Solubilization and degradation of perchloroethylene (PCE) in cationic and nonionic surfactant solutions

    Sivaram Harendra; Cumaraswamy Vipulanandan


    Solubilization of perchloroethylene (PCE) in a nonionic (Triton X-100) and a cationic (cetyltrimethylammonium bromide (CTAB)) surfactant solutions and the degradation of surfactant solubilized PCE using fine to nanosize Fe and bi-metallic Fe-Ni particles were investigated.Micelle partition coefficients (Km) and molar solubility ratio (MSR) for PCE in 10 g/L of surfactant solutions have been quantified and the solubility of PCE (100 mg/L in water) in the surfactant solutions increased by about ten fold.Of the two surfactants studied,Triton X-100 solubilized the higher amount of PCE per gram of surfactant.To degrade solubilized PCE,both iron and bimetallic Fe-Ni particles were used in continuously stirred batch reactors.The iron and bi-metallic particles were synthesized using the solution method and the particles were characterized using the SEM,EDS,TEM and XRD.The PCE solubilized up to 500 mg/L in both surfactant solutions were totally degraded at various rates by 200 g/L of bi-metallic Fe-Ni particles in less than 20 hr,which is the highest concentration of PCE degraded in the shortest time compared to data in the literature.The degradations of PCE solubilized in surfactant solutions were represented by nonlinear kinetic relationships which depended on the type of surfactant used for solubilizing the PCE.

  10. Phosphate Solubilization Potential of Rhizosphere Fungi Isolated from Plants in Jimma Zone, Southwest Ethiopia

    Elias, Firew


    Phosphorus (P) is one of the major bioelements limiting agricultural production. Phosphate solubilizing fungi play a noteworthy role in increasing the bioavailability of soil phosphates for plants. The present study was aimed at isolating and characterizing phosphate solubilizing fungi from different rhizospheres using both solid and liquid Pikovskaya (PVK) medium. A total of 359 fungal isolates were obtained from 150 rhizosphere soil samples of haricot bean, faba bean, cabbage, tomato, and sugarcane. Among the isolates, 167 (46.52%) solubilized inorganic phosphate. The isolated phosphate solubilizing fungi belonged to genera of Aspergillus (55.69%), Penicillium spp. (23.35%), and Fusarium (9.58%). Solubilization index (SI) ranged from 1.10 to 3.05. Isolates designated as JUHbF95 (Aspergillus sp.) and JUFbF59 (Penicillium sp.) solubilized maximum amount of P 728.77 μg·mL−1 and 514.44 μg mL−1, respectively, from TCP (tricalcium phosphate) after 15 days of incubation. The highest (363 μg mL−1) soluble-P was released from RP with the inoculation of JUHbF95 in the PVK broth after 10 days of incubation. The present study indicated the presence of diverse plant associated P-solubilizing fungi that may serve as potential biofertilizers. PMID:27688771

  11. Rock inhabiting potassium solubilizing bacteria from Kerala, India: characterization and possibility in chemical K fertilizer substitution.

    Anjanadevi, Indira Parameswaran; John, Neetha Soma; John, Kuzhivilayil Susan; Jeeva, Muthulekshmi Lajapathy; Misra, Raj Shekhar


    The role of rock inhabiting bacteria in potassium (K) solubilization from feldspar and their application in crop nutrition through substitution of fertilizer K was explored through the isolation of 36 different bacteria from rocks of a major hill station at Ponmudi in Thiruvananthapuram, Kerala, India. A comprehensive characterization of K solubilization from feldspar was achieved with these isolates which indicated that the K solubilizing efficiency increases with decrease in pH and increase in viscosity and viable cell count. Based on the level of K solubilization, two potent isolates were selected and identified as Bacillus subtilis ANctcri3 and Bacillus megaterium ANctcri7. Exopolysaccharide production, scanning electron microscopic and fourier transform infrared spectroscopic studies with these efficient strains conclusively depicted the role of low pH, increase in viscosity, and bacterial attachment in K solubilization. They were also found to be efficient in phosphorus (P) solubilization, indole acetic acid production as well as tolerant to wide range of physiological conditions. Moreover, the applicability of K containing rock powder as a carrier for K solubilizing bacteria was demonstrated. A field level evaluation on the yield of a high K demanding tuberous vegetable crop, elephant foot yam (Amorphophallus paeoniifolius (dennst.) nicolson) established the possibility of substituting chemical K fertilizer with these biofertilizer candidates successfully.

  12. Solubilization of octane in cationic surfactant-anionic polymer complexes: effect of polymer concentration and temperature.

    Zhang, Hui; Deng, Lingli; Zeeb, Benjamin; Weiss, Jochen


    Polymers may alter the ability of oppositely charged surfactant micelles to solubilize hydrophobic molecules depending on surfactant-polymer interactions. This study was conducted to investigate the effects of polymer concentration and temperature on the solubilization thermodynamics of an octane oil-in-water emulsion in mixtures of an anionic polymer (carboxymethyl cellulose) and cationic cetyltrimethylammonium bromide (CTAB) surfactant micelles using isothermal titration calorimetry (ITC). Results showed that the CTAB binding capacity of carboxymethyl cellulose increased with increasing temperature from 301 to 323 K, and correspondingly the thermodynamic behavior of octane solubilization in CTAB micelles, either in the absence or presence of polymer, was found to depend on temperature. The addition of carboxymethyl cellulose caused the solubilization in CTAB micelles to be less endothermic, and increased the solubilization capacity. Based on the phase separation model, the solubilization was suggested to be mainly driven by enthalpy gains. Results suggest that increasing concentrations of the anionic polymer gave rise to a larger Gibbs energy decrease and a larger unfavorable entropy increase for octane solubilization in cationic surfactant micelles.

  13. Empigen BB: a useful detergent for solubilization and biochemical analysis of keratins.

    Lowthert, L A; Ku, N O; Liao, J; Coulombe, P A; Omary, M B


    Intermediate filament (IF) proteins make up some of the most insoluble proteins known, and within the IF protein family, keratins are the least soluble. We compared the efficiency of nonionic, cationic, mixed nonionic and anionic, and zwitterionic detergents in solubilizing keratins from insect cells that express recombinant human keratins and from human colonic cell lines and normal keratinocytes. The cationic detergent cetyltrimethylammonium bromide was similar to the zwitterionic detergent Empigen BB in its ability to efficiently solubilize keratins, but the latter detergent was superior in that it maintained antibody reactivity and allowed for immunoprecipitation of the keratins. Although Nonidet-P40 partially solubilizes keratins, Empigen BB solubilizes a significant amount of keratins not solubilized by Nonidet-P40. In the case of vimentin, differences in solubilization efficiency among the detergents was not as dramatic as with keratins. Our results show that Empigen BB solubilizes a significant amount of epidermal and glandular keratins while preserving antigenicity. This detergent should prove useful for carrying out biochemical and molecular studies on these proteins and may be similarly beneficial for other IF proteins.

  14. Effect of carbon substrates on rock phosphate solubilization by bacteria from composts and macrofauna.

    Hameeda, B; Reddy, Y Harish Kumar; Rupela, O P; Kumar, G N; Reddy, Gopal


    Five of the 207 isolates from different composts, farm waste compost (FWC), rice straw compost (RSC), Gliricidia vermicompost (GVC), and macrofauna, showed rock phosphate (RP) solubilization in buffered medium in plate culture. When tested in RP broth medium, all five strains, Enterobacter cloacae EB 27, Serratia marcescens EB 67, Serratia sp. EB 75, Pseudomonas sp. CDB 35, and Pseudomonas sp. BWB 21, showed gluconic acid production and solubilized RP. Based on cellulose-degrading and P-solubilizing ability, two strains were selected for further studies. In the presence of different carbon sources, both strains showed a drop in pH and solubilized RP. P released was maximum with glucose (1212 and 522 micromol) and minimum with cellobiose (455 and 306 micromol) by S. marcescens EB 67 and Pseudomonas sp. CDB 35, respectively. Glucose dehydrogenase (GDH) activity was 63 and 77% with galactose and 35 and 46% with cellobiose when compared to glucose (100%) by EB 67 and CDB 35, respectively. Both strains solubilized RP in the presence of different crop residues. EB 67 and CDB 35 showed maximum cellulase activity (0.027 units) in the presence of rice straw and a mixture of rice straw and root. P solubilized from RP in the presence of pigeonpea root was 134 and 140 micromol with EB 67 and CDB 35. Significantly, these bacteria isolated from composts and macrofauna solubilized rock phosphate in the presence of various pure carbon substrates and crop residues and their importance in soil/rhizosphere conditions is discussed.

  15. Hepatic microsomal cytochromes P450 in mink fed Saginaw Bay carp (SBC)

    Melancon, M.J.; LeCaptain, L.; Rattner, B.A.; Heaton, S.; Aulerich, R.; Tillitt, D.; Stegeman, John J.; Woodin, B.


    Livers from mink fed diets containing 0% (n = 12), 10% (n = 11), 20% (n = 12) and 40% (n = 10) SBC for 6 months contained 0.1, 2.2, 3.6, and 6.3 ug/g total PCBs, respectively. Hepatic microsomes were prepared and assayed for protein, arylhydrocarbon hydroxylase (AHH), benzyloxyresorufin-O-dealkylase (BROD), ethoxy-ROD (ER0D), pentoxy-ROD (PROD), and ethoxycoumarin-OD (ECOD). Mink fed SBC had increased AHH, EROD, and ECOD (group means 2.2-3.4 X control means), decreased BROD and unchanged PROD (the latter 2 assays indicators for phenobarbital-type induction in mammals). Three samples from each group were examined by western blot using a polyclonal anti-P450llB antibody and a monoclonal anti-P450lA antibody (MAb 1-12-3). Mink fed SBC showed induction of a protein recognized by anti-P450lA (8 X control), but had little protein recognized by anti-P450IlB. The monooxygenase activities and western blot data give a consistent picture of MC-type but not PB-type induction in mink fed SBC.

  16. Mechanism of enhancement of prochymosin renaturation by solubilization of inclusion bodies at alkaline pH

    张治洲; 张渝英; 杨开宇


    The renaturation efficiency of recombinant prochymosin depends on not only the renaturation condi-tions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-contain-ing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency ( ~ 40% vs. ~ 20 % ). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.

  17. Kinetics of solubilization with Triton X-100 of egg-yolk lecithin bilayers containing cholesterol

    Hobai, S


    The titration solubilization of multilamellar egg-yolk lecithin liposomes (MLV-EYL) with Triton X-100 was studied by rectangular optical diffusimetric measurements as a function of cholesterol (Chol) concentration. It was determinated the variation of optic percentage diffu-sion (per mmol surfactant), DDif%/mmol TX-100, in the course of solubilization of MLV-EYL-Chol system with TX-100 10mM. The statistical analysis of the titration curves can reveal the contribution of cholesterol to the stability of phospholipid bilayer membranes. The solubilization of the lecithin-cholesterol mixtures, with a high cholesterol content, much more bile salt requires.

  18. EDTA-type Polymer Based on Diazacrown Ether as the Solubilizer of Barium Sulfate to Water

    Bo ZHOU; Jian Zhang LI; Chun Hong HE; Sheng Ying QIN


    The EDTA dianhydride reacted with diazacrown ethers to obtain the water-soluble EDTA-diazacrown ether polymers 1~3. The effects of crown ether ring in the polymer chains including its cavity size on the solubilization of barium sulfate to water were investigated by comparison with the crown ring-free analogue 4. The result shows that the polymer 2 is the efficient solubilizer of BaSO4 and the highest solubilization efficiency of the BaSO4 to water is up to 72.5%.

  19. Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

    Shi, Shan-Rong; Taylor, Clive R; Fowler, Carol B; Mason, Jeffrey T


    Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Solubilization of the O2(-)-forming activity responsible for the respiratory burst in human neutrophils.

    Gabig, T G; Kipnes, R S; Babior, B M


    On exposure to suitable activating agents, neutrophils sharply alter their oxygen metabolism, showing large increases in oxygen uptake, O2 and H2O2 production, and glucose consumption via the hexose monophosphate shunt. These metabolic alterations, which together are designated the "respiratory burst," are due to the activation of a system which catalyzes the reaction: 2O2 + NADPH leads to 2O2(-) + NADP. This O2(-)-forming system is found in a particulate fraction isolated from neutrophils which had been activated with opsonized zymosan. When these particles were treated with detergent under suitable conditions, the O2(-)-forming activity was released in a form which passed through a membrane filter capable of retaining species of Mr greater than 3000,000. Soluble O2(-)-forming activity was obtained from normal activated neutrophils, but not from normal resting neutrophils or from activated neutrophils obtained from patients with chronic granulomatous disease, an inherited condition in which the respiratory burst is defective. O2(-)production by the soluble system required a reduced pyridine nucleotide as electron donor, and showed a quadratic dependence on the concentration of the solubilized preparation.

  1. [A substrate-type induction of liver microsomal monooxygenases by phenobarbital].

    Tsyrlov, I B; Gromova, O A; Rivkind, N B; Vakulin, G M; Liakhovich, V V


    A possibility of step-wise induction of microsomal monooxygenases after injection of phenobarbital in the presence of 3-methylcholanthrene-caused induction was studied. It was found that the ratio of the high- and low-spin types of cytochrome, rather than the position of the CO-peak of its reduced form is a criterion for functional specificity of hemoprotein. Induction by phenobarbital appears possible under conditions when the inductor binding to microsomal hemoprotein is lacking, since cytochrome P-488 has no binding sites for phenobarbital. It is assumed that under microsomal monooxygenases induction by phenobarbital activation of genome and subsequent protein synthesis are operated by the substrate rather than by products of its primary metabolism in microsomes.

  2. Stimulatory effects of chlordiazepoxide, diazepam and oxazepam on the drug-metabolizing enzymes in microsomes.

    Jablońska, J K; Knobloch, K; Majka, J; Wiśniewska-Knypl, J M


    5 days' exposure of rats to daily doses of 400 mg/kg body wt. of chlordiazepoxide, diazepam and oxazepam stimulated the microsomal metabolism in the liver, as evidenced by acceleration of both p-hydroxylation of aniline and hydroxylation of benzene. The effect was accompanied by an increased concentration of liver microsomal protein and by the development of tolerance to the drugs. Similar effects were found after exposure of rats to lower doses of the drugs. The metabolism of aniline in vivo in rats treated with chlordiazepoxide was accelerated; this was correlated with development of tolerance to these drugs. It is suggested that both the stimulation of microsomal metabolism and the development of tolerance are associated with the induction of microsomal drug-metabolizing enzymes.

  3. Hepatic microsomal mixed-function oxidase system in rats pretreated with cicloxilic acid.

    Casini, A F; Comporti, M; Subissi, A; Murmann, W


    In a study of the hepatic microsomal drug metabolising system in rats, pretreatment with cis-2-hydroxy-2-phenyl-cyclohexanecarboxilic acid (cicloxilic acid) did not prolong pentobarbital sleeping time or decrease the cytochrome P 450 content of the liver microsomes. The drug interacted with the liver microsomes to yield a type II spectral change. Cicloxilic acid did not affect the metabolism of CCl4 in vivo, as judged by the covalent binding of 14C from 14CCl r to microsomal lipids, or delay the gastrointestinal absorption of CCl4, as judged by the concentration of free CCl4 in the liver. The protection afforded by cicloxilic acid against CCl4 liver damage is therefore due neither to inhibition of the metabolism nor to delayed absorption of the toxin.

  4. Does de novo synthesis of lysophosphatidylcholine occur in rat lung microsomes?

    Aarsman, A.J.; Bosch, H. van den


    Incubation of rat lung microsomes with CDP[Me-14C]choline resulted in formation of radioactive lysophosphatidylcholine and phosphatidylcholine. Evidence is provided which suggests that lysophosphatidylcholine formation cannot be ascribed completely to phospholipase A degradation of phosphatidylcholi

  5. Different enzyme kinetics of midazolam in recombinant CYP3A4 microsomes from human and insect sources.

    Christensen, Hege; Mathiesen, Liv; Postvoll, Lillian W; Winther, Bjørn; Molden, Espen


    In vitro drug metabolism techniques with human CYP c-DNA expressed systems are frequently used to predict human drug metabolism in vivo. The aim of this study was to compare midazolam enzyme kinetics in recombinant expressed CYP3A4 microsomes from human and insect cells. The amounts of 1'- hydroxymidazolam and 4-hydroxymidazolam formed in CYP3A4 microsomes from transfected human liver epithelial cells (T5-3A4 microsomes) and baculovirus-infected insect cells (with and without coexpressed cytochrome b(5)) were analysed by LC-MS. Enzyme kinetic parameters were estimated by nonlinear regression. Mean K(m) for the formation of 1'-hydroxymidazolam was 3- and 4-fold higher in T5-3A4 microsomes than in insect microsomes (pmicrosomes was reflected by significantly lower Cl(int) compared to insect microsomes (pmicrosomes displayed Michaelis-Menten kinetics, while insect microsomes showed substrate inhibition kinetics. The different enzyme kinetics of midazolam observed in recombinant CYP3A4 microsomes from human and insect sources, especially the substantially higher K(m) obtained in human microsomes compared to insect microsomes, should be further evaluated since it may have implications for correlations to in vivo situation.

  6. Kinetics of glyburide metabolism by hepatic and placental microsomes of human and baboon.

    Zharikova, Olga L; Ravindran, Selvan; Nanovskaya, Tatiana N; Hill, Ronald A; Hankins, Gary D V; Ahmed, Mahmoud S


    Glyburide (glibenclamide) is under investigation for treatment of gestational diabetes. Two metabolites of glyburide have been previously identified in patients, namely, 4-trans-(M1) and 3-cis-(M2) hydroxycyclohexyl glyburide. Recently, the metabolism of glyburide by microsomes of liver and placenta from humans and baboons revealed the formation of four additional metabolites: 4-cis-(M2a), 3-trans-(M3), and 2-trans-(M4) hydroxycyclohexyl glyburide, and ethyl-hydroxy glyburide (M5). The aim of this investigation was to determine the kinetics for the metabolism of glyburide by cytochrome P450 (CYP) isozymes of human and baboon placental and hepatic microsomes. The metabolism of glyburide by microsomes from the four organs revealed saturation kinetics and apparent K(m) values between 4 and 12 microM. However, the rates for formation of the metabolites varied between organs and species. M1 was the major metabolite (36% of total), formed by human hepatic microsomes with V(max) of 80+/-13 pmol mg protein(-1)min(-1), and together with M2, accounted for only 51% of the total. M5 was the major metabolite (87%) formed by human placental microsomes with V(max) of 11 pmol mg protein(-1)min(-1). In baboon liver, M5 had the highest rate of formation (V(max) 135+/-32 pmol mg protein(-1)min(-1), 39% of total), and in its placenta, was M4 (V(max) 0.7+/-0.1 pmol mg protein(-1)min(-1), 65%). The activity of human and baboon hepatic microsomes in metabolizing glyburide was similar, but the activity of human and baboon placental microsomes was 7% and 0.3% of their respective hepatic microsomes. The data obtained suggest that more than 1 CYP isozyme is responsible for catalyzing the hydroxylation of glyburide.

  7. Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes.

    Kobayashi, Kaoru; Kajiwara, Eri; Ishikawa, Masayuki; Oka, Hidenobu; Chiba, Kan


    Benzbromarone (BBR) is metabolized to 1'-hydroxy BBR and 6-hydroxy BBR in the liver. 6-Hydroxy BBR is further metabolized to 5,6-dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1'-hydroxy BBR and 6-hydroxy BBR and in the metabolism of 6-hydroxy BBR to 5,6-dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1'-hydroxy BBR. The formation rate of 1'-hydroxy BBR significantly correlated with testosterone 6β-hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1'-hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6-hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6-hydroxy BBR and diclofenac 4'-hydroxylation activity in 12 human liver microsomes. The formation of 6-hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6-dihydroxy BBR from 6-hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6-dihydroxy BBR was significantly correlated with diclofenac 4'-hydroxylation activity and phenacetin O-deethylation activity in 12 human liver microsomes. The formation of 5,6-dihydroxy BBR was inhibited with either tienilic acid or α-naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1'-hydroxy BBR and 6-hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6-dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes.

  8. Phospholipide turnover in microsomal membranes of the pancreas during enzyme secretion.



    After incubation of pigeon pancreas slices with P(32) and isolation of various fractions by differential centrifugation the deoxycholate extract of the microsome fraction was found to account for over half of the phospholipide P and over half of the P(32) incorporated into the phospholipides. The remaining phospholipide P and P(32) were fairly evenly distributed in the nuclei, zymogen granules, mitochondria, microsomal ribonucleoprotein particles, and the soluble fraction. When enzyme secretion was stimulated with acetylcholine about two-thirds of the increment in radioactivity in the total phospholipides was found in deoxycholate soluble components of the microsome fraction. The remainder of the increment was distributed in the other fractions. This indicates that the cellular component in which the increase in phospholipide turnover occurs on stimulation of secretion is a membranous structure. Evidence is presented which indicates that the increment in radioactivity in the non-microsomal fractions on stimulation of secretion is due to contamination of these fractions with fragments of the stimulated membranous structure. The distribution of P(32) radioactivity in each of the chromatographically separated phospholipides in the various fractions from unstimulated tissue paralleled the distribution of radioactivity in the total phospholipide fraction, indicating that individual phospholipides are not concentrated in different fractions but are associated together in the membranous structures of the microsome fraction. The major proportion of the stimulation of the turnover of the individual phospholipides also occurred in the microsome fraction. The distribution of radioactivity from glycerol-1-C(14) in the total phospholipides and in the individual phospholipides in the various fractions was similar to the distribution of P(32). In the microsome fraction acetylcholine stimulated the incorporation of glycerol-1-C(14) in each phospholipide which showed a stimulation

  9. Effect of x radiation on hydroperoxidase activity of rat liver microsomes

    Platonov, A.G.; Deev, L.I.


    The effect of single exposure to total-body x radiation on hydroperoxidase activity (HPA) of rat liver microsomes and levels of cytochromes P-450 and b/sub 5/ in them were studied. Cytochrome b/sub 5/ content was measured in view of data indicative of the possible involvement of this enzyme in breaking down hydroperoxides of the microsomal fraction of the liver.

  10. Production of humic substances through coal-solubilizing bacteria

    Nelson Valero


    Full Text Available In this paper, the production of humic substances (HS through the bacterial solubilization of low rank coal (LRC was evaluated. The evaluation was carried out by 19 bacterial strains isolated in microenvironments with high contents of coal wastes. The biotransformed LRC and the HS produced were quantified in vitro in a liquid growth medium. The humic acids (HA obtained from the most active bacterial strain were characterized via elemental composition (C, H, N, O, IR analyses, and the E4/E6 ratio; they were then compared with the HA extracted chemically using NaOH. There was LRC biotransformation ranged from 25 to 37%, and HS production ranged from 127 to 3100 mg.L-1. More activity was detected in the isolated strains of Bacillus mycoides, Microbacterium sp, Acinetobacter sp, and Enterobacter aerogenes. The HA produced by B. mycoides had an IR spectrum and an E4/E6 ratio similar to those of the HA extracted with NAOH, but their elemental composition and their degree of aromatic condensation was different. Results suggest that these bacteria can be used to exploit the LRC resulting from coal mining activities and thus produce HS in order to improve the content of humified organic matter in soils.

  11. [Phosphate solubilization of Aureobasidium pullulan F4 and its mechanism].

    Wang, Dan; Zhan, Jing; Sun, Qing-Ye


    The Aureobasidium pullulans F4 was isolated from the rhizosphere of Hippochaete ramosissimum in Tongguanshan mine wasteland in Tongling City, Anhui Province. Liquid culture was conducted with four kinds of phosphorus sources, calcium phosphate, aluminum phosphate, ferric phosphate and rock phosphate to determine the pH, dissolved phosphorus, phosphorus in the bacteria and organic acid in the solution. The results showed that the phosphate solubilization by A. pullulans F4 varied with phosphorus sources, which decreased in order of aluminum phosphate > ferric phosphate, calcium phosphate > rock phosphate. The amounts of dissolved phosphorus in the different treatments were all higher than 200 mg x L(-1). The pH of the medium dropped immediately in 48 h, and the aluminum phosphate and ferric phosphate treatments showed a greater decrease in pH than the calcium phosphate and rock phosphate treatments. The organic acid synthesized by A. pullulans F4 included oxalic acid, citric acid and tartaric acid, and oxalic acid, among which oxalic acid was the dominated component. The phosphate dissolving capacity of A. pullulans F4 showed no significant correlation with organic acid, but significantly correlated with the pH. The available phosphorus was significantly improved with the combined application of A. pullulans F4 and glucose, suggesting A. pullulans F4 was a potent candidate for remediation of copper mine wastelands.

  12. Production of humic substances through coal-solubilizing bacteria

    Valero, Nelson; Gómez, Liliana; Pantoja, Manuel; Ramírez, Ramiro


    In this paper, the production of humic substances (HS) through the bacterial solubilization of low rank coal (LRC) was evaluated. The evaluation was carried out by 19 bacterial strains isolated in microenvironments with high contents of coal wastes. The biotransformed LRC and the HS produced were quantified in vitro in a liquid growth medium. The humic acids (HA) obtained from the most active bacterial strain were characterized via elemental composition (C, H, N, O), IR analyses, and the E4/E6 ratio; they were then compared with the HA extracted chemically using NaOH. There was LRC biotransformation ranged from 25 to 37%, and HS production ranged from 127 to 3100 mg.L−1. More activity was detected in the isolated strains of Bacillus mycoides, Microbacterium sp, Acinetobacter sp, and Enterobacter aerogenes. The HA produced by B. mycoides had an IR spectrum and an E4/E6 ratio similar to those of the HA extracted with NAOH, but their elemental composition and their degree of aromatic condensation was different. Results suggest that these bacteria can be used to exploit the LRC resulting from coal mining activities and thus produce HS in order to improve the content of humified organic matter in soils. PMID:25477925

  13. Interactions between solubilized polymer molecules and blood components.

    Liu, Zonghua; Jiao, Yanpeng; Wang, Tong; Zhang, Yuanming; Xue, Wei


    In biomedical field, a variety of natural or synthetic polymers are being exponentially developed and used in vivo to improve human health. In practical applications, these biopolymers would enter blood circulation directly or indirectly, positively or passively, rapidly or slowly. Blood is a special tissue of human body, which fulfills many important missions to sustain normal metabolism. The contact with blood of the biopolymers, which are seen as foreign substances by the body, would be harmful or even instantaneously lethal, depending on the nature and the dose of the biopolymers administered. Therefore, it is critical to clearly understand the potential influences of the foreign polymers on blood before the polymers are applied from the lab to bedside. In this review, we discuss the recent studies on the interactions of foreign, solubilized polymer molecules (excluding formed polymer materials) with blood constituents (red blood cells, white blood cells, platelets, plasma proteins, etc.), to gain insight into the potential in vivo applications of the biopolymers in the biomedical field. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Solubilizer Selection of Compound Yinchen Injection Prescription%复方茵陈注射液处方中增溶剂的选择

    李正明; 袁海龙; 张诗龙; 周燕萍; 易毛; 张瑞新; 吴素体


    目的:选择增溶效果好而不良反应少的药用辅料.方法:分别采用泊洛沙姆188、吐温80作为增溶剂制备复方茵陈注射液,检查药物微粒数量、栀子苷含量、药液pH值、溶血与凝聚等指标变化.结果:2种复方茵陈注射液的栀子苷含量、pH值、微粒数、溶血与凝聚指标均合格.结论:泊洛沙姆188与吐温80具有同等增溶效果,可选择泊洛沙姆作为新一代增溶辅料.%OBJECTIVE: To select pharmaceutical adjuvant with sound solubilizing effect and little ADR. METHODS: Compound yinchen injection was prepared with poloxamer-188 and tween-80 as solubilizer. The number of drug microparticle, the content of jasminoidin, pH value of drug solution, hemolysis and coacervation were determined. RESULTS: The content of jasminoi-din, pH value, the number of drug microparticle, hemolysis and coacervation of 2 kinds of Compound yinchen injection were all qualified. CONCLUSION: Poloxamer-188 has the same solubilizing effects as tween-80. Poloxamer-188 is an appropriate choice in the new generation of solubilizing adjuvant.

  15. Microbial Efficacy of Phosphate Solubilization in Agro-Saline Soils of Various Areas of Sindh Region

    Agha Asad Noor


    Full Text Available Microorganisms are the most prominent entities for solubilization of phosphate in various soils of different areas of Sindh Province including Tando Muhammad Khan, Tando Allah Yar, Nawabshah, Rato Dero-Larkana, Shikarpur and Umer Kot. These soils, having varying concentrations of chemicals, different climatic conditions, pH and varying numbers of microorganisms for PSA (Phosphate Solubilization Activity. This presentation shows the isolation of different fungi and bacteria capable Psa including fungi (Fusarium sp. Aspergillus sp. Penicillium sp. and Rhizopus sp. and bacteria (Bacillus sp. Pseudomonas sp. and Arthrobacter sp.. From the observations, it was revealed that fungi Aspergillus sp. and Bacillus sp. showed greater phosphate solubilization activity as compared to other fungi and bacteria showing 60 and 53.33% Psa (Phosphate Solubilizing Activity respectively

  16. Isolation and characterization of phosphate-solubilizing bacteria from seagrass rhizosphere soil

    Ghosh, Upasana; Subhashini, Ponnambalam; Dilipan, Elangovan; Raja, Subramanian; Thangaradjou, Thirunavukarassu; Kannan, Lakshmanan


    Phosphate-solubilizing bacterial strains (6 Nos.) were isolated from the rhizosphere soils of two seagrasses ( Halophila ovalis (R. Br.) Hook and Halodule pinifolia (Miki) Hartog) in the Vellar estuary. Experimental studies found that the strain PSSG6 was effective in phosphate solubilization with Phosphate Solubilization efficiency index E = 375 ± 8.54, followed by the strain PSSG5 with Phosphate Solubilization efficiency index E = 275 ± 27.3. Of the 6 strains isolated, the strains PSSG4 and PSSG5 belonged to the genus Bacillus, and PSSG1, PSSG2 and PSSG3 were identified as Citrobacter sp., Shigella sp., and Klebsiella sp., respectively, by conventional method, and PSSG6 was identified as Bacillus circulans using conventional and molecular methods.

  17. Identification and characterization of Gluconacetobacter diazotrophicus mutants defective in the solubilization of phosphorus and zinc.

    Intorne, Aline C; de Oliveira, Marcos Vinicius V; Lima, Mariana L; da Silva, Juliana F; Olivares, Fábio L; de Souza Filho, Gonçalo Apolinário


    Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca(5)(PO(4))(3)OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus.

  18. Isolation and Characterization of Phosphate-Solubilizing Bacteria from Seagrass Rhizosphere Soil

    Upasana Ghosh; Ponnambalam Subhashini; Elangovan Dilipan; Subramanian Raja; Thirunavukarassu Thangaradjou; Lakshmanan Kannan


    Phosphate-solubilizing bacterial strains (6 Nos.) were isolated from the rhizosphere soils of two seagrasses (Halophila ovalis (R.Br.) Hook and Halodule pinifolia (Miki) Hartog) in the Vellar estuary.Experimental studies found that the strain PSSG6was effective in phosphate solubilization with Phosphate Solubilization efficiency index E=375 ± 8.54,followed by the strain PSSG5with Phosphate Solubilization efficiency index E=275 ± 27.3.Of the 6 strains isolated,the strains PSSG4 and PSSG5 belonged to the genus Bacillus,and PSSG1,PSSG2 and PSSG3 were identified as Citrobacter sp.,Shigella sp.,and Klebsiella sp.,respectively,by conventional method,and PSSG6 was identified as Bacillus circulans using conventional and molecular methods.


    Although surfactants have received considerable attention as a potential means for enhancing the recovery of organic compounds from the subsurface, only limited information is available regarding the micellar solubilization of common groundwater contaminants by nonionic surfactan...

  20. Mechanisms of coal solubilization by the deuteromycetes Trichoderma atroviride and Fusarium oxysporum

    Hoelker, U.; Ludwig, S.; Scheel, T.; Hoefer, M. [Bonn Univ. (Germany). Botanisches Inst. und Botanischer Garten


    Three different mechanisms can be envisaged that are used by fungi to solubilize coal: the production of alkaline substances, the extrusion of chelators and, of special interest in the scope of biotechnology, the action of enzymes. Whether these mechanisms are operating separately or in various combinations has not yet been finally assessed. The two deuteromycetes Fusarium oxysporum and Trichoderma atroviride solubilize coal by synergistic effects of various different mechanisms depending on the cell metabolism. F. oxysporum seems to solubilize coal by increasing the pH of the mycelial surroundings and by the action of chelators induced during growth in glutamate-containing media (without involvement of enzymes). T. atroviride, on the other hand, appears to use, in addition to an alkaline pH and a high chelator activity, at least two classes of enzyme activity to attack coal: hydrolytic activity for coal solubilization and ligninolytic activity for degradation of humic acids. (orig.)

  1. Expanding role of microsomal enzyme induction, and its implications for clinical chemistry

    Goldberg, D.M.


    Microsomal enzyme induction, a term denoting the ability of the substrate for a microsomal enzyme to enhance the activity of that enzyme and frequently of related enzymes, has been demonstrated in a wide range of tissues, notably the liver, placenta, small intestinal muccosa, and peripheral lymphocytes. The major agents that cause microsomal enzyme induction are drugs and xenobiotics. Factors modulating the extent of enzyme induction by a given agent include age and nutrition, and wide species variations are encountered with different inducing agents. Markers for microsomal enzyme induction include determination of the plasma half-life for conveniently measured drugs, and the measurement of endogenous metabolites such as 6;-hydroxycortisol and D-glucaric acid in 24-h urine collections. While these are valuable for monitoring enzyme induction in healthy patients, they are altered in certain forms of liver disease, and results must then be interpreted with caution. Microsomal enzyme induction may interfere with reference values, particularly for membrane-bound enzymes, in otherwise healthy populations, and may play a role in metabolic bone disease, drug interactions, carcinogenesis, and hypertriglyceridemia. Drug therapy of the neonatal and congenital hyperbilirubinemias has been inspired by the mechanism of hepatic microsomal enzyme induction, and ''markers'' for enzyme induction can be used to monitor drug compliance. The activity of serum <-glutamyltransferase seems to be especially valuable for this purpose.

  2. Involvement of CYP2B6 in the biotransformation of propofol by human liver microsomes

    TANG Bing; WANG Jun-ke; FENG Wan-yu


    Objective To determine whether the cytochrome P4502B6 (CYP2B6) is involved in the oxidation of propofol by human liver microsomes. Methods The change of propofol concentration in an incubation mixture with human liver microsomes was monitored by the high performance liquid chromatography (HPLC), in order to calculate the rate constants of metabolism of propofol. The correlation between the rate constants and the rate of metabolism of CYP2B6 selective substrate bupropion, and the effect of two different CYP2B6 specific inhibitors on the propofol metabolism were examined. Results The mean rate constant of propofol metabolism by liver microsomes obtained from twelve individuals was 3.9 (95 % confidence intervals 3.3, 4.5) nmol·min-1·mg-1 protein. The rate constants of propofol metabolism by liver microsomes were significantly correlated with bupropion hydroxylation (r=0.888, P<0.001). Both selective chemical inhibitors of CYP2B6, orphenadrine and N, N′, N″-triethylenethiophosphoramide (thioTEPA), reduced the rate constants of propofol metabolism by 37.596 (P<0.001) and 42.796 (P<0.001)in liver microsomes, respectively. Conclusions CYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes.

  3. Stereoselective degradation of metalaxyl and its enantiomers in rat and rabbit hepatic microsomes in vitro.

    Zhang, Ping; Shen, Zhigang; Xu, Xinyuan; Zhu, Wentao; Dang, Ziheng; Wang, Xinru; Liu, Donghui; Zhou, Zhiqiang


    The stereoselective degradations of racemate metalaxyl (rac-MX) and its single enantiomers in rat and rabbit hepatic microsomes were assayed by a chiral high-performance liquid chromatography method. The t(1/2) of (+)-S-MX in rat liver microsomes was between 7-8 min tested by rac-MX and the individual (+)-S-enantiomer, respectively, and that for (-)-R-MX was 15-16 min. In contrast, t(1/2) in rabbit liver microsomes was much longer and showed great difference when using racemate and single enantiomer, which was similar to the results of in vivo study. The enantioselectivity in rat hepatic microsomes was more evident and the degradations of MX enantiomers in rat and rabbit hepatic microsomes were Nicotinamide adenine dinucleotide phosphate-dependent. Michaelis constant (K(m)) and intrinsic metabolic clearance (CL(int)) of (+)-S-MX were larger than that of (-)-R-MX and there was no chiral inversion from (+)-S-MX to (-)-R-MX or vice versa in both rat and rabbit hepatic microsomes.

  4. Solubilization and quantification of lycopene in aqueous media in the form of cyclodextrin binary systems.

    Vertzoni, Maria; Kartezini, Theodora; Reppas, Christos; Archontaki, Helen; Valsami, Georgia


    An optimized kneading method for the preparation of lycopene-cyclodextrin binary systems was developed leading to solubilization of lycopene in water and 5% (w/v) dextrose solution. Lycopene quantification in the prepared binary systems was performed by a developed spectrometric method that followed a successful single-step extraction with dichloromethane. Storage stability characteristics of the binary systems were studied at 4 degrees C in solution and at -20 degrees C in the lyophilized products. Lycopene content was monitored at lambda(max)=482 nm, the limit of detection was 0.41 microg/ml and relative standard deviation was less than 3.1%. The results obtained with the spectrometric method were confirmed by a HPLC method. In the presence of cyclodextrins, lycopene concentration in water was 8.0+/-1.0, 27.1+/-3.2 and 16.0+/-2.2 microg/ml for beta-CD, HP-beta-CD and Me-beta-CD, respectively. In 5% (w/v) aqueous dextrose solutions the corresponding values were 16.0+/-1.8, 48.0+/-5.1 and 4.0+/-0.5 microg/ml, respectively. At 4 degrees C, storage stability of lycopene-cyclodextrin binary systems in water or 5% (w/v) aqueous dextrose solutions, was limited (t(1/2)=1-4 days). Addition of the antioxidant sodium metabisulfite increased the stability of lycopene-HP-beta-CD binary system in water. At -20 degrees C, the lyophilized lycopene-cyclodextrin binary systems were stable for at least 2 weeks.

  5. Enzymatic solubilization of a pectinaceous dietary fiber fraction from potato pulp: Optimization of the fiber extraction process

    Meyer, Anne S.; Dam, Birgitte P; Lærke, Helle N.


    Upgrading of potato pulp, a byproduct stream from industrial manufacture of potato starch, is important for the continued economic competitiveness of the potato starch industry. The major part of potato pulp consists of the tuber plant cell wall material which is particularly rich in galactan...... branched rhamnogalacturonan I type pectin. In the work reported here, the release of high-molecular weight pectinaceous dietary fiber polysaccharides from starch free potato pulp was accomplished by use of a multicomponent pectinase preparation from Aspergillus aculeatus (Viscozyme® L). The enzyme reaction...... that they were rich in galactose and uronic acid indicating that the solubilized fibers were mainly made up of galactan branched rhamnogalacturonan I type pectin polymers....

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

    Carmem Dickow Cardoso


    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. Phosphate-solubilizing microbes and their occurrence in the rhizospheres of Piper betel in Karnataka, India



    Low phosphate solubility is one of the most important factors limiting the plant growth in Indian soils. Many microorganisms can enhance phosphate solubility, but little is known about the magnitude of their phosphorus-solubilizing ability. The native populations of phosphate-solubilizing bacteria and fungi were studied in different rhizospheric soil samples obtained from betel vine plants (Piper betel L.) in order to compare the results. The present study focuses on the phosphate-solubilizin...

  8. Diversity and phosphate solubilization by bacteria isolated from Laki Island coastal ecosystem



    Widawati S (2011) Diversity and phosphate solubilization by bacteria isolated from Laki Island coastal ecosystem. Biodiversitas 12: 17-21. Soil, water, sand, and plant rhizosphere samples collected from coastal ecosystem of Laki Island-Jakarta were screened for phosphate solubilizing bacteria (PSB). While the population was dependent on the cultivation media and the sample type, the highest bacterial population was observed in the rhizosphere of Ipomea aquatica. The PSB strains isolated from ...

  9. Performance of pineapple slips inoculated with diazotrophic phosphate-solubilizing bacteria and rock phosphate

    Lílian Estrela Borges Baldotto; Marihus Altoé Baldotto; Fábio Lopes Olivares; Adriane Nunes de Souza


    Besides fixing N2, some diazotrophic bacteria or diazotrophs, also synthesize organic acids and are able to solubilize rock phosphates, increasing the availability of P for plants. The application of these bacteria to pineapple leaf axils in combination with rock phosphate could increase N and P availability for the crop, due to the bacterial activity of biological nitrogen fixation and phosphate solubilization. The objectives of this study were: (i) to select and characterize diazotrophs abl...

  10. Selection and evaluation of phosphate-solubilizing bacteria from grapevine rhizospheres for use as biofertilizers

    Min Liu; Xu Liu; Bao-Sen Cheng; Xue-Lei Ma; Xiao-Tong Lyu; Xian-Fang Zhao; Yan-Lun Ju; Zhuo Min; Yu-Lin Fang


    Phosphate-solubilizing bacteria (PSB) have the ability to solubilize insoluble phosphorus (P) and release soluble P. Extensive research has been performed with respect to PSB isolation from the rhizospheres of various plants, but little is known about the prevalence of PSB in the grapevine rhizosphere. In this study, we aimed to isolate and identify PSB from the grapevine rhizosphere in five vineyards of Northwest China, to characterize their plant-growth-promoting (PGP) traits, evaluate the ...

  11. Phosphate-Solubilizing and -Mineralizing Abilities of Bacteria Isolated from Soils

    TAO Guang-Can; TIAN Shu-Jun; CAI Miao-Ying; XIE Guang-Hui


    Microorganisms capable of solubilizing and mineralizing phosphorus (P) pools in soils are considered vital in promoting P bioavailability. The study was conducted to screen and isolate inorganic P-solubilizing bacteria (IPSB) and organic P-mineralizing bacteria (OPMB) in soils taken from subtropical flooded and temperate non-flooded soils, and to compare inorganic P-solubilizing and organic P-solubilizing abilities between IPSB and OPMB. Ten OPMB strains were isolated and identified as Bacillus cereus and Bacillus megaterium, and five IPSB strains as B. megaterium, Burkholderia caryophylli,Pseudomonas ciehorii, and Pseudomonas syringae. P-solubilizing and -mineralizing abilities of the strains were measured using the methods taking cellular P into account. The IPSB strains exhibited inorganic P-sohibilizing abilities ranging between 25.4-41.7 μg P mL-1 and organic P-mineralizing abilities between 8.2-17.8 μg P mL-1. Each of the OPMB strains also exhibited both solubilizing and mineralizing abilities varying from 4.4 to 26.5 μg P mL-1 and from 13.8 to 62.8 μg P mL-1, respectively. For both IPSB and OPMB strains, most of the P mineralized from the organic P source was incorporated into the bacterial cells as cellular P. A significantly negative linear correlation (P < 0.05) was found between culture pH and P solubilized from inorganic P by OPMB strains. The results suggested that P solubilization and mineralization could coexist in the same bacterial strain.

  12. The mechanism of detergent solubilization of liposomes and protein-containing membranes.

    Kragh-Hansen, U; le Maire, M; Møller, J V


    The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentr...

  13. Diversity and phosphate solubilization by bacteria isolated from Laki Island coastal ecosystem



    Full Text Available Widawati S (2011 Diversity and phosphate solubilization by bacteria isolated from Laki Island coastal ecosystem. Biodiversitas 12: 17-21. Soil, water, sand, and plant rhizosphere samples collected from coastal ecosystem of Laki Island-Jakarta were screened for phosphate solubilizing bacteria (PSB. While the population was dependent on the cultivation media and the sample type, the highest bacterial population was observed in the rhizosphere of Ipomea aquatica. The PSB strains isolated from the sample registered 18.59 g-1L-1, 18.31 g-1L-1, and 5.68 g-1L-1 of calcium phosphate (Ca-P, Al-P and rock phosphate solubilization after 7-days. Phosphate solubilizing capacity was the highest in the Ca-P medium. Two strains, 13 and 14, registered highest Phosphomonoesterase activities (2.01 µgNP.g-1.h-1 and 1.85NP µg.g-1.h-1 were identified as Serattia marcescens, and Pseudomonas fluorescense, respectively. Both strains were isolated from the crops of Amaranthus hybridus and I. aquatica, respectively, which are commonly observed in coastal ecosystems. The presence of phosphate solubilizing microorganisms and their ability to solubilize various types of phosphate species are indicative of the important role of both species of bacteria in the biogeochemical cycle of phosphorus and the plant growth in coastal ecosystems.

  14. Biochemical analysis and solubilization of central alpha 1-adrenoceptors in the rat

    Guicheney, P.; Rappaport, A.; Meyer, P.


    To study the structure and the molecular mechanisms of action of brain alpha-adrenoceptors, their solubilization was undertaken. alpha 1-Adrenoceptors were first successfully solubilized from fresh rat brain membranes by treatment with 0.3 percent deoxycholate, after prelabeling of the binding site by the highly specific tritiated antagonist /sup 3/H-prazosin. The complex thus solubilized underwent a rapid loss of activity at 25 degrees C. Direct solubilization of brain alpha 1-adrenoceptors was obtained by treatment with a new zwitterionic derivative of cholic acid (CHAPS) at a concentration of 5 to 10 mM. The soluble complex was detected by precipitation by polyethylene glycol 6,000 with gamma globulin as a carrier. Binding of /sup 3/H-prazosin at 25 degrees C was rapid; at 4 degrees C the steady state was obtained within two hours and remained unchanged for at least six hours. The affinity of the soluble binding site, determined by Scatchard analysis (congruent to 0.6 nM), varied with the concentration of detergent. Specificity of the membrane-bound receptor was preserved as demonstrated by incubation in the presence of alpha 1- and alpha 2-antagonists at various concentrations (by order of potency: prazosin greater than phentolamine greater than yohimbine). Stereoselectivity was also retained in the solubilized binding protein. The solubilization of an active brain alpha 1-adrenoceptor will allow further investigation at the molecular level.

  15. Ethyl methanesulfonate mutagenesis-enhanced mineral phosphate solubilization by groundnut-associated Serratia marcescens GPS-5.

    Tripura, Chaturvedula; Sashidhar, Burla; Podile, Appa Rao


    Twenty-three bacterial isolates were screened for their mineral phosphate-solubilizing (MPS) ability on Pikovskaya and National Botanical Research Institute's phosphate (NBRIP) agar. The majority of the isolates exhibited a strong ability to solubilize hydroxyapatite in both solid and liquid media. The solubilization in liquid medium corresponded with a decrease in the pH of the medium. Serratia marcescens GPS-5, known for its biocontrol of late leaf spot in groundnut, emerged as the best solubilizer. S. marcescens GPS-5 was subjected to ethyl methanesulfonate (EMS) mutagenesis, and a total of 1700 mutants, resulting after 45 minutes of exposure, were screened on buffered NBRIP medium for alterations in MPS ability compared with that of the wild type. Seven mutants with increased (increased-MPS mutants) and 6 mutants with decreased (decreased-MPS mutants) MPS ability were isolated. All seven increased-MPS mutants were efficient at solubilizing phosphate in both solid and liquid NBRIP medium. Among the increased-MPS mutants, EMS XVIII Sm-35 showed the maximum (40%) increase in the amount of phosphate released in liquid medium compared with wild-type S. marcescens GPS-5, therefore, it would be a useful microbial inoculant in groundnut cultivation. EMS III Sm W, a nonpigmented mutant, showed the lowest solubilization of phosphate among the 6 decreased-MPS mutants.

  16. Solubilization of Waste Activated Sludge and Nitrogenous Compounds Transformation During Solubilization by Thermophilic Enzyme (S-TE) Process.

    Yang, Qi; Luo, Kun; Li, Xiao-ming; Zhong, Yu; Chen, Hong-bo; Yang, Guo-jing; Shi, Yan-wei; Zeng, Guang-ming


    A representative thermophilic bacterial strain (AT06-1) capable of secreting protease was isolated from thermophilic aerobic digestion reactor, and 16S rRNA gene analysis indicated that it was Bacillus sp. The isolated strain was inoculated in waste activated sludge (WAS) to evaluate the performance of solubilization by thermophilic enzyme (S-TE) process under aerobic or microaerobic conditions at different temperatures (55-70 °C). Results showed that the inoculation of specific thermophilic strain significantly affected the volatile suspended solids (VSS) removal. At the optimal temperature of 65 °C, the maximum VSS removal of 43.6 % and highest SCOD of 4475 mg/L was achieved during microaerobic S-TE process. Compared to the noninoculation, more soluble protein was released during S-TE process due to the higher protease activity associated with the protein hydrolysis originated from cell lysis. The protease activity at aerobic and microaerobic S-TE process was respectively 1.73 and 1.88 times that of the noninoculation. Ammonia was the end nitrogenous compound of protein hydrolysis during S-TE process, which was stripped from the digestion system through continuous aeration.

  17. Four new koumine metabolites in rat liver microsomes.

    Zhang, Lin; Du, Lan; Lv, Wen-Wen; Zhao, Qing-Chun; Hua, Wei; An, Ye; Guo, Tao; Wu, Li-Jun


    Four new metabolites M-1 [1,2,18,19-tetradehydro-4-demethyl-3,17-epoxy-7,20(2H,19H)-cyclovobasan], M-2 [1,2,4,21,18,19-hexadehydro-4-demethyl-3,17-epoxy-7,20(2H,19H)-cyclovobasan], M-3 [1,2,18,19-tetradehydro-4-demethyl-4-formaldehyde-3,17-epoxy-7,20(2H,19H)-cyclovobasan], and M-4 [1,2,4,21,18,19-hexadehydro-4-demethyl-4-oxy-3,17-epoxy-7,20(2H,19H)-cyclovobasan] were isolated from the chloroform extract of koumine incubated with phenobarbital-treated rat liver microsomes. The structures of M-1, M-2, M-3, and M-4 were elucidated by spectroscopic methods including ESI-TOF-MS, 1D, and 2D NMR experiments. The metabolic pathway of koumine was proposed. The cytotoxic activities between koumine and its metabolites were also compared in the A549 cell line.

  18. Stereoselective degradation of tebuconazole in rat liver microsomes.

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


    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).

  19. Impact of Micellar Surfactant on Supersaturation and Insight into Solubilization Mechanisms in Supersaturated Solutions of Atazanavir.

    Indulkar, Anura S; Mo, Huaping; Gao, Yi; Raina, Shweta A; Zhang, Geoff G Z; Taylor, Lynne S


    The goals of this study were to determine: 1) the impact of surfactants on the "amorphous solubility"; 2) the thermodynamic supersaturation in the presence of surfactant micelles; 3) the mechanism of solute solubilization by surfactant micelles in supersaturated solutions. The crystalline and amorphous solubility of atazanavir was determined in the presence of varying concentrations of micellar sodium dodecyl sulfate (SDS). Flux measurements, using a side-by-side diffusion cell, were employed to determine the free and micellar-bound drug concentrations. The solubilization mechanism as a function of atazanavir concentration was probed using fluorescence spectroscopy. Pulsed gradient spin-echo proton nuclear magnetic resonance (PGSE-NMR) spectroscopy was used to determine the change in micelle size with a change in drug concentration. Changes in the micelle/water partition coefficient, K m/w , as a function of atazanavir concentration led to erroneous estimates of the supersaturation when using concentration ratios. In contrast, determining the free drug concentration using flux measurements enabled improved determination of the thermodynamic supersaturation in the presence of micelles. Fluorescence spectroscopic studies suggested that K m/w changed based on the location of atazanavir solubilization which in turn changed with concentration. Thus, at a concentration equivalent to the crystalline solubility, atazanavir is solubilized by adsorption at the micelle corona, whereas in highly supersaturated solutions it is also solubilized in the micellar core. This difference in solubilization mechanism can lead to a breakdown in the prediction of amorphous solubility in the presence of SDS as well as challenges with determining supersaturation. PGSE-NMR suggested that the size of the SDS micelle is not impacted at the crystalline solubility of the drug but increases when the drug concentration reaches the amorphous solubility, in agreement with the proposed changes in

  20. [Isolation and properties of a preparation of arterial collagen solubilized by pretreatment with alkali (author's transl)].

    Henkel, W


    The acid-soluble, highly cross-linked aorta collagen, of which about 30% can be converted into a soluble form by alkali treatment, followed by extraction with aetic acid, was obtained predominantly in the form of monomeric, helical molecules, as indicated by the value for the intrinsic viscosity and its behaviour in sodium dodecylsulphate disc electrophoresis. Apart from decreased values for tyrosine (0.26%), arginine (4.4%) and aspartic acid (3.9%), the amino acid composition of the aorta collagen fraction was similar to that of the acid-soluble calf skin collagen. This finding, together with the cyanogen bromide peptide pattern, shows that the collagen extracted from the artery is predominantly type I. Treatment with alkali probably shortens the alpha1-CB6-peptide by about 45 amino acids. The collagen extracted from artery was compared with acid soluble skin collagen by sodium dodecylsulphate polyacrylamide electrophoresis. The arterial collagen showed a marked increase in the rations alpha1 to alpha2 (4:1), alpha to beta (3:1) and beta11 to beta12 (2.5:1). Compared with acid soluble skin collagen, the aorta collagen contained twice as much galactose and glucose (13.5 and 9.6 nmol/mg protein respectively), which are bound to hydroxylysine. 50% of the hydroxylysine residues are unsubstituted, 15% are present as galactosyl hydroxylysine, and 35% as glucosyl-galactosyl hydroxylysine. On the basis of its reported properties, arterial collagen obtained by the method of Fujii appears to be a suitable substrate for the study of the enzymic synthesis and enzymic degradation of hydroxylysine glycosides of native arterial collagen.

  1. 2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is enantioselectively oxidized to hydroxylated metabolites by rat liver microsomes.

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


    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 enzyme. 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 atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer did not 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 PCBs and their metabolites in wildlife

  2. Metabolism of ketoconazole and deacetylated ketoconazole by rat hepatic microsomes and flavin-containing monooxygenases.

    Rodriguez, R J; Acosta, D


    Ketoconazole (KT) has been reported to cause hepatotoxicity, which is probably not mediated through an immunoallergic mechanism. Although KT is extensively metabolized by hepatic microsomal enzymes, the nature, route of formation, and toxicity of suspected metabolites are largely unknown. Recent reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice, which, like lipophilic 4-alkylpiperazines, is susceptible to successive oxidative attacks on the N-1 position producing ring-opened dialdehydes. The rate of formation of DAK from hepatic rat microsomal incubations of KT was determined by HPLC. The rate of disappearance for KT was almost equal to the rate of DAK formation: 5.96 and 5.88 microM/hr, respectively. Also, the potential bioactivation of DAK was evaluated by measuring substrate activity of DAK with purified pig liver flavin-containing monooxygenase (FMO) and rat liver microsomes. Activity was measured by following DAK-dependent oxygen uptake polarographically at 37 degrees C in pyrophosphate buffer (pH 8.8) containing the glucose-6-phosphate NADPH-generating system. The K(M)'s of DAK were 34.6 and 77.4 microM for the purified FMO and rat microsomal FMO, respectively. Lastly, DAK was found to be metabolized by an NADPH-dependent rat liver microsomal monooxygenases at pH 8.8 to two metabolites as determined by HPLC. Heat inactivation of rat liver microsomal FMO abolished the formation of these metabolites from DAK. SKF-525A and anti-rat NADPH cytochrome P450 reductase did not inhibit this reaction. These results suggest that deacetylation of KT yields a major product, DAK, for further metabolism by microsomal monooxygenases that seem to be FMO-related.

  3. Potentiality of Acidithiobacillus thiooxidans in Microbial Solubilization of Phosphate Mine Tailings

    S Dhakar


    Full Text Available This paper deals with the solubilization behavior of the tailings produced by the floatation of a complex low grade phosphate ore. The composition of the tailings was essentially dolomite (52.04% with minor amounts of phosphate, iron and aluminium oxides (10.4 and 0.5% respectively. The presence of these products created uncontrolled land pollution and severely affected groundwater. An initiative has been taken up for utilization of this waste to generate an eco-friendly product. First step towards this panorama is incorporation of suitable microorganisms for the biodegradation of this effluent. Sulphur oxidizing bacteria Acidithiobacillus thiooxidans produces sulphuric acid which neutralizes the dolomitic tailings and convert it into plant available forms. The solubilization activity was tested in sulphur medium with 5, 10, 15 and 20% concentration of tailings. The solubilization is graded on the basis of pH, Electrical conductivity (EC, soluble calcium and magnesium and soluble phosphate. The results from ex-situ experiments showed that the treatment with 15% tailings ended with highest solubilization. The values of pH, EC, soluble calcium and magnesium and soluble phosphate for this treatment were 4.92, 31.6 dS/m, 10.8 mL EDTA and 17.24 µg/mL respectively. Also, the results proved that sulphur oxidizing bacteria Acidithiobacillus thiooxidans is capable of solubilizing dolomitic tailings from the Jhamarkotra mines. Finally, an important factor taken into account was solubilization of residual phosphate along with dolomite in the tailings. This combined action affects the solubilization behaviour of the residue, which was also showed successfully with the assayed laboratory studies.

  4. Mineral phosphate solubilization by wild type and radiation induced mutants of pantoea dispersa and pantoea terrae

    Murugesan, Senthilkumar; Lee, Young Keun; Kim, Jung Hun [Korea Atomic Energy Research Institte, Jeongeup (Korea, Republic of)


    Three mineral phosphate solubilizing (MPS) bacteria where isolated from rhizosphere soil samples of common bean and weed plants. 16S rDNA analysis indicated that the isolate P2 and P3 are closely related to Pantoea dispersa while isolate P4 is closely related to Pantoea terrae. Islates P2 and P3 recorded 381.60 {mu}g ml{sup -1} of tricalcium phosphate (TCP) solubilization respectively on 3 days incubation. Isolate P4 recorded the TCP solubilization of 215.85 {mu}g ml{sup -1} and the pH was dropped to 4.44 on 24 h incubation. Further incubation of P4 sharply decreased the available phosphorous to 28.94 {mu}g ml{sup -1} and pH level was raised to 6.32. Gamma radiation induced mutagenesis was carried out at LD{sub 99} dose of the wild type strains. The total of 14 mutant clones with enhanced MPS activity and 4 clones with decreased activity were selected based on solubilization index (SI) and phosphate solubilization assay. Mutant P2-M1 recorded the highest P-solubilizing potential among any other wild or mutnat clones by releasing 504.21 {mu}g ml{sup -1} of phosphorous i.e. 35% higher than its wild type by the end of day 5. A comparative evaluation of TCP solubilization by wild type isolates of Pantoea and their mutants, led to select three MPS mutant clones such as P2-M1, P3-M2 and P3-M4 with a potential to release >471.67 {mu}g ml{sup {sub {sup 1}}} of phosphorous from TCP. These over expressing mutant clones are considered as suitable candidates for biofertilization.

  5. Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil

    Narayanan Kannan


    Full Text Available Abstract Background Phosphorus is an essential macronutrient for the growth of plants. However, in most soils a large portion of phosphorus becomes insoluble and therefore, unavailable to plants. Knowledge on biodiversity of phosphate-solubilizing fluorescent pseudomonads is essential to understand their ecological role and their utilization in sustainable agriculture. Results Of 443 fluorescent pseudomonad strains tested, 80 strains (18% showed positive for the solubilization of tri-calcium phosphate (Ca3(PO42 by the formation of visible dissolution halos on Pikovskaya's agar. These phosphate solubilizing strains showed high variability in utilizing various carbon sources. Numerical taxonomy of the phosphate solubilizing strains based on their carbon source utilization profiles resulted into three major phenons at a 0.76 similarity coefficient level. Genotypic analyses of strains by BOX (bacterial repetitive BOX element-polymerase chain reaction (PCR resulted into three distinct genomic clusters and 26 distinct BOX profiles at a 80% similarity level. On the basis of phenotypic characterization and 16S rRNA gene phylogenetic analyses strains were identified as Pseudomonas aeruginosa, P. mosselii, P. monteilii, P. plecoglossicida, P. putida, P. fulva and P. fluorescens. These phosphate solubilizing strains also showed the production of plant growth promoting enzymes, hormones and exhibited antagonism against phytopathogenic fungi that attack on various crops. Gene specific primers have identified the putative antibiotic producing strains. These putative strains were grown in fermentation media and production of antibiotics was confirmed by thin layer chromatography (TLC and high performance liquid chromatography (HPLC. Conclusion Present study revealed a high degree of functional and genetic diversity among the phosphate solubilizing fluorescent pseudomonad bacteria. Due to their innate potential of producing an array of plant growth promoting

  6. Solubilization of Polycyclic Aromatic Hydrocarbons by Single and Binary Mixed Rhamnolipid-Sophorolipid Biosurfactants.

    Song, Dandan; Liang, Shengkang; Yan, Lele; Shang, Yujun; Wang, Xiuli


    Biosurfactants are promising additives for surfactant enhanced remediation (SER) technologies due to their low toxicity and high biodegradability. To develop green and efficient additives for SER, the aqueous solubility enhancements of polycyclic aromatic hydrocarbons (PAHs; naphthalene, phenanthrene, and pyrene) by rhamnolipid (RL) and sophorolipid (SL) biosurfactants were investigated in single and binary mixed systems. The solubilization capacities were quantified in terms of the solubility enhancement factor, molar solubilization ratio (MSR), and micelle-water partition coefficient (). Rughbin's model was applied to evaluate the interaction parameters (β) in the mixed RL-SL micelles. The solubility of the PAHs increased linearly with the glycolipid concentration above the critical micelle concentration (CMC) in both single and mixed systems. Binary RL-SL mixtures exhibited greater solubilization than individual glycolipids. At a SL molar fraction of 0.7 to 0.8, the solubilization capacity was the greatest, and the MSR and reached their maximum values, and β values became positive. These results suggest that the two biosurfactants act synergistically to increase the solubility of the PAHs. The solubilization capacity of the RL-SL mixtures increased with increasing temperature and decreased with increasing salinity. The aqueous solubility of phenanthrene reached a maximum value at pH of 5.5. Moreover, the mixed RL-SL systems exhibited a strong ability to solubilize PAHs, even in the presence of heavy metal ions. These mixed biosurfactant systems have the potential to improve the performance of SER technologies using biosurfactants to solubilize hydrophobic organic contaminants by decreasing the applied biosurfactant concentration, which reduces the costs of remediation.

  7. Optimized lysis buffer reagents for solubilization and preservation of proteins from cells and tissues.

    Hwang, Byeong Hee; Tsai, Kenneth Y; Mitragotri, Samir


    Reagents that facilitate solubilization of cells and tissues while preserving the biological activity of their constituents play a major role in various applications including drug delivery. Such reagents are necessary for the accurate determination of cellular and tissue concentrations of proteins, peptides, and nucleic acids, and to measure therapeutic efficacy of drug delivery technologies. Surfactant-based reagents are commonly used for this purpose; however, their utility is marred either by limited ability to solubilize or tendency to denature the proteins during solubilization. Here, we report on the screening and identification of combinations of nonionic and zwitterionic surfactants that possess excellent ability to solubilize mechanically strong and elastic tissues such as skin, while preserving its protein constituents. The leading combination, comprising an equi-mass mixture of 3-(N,N-dimethyl myristyl ammonio) propanesulfonate (TPS, CAS number:14933-09-6) and polyoxyethylene(10) cetyl ether (Brij® C10, CAS number: 9004-95-9) with a total surfactant concentration 0.5 % w/v, solubilized keratinocytes and preserved the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme in its extracts at room temperature for 7 days. The ability of this mixture to preserve GAPDH activity far exceeded that of a commonly used reagent, Triton-X100. The same mixture also helped solubilize mouse skin to extract proteins and maintain detectable activity of GAPDH in the extract for 1 day. Several other mixtures of nonionic and zwitterionic surfactants were studied. These mixtures provide new reagents for solubilization of cells and tissues for research as well as technological applications.

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

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


    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 NAD(P)H: 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

  9. Immunolocalization of a microsomal prostaglandin E synthase in rabbit kidney.

    Fuson, Amanda L; Komlosi, Peter; Unlap, Tino M; Bell, P Darwin; Peti-Peterdi, János


    PGE2, the major cyclooxygenase (COX) metabolite of arachidonic acid, is an important paracrine regulator of numerous tubular and vascular functions in the kidney. To date, COX activity has been considered the key step in prostaglandin synthesis and is well characterized. However, much less is known about the recently cloned microsomal PGE2 synthase (mPGES), the terminal enzyme of PGE2 synthesis, which converts COX-derived PGH2 to the biologically important PGE2. Present studies provide the detailed localization of mPGES protein in the rabbit kidney using immunohistochemistry. In the cortex, strong mPGES labeling was found in the macula densa (MD) and principal cells of the connecting segment and cortical collecting tubule but not in intercalated cells. The medulla was abundant in mPGES-positive structures, with heavy labeling in the collecting duct system. In descending thin limbs and renal medullary interstitial cells, mPGES expression was less intense, and it was below the limits of detection in the vasa recta. Expression of MD mPGES, similarly to COX-2, was greatly increased in response to low-salt diet and angiotensin I-converting enzyme inhibition by captopril. These findings suggest autocrine regulation of renal salt and water transport by PGE2 in descending thin limb and collecting tubule and a paracrine effect of PGE2 on the glomerular and medullary vasculature. Similar to other organs, mPGES in the kidney is an inducible enzyme and may be similarly regulated and acts in concert with COX-2.

  10. Isolation and phosphate-solubilizing ability of a fungus, Penicillium sp. from soil of an alum mine.

    Chai, Bo; Wu, Yan; Liu, Pengming; Liu, Biao; Gao, Meiying


    The use of microorganisms to solubilize elemental phosphorus from insoluble rock phosphate is a promising method to greatly reduce not only environmental pollution but also production costs. Phosphate-solubilizing microorganisms were isolated from soils in China, and a fungus strain (PSM11-5) from a soil sample from an alum mine, with the highest phosphate solubilization potential, was selected and identified as a Penicillium sp. Strain PSM11-5 could grow in buffered medium with pH values between 3.0 and 8.0 and showed phosphate solubilizing activity at pH values from 5.0 to 8.0. It also exhibited a degree of tolerance to the heavy metal ions, Cd(2+), Co(2+), and Cr(6+). PSM11-5 could rapidly solubilize tricalcium phosphate, and a high phosphate-solubilizing efficiency of 98% was achieved in an optimized medium. The strain could solubilize rock phosphate and aluminum phosphate with a solubilizing efficiency of approximately 74.5%, but did not solubilize iron phosphate. Solubilization of phosphate depended on acidification. Analysis of PSM11-5 culture supernatants by capillary electrophoresis showed that tricalcium phosphate was solubilized to PO(4) (3-) and Ca(2+) , and that the organic acid produced by the fungus was mainly gluconic acid at approximately ca. 13 g l(-1). In addition, PSM11-5 produced ca. 830 mg l(-1) of citric acid when it was used to solubilize rock phosphate. These excellent properties of strain PSM11-5 suggest that the fungus has potential for agricultural and industrial utilization.

  11. Phosphate solubilization and multiple plant growth promoting properties of rhizobacteria isolated from chickpea (Cicer aeritinum L.) producing areas of Ethiopia

    Midekssa, Mulissa J.; Löscher, Carolin; Ruth A Schmitz; Assefa, Fassil


    Chickpea is one of the major legume crops widely grown in Ethiopia. The low availability of phosphorus in soil is among the stresses that constrain the production of this crop in the country. However, there are rhizobacteria capable of solubilizing insoluble forms of phosphorus in soil and make it available to the plant. Thus, this study was aimed at isolation and characterization of phosphate solubilizing bacteria from chickpea rhizosphere. Fifty phosphate solubilizing bacterial strains were...

  12. Comparison of bile salt/phosphatidylcholine mixed micelles in solubilization to sterols and stability.

    Guo, Qin; Cai, Jie; Li, Pengyu; Xu, Dongling; Ni, Xiaomin; Wen, Hui; Liu, Dan; Lin, Suizhen; Hu, Haiyan


    Androst-3β,5α,6β-triol (Triol) is a promising neuroprotective agent, but its poor solubility restricts its development into parenteral preparations. In this study, Triol is significantly solubilized by bile salt/phosphatidylcholine mixed micelles (BS/PC-MM). All BS/PC-MM systems are tested to remarkably improve the drug solubility with various stabilities after drug loading. Among them, the sodium glycocholate (SGC)/egg phosphatidylcholine (EPC) system with 2:1 ratio in weight and the total concentration of SGC and EPC of 100 mg/mL is proved to produce stable mixed micelles with high drug loading. It is found that the stability of drug-loaded mixed micelles is quite different, which might be related to the change in critical micelle concentration (CMC) after incorporating drugs. SGC/EPC and SGC/soya phosphatidylcholine (SPC) remain transparent under accelerated conditions and manifest a decreased CMC (dropping from 0.105 to 0.056 mg/mL and from 0.067 to 0.024 mg/mL, respectively). In contrast, swine bile acid-sodium salt (SBA-Na)/PC and sodium deoxycholate (SDC)/PC are accompanied by drug precipitation and reached the maximum CMC on the first and the third days, respectively. Interestingly, the variation of CMC under accelerated testing conditions highly matches the drug-precipitating event in the primary stability experiment. In brief, the bile salt/phosphatidylcholine system exists as a potential strategy of improving sterol drug solubility. CMC variation under accelerated testing conditions might be a simple and easy method to predict the stability of drug-loaded mixed micelles.

  13. Targeted label-free approach for quantification of epoxide hydrolase and glutathione transferases in microsomes.

    Song, Wei; Yu, Longjiang; Peng, Zhihong


    The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography-tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1 pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.

  14. Metabolism of 17alpha-hydroxyprogesterone caproate by hepatic and placental microsomes of human and baboons.

    Yan, Ru; Nanovskaya, Tatiana N; Zharikova, Olga L; Mattison, Donald R; Hankins, Gary D V; Ahmed, Mahmoud S


    Recent data from our laboratory revealed the formation of an unknown metabolite of 17 hydroxyprogesterone caproate (17-HPC), used for treatment of preterm deliveries, during its perfusion across the dually perfused human placental lobule. Previously, we demonstrated that the drug is not hydrolyzed, neither in vivo nor in vitro, to progesterone and caproate. Therefore, the hypothesis for this investigation is that 17-HPC is actively metabolized by human and baboon (Papio cynocephalus) hepatic and placental microsomes. Baboon hepatic and placental microsomes were investigated to validate the nonhuman primate as an animal model for drug use during pregnancy. Data presented here indicate that human and baboon hepatic microsomes formed several mono-, di-, and tri-hydroxylated derivatives of 17-HPC. However, microsomes of human and baboon placentas metabolized 17-HPC to its mono-hydroxylated derivatives only in quantities that were a fraction of those formed by their respective livers, except for two metabolites (M16' and M17') that are unique for placenta and contributed to 25% and 75% of the total metabolites formed by human and baboon, respectively. The amounts of metabolites formed, relative to each other, by human and baboon microsomes were different suggesting that the affinity of 17-HPC to CYP enzymes and their activity could be species-dependent.

  15. Determination of microsomal CYP2A6 activity by high performance liquid chromatography

    Lavhekar S


    Full Text Available Coumarin has been reported to be a reliable probe for the determination of human microsomal CYP2A6 activity. Coumarin is converted to 7-hydroxycoumarin by CYP2A6. A high pressure liquid chromatographic assay for the estimation of CYP2A6 activity in microsomes was evaluated. A RP C-18 Novapak Waters (15 cm x 3.9 mm, 5 µm column was used for the assay. The mobile phase composition was methanol : 1% glacial acetic acid (35:65 v/v (pH~3.1, with a flow rate of 0.6 ml/min, injection volume of 100 µl and detection at 320 nm. The retention times for coumarin and 7-hydroxycoumarin were 8.7 min and 5.3 min, respectively. The limit of detection (LOD was 0.05 µM, and the limit of quantitation (LOQ was 0.1 µM, for 7-hydroxycoumarin. The percent coefficient of variation associated with 7-hydroxycoumarin determination after duplicate estimation was found to be in the range 0.03 to 3.6% for buffer matrix and 0.1 to 6.5% for microsomal matrix. The mean rate of 7-hydroxycoumarin formation in guinea pig liver microsomes was 0.084 nmol/min/nmol P450. Coumarin 7-hydroxylase activity was absent in rat liver microsomes. No interference was observed from incubation mixture components.

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

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


    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.

  17. Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation.

    Abas, Lindy; Luschnig, Christian


    Isolation of a microsomal membrane fraction is a common procedure in studies involving membrane proteins. By conventional definition, microsomal membranes are collected by centrifugation of a postmitochondrial fraction at 100,000g in an ultracentrifuge, a method originally developed for large amounts of mammalian tissue. We present a method for isolating microsomal-type membranes from small amounts of Arabidopsis thaliana plant material that does not rely on ultracentrifugation but instead uses the lower relative centrifugal force (21,000g) of a microcentrifuge. We show that the 21,000g pellet is equivalent to that obtained at 100,000g and that it contains all of the membrane fractions expected in a conventional microsomal fraction. Our method incorporates specific manipulation of sample density throughout the procedure, with minimal preclearance, minimal volumes of extraction buffer, and minimal sedimentation pathlength. These features allow maximal membrane yields, enabling membrane isolation from limited amounts of material. We further demonstrate that conventional ultracentrifuge-based protocols give submaximal yields due to losses during early stages of the procedure; that is, extensive amounts of microsomal-type membranes can sediment prematurely during the typical preclearance steps. Our protocol avoids such losses, thereby ensuring maximal yield and a representative total membrane fraction. The principles of our method can be adapted for nonplant material.

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

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


    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.

  19. Bio-liquefaction/solubilization of low-rank Turkish lignites and characterization of the products

    Yesim Basaran; Adil Denizli; Billur Sakintuna; Alpay Taralp; Yuda Yurum [Hacettepe University, Ankara (Turkey). Department of Environmental Sciences


    The effect of some white-rot fungi on the bio-liquefaction/solubilization of two low-rank Turkish coals and the chemical composition of the liquid products and the microbial mechanisms of coal conversion were investigated. Turkish Elbistan and Beypazari lignites were used in this study. The white-rot fungi received from various laboratories used in the bio-liquefaction/solubilization of the lignites were Pleurotus sajor-caju, Pleurotus sapidus, Pleurotus florida, Pleurotus ostreatus, Phanerochaete chrysosporium, and Coriolus versicolor. FT-IR spectra of raw and treated coal samples were measured, and bio-liquefied/solubilized coal samples were investigated by FT-IR and LC-MS techniques. The Coriolus versicolor fungus was determined to be most effective in bio-liquefying/solubilizing nitric acid-treated Elbistan lignite. In contrast, raw and nitric acid-treated Beypazari lignite seemed to be unaffected by the action of any kind of white-rot fungi. The liquid chromatogram of the water-soluble bio-liquefied/solubilized product contained four major peaks. Corresponding mass spectra of each peak indicated the presence of very complicated structures. 17 refs., 9 figs., 2 tabs.

  20. Phosphate solubilizing ability of Emericella nidulans strain V1 isolated from vermicompost.

    Bhattacharya, Satya Sunder; Barman, Soma; Ghosh, Ranjan; Duary, Raj Kumar; Goswami, Linee; Mandal, Narayan C


    Phosphorus is one of the key factors that regulate soil fertility. Its deficiencies in soil are largely replenished by chemical fertilizers. The present study was aimed to isolate efficient phosphate solubilizing fungal strains from Eisenia fetida vermicompost. Out of total 30 fungal strains the most efficient phosphate solubilizing one was Emericella (Aspergillus) nidulans V1 (MTCC 11044), identified by custom sequencing of beta-tubulin gene and BLAST analysis. This strain solubilized 13 to 36% phosphate from four different rock phosphates. After three days of incubation of isolated culture with black Mussorie phosphate rock, the highest percentage of phosphate solubilization was 35.5 +/- 1.01 with a pH drop of 4.2 +/- 0.09. Kinetics of solubilization and acid production showed a linear relationship until day five of incubation. Interestingly, from zero to tenth day of incubation, solubility of soil phosphate increased gradually from 4.31 +/- 1.57 to 13.65 +/- 1.82 (mg kg(-1)) recording a maximum of 21.23 +/- 0.54 on day 45 in respect of the V1 isolate. Further, enhanced phosphorus uptake by Phaseolus plants with significant pod yield due to soil inoculation of Emericella nidulans V1 (MTCC 11044), demonstrated its prospect as an effective biofertilizer for plant growth.

  1. Solubilization of thiamine triphosphatase from the electric organ of Electrophorus electricus.

    Bettendorff, L; Longrée, I; Wins, P; Schoffeniels, E


    The membrane-associated, anion-regulated thiamine triphosphatase from Electrophorus electricus electric organ can be solubilized by various neutral detergents. Polyoxyethylene ethers are the most effective. Anionic detergents readily inactivate the enzyme. A 6.4-fold increase in specific activity is obtained by successive treatment of crude membranes with octanoyl-N-methylglucamide, which solubilized other proteins, and Lubrol-PX with releases 60% of the thiamine triphosphatase (TTPase) activity. Solubilization by Lubrol-PX rapidly modifies kinetic parameters. The Km, Vmax and pH optimum are decreased. However, the solubilized TTPase may be kept at 0 degrees C for many hours without further change in specific activity. At 35 degrees C, the half-life is still 83 min at pH 5.0, but denaturation becomes rapid at pH greater than or equal to 7. Solubilization modifies anion effects on TTPase activity. The activating effect of nitrate is nearly lost, while inhibition by sulfate is no longer time-dependent.

  2. Solubilization of Rock Phosphate in Liquid Culture by Fungal Isolates from Rhizosphere Soil

    WANG Guang-Hua; ZHOU De-Rui; YANG Qian; JIN Jian; LIU Xiao-Bing


    Rock phosphate (RP) is a low efficiency P fertilizer that is directly applied to the soil and can be solubilized by phosphate-solubilizing microorganisms (PSMs) in fermentation or soil conditions. This study investigated dynamic solubilization of 2 concentrations of rock phosphate in a liquid culture with different dosages of glucose by two fungal isolates,Aspergillus niger P39 and Penicillium oxalicum P66, from soybean and wheat rhizosphere soil. Although during the 20day culture period A. niger P39 had a stronger ability to acidify the culture media than P. oxalicum P66, soluble P concentrations at glucose dosages of 30 and 50 gL-1 with RP of 15 g L-1 in the culture solution were much higher by P. oxalicum P66. The greater effectiveness of P. oxalicum P66 compared to A. niger P39 in the solubilization of RP was strongly associated with the production of organic acids. This study suggested that for RP solubilization the type rather than the concentration of PSM-produced organic acids was more important.

  3. Solubilization properties of polycyclic aromatic hydrocarbons by saponin, a plant-derived biosurfactant.

    Zhou, Wenjun; Yang, Juanjuan; Lou, Linjie; Zhu, Lizhong


    The enhanced solubilization of polycyclic aromatic hydrocarbons (PAHs) by saponin, a plant-derived non-ionic biosurfactant, was investigated. The results indicated that the solubilization capabilities of saponin for PAHs were greater than some representative synthetic non-ionic surfactants and showed strong dependence on solution pH and ionic strength. The molar solubilization ratio (MSR) of saponin for phenanthrene was about 3-6 times of those of the synthetic non-ionic surfactants, and decreased by about 70% with the increase of solution pH from 4.0 to 8.0, but increased by approximately 1 times with NaCl concentration increased from 0.01 to 1.0 M. Heavy metal ions can enhance saponin solubilization for phenanthrene and the corresponding MSR values increased by about 25% with the presence of 0.01 M of Cd2+ or Zn2+. Saponin is more effective in enhancing PAHs solubilization than synthetic non-ionic surfactants and has potential application in removing organic pollutants from contaminated soils.

  4. Enhancing uranium solubilization in soils by citrate, EDTA, and EDDS chelating amendments.

    Lozano, J C; Blanco Rodríguez, P; Tomé, F Vera; Calvo, C Prieto


    A systematic study was made of the effects of three soil amendments on the solubilization of uranium from a granitic soil. The aim was to optimize solubilization so as to enhance bioavailability for the purposes of remediation. The three amendments tested were with citrate, EDTA, and EDDS as chelating agents. The effects of pH, chelator concentration, and leaching time were studied. The most important factor in uranium solubilization was found to be the pH. In the absence of chelating agents, the greatest solubilization was obtained for alkaline conditions, with values representing about 15% of the total uranium activity in the bulk soil. There were major differences in uranium solubilization between the different amendments. The citrate treatment was the most efficient at acidic pH, particularly with the greatest concentration of citrate tested (50 mmol kg(-1)) after 6 days of treatment. Under these conditions, the uranium concentration in solution was greater by a factor of 356 than in the control suspension, and represented some 63% of the uranium concentration in the bulk soil. Under alkaline conditions, the EDTA and EDDS treatments gave the greatest uranium activity concentrations in solution, but these concentrations were much lower than those with the citrate amendment, and were not very different from the control results. The uranium extraction yield with EDDS amendment was greater than with EDTA.

  5. Microbial Phosphorus Solubilization and Its Potential for Use in Sustainable Agriculture

    Alori, Elizabeth T.; Glick, Bernard R.; Babalola, Olubukola O.


    The use of excess conventional Phosphorus (P) fertilizers to improve agricultural productivity, in order to meet constantly increasing global food demand, potentially causes surface and ground water pollution, waterway eutrophication, soil fertility depletion, and accumulation of toxic elements such as high concentration of selenium (Se), arsenic (As) in the soil. Quite a number of soil microorganisms are capable of solubilizing/mineralizing insoluble soil phosphate to release soluble P and making it available to plants. These microorganisms improve the growth and yield of a wide variety of crops. Thus, inoculating seeds/crops/soil with Phosphate Solubilizing Microorganisms (PSM) is a promising strategy to improve world food production without causing any environmental hazard. Despite their great significance in soil fertility improvement, phosphorus-solubilizing microorganisms have yet to replace conventional chemical fertilizers in commercial agriculture. A better understanding of recent developments in PSM functional diversity, colonizing ability, mode of actions and judicious application should facilitate their use as reliable components of sustainable agricultural systems. In this review, we discussed various soil microorganisms that have the ability to solubilize phosphorus and hence have the potential to be used as bio fertilizers. The mechanisms of inorganic phosphate solubilization by PSM and the mechanisms of organic phosphorus mineralization are highlighted together with some factors that determine the success of this technology. Finally we provide some indications that the use of PSM will promote sustainable agriculture and conclude that this technology is ready for commercial exploitation in various regions worldwide. PMID:28626450

  6. Phosphorus solubilization and plant growth enhancement by arsenic-resistant bacteria.

    Ghosh, Piyasa; Rathinasabapathi, Bala; Ma, Lena Q


    Phosphorus is an essential nutrient, which is limited in most soils. The P solubilization and growth enhancement ability of seven arsenic-resistant bacteria (ARB), which were isolated from arsenic hyperaccumulator Pteris vittata, was investigated. Siderophore-producing ARB (PG4, 5, 6, 9, 10, 12 and 16) were effective in solubilizing P from inorganic minerals FePO4 and phosphate rock, and organic phytate. To reduce bacterial P uptake we used filter-sterilized Hoagland medium containing siderophores or phytase produced by PG12 or PG6 to grow tomato plants supplied with FePO4 or phytate. To confirm that siderophores were responsible for P release, we compared the mutants of siderophore-producing bacterium Pseudomonas fluorescens Pf5 (PchA) impaired in siderophore production with the wild type and test strains. After 7d of growth, mutant PchA solubilized 10-times less P than strain PG12, which increased tomato root biomass by 1.7 times. For phytate solubilization by PG6, tomato shoot biomass increased by 44% than control bacterium Pseudomonas chlororaphis. P solubilization by ARB from P. vittata may be useful in enhancing plant growth and nutrition in other crop plants.

  7. Microbial Phosphorus Solubilization and Its Potential for Use in Sustainable Agriculture

    Elizabeth T. Alori


    Full Text Available The use of excess conventional Phosphorus (P fertilizers to improve agricultural productivity, in order to meet constantly increasing global food demand, potentially causes surface and ground water pollution, waterway eutrophication, soil fertility depletion, and accumulation of toxic elements such as high concentration of selenium (Se, arsenic (As in the soil. Quite a number of soil microorganisms are capable of solubilizing/mineralizing insoluble soil phosphate to release soluble P and making it available to plants. These microorganisms improve the growth and yield of a wide variety of crops. Thus, inoculating seeds/crops/soil with Phosphate Solubilizing Microorganisms (PSM is a promising strategy to improve world food production without causing any environmental hazard. Despite their great significance in soil fertility improvement, phosphorus-solubilizing microorganisms have yet to replace conventional chemical fertilizers in commercial agriculture. A better understanding of recent developments in PSM functional diversity, colonizing ability, mode of actions and judicious application should facilitate their use as reliable components of sustainable agricultural systems. In this review, we discussed various soil microorganisms that have the ability to solubilize phosphorus and hence have the potential to be used as bio fertilizers. The mechanisms of inorganic phosphate solubilization by PSM and the mechanisms of organic phosphorus mineralization are highlighted together with some factors that determine the success of this technology. Finally we provide some indications that the use of PSM will promote sustainable agriculture and conclude that this technology is ready for commercial exploitation in various regions worldwide.

  8. Solubilization of octane in electrostatically-formed surfactant-polymer complexes.

    Zhang, Hui; Zeeb, Benjamin; Salminen, Hanna; Feng, Fengqin; Weiss, Jochen


    Polymers can be used to modulate the stability and functionality of surfactant micelles. The purpose of this study was to investigate the solubilization of an octane oil-in-water emulsion in mixtures of an anionic polymer (carboxymethyl cellulose) and anionic sodium dodecylsulphate (SDS), nonionic polyoxyethylene sorbitan monooleate (Tween 80) and cationic cetyltrimethylammonium bromide (CTAB) surfactant micelles using dynamic light scattering, microelectrophoresis and turbidity measurements. The results showed that the addition of anionic carboxymethyl cellulose accelerated octane solubilization in cationic CTAB and CTAB-Tween 80 micelles, but did not affect the solubilization behaviors of micelles that were nonionic and anionic. The surfactant-polymer interactions were also studied using isothermal titration calorimetry (ITC) to characterize different physiochemical interaction regions depending on surfactant concentration in surfactant-polymer systems. Upon octane solubilization in CTAB-carboxymethyl cellulose mixtures, shape transitions of polymer-micelle complexes may have taken place that altered light scattering behavior. Based on these results, we suggest a mechanism for oil solubilization in electrostatically-formed surfactant-polymer complexes.

  9. The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris.

    Klug, Lisa; Tarazona, Pablo; Gruber, Clemens; Grillitsch, Karlheinz; Gasser, Brigitte; Trötzmüller, Martin; Köfeler, Harald; Leitner, Erich; Feussner, Ivo; Mattanovich, Diethard; Altmann, Friedrich; Daum, Günther


    The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.

  10. Characterization of triptolide hydroxylation by cytochrome P450 in human and rat liver microsomes.

    Li, W; Liu, Y; He, Y-Q; Zhang, J-W; Gao, Y; Ge, G-B; Liu, H-X; Huo, H; Liu, H-T; Wang, L-M; Sun, J; Wang, Q; Yang, L


    Triptolide, the primary active component of a traditional Chinese medicine Tripterygium wilfordii Hook F, has a wide range of pharmacological activities. In the present study, the metabolism of triptolide by cytochrome P450s was investigated in human and rat liver microsomes. Triptolide was converted to four metabolites (M-1, M-2, M-3, and M-4) in rat liver microsomes and three (M-2, M-3, and M-4) in human liver microsomes. All the products were identified as mono-hydroxylated triptolides by liquid chromatography-mass spectrometry (LC-MS). The studies with chemical selective inhibitors, complementary DNA-expressed human cytochrome P450s, correlation analysis, and enzyme kinetics were also conducted. The results demonstrate that CYP3A4 and CYP2C19 could be involved in the metabolism of triptolide in human liver, and that CYP3A4 was the primary isoform responsible for its hydroxylation.

  11. Study of the mechanisms by which microorganisms solubilize and/or liquefy Spanish coals

    Laborda, F.; Fernandez, M.; Luna, N.; Monistrol, I.F. [Campus Universitario Alcala de Henares (Spain). Dept. de Microbiologia y Parasitologia


    Cell-free culture filtrates of fungi were tested on their capacity to solubilize different Spanish coals. Two kinds of cell-free culture filtrates were used: a non-coal induced cell-free culture filtrate (NCI) and a coal induced cell-free culture filtrate (CI). Either of them was able to solubilize hard coal, subbituminous coal and most efficiently lignite. In most of the culture filtrates the measured pH values were approximately neutral, even acidic in some cases. The agent(s) for coal solubilization that was present in NCI was heat stable (100{degree}C) but the active substance(s) in CI was thermosensitive. Microorganisms were observed by electron microscopy to grow on the coal surface and cover it. A fibrilar extracelluar polymer which could facilitate not only the adhesion to the coal particle but microbial attack too, was produced by the microorganisms. 11 refs., 8 figs., 3 tabs.

  12. Rapid room temperature solubilization and depolymerization of polymeric lignin at high loadings

    Sun, Jian; Dutta, Tanmoy; Parthasarathi, Ramakrishnan; Kim, Kwang Ho; Tolic, Nikola; Chu, Rosalie K.; Isern, Nancy G.; Cort, John R.; Simmons, Blake A.; Singh, Seema


    The relatively poor solubility of lignin in most pretreatment solvents remains one of the biggest challegnes in lignin valorization to improve overall biorefinery economics. In this work, rapid room temperature solubilization of lignin at high solid loadings (>30 wt%) can be easily achieved in a single step using ethylene glycol (EG). The solubilized lignin can be rapidly and quantitively recovered with the addtion of ethanol. The computational and nuclear magnetic resonance (NMR) spectroscopic studies confirm that strong hydrogen bond interactions between EG and the free hydroxyl groups present in lignin contribute to the lignin dissolution. In addition, hydrogen peroxide mediated depolymerization of dissolved lignin at low temperature (80 oC) was tested and the effect of EG molecules on depolymerization of ligin was also theoritically studied. The findings of this work provide mechanistic insights of hydrogen bond interactions in high lignin solubilization and valorization.

  13. Solubilization and Interaction Studies of Bile Salts with Surfactants and Drugs: a Review.

    Malik, Nisar Ahmad


    In this review, bile salt, bile salt-surfactant, and bile salt-drug interactions and their solubilization studies are mainly focused. Usefulness of bile salts in digestion, absorption, and excretion of various compounds and their rare properties in ordering the shape and size of the micelles owing to the presence of hydrophobic and hydrophilic faces are taken into consideration while compiling this review. Bile salts as potential bio-surfactants to solubilize drugs of interest are also highlighted. This review will give an insight into the selection of drugs in different applications as their properties get modified by interaction with bile salts, thus influencing their solution behavior which, in turn, modifies the phase-forming behavior, microemulsion, and clouding phenomenon, besides solubilization. Finally, their future perspectives are taken into consideration to assess their possible uses as bio-surfactants without side effects to human beings.

  14. Assessing the Zinc solubilization ability of Gluconacetobacter diazotrophicus in maize rhizosphere using labelled (65)Zn compounds.

    Sarathambal, C; Thangaraju, M; Paulraj, C; Gomathy, M


    Solubilization of insoluble zinc compounds like ZnCO(3) and ZnO by G. diazotrophicus was confirmed using radiotracers. The zinc compounds (ZnCO(3) and ZnO) were tagged with (65)Zn. (65)ZnCO(3) and (65)ZnO was effectively solubilized and the uptake of zn by the plants also more in G. diazotrophicus inoculated treatments compared to the uninoculated treatments. Three types of soils (Zn deficientsterile, Zn deficient-unsterile, and Zn sufficient-sterile) were used in experiment. Among the three soils, Zn deficient-unsterile soil registered maximum zinc solubilization compared to other two soils. This may be due to other soil microorganisms in unsterile soil. Application of ZnO with G. diazotrophicus showed better uptake of the nutrient.

  15. Concomitant rock phosphate dissolution and lead immobilization by phosphate solubilizing bacteria (Enterobacter sp.).

    Park, Jin Hee; Bolan, Nanthi; Megharaj, Mallavarapu; Naidu, Ravi


    This paper examines the potential value of phosphate solubilizing bacteria (Enterobacter cloacae) in the dissolution of rock phosphate (RP) and subsequent immobilization of lead (Pb) in both bacterial growth medium and soils. Enterobacter sp. showed resistance to Pb and the bacterium solubilized 17.5% of RP in the growth medium. Enterobacter sp. did not enhance Pb immobilization in solution because of acidification of bacterial medium, thereby inhibiting the formation of P-induced Pb precipitation. However, in the case of soil, Enterobacter sp. increased Pb immobilization by 6.98, 25.6 and 32.0% with the RP level of 200, 800 and 1600 mg P/kg, respectively. The immobilization of Pb in Pb-spiked soils was attributed to pyromorphite formation as indicated by XRD analysis. Inoculation of phosphate solubilizing bacteria with RP in soil can be used as an alternative technique to soluble P compounds which can cause eutrophication of surface water.

  16. EPS solubilization treatment by applying the biosurfactant rhamnolipid to reduce clogging in constructed wetlands.

    Du, Mingpu; Xu, Dong; Trinh, Xuantung; Liu, Shuangyuan; Wang, Mei; Zhang, Yi; Wu, Junmei; Zhou, Qiaohong; Wu, Zhenbin


    Application of extracellular polymeric substances (EPS) solubilization treatment with biosurfactant rhamnolipid (RL) to reduce clogging in constructed wetlands was first conducted in this study. The results showed significant improvement in the solubilization and dispersion of clogging matter following the treatment. And RL dosage of 0.09-0.15g/L altered microbial group make-up and had an overall positive effect on the growth of microorganisms. Moreover, RL was found to enhance EPS dissolution and dispersion, which was beneficial for the release of enzymes embedded in the EPS, and resulted in enhanced pollutant removal. The treatment had no apparent detrimental effect on wetland plants. Our results indicate that the optimum dosage of RL is 0.12g/L, and that the approach provides a promising and moderate option to reverse wetland clogging through RL-mediated solubilization treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Species and sex differences in propofol glucuronidation in liver microsomes of humans, monkeys, rats and mice.

    Mukai, M; Isobe, T; Okada, K; Murata, M; Shigeyama, M; Hanioka, N


    Propofol (2,6-diisopropylphenol) is a short-acting anesthetic commonly used in clinical practice, and is rapidly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, propofol glucuronidation was examined in the liver microsomes of male and female humans, monkeys, rats, and mice. The kinetics of propofol glucuronidation by liver microsomes fit the substrate inhibition model for humans and mice, the Hill model for monkeys, and the isoenzyme (biphasic) model for rats. The K(m), V(max), and CL(int) values of human liver microsomes were 50 μM, 5.6 nmol/min/mg protein, and 110 μL/min/mg protein, respectively, for males, and 46 μM, 6.0 nmol/min/mg protein, and 130 μL/min/mg protein, respectively, for females. The rank order of the CL(int) or CL(max) (in vitro clearance) values of liver microsomes was mice humans > monkeys > rats (high-affinity phase) rats (low-affinity phase) in both males and females. Although no significant sex differences were observed in the values of kinetic parameters in any animal species, the in vitro clearance values of liver microsomes were males females in monkeys, rats (high-affinity phase), and mice. These results demonstrated that the kinetic profile of propofol glucuronidation by liver microsomes markedly differed among humans, monkeys, rats, and mice, and suggest that species and sex differences exist in the roles of UGT isoform(s), including UGT1A9, involved in its metabolism.

  18. Fatty acyl-CoA esters inhibit glucose-6-phosphatase in rat liver microsomes.

    Fulceri, R; Gamberucci, A; Scott, H M; Giunti, R; Burchell, A; Benedetti, A


    In native rat liver microsomes glucose 6-phosphatase activity is dependent not only on the activity of the glucose-6-phosphatase enzyme (which is lumenal) but also on the transport of glucose-6-phosphate, phosphate and glucose through the respective translocases T1, T2 and T3. By using enzymic assay techniques, palmitoyl-CoA or CoA was found to inhibit glucose-6-phosphatase activity in intact microsomes. The effect of CoA required ATP and fatty acids to form fatty acyl esters. Increasing concentrations (2-50 microM) of CoA (plus ATP and 20 microM added palmitic acid) or of palmitoyl-CoA progressively decreased glucose-6-phosphatase activity to 50% of the control value. The inhibition lowered the Vmax without significantly changing the Km. A non-hydrolysable analogue of palmitoyl-CoA also inhibited, demonstrating that binding of palmitoyl-CoA rather than hydrolysis produces the inhibition. Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer demonstrated that palmitoyl-CoA alone or CoA plus ATP and palmitic acid altered the microsomal permeability to glucose 6-phosphate, but not to glucose or phosphate, indicating that T1 is the site of palmitoyl-CoA binding and inhibition of glucose-6-phosphatase activity in native microsomes. The type of inhibition found suggests that liver microsomes may comprise vesicles heterogeneous with respect to glucose-6-phosphate translocase(s), i.e. sensitive or insensitive to fatty acid ester inhibition. PMID:7733874

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

    Menéndez R.


    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.

  20. Effects of thiol antioxidants on the atropselective oxidation of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) by rat liver microsomes.

    Wu, Xianai; Lehmler, Hans-Joachim


    Chiral polychlorinated biphenyl (PCB) congeners, such as PCB 136, are atropselectively metabolized to various hydroxylated PCB metabolites (HO-PCBs). The present study investigates the effect of two thiol antioxidants, glutathione and N-acetyl-cysteine (NAC), on profiles and chiral signatures of PCB 136 and its HO-PCB metabolites in rat liver microsomal incubations. Liver microsomes prepared from rats pretreated with phenobarbital were incubated with PCB 136 (5 μM) in the presence of the respective antioxidant (0-10 mM), and levels and chiral signatures of PCB 136 and its HO-PCB metabolites were determined. Three metabolites, 5-136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol), 4-136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol), and 4,5-136 (2,2',3,3',6,6'-hexachlorobiphenyl-4,5-diol), were detected in all incubations, with 5-136 being the major metabolite. Compared to microsomal incubations without antioxidant, levels of 4,5-136 increased with increasing antioxidant concentration, whereas levels of PCB 136 and both mono-HO-PCBs were not affected by the presence of either antioxidant. PCB 136, 4-136, and 5-136 displayed significant atropisomeric enrichment; however, the direction and extent of the atropisomeric enrichment was not altered in the presence of an antioxidant. Because 4,5-136 can either be conjugated to a sulfate or glucuronide metabolite that is readily excreted or further oxidized a potentially toxic PCB 136 quinone, the effect of both thiol antioxidants on 4,5-136 formation suggests that disruptions of glutathione homeostasis may alter the balance between both metabolic pathways and, thus, PCB 136 toxicity in vivo.


    Samikan Krishnakumar


    Full Text Available Indigenous rhizosphere soil samples were collected during study period (October 2011 – March 2012 of different crop plant from Thiruvannamalai District, Tamilnadu, India for the enumeration of Phosphate solubilizing microorganisms (PSMs. Efficient phosphate solubilizing bacteria, fungi and heterotrophic bacteria were enumerated. Maximum heterotrophic bacterial populations (19.4 X105, phosphate solubilizing bacteria (4.7 X 105 were recorded in the month of February and phosphate solubilizing fungi (3.9 X 102 were documented in the month of December in rhizosphere soil of ground nut. Minimum bacterial populations (14.3 X 105 were observed in rhizosphere soil of chilli in the month of March. Lowest phosphate solubilizing bacteria (1.2 X105 and phosphate solubilzing fungi (1.2 X 102 were observed in rhizosphere soil of paddy during the month of October. Phosphate solubilizing bacteria Pseudomonassp. - BS1, Bacillus sp. – BS2, Micrococcus sp. – BS3 and fungi Aspergillus sp. – FS1, Penicillium sp. – FS2.and Trichoderma sp. – FS3 were identified. Pseudomonas sp. - BS1. exhibited maximum solubilizing efficiency (SE and solubilizing index (SI of 300.0 and 4.0 respectively. In fungi Aspergillus sp. – FS1 showed a maximum solubilizing efficiency (SE and solubilizing index(SI of 283.3 and 3.8 respectively. Antagonistic activity of P-solubilizing Pseudomonassp. - BS1 was deliberated against selected fungal plant pathogens. Among pathogens studied Aspergillus sp. showed a maximum inhibition activity (16 mm and minimum activity (12 mm was observed against Fusarium sp. Moreover inhibition efficiency (IE and inhibition index (II of Pseudomonas sp. - BS1. also calculated base on the antagonistic activity. Aspergillus sp. exhibited highest inhibition efficiency and inhibition index of 166.6 and 3.6 respectively.

  2. Organic acid production in vitro and plant growth promotion in maize under controlled environment by phosphate-solubilizing fluorescent Pseudomonas

    Vyas Pratibha


    Full Text Available Abstract Background Phosphorus deficiency is a major constraint to crop production due to rapid binding of the applied phosphorus into fixed forms not available to the plants. Microbial solubilization of inorganic phosphates has been attributed mainly to the production of organic acids. Phosphate-solubilizing microorganisms enhance plant growth under conditions of poor phosphorus availability by solubilizing insoluble phosphates in the soil. This paper describes the production of organic acids during inorganic phosphate solubilization and influence on plant growth as a function of phosphate solubilization by fluorescent Pseudomonas. Results Nineteen phosphate-solubilizing fluorescent Pseudomonas strains of P. fluorescens, P. poae, P. trivialis, and Pseudomonas spp. produced gluconic acid, oxalic acid, 2-ketogluconic acid, lactic acid, succinic acid, formic acid, citric acid and malic acid in the culture filtrates during the solubilization of tricalcium phosphate, Mussoorie rock phosphate, Udaipur rock phosphate and North Carolina rock phosphate. The strains differed quantitatively and qualitatively in the production of organic acids during solubilization of phosphate substrates. Cluster analysis based on organic acid profiling revealed inter-species and intra-species variation in organic acids produced by Pseudomonas strains. The phosphate-solubilizing bacterial treatments P. trivialis BIHB 745, P. trivialis BIHB 747, Pseudomonas sp. BIHB 756 and P. poae BIHB 808 resulted in significantly higher or statistically at par growth and total N, P and K content over single super phosphate treatment in maize. These treatments also significantly affected pH, organic matter, and N, P, and K content of the soil. Conclusion The results implied that organic acid production by Pseudomonas strains is independent of their genetic relatedness and each strain has its own ability of producing organic acids during the solubilization of inorganic phosphates

  3. Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues.

    Ozaki, Hitomi; Sugihara, Kazumi; Watanabe, Yoko; Fujino, Chieri; Uramaru, Naoto; Sone, Tomomichi; Ohta, Shigeru; Kitamura, Shigeyuki


    Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure-metabolic activity relationship was examined. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.

  4. Impact of Solubilizing Additives on Supersaturation and Membrane Transport of Drugs.

    Raina, Shweta A; Zhang, Geoff G Z; Alonzo, David E; Wu, Jianwei; Zhu, Donghua; Catron, Nathaniel D; Gao, Yi; Taylor, Lynne S


    Many enabling formulations give rise to supersaturated solutions wherein the solute possesses higher thermodynamic activity gradients than the solute in a saturated solution. Since flux across a membrane is driven by solute activity rather than concentration, understanding how solute thermodynamic activity varies with solution composition, particularly in the presence of solubilizing additives, is important in the context of passive absorption. In this study, a side-by-side diffusion cell was used to evaluate solute flux for solutions of nifedipine and felodipine in the absence and presence of different solubilizing additives at various solute concentrations. At a given solute concentration above the equilibrium solubility, it was observed that the solubilizing additives could reduce the membrane flux, indicating that the extent of supersaturation can be reduced. However, the flux could be increased back to the same maximum value (which was determined by the concentration where liquid-liquid phase separation (LLPS) occurred) by increasing the total solute concentration. Qualitatively, the shape of the curves of solute flux through membrane as a function of total solute concentration is the same in the absence and presence of solubilizing additives. Quantitatively, however, LLPS occurs at higher solute concentrations in the presence of solubilizing additives. Moreover, the ratios of the LLPS onset concentration and equilibrium solubility vary significantly in the absence and presence of additives. These findings clearly point out the flaws in using solute concentration in estimating solute activity or supersaturation, and reaffirm the use of flux measurements to understand supersaturated systems. Clear differentiation between solubilization and supersaturation, as well as thorough understanding of their respective impacts on membrane transport kinetics is important for the rational design of enabling formulations for poorly soluble compounds.

  5. Solubilization of pentanol by cationic surfactants and binary mixtures of cationic surfactants

    Morgan, M.E.


    The research reported here has included studies of the solubilization of pentanol in hexadecylpyridinium chloride (CPC), trimethyletetradecylammonium chloride (C{sub 14}Cl), benzyldimethyltetradecylammonium chloride (C{sub 14}BzCl), benzyldimethylhexadecylpyridinium chloride (C{sub 16}BzCl), hexadecyltrimethylammonium bromide (CTAB), and binary mixtures of CPC + C{sub 16}BzCl and C{sub 14}Cl + C{sub 14}BzCl. Rather than using calorimetric methods, this project will employ headspace chromatography to measure solubilization of pentanol over a wide range of solute concentrations. While not yielding as much thermodynamic data as calorimetry, headspace chromatography is a more direct measure of the extent of solubilization. Using headspace chromatography, is a more direct measure of the extent of solubilization. Using headspace chromatography, this study will seek to determine whether strongly synergistic mixture ratios exist in the case of binary cationic surfactant systems. There are two equilibria in the pentanol-water-surfactant system: (1) The pentanol solubilized in micelles is in equilibrium with the monomeric pentanol in solution, and (2) the monomeric pentanol is in equilibrium with the pentanol in the vapor above the solution. To establish the link between the two equilibria, a sample of the vapor above pure liquid pentanol must be collected, in order to find the activity of pentanol in solution. Also, a calibration curve for various concentrations of pentanol in solution. From this type of data it is possible to infer both the concentration of pentanol solubilized in micelles and the concentrations of pentanol in the ``bulk`` solution outside the micelles. The method is equally applicable to systems containing a single surfactant as well as mixtures of surfactants.

  6. Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins.

    Mathias, Rommel A; Chen, Yuan-Shou; Kapp, Eugene A; Greening, David W; Mathivanan, Suresh; Simpson, Richard J


    Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC-MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC-MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and

  7. The effect of alpha-tocopherol on lipid peroxidation of microsomes and mitochondria from rat testis.

    Gavazza, M B; Catalá, A


    The testis is a remarkably active metabolic organ; hence it is suitable not only for studies of lipid metabolism in the organ itself but also for the study of lipid peroxidation processes in general. The content of fatty acids in testis is high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. Studies were carried out to evaluate the effect of alpha-tocopherol in vitro on ascorbate-Fe(++) lipid peroxidation of rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition were used as an index of the oxidative destruction of lipids. Special attention was paid to the changes produced on the highly PUFA [C20:4 n6] and [C22:5 n6]. Lipid peroxidation of testis microsomes or mitochondria induced a significant decrease of both fatty acids. Total chemiluminescence was similar in both kinds of organelles when the peroxidized without (control) and with ascorbate-Fe(++) (peroxidized) groups were compared. Arachidonic acid was protected more efficiently than docosapentaenoic acid at all alpha-tocopherol concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbate-Fe(++). The maximal percentage of inhibition in both organelles was approximately 70%; corresponding to an alpha-tocopherol concentration between 1 and 0.25 mM. IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.144 mM) than mitochondria (0.078 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6]+[C22:5 n6] in microsomes that in mitochondria. It is proposed that the vulnerability to lipid peroxidation of rat testis microsomes and mitochondria is different because of the different proportion of PUFA in these organelles The peroxidizability index (PI) was positively correlated with the level of long chain fatty acids. The

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

    Rasmussen, Eva


    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...... 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...

  9. The solubilization of fatty acids in systems based on block copolymers and nonionic surfactants

    Mirgorodskaya, A. B.; Yatskevich, E. I.; Zakharova, L. Ya.


    The solubilizing action of micellar, microemulsion, and polymer-colloid systems formed on the basis of biologically compatible amphiphilic polymers and nonionic surfactants on capric, lauric, palmitic, and stearic acids was characterized quantitatively. Systems based on micelle forming oxyethyl compounds increased the solubility of fatty acids by more than an order of magnitude. Acid molecules incorporated into micelles increased their size and caused structural changes. Solubilization was accompanied by complete or partial destruction of intrinsic acid associates and an increase in their p K a by 1.5-2 units compared with water.

  10. Novel Application of Hydrotropic Solubilizing Additives in the Estimation of Aspirin in Tablets

    Maheshwari, R. K.; Saxena, M.; Gahlot, M.; Chaki, R.; Kinariwala, M.; Jagwani, Y.


    Highly concentrated aqueous solutions of various hydrotropic agents like sodium benzoate, sodium salicylate, sodium acetate, sodium citrate, nicotinamide and sodium ascorbate have been observed to enhance aqueous solubilities of a large number of poorly water-soluble drugs. In the present investigation hydrotropic solubilization technique has been employed to solubilize poorly water-soluble aspirin (analgesic and antipyretic drug) by 0.5 M ibuprofen sodium solution to carry out titrimetric analysis of aspirin in tablet dosage form. Results of analysis by proposed method and Phamacopoeial method are very comparable. Proposed method is new, rapid, simple, accurate, and reproducible. Statistical data proved the accuracy, reproducibility and the precision of proposed method. PMID:21695002

  11. Influence of nonionic surfactant on the solubilization and biodegradation of phenanthrene

    YANG Jian-gang; LIU Xiang; LONG Tao; YU Gang; PENG She; ZHENG Liu


    Phenanthrene was solubilized in two different nonionic surfactants, Tween80 and Triton X-100. The bioavailability of phenanthrene to the bacteria isolated from the petroleum contaminated soils was studied based on the rotary flasks experiments. The results showed that the concentration of nonionic surfactants above the critical micelle concentration(CMC) can increase the solubility of phenanthrene in water and were innoxious to the phenanthrene-degrading bacteria; phenanthrene solubilized in the micelles of Tween80 was bioavailable and biodegradable. The research demonstrated the potential of surfactant-enhanced bioremediation of soils contaminated by hydrophobic organic compounds( HOCs).

  12. Enantioselective S-oxygenation of para-methoxyphenyl-1,3-dithiolane by various tissue preparations: effect of estradiol.

    Cashman, J R; Olsen, L D; Lambert, C E; Presas, M J


    Liver, kidney, and lung microsomes prepared from nonpretreated female Sprague-Dawley rats catalyze the NADPH- and oxygen-dependent S-oxygenation of para-methoxyphenyl-1,3-dithiolane. Studies on the biochemical mechanism of dithiolane S-oxygenation in liver, kidney, and lung microsomes suggest that this reaction is catalyzed in a diastereoselective and enantioselective fashion by the flavin-containing monooxygenase and, to a lesser extent, the cytochromes P-450. This conclusion is based on results examining the effects of selective cytochrome P-450 inhibitors and positive effectors, microsome heat-inactivation treatment, and alternate substrates for the flavin-containing monooxygenase. Liver and kidney microsomes prepared from ovarectomized female rats tended to have decreased S-oxygenase activity, compared with nonpretreated female rats, whereas ovarectomized rats pretreated with estradiol had markedly lower S-oxygenase activity. In contrast, lung microsomal S-oxygenase activity, which is low in pulmonary microsomes from nonpretreated female rats, increases 2-4-fold after ovariectomization and estradiol pretreatment. In female Sprague-Dawley rats, estradiol pretreatment is mainly responsible for the large decrease (or increase) in S-oxygenase activity observed in the tissues examined, although it is unlikely that estradiol alone controls flavin-containing monooxygenase S-oxygenase activity.

  13. Isolation of phosphatase-producing phosphate solubilizing bacteria from Loriya hot spring: Investigation of phosphate solubilizing in the presence of different parameters

    Maryam Parhamfar


    Full Text Available Introduction: Biofertilizers are the microorganisms that can convert useless nutrient to usable compounds. Unlike fertilizer, cost of biofertilizer production is low and doesn’t produce ecosystem pollution. Phosphate fertilizers can be replaced by phosphate biofertilizer to produce improvement. So, it is necessary to screen the climate-compatible phosphate solubilizing bacteria. Materials and methods: In this project samples were picked up from Loriya hot spring, which are located in Jiroft. Samples were incubated in PKV medium for 3 days. Screening of phosphate solubilizing bacteria was performed on the specific media, based on clear area diameter. The best bacterium was identified based on 16s rDNA gene. Phosphate solubilizing activity of this strain was considered in different carbon, nitrogen, phosphate and pH sources. Results: Sequence alignment and phylogenetic tree results show that B. sp. LOR033 is closely related to Bacillus licheniformis, with 97% homology. In addition, results show that maximum enzyme production was performed after 2 days that incubation pH was decreased simultaneously when the time was increased. Carbon sources investigation show that glucose is the most appropriate in enzyme production and phosphate releasing. Furthermore, results show that the optimum initial pH for phytase production was pH5.0. Different phosphate sources show that tricalcium phosphate has the suitable effect on enzyme activity in three days of incubation. Discussion and conclusion: Phosphatase enzyme production capacity, growth in acidic pH and phosphate solubilizing potential in different salt and phosphate sources show that this strain has considerable importance as biofertilizers.

  14. Evaluation for rock phosphate solubilization in fermentation and soil-plant system using a stress-tolerant phosphate-solubilizing Aspergillus niger WHAK1.

    Xiao, Chunqiao; Zhang, Huaxiang; Fang, Yujuan; Chi, Ruan


    A strain WHAK1, identified as Aspergillus niger, was isolated from Yichang phosphate mines in Hubei province of China. The fungus developed a phosphate solubilization zone on modified National Botanical Research Institute's phosphate growth (NBRIP) agar medium, supplemented with tricalcium phosphate. The fungus was applied in a repeated-batch fermentation process in order to test its effect on solubilization of rock phosphate (RP). The results showed that A. niger WHAK1 could effectively solubilize RP in NBRIP liquid medium and released soluble phosphate in the broth, which can be illustrated by the observation of scanning electron microscope, energy-dispersive X-ray microanalysis, and Fourier transform infrared spectroscopy. Acidification of the broth seemed to be the major mechanism for RP solubilization by the fungus. Indeed, multiple organic acids (mainly gluconic acid) were detected in the broth by high-performance liquid chromatography analysis. These organic acids caused a significant drop of pH and an obvious rise of titratable acidity in the broth. The fungus also exhibited high levels of tolerance against temperature, pH, salinity, and desiccation stresses, although a significant decline in the fungal growth and release of soluble phosphate was marked under increasing intensity of stress parameters. Further, the fungus was introduced into the soil supplemented with RP to analyze its effect on plant growth and phosphate uptake of wheat plants. The result revealed that inoculation of A. niger WHAK1 significantly increased the growth and phosphate uptake of wheat plants in the RP-amended soil compared to the control soil.

  15. The population of phosphate solubilizing bacteria (PSB from Cikaniki, Botol Mountain, and Ciptarasa Area, and the ability of PSB to solubilize insoluble P in solid pikovskaya medium



    Full Text Available Phosphate solubilizing bacteria (PSB were collected from plant rhizosphere at Cikaniki (1100 m asl., soil at Botol Mountain (1000, 1500, and 1800 m asl., and Ciptarasa (600, 1000, and 1500 m asl., area at Gunung Halimun National Park (GHNP. The soil ware collected randomly from 3 areas and taken from 0-15 cm depth in the plants rhizosphere at Cikaniki and forest floor soil in Gunung Botol and Ciptarasa. The result showed that the difference of elevation area, soil pH, forest vegetation, and microbial habitat (rhizosphere and forest floor were not the inhabitation factors of the biodiversity of PSB and their ability to solubilize insoluble phosphate, but the inhabitation factors on the growth of the PSB population. The highest population of PSB at GHNP was founded in the plant rhizosphere of Altingia exelsa Norona and Schima wallichii (Dc. Korth (107sel/g soil at Cikaniki and in the forest floor soil (108sel/g soil at Botol Mountain (1000 m asl.. Pseudomonas sp., Bacillus sp., Bacillus megaterium, and Chromobacterium sp. dominated Cikaniki, Botol Mountain and Ciptarasa area. Those isolates could solubilize insoluble phosphate on solid Pikovskaya medium with the range of diameter is 1.5-2.5 cm.

  16. Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with Human Liver Microsomes.

    Choi, Jong Min; Oh, Soo Jin; Lee, Ji-Yoon; Jeon, Jang Su; Ryu, Chang Seon; Kim, Young-Mi; Lee, Kiho; Kim, Sang Kyum


    Drug-induced liver injury (DILI) via metabolic activation by drug-metabolizing enzymes, especially cytochrome P450 (CYP), is a major cause of drug failure and drug withdrawal. In this study, an in vitro model using HepG2 cells in combination with human liver microsomes was developed for the prediction of DILI. The cytotoxicity of cyclophosphamide, a model drug for bioactivation, was augmented in HepG2 cells cultured with microsomes in a manner dependent on exposure time, microsomal protein concentration, and NADPH. Experiments using pan- or isoform-selective CYP inhibitors showed that CYP2B6 and CYP3A4 are responsible for the bioactivation of cyclophosphamide. In a metabolite identification study employing LC-ESI-QTrap and LC-ESI-QTOF, cyclophosphamide metabolites including phosphoramide mustard, a toxic metabolite, were detected in HepG2 cells cultured with microsomes, but not without microsomes. The cytotoxic effects of acetaminophen and diclofenac were also potentiated by microsomes. The potentiation of acetaminophen cytotoxicity was dependent on CYP-dependent metabolism, and the augmentation of diclofenac cytotoxicity was not mediated by either CYP- or UDP-glucuronosyltransferase-dependent metabolism. The cytotoxic effects of leflunomide, nefazodone, and bakuchiol were attenuated by microsomes. The detoxication of leflunomide by microsomes was attributed to mainly CYP3A4-dependent metabolism. The protective effect of microsomes against nefazodone cytotoxicity was dependent on both CYP-mediated metabolism and nonspecific protein binding. Nonspecific protein binding but not CYP-dependent metabolism played a critical role in the attenuation of bakuchiol cytotoxicity. The present study suggests that HepG2 cells cultured with human liver microsomes can be a reliable model in which to predict DILI via bioactivation by drug metabolizing enzymes.

  17. Development of QSAR models for microsomal stability: identification of good and bad structural features for rat, human and mouse microsomal stability.

    Hu, Yongbo; Unwalla, Ray; Denny, R Aldrin; Bikker, Jack; Di, Li; Humblet, Christine


    High throughput microsomal stability assays have been widely implemented in drug discovery and many companies have accumulated experimental measurements for thousands of compounds. Such datasets have been used to develop in silico models to predict metabolic stability and guide the selection of promising candidates for synthesis. This approach has proven most effective when selecting compounds from proposed virtual libraries prior to synthesis. However, these models are not easily interpretable at the structural level, and thus provide little insight to guide traditional synthetic efforts. We have developed global classification models of rat, mouse and human liver microsomal stability using in-house data. These models were built with FCFP_6 fingerprints using a Naïve Bayesian classifier within Pipeline Pilot. The test sets were correctly classified as stable or unstable with satisfying accuracies of 78, 77 and 75% for rat, human and mouse models, respectively. The prediction confidence was assigned using the Bayesian score to assess the applicability of the models. Using the resulting models, we developed a novel data mining strategy to identify structural features associated with good and bad microsomal stability. We also used this approach to identify structural features which are good for one species but bad for another. With these findings, the structure-metabolism relationships are likely to be understood faster and earlier in drug discovery.

  18. Gelation properties of spent duck meat surimi-like material produced using acid-alkaline solubilization methods.

    Nurkhoeriyati, T; Huda, N; Ahmad, R


    The gelation properties of spent duck meat surimi-like material produced using acid solubilization (ACS) or alkaline solubilization (ALS) were studied and compared with conventionally processed (CON) surimi-like material. The ACS process yielded the highest protein recovery (P gelation and color properties of spent duck and possibly applied for other high fat raw material.

  19. Effects of halophenol LM49 on microsomal CYP450 in Rats%卤酚化合物LM49对大鼠肝微粒体细胞色素P450的影响

    白玉; 张丽锋; 冯秀娥; 梁泰刚; 李青山


    目的 研究LM49对大鼠肝微粒体蛋白及细胞色素P450含量的影响.方法 将大鼠分成空白对照组、溶剂对照组、阳性对照组(苯巴比妥组、地塞米松组、β-萘黄酮组)、LM49低、中、高剂量组.给药后采用超速离心法制备大鼠肝微粒体;BCA法测定大鼠肝微粒体蛋白浓度;Omura and Sato法测定大鼠肝微粒体细胞色素P450的含量.结果 给予不同剂量的LM49后,大鼠肝微粒体的蛋白及细胞色素P450含量均明显降低.低、中、高剂量组与对照组比较差异有统计学意义(P<0.05).结论 LM49对大鼠肝微粒体细胞色素P450具有一定的抑制作用,可能引起肝药酶对某些药物代谢的改变.%Objective To determine the effects of LM49 on microsomal protein and CYP450 in rats. Methods Rats were assigned into blank control group, solvent control group, positive control groups (phenobarbital, dexametha-sone and (3-nephthoflavone) and LM49-treated (low, medium and high dosage) groups, respectively. Hepatic micro-somes were prepared using ultracentrifuge after administration of interventions. The concentration of microsomal proteins was determined using BAG approach, and the content of microsomal CYP450 was assayed via Omura and Sato method. Results Marked reduction in the concentration of microsomal proteins and CYP450 was associated with administration of LM49 with various (low, medium and high) dosages as compared with control group (all P<0.05). Conclusion LM49 inhibits hepatic microsomal CYP450 and may result in changes of metabolism of certain medicines induced by liver drug enzymes in rats.

  20. Evidence that beta-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin.

    Cook, L; Nagi, M N; Suneja, S K; Hand, A R; Cinti, D L


    The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC, believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase. Images Fig. 4. Fig. 7. Fig. 8. PMID:1417796

  1. 4-Hydroxyestradiol is conjugated with thiols primarily at C-2: evidence from regiospecific displacement of tritium by rat liver microsomes or tyrosinase

    Jellinck, P.H.


    4-Hydroxyestradiol bearing a 3H label specifically at C-2 was prepared chemically and incubated with male rat liver microsomes or mushroom tyrosinase. A very high proportion (80-90%) of the 3H was displaced from the labeled steroid when either glutathione or N-acetylcysteine was present, and tyrosinase was shown not to require NADPH as cofactor for this reaction. In either case, only negligible amounts (less than 3%) of the 3H radioactivity were found associated with water-soluble adducts in contrast to 3H-labeled 2-hydroxyestradiol, which gave rise to about 25% of such products. The effect of ascorbic acid on the microsomal reaction with regiospecifically labeled estradiol, 2-hydroxyestradiol, and 4-hydroxyestradiol was also investigated, and the results are discussed in terms of the reactivity at different carbon atoms in ring A of the catechol estrogens. All the evidence points to conjugation of 4-hydroxyestradiol with glutathione or N-acetylcysteine at C-2 but not C-1 of this highly reactive catechol estrogen. Measuring the displacement of 3H as 3H2O from specific positions in the steroid ring provides a useful and sensitive method to assess the formation of adducts in cases where their isolation and characterization is particularly difficult.

  2. Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide.

    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


    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.

  3. Metabolism and metabolic inhibition of gamboglc acid in rat liver microsomes

    Yi-tong LIU; Kun HAO; Xiao-quan LIU; Guang-Ji WANG


    Aim: To study the metabolism of gambogic acid (GA) and the effects of selective cytochrome P-450 (CYP450) inhibitors on the metabolism of GA in rat liver microsomes in vitro. Methods: Rat liver micrp,so,rn,e$ were used to perform metabolism studies. Various selective CYP450 inhibitors were used to investigate their effects on the metabolism of GA and the principal CYP450 isoform involved in the formation of major metabolite M1 in rat liver microsomes. Types of inhibition in an enzyme kinetics model were used to model the interaction. Results: GA was rapidly metabolized to two phase Ⅰ metabolites,, M1 and M2, in rat liver microsomes. M1 and M2 were tentatively presumed to be the hydration metabolite and epoxide metabolite of GA, respectively. α-Naphthoflavone uncompetitively inhibited the formation of M1 while ketoconazole, sulfophenazole, diethyl dithiocarbamate and quinidine had little or no inhibitory effects on the formation of M1. Conclusion: GA is rapidly metabolized in rat liver microsomes and M1 is crucial for the elimination of GA. Cytochrome P-450 1A2 is the major rat CYP involved in the metabolism of GA.

  4. Mutagenic Potential of DIGL-RO Solid Propellant in the Ames Salmonella/ Mammalian Microsome Mutagenicity Test


    303913 11. TITLE (Include Security Clasification ) Mutagenic Potential of DIGL-RP Solid Propellant in the Ames Salmonella/Mammalian Microsome protein /plate. After all the ingredients were added, the top agar was mixed, then overlaid on MGA plates. These plates contained 2% glucose and

  5. Comparative azo reductase activity of red azo dyes through caecal and hepatic microsomal fraction in rats.

    Singh, S; Das, M; Khanna, S K


    In order to study the rate of formation of toxic aromatic amines, anaerobic reduction of four red azo dyes viz. amaranth, carmoisine, fast Red E and ponceau 4R was investigated by incubating caecal content and hepatic microsomal fraction of rats with 37.5 microM concentration of dyes in sodium phosphate buffer pH 7.4 using NADPH generating system, glucose oxidase system and nitrogen as the gaseous phase. Caecal suspension exhibited higher azo reductase activity than that of hepatic microsomal fraction using any of the 4 azo dyes. Caecal microbes showed maximal azo reductase activity when ponceau 4R was used as a substrate followed by fast Red E and carmoisine, while with amaranth the activity was minimum. Similarly ponceau 4 R exhibited maximum hepatic microsomal azo reductase activity followed by fast Red E and carmoisine whereas, amaranth had minimum activity. Caecal flora possessed almost 17 fold higher degradative capability of ponceau 4 R and fast Red E colourants than the hepatic microsomal fraction. The higher reductive ability through caecal flora for ponceau 4R and fast Red E signifies the formation of more aromatic amines which may be re-absorbed through the intestine to be either eliminated through urine as conjugates or retained in the target tissues to elicit toxic effects.

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

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


    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...

  7. Isolation and structural elucidation of tiamulin metabolites formed in liver microsomes of pigs

    Lykkeberg, Anne Kruse; Cornett, Claus; Halling-Sørensen, Bent;


    Although the antimicrobial tiamulin is extensively metabolized in pigs, the metabolism is not well investigated. In this work the NADPH dependent metabolism of tiamulin in liver microsomes from pigs has been studied. The tiamulin metabolites formed in the incubations were analysed using LC...

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

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


    The biosynthesis of small-intestinal aminopeptidase N (EC was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rat......The biosynthesis of small-intestinal aminopeptidase N (EC was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane......-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...... 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...


    Metabolism of two triazole-containing antifungal azoles was studied using expressed human and rat cytochrome P450s (CYP) and liver microsomes. Substrate depletion methods were used due to the complex array of metabolites produced from myclobutanil and triadimefon. Myclobutanil wa...

  10. In vitro metabolic study of Rhizoma coptidis extract using liver microsomes immobilized on magnetic nanoparticles.

    Xue, Ying; Xiong, Jing; Shi, Hai-Li; Liu, Yi-Ming; Qing, Lin-Sen; Liao, Xun


    Although metabolic study of individual active compounds isolated from herbal plants has been intensive, it cannot truly reflect the fate of herbs because the herbal extracts in use have many constituents. To address this problem, whole extracts of herbs should be investigated. Microsomes have been heavily used in the in vitro metabolic study of drugs, and various materials have been used to immobilize microsomes to develop highly effective and reusable bioreactors in this field. In this work, rat liver microsomes were immobilized on magnetic nanoparticles (LMMNPs) to develop a highly active and recoverable nanoparticle bioreactor. Using this bioreactor, we investigated the in vitro metabolism of Rhizoma coptidis extract. Incubation of berberine, a major active ingredient of R. coptidis, with LMMNPs for 20 min produced two metabolites, i.e., demethyleneberberine and thalifendine, at high levels. From a comparison of the time courses of thalifendine formation obtained by ultraperformance liquid chromatography-mass spectrometry analysis, it was found that LMMNPs had a higher biological activity than free liver microsomes in metabolizing berberine. Further, the activity of LMMNPs remained almost unchanged after six consecutive uses in the incubation tests. Metabolism of R. coptidis extracts by LMMNPs was studied. The same two metabolites of berberine, i.e., demethyleneberberine and thalifendine, were detected. After a thorough study seeking support for this observation, it was found that demethyleneberberine was the common metabolite of five protoberberine-type alkaloids present in R. coptidis extract, including palmatine, jatrorrhizine, columbanine, epiberberine, and berberine.

  11. Glutathione delays varies as-tocopherol oxidation and subsequent lipid peroxidation in rat liver microsomes

    Robey, S.; Mavis, R.


    A method has been developed for in vitro trace radiolabeling of rat liver microsomes with /sup 3/H-..cap alpha..-tocopherol (..cap alpha..T*) which allows virtually complete oxidation of the ..cap alpha..T* under oxidizing conditions. The supernatant of a 16,000 xg centrifugation of homogenized rat liver, containing the cytosolic rat liver vitamin E (VE) transfer protein, was incubated with an ethanolic solution of ..cap alpha..T* for 10 minutes at 37/sup 0/C. Labeled microsomes were collected in the washed 100,000 xg pellet. Microsomes were then incubated with 30 Fe/sup 2 +/ in an NADPH-generating system, and both production of malondialdehyde (MDA) (a product of lipid peroxidation) and oxidation of ..cap alpha..T* were monitored over a time course in the presence and absence of glutathione (GSH). The results indicate virtually complete oxidation of ..cap alpha..T* precedes significant membrane lipid peroxidation, and that addition of 5 mM GSH delays both ..cap alpha..T* oxidation and subsequent MDA production. This suggests that the previously observed VE-dependent heat labile inhibition of microsomal lipid peroxidation by GSH involves maintaining membrane levels of ..cap alpha..-tocopherol.

  12. Characterization and biosynthesis of cytochrome b5 in rat liver microsomes

    Sargent, J. R.; Vadlamudi, B. P.


    1. Cytochrome b5 is released from rat liver microsomes by both proteolytic enzymes and by treatments that disrupt phospholipids. Cytochrome P-420 is only released to a marked extent by treatments that disrupt phospholipids. 2. Cytochrome b5 was isolated in a pure state from both the rough and smooth fractions of rat liver microsomes after treatment with trypsin, and was shown to contain two cytochrome components with identical spectral properties. 3. Amino acid analyses of the two components are presented, together with peptide `fingerprint' patterns of tryptic digests of the two components. 4. Studies based on the direct isolation of cytochrome b5 after administration of a single dose of radioactive amino acid to rats demonstrate that the cytochrome is synthesized initially in the rough fraction of microsomes and only subsequently appears in the smooth fraction. 5. Isolated rat liver microsomes are capable of incorporating radioactive amino acids into cytochrome b5 under standard conditions. 6. Under these conditions the amino acid is incorporated into peptide linkage in the cytochrome. PMID:16742610

  13. Metabolism of kadsurenone and 9,10-dihydrokadsurenone in rhesus monkeys and rat liver microsomes

    Thompson, K.L.; Chang, M.N.; Chabala, J.C.; Chiu, S.H.; Eline, D.; Hucker, H.B.; Sweeney, B.M.; White, S.D.; Arison, B.H.; Smith, J.L.


    The metabolism of the PAF antagonists kadsurenone and tritium-labeled 9,10-dihydrokadsurenone was studied in rhesus monkeys and rat liver microsomes. The monkey metabolites of the two drugs were isolated as their glucuronide conjugates from the urine of iv dosed males. The metabolites from both monkey and microsomal metabolism were purified by reverse phase HPLC and identified by spectral (NMR, UV, and mass spectrometric) analysis. The principal pathway of biotransformation of the tritium-labeled 9,10-dihydrokadsurenone in monkeys was hydroxylation of the C-5 propyl side chain to give two metabolites, 10-hydroxy-9,10-dihydrokadsurenone and 9-hydroxy-9,10-dihydrokadsurenone. These compounds were excreted as glucuronides. Microsomal incubation of tritium-labeled 9,10-dihydrokadsurenone yielded the 10-, 9-, and 8-hydroxy-9,10-dihydrokadsurenone as major metabolites. Kadsurenone was also metabolized at the C-5 side chain, an allyl group. The monoglucuronide of 9,10-dihydroxykadsurenone was isolated from monkey urine. Spectral analysis was not definitive as to the site of conjugation, and the structure of the metabolite was assigned as the C-10 conjugate. A major metabolite of rat liver microsomal incubation of kadsurenone was 9,10-dihydroxykadsurenone.


    Chauveau, J.; Moulé, Y.; Rouiller, C.; Schneebeli, J.


    Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry. PMID:13878497

  15. Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis.

    Reddy, Vaddi Damodara; Padmavathi, Pannuru; Hymavathi, Reddyvari; Maturu, Paramahamsa; Varadacharyulu, N Ch


    The protective effect of Emblica officinalis fruit extract (EFE) against alcohol-induced oxidative damage in liver microsomes was investigated in rats. EFE (250mg/kg b.wt/day) and alcohol (5g/kg b.wt/day, 20%, w/v) were administered orally to animals for 60 days. Alcohol administration significantly increased lipid peroxidation, protein carbonyls with decreased sulfhydryl groups in microsomes, which were significantly restored to normal levels in EFE and alcohol co-administered rats. Alcohol administration also markedly decreased the levels of reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in the liver microsomes, which were prevented with EFE administration. Further, alcohol administration significantly increased the activities of cytochrome P-450, Na(+)/K(+) and Mg(2+) ATPases and also membrane fluidity. But, administration of EFE along with alcohol restored the all above enzyme activities and membrane fluidity to normal level. Thus, EFE showed protective effects against alcohol-induced oxidative damage by possibly reducing the rate of lipid peroxidation and restoring the various membrane bound and antioxidant enzyme activities to normal levels, and also by protecting the membrane integrity in rat liver microsomes. In conclusion, the polyphenolic compounds including flavonoid and tannoid compounds present in EFE might be playing a major role against alcohol-induced oxidative stress in rats.

  16. Oxidation of carbon tetrachloride, bromotrichloromethane, and carbon tetrabromide by rat liver microsomes to electrophilic halogens

    Mico, B.A. (National Inst. of General Medical Sciences, Bethesda, MD); Branchflower, R.V.; Pohl, L.R.; Pudzianowski, A.T.; Loew, G.H.


    In order to determine whether CCl/sub 4/, CBrCl/sub 3/, CBr/sub 4/ or CHCl/sub 3/ undergo oxidative metabolism to electrophilic halogens by liver microsomes, they were incubated with liver microsomes from phenobarbital pretreated rats in the presence of NADPH and 2,6-dimethylphenol. The analysis of the reaction mixtures by capillary gas chromatography mass spectrometry revealed that 4-chloro-2,6-dimethylphenol was a metabolite of CCl/sub 4/ and CBrCl/sub 3/ whereas 4-bromo-2,6-dimethylphenol was a metabolite of CBr/sub 4/. The formation of the metabolites was significantly decreased when the reactions were conducted with heat denatured microsomes, in the absence of NADPH or under an atmosphere of N/sub 2/. These results indicate that the chlorines of CBrCl/sub 3/ and CCl/sub 4/ and the bromines of CBr/sub 4/ are oxidatively metabolized by rat liver microsomes to electrophilic and potentially toxic metabolites.

  17. Development of vitamin D/sub 3/ 25-hydroxylase activity in rat liver microsomes

    Thierry-Palmer, M.; Cullins, S.; Rashada, S.; Gray, T.K.; Free, A.


    The authors have determined the ontogeny of vitamin D/sub 3/ 25-hydroxylase activity in rat liver microsomes. Microsomes from fetuses, neonates, and their mothers were incubated with 44 nM /sup 3/H-vitamin D/sub 3/ in the presence of an NADPH generating system, oxygen, KCl, and MgCl/sub 2/. Lipid extracts of the incubation samples were partially purified by thin-layer chromatography. Tritiated 25-hydroxy vitamin D/sub 3/ (250HD/sub 3/) was analyzed by high-pressure liquid chromatography using 94/6 hexane/isopropanol. Production rate for 250HD/sub 3/ in the mothers ranged from 0.22 to 0.30 pmol/mg protein/hr. Activities in the fetuses and neonates were 2.1, 12.9, 32.0, 35.8, and 71.0% of that of their mothers at -3, 0, 2, 7, and 15 days of age. The cytosolic fraction protected the substrate from degradation, stimulated the vitamin D/sub 3/ 25-hydroxylase reaction in neonates and mothers (1.4 to 1.7 fold increase), and was absolutely required for 25-hydroxylase activity in fetuses. These data suggest that microsomal vitamin D/sub 3/ 25-hydroxylase activity develops slowly and approaches full activity near the weaning stage. A cytosolic factor present as early as -3 days of age stimulates the activity of the microsomal vitamin D/sub 3/ 25-hydroxylase.

  18. Biphenyl metabolism by rat liver microsomes. Regioselective effects of inducers, inhibitors, and solvents

    Haugen, D.A.


    The effects of the inducers phenobarbital and 3-methylcholanthrene, the inhibitors 7,8-benzoflavone and 1-benzyl-imidazole, and the solvents methanol, acetone, and dimethyl sulfoxide on the 2-, 3-, and 4-hydroxylation of biphenyl and the O-de-ethylation of 7-ethoxycoumarin by rat liver microsomes were examined. Phenobarbital pretreatment primarily induced 2- and 3-hydroxylation, the latter most dramatically. 3-Methylcholanthrene pretreatment induced 2- and 3-hydroxylation to similar extents. The inhibitors and solvents had regioselective effects on biphenyl metabolism that were characteristic of the uninduced, phenobarbital-induced, and 3-methylcholanthrene-induced microsomes. The presence of multiple forms of cytochrome P-450 in uninduced microsomes is indicated by the regioselective effects of the solvents and the inhibitors. The 3-methylcholanthrene-dependent increases in 2- and 3-hydroxylation appear due to induction of a single form of cytochrome P-450, as indicated by similar dose-response relationships and similar changes in sensitivitty to the inhibitors. The phenobarbital-dependent increases in 2- and 3-hydroxylation appear due to the induction of two forms of cytochrome P-450, as indicated by different changes in sensitivity to the effects of dimethyl sulfoxide and 7,8-benzoflavone. The results indicate that examination of the regioselectivity of biphenyl metabolism is a useful approach for characterizing microsomal mono-oxygenases, and they suggest that the approach may also be useful in the characterization of purified mono-oxygenase systems. (JMT)

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

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


    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, p

  20. The acylation of 1-acylglycero-3-phosphorylcholines by rat-liver microsomes

    Bosch, H. van den; Golde, L.M.G. van; Eibl, H.; Deenen, L.L.M. van


    1. 1. The transfer of acyl groups from acyl-coenzyme A derivatives to phosphatidylcholine by rat-liver microsomes was found to be significantly stimulated by the addition of synthetic 1-acylglycero-3-phosphorylcholines. Unsaturated acyl chains were transferred in preference to saturated ones, partic

  1. Effect of paraquat on microsomal lipid peroxidation in vitro and in vivo. [Rats, rabbits, man, mice

    Kornbrust, D.J.; Mavis, R.D.


    Rat lung and liver microsomes did not undergo lipid peroxidation in the absence of iron when incubated with NADPH and concentrations of paraquat ranging from 10/sup -7/ to 10/sup -2/ M. Paraquat also did not stimulate rat liver and lung microsomal peroxidation induced by added iron and NADPH, and was inhibitory at concentrations above 10 Similarly, no stimulation of peroxidation was produced by paraquat in rabbit or human lung microsomes; however, under similar conditions, paraquat enhanced NADPH/iron-dependent peroxidation in mouse lung and liver microsomes obtained from rats sacrificed at 12, 18, and 24 hr following a lethal dose of paraquat (50 mg/kg, ip), there was no loss of vitamin E or increase in susceptibility to in vitro peroxidation which would be expected if lipid peroxidation had occurred in vivo although extensive lung damage developed during this time period. These results indicate that paraquat does not cause pulmonary toxicity by initiating peroxidation of lung lipids, and the decrease of palmitate in paraquat-damaged lungs is consistent with inhibition of fatty acid synthesis as an early event in the pathogenesis of paraquat toxicity.

  2. Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

    UDP-glucuronosyltransferase (UGT) activity toward the flavonoid quercetin and UGT protein were characterized in 3 equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats, n=8/age using villin to control for enterocyte content. SI microsomal intrinsic clearance of quercetin...

  3. Age-related increases in F344 rat intestine microsomal quercetin glucuronidation

    The objective of this study was to establish the extent age modifies intestinal quercetin glucuronidation capacity. Pooled microsomal fractions of three equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats (n=8/group) were employed to model the enzyme kinetics of UDP-gl...

  4. CYP3A4 mediated in vitro metabolism of vinflunine in human liver microsomes

    Xiao-ping ZHAO; Jiao ZHONG; Xiao-quan LIU; Guang-ji WANG


    Aim: To study the metabolism of vinflunine and the effects of selective cyto-chrome P-450 (CYP450) inhibitors on the metabolism of vinflunine in human liver microsomes. Methods: Individual selective CYP450 inhibitors were used to inves-tigate their effects on the metabolism of vinflunine and the principal CYP450 isoform involved in the formation of metabolites M1 and M2 in human liver microsomes.Results: Vinflunine was rapidly metabolized to 2 metabolites: M1 and M2 in human liver microsomes. M1 and M2 were tentatively presumed to be the N-oxide metabo-lite or hydroxylated metabolite and epoxide metabolite of vinflunine, respectively. Ketoconazole uncompetitively inhibited the formation of M1, and competitively inhibited the formation of M2, while α-naphthoflavone, sulfaphenazole, diethyl dithiocarbamate, tranylcypromine and quinidine had little or no inhibitory effect on the formation of M1 and M2. Conclusion: Vinflunine is rapidly metabolized in human liver microsomes, and CYP3A4 is the major human CYP450 involved in the metabolism of vinflunine.

  5. Pressure injection of solubilized benomyl for prevention and cure of oak wilt

    Thomas W. Jones; Garold F. Gregory; Percy McWain


    A preliminary evaluation of the effectiveness of injecting solubilized benomyl into oaks for prevention or cure of oak wilt disease is presented. Symptom development was greatly reduced or prevented in trees injected with fungicide before inoculation. Symptom development was markedly arrested in diseased trees by fungicide injected before more than 10 percent of the...

  6. Enhanced solubilization of iron and calcium phosphates by Aspergillus niger by the addition of alcohols

    Cinthya Babá Barroso


    Full Text Available The present study deals with the effect of increased concentrations of alcohols (methanol and ethanol on the solubilization of FePO4 and CaHPO4 by Aspergillus niger. The efficiency of solubilization (ES of inorganic phosphates, fungal growth, acid production and pH variation were determined. The concentrations of alcohols that most favored the solubilization of phosphates were 4% methanol and 3% ethanol. The ES of phosphates by the fungus in media containing methanol ranged from 11-96% and 0.4-87% for ethanol. The stimulation or inhibition of solubilization was dependent on greater or lesser fungus growth, acid production and decreased pH. These responses were supported by the correlation between the amount of phosphate dissolved and fungal growth (0.630** to 0.831**, the production of acids (0.529* to 0.989** and a decrease in pH (-0.755** to -0.962**. Thus, the addition of alcohols facilitated insoluble phosphates dissolution during the fungus growth.

  7. Block-Copolymer-Assisted Solubilization of Carbon Nanotubes and Exfoliation Monitoring Through Viscosity

    Cotiuga, Irina; Picchioni, Francesco; Agarwal, Uday S.; Wouters, Daan; Loos, Joachim; Lemstra, Piet J.


    The use of the block copolymers polystyrene-block-poly(ethylene oxide) and poly(methyl methacrylate)-block-poly(ethylene oxide) is described to assist the direct solubilization of single-walled carbon nanotubes (SWNTs) into water under ultrasonic irradiation. As compared to surfactants and homopolym

  8. Sub-CMC solubilization of dodecane by rhamnolipid in saturated porous media

    Zhong, Hua; Zhang, Hui; Liu, Zhifeng; Yang, Xin; Brusseau, Mark L.; Zeng, Guangming


    Experiments were conducted with a two-dimensional flow cell to examine the effect of monorhamnolipid surfactant at sub-CMC concentrations on solubilization of dodecane in porous media under dynamic flow conditions. Quartz sand was used as the porous medium and artificial groundwater was used as the background solution. The effectiveness of the monorhamnolipid was compared to that of SDBS, Triton X-100, and ethanol. The results demonstrated the enhancement of dodecane solubility by monorhamnolipid surfactant at concentrations lower than CMC. The concentrations (50-210 μM) are sufficiently low that they do not cause mobilization of the dodecane. Retention of rhamnolipid in the porous medium and detection of nano-size aggregates in the effluent show that the solubilization is based on a sub-CMC aggregate-formation mechanism, which is significantly stronger than the solubilization caused by the co-solvent effect. The rhamnolipid biosurfactant is more efficient for the solubilization compared to the synthetic surfactants. These results indicate a strategy of employing low concentrations of rhamnolipid for surfactant-enhanced aquifer remediation (SEAR), which may overcome the drawbacks of using surfactants at hyper-CMC concentrations.

  9. Solubilization and Transformation of Insoluble Zinc Compounds by Fungi Isolated from a Zinc Mine

    Thanawat Sutjaritvorakul


    Full Text Available Fungi were isolated from zinc-containing rocks and mining soil. They were screened for the ability to solubilize and transform three insoluble zinc compounds: ZnO, Zn3(PO4, and ZnCO3. Fungi were plated on potato dextrose agar (PDA medium which was supplemented with 0.5% (w/v of insoluble zinc compounds. Of the strains tested, four fungal isolates showed the highest efficiency for solubilizing all the insoluble zinc compounds, producing clearing zone diameters > 40 mm. These were identified as a Phomopsis spp., Aspergillus sp.1, Aspergillus sp.2, and Aspergillus niger. Zinc oxide was the most easily solubilized compound and it was found that 87%, 52%, and 61% of the tested fungi (23 isolates were able to solubilize zinc oxide, zinc phosphate, and zinc carbonate, respectively. Precipitation of zinc-containing crystals was observed in zinc oxide-containing agar medium underneath colonies of Aspergillus sp.1, and these were identified as zinc oxalate. It is suggested that these kinds of fungi have the potential application in bioremediation practices for heavy metal contaminated soils.

  10. Different behaviours in the solubilization of polycyclic aromatic hydrocarbons in water induced by mixed surfactant solutions.

    Sales, Pablo S; de Rossi, Rita H; Fernández, Mariana A


    Water solubility of polycyclic aromatic hydrocarbons (PAHs), viz, naphthalene and phenanthrene, in micellar solutions at 25°C was investigated, using two series of different binary mixtures of anionic and nonionic surfactants. Tween 80 and Brij-35 were used as nonionic surfactants whereas fatty acids or amphiphilic cyclodextrins (Mod-β-CD) synthesized in our laboratory were used as anionic ones. Solubilization capacity has been quantified in terms of the molar solubilization ratio and the micelle-water partition coefficient, using UV-visible spectrophotometry. Anionic surfactants exhibited less solubilization capacity than nonionics. The mixtures between Tween 80 and Mod-β-CD did not show synergism to increase the solubilization of PAHs. On the other hand, the mixtures formed by Tween 80 and fatty acids at all mole fractions studied produced higher enhancements of the solubility of naphthalene than the individual surfactants. The critical micellar concentration of the mixtures of Tween 80/sodium laurate was determined by surface tension measurements and spectrofluorimetry using pyrene as probe. The system is characterized by a negative interaction parameter (β) indicating attractive interactions between both surfactants in the range of the compositions studied. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. [Solubilization of nitrobenzene in micellar solutions of Tween 80 and inorganic salts].

    Li, Sui; Zhao, Yong-sheng; Xu, Wei; Dai, Ning


    The solubilization of nitrobenzene by a nonionic surfactant Tween 80 was investigated at 10 degrees C. Experimental results indicated that the solubility of nitrobenzene in water was greatly enhanced by Tween 80 at surfactant concentration above CMC(critical micelle concentration) and a linear relationship was obtained between surfactant concentration and nitrobenzene concentration from the solubility curve. The molar solubilization ratio (MSR) value was 5.093 and IgKm was 3.499. The solubilization was attributed to the ethoxylation group in Tween 80 micellar. Effect of four inorganic salts such as NaCl, KCl, CaCl2 , MgCl2 on water solubilities of nitrobenzene in Tween 80 micellar solutions was also investigated by a matrix of batch experiments. Mix the Tween 80-inorganic salts at the total mass ratios of 2:1, 5:1 and 10:1. The results show that the inorganic salts at a high concentration( > or = 500 mg x L(-1)) can enhance the solubilization capacities of Tween 80 micellar solution and increase the value of MSR and IgKm . Because of the salting-out effect between the micellar of Tween 80 and inorganic salts, the volume of micelle turns bigger, which may provide larger solubility volume for nitrobenzene. The mixture of nonionic surfactant and inorganic salts can be used in subsurface remediation as a flushing solution.

  12. "Mixed-solvency approach" - Boon for solubilization of poorly water-soluble drugs

    Maheshwari R


    Full Text Available Based on a large number of experiments on solubilization of poorly water-soluble drugs, the author is of the opinion that hydrotropy is another type of cosolvency and all water-soluble substances whether liquids, solids, or gases may act as solubilizers for poorly water-soluble drugs. In the present investigation, a mixed-solvency approach has been utilized for solubility enhancement of poorly water-soluble drug, salicylic acid (as a model drug. Sixteen blends (having total 40% w/v strength of solubilizers containing various solubilizers among the commonly used hydrotropes (urea and sodium citrate, cosolvents (glycerin, propylene glycol, PEG 300 and PEG 400, and water-soluble solids (PEG 4000 and PEG 6000 were made to study the influence on solubility of salicylic acid. Twelve blends were found to increase the solubility of salicylic acid, synergistically. This approach shall prove a boon in pharmaceutical field to develop various formulations of poorly water-soluble drugs by combining various water-soluble excipients in safe concentrations to give a strong solution (say 25% w/v or so to produce a desirable aqueous solubility of poorly water-soluble drugs. In the present investigation, the mixed-solvency approach has been employed to analyze salicylic acid in the bulk drug sample (using six blends precluding the use of organic solvents (a way to green chemistry.

  13. Performance of pineapple slips inoculated with diazotrophic phosphate-solubilizing bacteria and rock phosphate

    Lílian Estrela Borges Baldotto


    Full Text Available Besides fixing N2, some diazotrophic bacteria or diazotrophs, also synthesize organic acids and are able to solubilize rock phosphates, increasing the availability of P for plants. The application of these bacteria to pineapple leaf axils in combination with rock phosphate could increase N and P availability for the crop, due to the bacterial activity of biological nitrogen fixation and phosphate solubilization. The objectives of this study were: (i to select and characterize diazotrophs able to solubilize phosphates in vitro and (ii evaluate the initial performance of the pineapple cultivars Imperial and Pérola in response to inoculation with selected bacteria in combination with rock phosphate. The experiments were conducted at Universidade Estadual do Norte Fluminense Darcy Ribeiro, in 2009. In the treatments with bacteria the leaf contents of N, P and K were higher than those of the controls, followed by an increase in plant growth. These results indicate that the combined application of diazotrophic phosphate-solubilizing bacteria Burkholderia together with Araxá rock phosphate can be used to improve the initial performance of pineapple slips.

  14. Lignin solubilization and aqueous phase reforming for the production of aromatic chemicals and hydrogen

    Zakzeski, J.; Weckhuysen, B.M.


    The solubilization and aqueous phase reforming of lignin, including kraft, soda, and alcell lignin along with sugarcane bagasse, at low temperatures (T≤498 K) and pressures (P≤29 bar) is reported for the first time for the production of aromatic chemicals and hydrogen. Analysis of lignin model compo

  15. Ability of rhizobacteria of valerian in phosphate solubilization and their symbiotic efficiency

    B. S. Ghods-Alavi


    Full Text Available Phosphorus (P and nitrogen (N are the most essential nutrients for plant growth. Deficiency of these elements causes a considerable loss of crop yield. Nowadays, using of biofertilizers in cultivation of strategic crops, such as greenhouse crops, is very important because of resolving the P and N deficiency. This research was carried out to evaluate the potential of rhizobacteria of valerian (as a medicinal plant in phosphate solubilization, N fixation, as well as salinity tolerance in the root zone, in laboratory conditions. To do so, 40 bacteria were isolated from the rhizosphere of valerian, and their mineral-phosphate solubilizing activities were measured on solid and liquid Sperber media. Besides, N-fixation ability of the isolates in symbiosis with wheat was evaluated under greenhouse conditions. Results showed that 77.5% of the isolates had the ability to solubilize mineral phosphate. Two isolates belonging to Pseudomonas and Xanthomonas genera and two isolates belonging to Pseudomonas genus had the maximum ability for phosphate solubilization in solid and liquid media, respectively. Among the isolated bacteria, one isolate belonging to Pseudomonas genus, showed the highest symbiosis efficiency (132%. Furthermore, results of the salinity-tolerance test of the bacteria revealed that all of the isolated bacteria could tolerate 500 mM NaCl concentration and two isolates (belonging to Pseudomonas genus were able to tolerate salinity of 1000 mM NaCl. Overall, it can be concluded that rhizobacteria of valerian have high potential to be used as biofertilizer.

  16. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R;


    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Ma...

  17. Role of phosphate solubilizing bacteria on rock phosphate solubility and growth of aerobic rice.

    Panhwar, Q A; Radziah, O; Zaharah, A R; Sariah, M; Razi, I Mohd


    Use of phosphate-solubilizing bacteria (PSB) as inoculants has concurrently increased phosphorous uptake in plants and improved yields in several crop species. The ability of PSB to improve growth of aerobic rice (Oryza sativa L.) through enhanced phosphorus (P) uptake from Christmas island rock phosphate (RP) was studied in glasshouse experiments. Two isolated PSB strains; Bacillus spp. PSB9 and PSB16, were evaluated with RP treatments at 0, 30 and 60 kg ha(-1). Surface sterilized seeds of aerobic rice were planted in plastic pots containing 3 kg soil and the effect of treatments incorporated at planting were observed over 60 days of growth. The isolated PSB strains (PSB9 and PSB16) solubilized significantly high amounts of P (20.05-24.08 mg kg(-1)) compared to non-inoculated (19-23.10 mg kg(-1)) treatments. Significantly higher P solubilization (24.08 mg kg(-1)) and plant P uptake (5.31 mg plant(-1)) was observed with the PSB16 strain at the highest P level of 60 kg ha(-1). The higher amounts of soluble P in the soil solution increased P uptake in plants and resulted in higher plant biomass (21.48 g plant(-1)). PSB strains also increased plant height (80 cm) and improved root morphology in aerobic rice. The results showed that inoculation of aerobic rice with PSB improved phosphate solubilizing activity of incorporated RP.

  18. Lignin solubilization and aqueous phase reforming for the production of aromatic chemicals and hydrogen

    Zakzeski, J.; Weckhuysen, B.M.


    The solubilization and aqueous phase reforming of lignin, including kraft, soda, and alcell lignin along with sugarcane bagasse, at low temperatures (T≤498 K) and pressures (P≤29 bar) is reported for the first time for the production of aromatic chemicals and hydrogen. Analysis of lignin model

  19. Selection of phosphorus solubilizing bacteria with biocontrol potential for growth in phosphorus rich animal bone charcoal

    Postma, J.; Nijhuis, E.H.; Sommeus, E.


    Bacteria with the ability to solubilize phosphorus (P) and to improve plant health were selected and tested for growth and survival in P-rich animal bone charcoal (ABC). ABC is suggested to be suitable as a carrier for biocontrol agents, offering them a protected niche as well as delivering phosphat

  20. Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process

    Freydell, E.J.; Wielen, van der L.A.M.; Eppink, M.H.M.; Ottens, M.


    Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be ref

  1. Kinetics of degradation of surfactant-solubilized fluoranthene by a Sphingomonas paucimobilis

    Willumsen, P.A. (National Environmental Research Institute, Roskilde (Denmark). Dept. of Marine Ecology and Microbiology); Arvin, E. (Technical Univ. of Denmark, Lyngby (Denmark). Dept. of Environmental Science and Engineering)


    To achieve a better quantitative understanding of the stimulating or inhibiting effect of surfactants on the metabolism of polycyclic aromatic hydrocarbons (PAHs), a biodegradation model describing solubilization, bioavailability, and biodegradation of crystalline fluoranthene is proposed and used to model experimental data. The degradation was investigated in batch systems containing the PAH-degrading bacterium Sphingomonas paucimobilis strain EPA505, the nonionic surfactant Triton X-100, and a fluoranthene-amended liquid mineral salts medium. Surfactant-enhanced biodegradation is complex; however, the biodegradation model predicted fluoranthene disappearance and the initial mineralization well. Surfactant-amendment did increase fluoranthene mineralization rates by strain EPA505; however, the increases were not proportional to the rates of fluoranthene solubilization. The surfactant clearly influenced the microbial PAH metabolism as indicated by a rapid accumulation of colored products and by a surfactant -related decreased in the overall extent of fluoranthene mineralization. Model estimations of the bioavailability of micelle-solubilized fluoranthene, the relatively fast fluoranthene disappearance, and the accumulation of extracellular compounds in the degradation system suggest that low availability of micellar fluoranthene is not the only factor controlling surfactant-enhanced biodegradation. Also factors such as the extent of accumulation and bioavailability of the PAH metabolites and the crystalline solubilization rate in the presence of surfactants may determine the overall effect of surfactant-enhanced biodegradation of high molecular weight PAHs.

  2. Comparison of Yeast Cell Protein Solubilization Procedures for Two-dimensional Electrophoresis

    Harder, A; Wildgruber, R; Nawrocki, A;


    Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high ...

  3. Structural analysis of bacteriorhodopsin solubilized by lipid-like phosphocholine biosurfactants with varying micelle concentrations.

    Wang, Xiaoqiang; Huang, Haihong; Sun, Chenghao; Huang, Fang


    Surfactants that can provide a more natural substitute for lipid bilayers are important in the purification and in vitro study of membrane proteins. Here we investigate the structural response of a model membrane protein, bacteriorhodopsin (BR), to phosphocholine biosurfactants. Phosphocholine biosurfactants are a type of biomimetic amphiphile that are similar to phospholipids, in which membrane proteins are commonly embedded. Multiple spectroscopic and zeta potential measurements are employed to characterize the conformational change, secondary and tertiary structure, oligomeric status, surface charge distribution and the structural stability of BR solubilized with phosphocholine biosurfactants of varying tail length. The process of phosphocholine micelle formation is found to facilitate the solubilization of BR, and for long-chain phosphocholines, concentrations much higher than their critical micelle concentrations achieve good solubilization. Phosphocholine biosurfactants are shown to be mild compared with the ionic surfactant SDS or CTAB, and tend to preserve membrane protein structure during solubilization, especially at low micelle concentrations, by virtue of their phospholipid-like zwitterionic head groups. The increase of alkyl chain length is shown to obviously enhance the capability of phosphocholine biosurfactants to stabilize BR. The underlying mechanism for the favorable actions of phosphocholine biosurfactant is also discussed.

  4. Genome Sequence of Selenium-Solubilizing Bacterium Caulobacter vibrioides T5M6

    Wang, Yihua; Qin, Yanan; Kot, Witold


    Caulobacter vibrioides T5M6 is a Gram-negative strain that strongly solubilizes selenium (Se) mineral into Se(IV) and was isolated from a selenium mining area in Enshi, southwest China. This strain produces the phytohormone IAA and promotes plant growth. Here we present the genome of this strain ...

  5. Exploring the Arabidopsis Proteome: Influence of Protein Solubilization Buffers on Proteome Coverage

    Marondedze, Claudius


    The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

  6. Effect of HPMC concentration on β-cyclodextrin solubilization of norfloxacin.

    Loh, Gabriel Onn Kit; Tan, Yvonne Tze Fung; Peh, Kok Khiang


    The effect of hydroxypropyl methylcellulose (HPMC) concentration on β-cyclodextrin (β-CD) solubilization of norfloxacin was examined. The solubility and dissolution of norfloxacin/β-CD and norfloxacin/β-CD/HPMC inclusion complexes were studied. The presence of β-CD increased significantly the solubility and dissolution of norfloxacin. The addition of HPMC until 5% (w/w) improved the solubilization of norfloxacin but further addition above 5% (w/w), decreased norfloxacin solubilization. Fourier transformed Infra-red (FTIR) showed that norfloxacin was successfully included into β-CD. Differential scanning calorimetry (DSC) showed that the norfloxacin endothermic peak shifted to a lower temperature with reduced intensity indicating the formation of inclusion complex. The addition of HPMC reduced further the intensity of norfloxacin endothermic peak. Most of the sharp and intense peaks of norfloxacin disappeared with the addition of HPMC. In conclusion, the concentration of hydrophilic polymer used to enhance β-CD solubilization of poorly soluble drugs is very critical. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Effect of benzo(a)pyrene exposure on fluoranthene metabolism by mouse adipose tissue microsomes.

    Huderson, Ashley C; Harris, Deacqunita L; Niaz, Mohammad S; Ramesh, Aramandla


    The present study has been undertaken to examine whether exposure to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound, influences the metabolism of fluoranthene (FLA), another PAH compound. Microsomes were isolated from the adipose tissue of mice that received 50 microg/kg BaP and incubated with FLA (3 microM) alone; FLA in combination with BaP at equimolar concentrations, and a control group that received nothing. Post-incubation, samples were extracted with ethyl acetate and analyzed for FLA metabolites by reverse-phase HPLC with fluorescence detection. The rate of FLA metabolism (pmol of metabolite/min/mg protein) was increased when microsomes from BaP-treated mice were exposed to FLA alone and FLA in combination with BaP, compared to controls. On the other hand, the difference in FLA metabolic rate between microsomes that were exposed to FLA + BaP was higher than the ones that received FLA. The microsomes from BaP-pre-treated mice produced a considerably higher proportion of FLA 2, 3-diol, and 2, 3 D FLA when microsomes were incubated with FLA. There were no differences in the FLA metabolite types formed when BaP-pre-treated mice were co-incubated with BaP and FLA than with FLA alone. The enhanced biotransformation of FLA as a result of prior and concomitant exposure to BaP may have implications for assessment of risks arising from human exposure to PAH mixtures.

  8. In vitro glucuronidation kinetics of deoxynivalenol by human and animal microsomes and recombinant human UGT enzymes.

    Maul, Ronald; Warth, Benedikt; Schebb, Nils Helge; Krska, Rudolf; Koch, Matthias; Sulyok, Michael


    The mycotoxin deoxynivalenol (DON), formed by Fusarium species, is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon ingestion, the majority of the toxin is excreted by humans and animal species as glucuronide conjugate. First in vitro data indicated that DON phase II metabolism is strongly species dependent. However, kinetic data on the in vitro metabolism as well as investigations on the specific enzymes responsible for DON glucuronidation in human are lacking. In the present study, the DON metabolism was investigated using human microsomal fractions and uridine-diphosphoglucuronyltransferases (UGTs) as well as liver microsomes from five animal species. Only two of the twelve tested human recombinant UGTs led to the formation of DON glucuronides with a different regiospecificity. UGT2B4 predominantly catalyzed the formation of DON-15-O-glucuronide (DON-15GlcA), while for UGT2B7 the DON-3-O-glucuronide (DON-3GlcA) metabolite prevailed. For human UGTs, liver, and intestinal microsomes, the glucuronidation activities were low. The estimated apparent intrinsic clearance (Clapp,int) for all human UGT as well as tissue homogenates was microsomes, moderate Clapp,int between 1.5 and 10 mL/min mg protein were calculated for carp, trout, and porcine liver. An elevated glucuronidation activity was detected for rat and bovine liver microsomes leading to Clapp,int between 20 and 80 mL/min mg protein. The obtained in vitro data points out that none of the animal models is suitable for estimating the human DON metabolism with respect to the metabolite pattern and formation rate.

  9. Estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes.

    Kitamura, Shigeyuki; Ohmegi, Motoko; Sanoh, Seigo; Sugihara, Kazumi; Yoshihara, Shin'ichi; Fujimoto, Nariaki; Ohta, Shigeru


    In this study we examined estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes. Trans-1,2-diphenylcyclobutane (TCB), cis-1,2-diphenylcyclobutane (CCB), 1,3-diphenylpropane, 2,4-diphenyl-1-butene, 2,4,6-triphenyl-1-hexene, and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin were negative in the yeast estrogen screening assay and estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, TCB exhibited estrogenic activity after incubation with liver microsomes of phenobarbital-treated rats in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Minor activity was observed when liver microsomes of untreated or 3-methylcholanthrene-treated rats were used instead of those from phenobarbital-treated rats. CCB, 1,3-diphenylpropane, and 2,4-diphenyl-1-butene also exhibited estrogenic activity after metabolic activation by liver microsomes, but the activity was lower than that of TCB. 2,4,6-Triphenyl-1-hexene and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin did not show estrogenic activity after such incubation. When TCB was incubated with liver microsomes of phenobarbital-treated rats in the presence of NADPH, three metabolites were detected by high-performance liquid chromatography (HPLC). One metabolite isolated by HPLC exhibited a significant estrogenic activity. The active metabolite was identified as trans-1-(4-hydroxyphenyl)-2-phenylcyclobutane by mass and nuclear magnetic resonance spectral analysis. These results suggest that the estrogenic activity of TCB was caused by the formation of the 4-hydroxylated metabolite. PMID:12611662

  10. Metabolism of the major Echinacea alkylamide N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomes.

    Toselli, F; Matthias, A; Bone, K M; Gillam, E M J; Lehmann, R P


    Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N-dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N-dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic-metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified.

  11. Metabolic activation of the olfactory toxicant, dichlobenil, in rat olfactory microsomes: comparative studies with p-nitrophenol.

    Eriksson, C; Brittebo, E B


    The tissue-specific toxicity of the herbicide, dichlobenil (2,6-dichlorobenzonitrile), in the olfactory mucosa is related to a cytochrome P450 (P450)-dependent metabolism, depletion of glutathione and covalent binding of metabolites. Pretreatment of mice with diethyldithiocarbamate (DEDTC) protected against the dichlobenil-induced necrosis. Addition of DEDTC abolished the covalent binding of [14C]-dichlobenil to rat olfactory microsomes, whereas P4502E1-substrates such as ethanol, acetone or p-nitrophenol (NP) had no effect. The NP-hydroxylation in olfactory microsomes was > 6 times higher than that in liver microsomes and was markedly decreased following addition of dichlobenil, DEDTC or metyrapone. In liver microsomes of acetone-treated rats the NP-hydroxylation was markedly decreased following addition of DEDTC, whereas metyrapone and dichlobenil had no effect. In acetone-treated rats, the NP-hydroxylation and the metabolic activation of [14C]-dichlobenil in olfactory microsomes were decreased to 50 and 73% of untreated controls, respectively, whereas in liver microsomes these activities increased > 6 and 3.5-fold, respectively. An antibody to P4502E1 had no effect on the NP-hydroxylation or metabolic activation of [14C]-dichlobenil in olfactory microsomes, whereas the NP-hydroxylation in liver microsomes of acetone-treated rats was markedly decreased. In conclusion, the results do not support a major role for P4502E1 in the metabolic activation of dichlobenil or hydroxylation of NP in rat olfactory microsomes and suggest that these catalytic activities in the olfactory mucosa may represent a common form of P450.

  12. Hydrolytic metabolism of phenyl and benzyl salicylates, fragrances and flavoring agents in foods, by microsomes of rat and human tissues.

    Ozaki, Hitomi; Sugihara, Kazumi; Tamura, Yuki; Fujino, Chieri; Watanabe, Yoko; Uramaru, Naoto; Sone, Tomomichi; Ohta, Shigeru; Kitamura, Shigeyuki


    Salicylates are used as fragrance and flavor ingredients for foods, as UV absorbers and as medicines. Here, we examined the hydrolytic metabolism of phenyl and benzyl salicylates by various tissue microsomes and plasma of rats, and by human liver and small-intestinal microsomes. Both salicylates were readily hydrolyzed by tissue microsomes, predominantly in small intestine, followed by liver, although phenyl salicylate was much more rapidly hydrolyzed than benzyl salicylate. The liver and small-intestinal microsomal hydrolase activities were completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Phenyl salicylate-hydrolyzing activity was co-eluted with carboxylesterase activity by anion exchange column chromatography of the Triton X-100 extracts of liver and small-intestinal microsomes. Expression of rat liver and small-intestinal isoforms of carboxylesterase, Ces1e and Ces2c (AB010632), in COS cells resulted in significant phenyl salicylate-hydrolyzing activities with the same specific activities as those of liver and small-intestinal microsomes, respectively. Human small-intestinal microsomes also exhibited higher hydrolyzing activity than liver microsomes towards these salicylates. Human CES1 and CES2 isozymes expressed in COS cells both readily hydrolyzed phenyl salicylate, but the activity of CES2 was higher than that of CES1. These results indicate that significant amounts of salicylic acid might be formed by microsomal hydrolysis of phenyl and benzyl salicylates in vivo. The possible pharmacological and toxicological effects of salicylic acid released from salicylates present in commercial products should be considered.

  13. The effect of the bay-region 12-methyl group on the stereoselective metabolism at the K-region of 7,12-dimethylbenz[a]anthracene by rat liver microsomes.

    Yang, S K; Fu, P P


    The enantiomers of a trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by rat liver microsomes (microsomal fractions) were resolved by chiral stationary-phase high-performance liquid chromatography. The major 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol enantiomer and its hydrogenation product 5,6,8,9,10,11-hexahydro-trans-5,6-diol were found to have 5S,6S absolute configurations by the exciton chirality c.d. method. The R,R/S,S enantiomer ratios of 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by liver microsomes from untreated, 3-methylcholanthrene-treated and phenobarbital-treated male Sprague-Dawley rats were found to be 11:89, 6:94, and 5:95 respectively. These findings and those reported previously on the metabolic formations of trans-5,6-dihydrodiols from 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene suggest that the 12-methyl group in 7,12-dimethylbenz[a]anthracene plays an important role in determining the stereoselective metabolism at the K-region 5,6-double bond. Furthermore, the finding that formation of 5S,6S-dihydrodiol as the predominant enantiomer was not significantly affected by the isoenzymic composition of cytochrome P-450 present in microsomes prepared from the livers of the rats pretreated with the different inducing agents indicates that the stereoselectivity depends on the substrate metabolized rather than on the precise nature of the metabolizing-enzyme system. PMID:6439187

  14. Multifunctional properties of phosphate-solubilizing microorganisms grown on agro-industrial wastes in fermentation and soil conditions.

    Vassileva, Maria; Serrano, Mercedes; Bravo, Vicente; Jurado, Encarnación; Nikolaeva, Iana; Martos, Vanessa; Vassilev, Nikolay


    One of the most studied approaches in solubilization of insoluble phosphates is the biological treatment of rock phosphates. In recent years, various techniques for rock phosphate solubilization have been proposed, with increasing emphasis on application of P-solubilizing microorganisms. The P-solubilizing activity is determined by the microbial biochemical ability to produce and release metabolites with metal-chelating functions. In a number of studies, we have shown that agro-industrial wastes can be efficiently used as substrates in solubilization of phosphate rocks. These processes were carried out employing various technologies including solid-state and submerged fermentations including immobilized cells. The review paper deals critically with several novel trends in exploring various properties of the above microbial/agro-wastes/rock phosphate systems. The major idea is to describe how a single P-solubilizing microorganism manifests wide range of metabolic abilities in different environments. In fermentation conditions, P-solubilizing microorganisms were found to produce various enzymes, siderophores, and plant hormones. Further introduction of the resulting biotechnological products into soil-plant systems resulted in significantly higher plant growth, enhanced soil properties, and biological (including biocontrol) activity. Application of these bio-products in bioremediation of disturbed (heavy metal contaminated and desertified) soils is based on another important part of their multifunctional properties.

  15. Drug activity screening based on microsomes-hydrogel system in predicting metabolism induced antitumor effect of oroxylin A.

    Yang, Huiying; Li, Jianfeng; Zheng, Yuanting; Zhou, Lu; Tong, Shanshan; Zhao, Bei; Cai, Weimin


    A novel microsomes-hydrogel added cell culture system (MHCCS) was employed in the antitumor activity screening of natural compounds, aiming to achieve drug screening with better in vivo correlation, higher initiative to explore the potential active metabolites, and investigation of the antitumor mechanism from the perspective of metabolism. MTT assay and cell apoptosis detection showed that test drug oroxylin A (OA) had enhanced cytotoxicity and wogonin (W) with reduced cytotoxicity on MCF-7 cell line upon MHCCS incubation. In vivo antitumor evaluations also demonstrated that OA induced higher tumor inhibition than W at the same dosage. To explore the reasons, nine major metabolites of OA were separated and collected through UPLC-Q-TOF and semi-preparative HPLC. Metabolites M318 exhibited higher cytotoxicity than OA and other metabolites by MTT assay. (1)H NMR spectrums, HPLC and TOF MS/MS results revealed that OA was catalyzed into its active metabolite M318 via a ring-opening reaction. M318 induced significant cell apoptosis and S-phase arrest through affecting tumor survival related genes after mechanism study. In conclusion, our MHCCS could be a useful tool for drug activity screening from a perspective of metabolism.

  16. Rapid metabolite discovery, identification, and accurate comparison of the stereoselective metabolism of metalaxyl in rat hepatic microsomes.

    Wang, Xinru; Qiu, Jing; Xu, Peng; Zhang, Ping; Wang, Yao; Zhou, Zhiqiang; Zhu, Wentao


    Metabolite identification and quantitation impose great challenges on risk assessment of agrochemicals, as many metabolite standards are generally unavailable. In this study, metalaxyl metabolites were identified by time-of-flight mass spectrometry and semiquantified by triple quadrupole tandem mass spectrometry with self-prepared (13)C-labeled metalaxyl metabolites as internal standards. Such methodology was employed to characterize the stereoselective metabolism of metalaxyl in rat hepatic microsomes successfully. Metabolites derived from hydroxylation, demethylation, and didemethylation were identified and semiquantified. The results indicated that (+)-S-metalaxyl eliminated preferentially as the enantiomer fraction was 0.32 after 60 min incubation. The amounts of hydroxymetalaxyl and demethylmetalaxyl derived from (-)-R-metalaxyl were 1.76 and 1.82 times higher than that of (+)-S-metalaxyl, whereas didemethylmetalaxyl derived from (+)-S-metalaxyl was 1.44 times larger than that from (-)-R-metalaxyl. This study highlights a new quantitation approach for stereoselective metabolism of chiral agrochemicals and provides more knowledge on metalaxyl risk assessment.

  17. Elicitor-induced biosynthesis of psoralens in Ammi majus L. suspension cultures. Microsomal conversion of demethylsuberosin into (+)marmesin and psoralen.

    Hamerski, D; Matern, U


    Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3-14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase. Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome-P450-dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and O-prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.

  18. A New Protocol for Solubilization, Refolding and Purification of Recombinant Human Granulocyte Colony-stimulating Factor in Inclusion Bodies

    Jia Hua LIU; Chao Zhan WANG; Xin Du GENG


    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4×107 IU/mg.

  19. Covalent and non-covalent functionalization and solubilization of double-walled carbon nanotubes in nonpolar and aqueous media

    L S Panchakarla; A Govindaraj


    Double-walled carbon nanotubes (DWNTs) have been functionalized by both covalent and non-covalent means. Covalent functionalization has been carried out by attaching an aliphatic amide function to DWNTs which enable solubilization in non-polar solvents. Solubilization in non-polar solvents has also been accomplished by non-covalent functionalization by using 1-pyrenebutanoicacid succinimidyl ester (PYBS). Non-covalent functionalization of DWNTs has been carried out by using polyethylene glycol (PEG) and polyoxyethylene(40)nonylphenyl ether (IGPAL), both of which enable solubilization in aqueous media. These functionalized DWNTs have been characterized by transmission electron microscopy, IR and Raman spectroscopy.

  20. Hydrotrope-Solubilization Action of Urea in CTAB/n-C5H11-OH/H2 O System

    GUO Rong; ZOU Ai-Hua; YANG Han-Min


    Urea can enhance the aqueous solubility of surfactant CTAB (hexadecyltrimethylammonium bromide) when it shows the hydrotrope action.It will show the hydrotrope-solubilization action when the solubilized amount microemulsion and that of n-C5H11OH in O/W increased The mechanism of the hydrotrope-solubilization ac-tion of urea is the increase of the stability of W/O and O/Wmicroemulsion and structural transition from the lamellar liq-uid crystalline phase to the bicontinuous structure.

  1. Large scale solubilization of coal and bioconversion to utilizable energy. Quarterly report, October 1--December 31, 1996

    Mishra, N.C.


    In order to develop a system for a large scale coal solubilization and its bioconversion to utilizable fuel, the author plans to clone the genes encoding Neurospora protein that facilitate depolymerization of coal. He also plans to use desulfurizing bacteria to remove the sulfur in situ and use other microorganisms to convert biosolubilized coal into utilizable energy following an approach utilizing several microorganisms. In addition the product of coal solubilized by fungus will be characterized to determine their chemical nature and the mechanism of reaction catalyzed by fungal product during in vivo and in vitro solubilization by the fungus or purified fungal protein.

  2. Large scale solubilization of coal and bioconversion to utilizable energy. Fifth quarterly technical report, January 1, 1995--March 31, 1995

    Mishra, N.C.


    In order to develop a system for a large scale coal solubilization and its bioconversion to utilizable fuel, we plan to clone the genes encoding Neurospora protein that facilitate depolymerization of coal. We also plan to use desulfurizing bacteria to remove the sulfur in situ and use other microorganisms to convert biosolubilized coal into utilizable energy following an approach utilizing several microorganisms. In addition the product of coal solubilized by fungus will be characterized to determine their chemical nature and the mechanism of reaction catalyzed by fungal product during in vivo and in vitro solubilization by the fungus or purified fungal protein.

  3. Calcium Alginate and Calcium Alginate-Chitosan Beads Containing Celecoxib Solubilized in a Self-Emulsifying Phase

    Segale, Lorena; Giovannelli, Lorella; Mannina, Paolo; Pattarino, Franco


    In this work alginate and alginate-chitosan beads containing celecoxib solubilized into a self-emulsifying phase were developed in order to obtain a drug delivery system for oral administration, able to delay the drug release in acidic environment and to promote it in the intestinal compartment. The rationale of this work was linked to the desire to improve celecoxib therapeutic effectiveness reducing its gastric adverse effects and to favor its use in the prophylaxis of colon cancer and as adjuvant in the therapy of familial polyposis. The systems were prepared by ionotropic gelation using needles with different diameters (400 and 600 μm). Morphology, particle size, swelling behavior, and in vitro drug release performance of the beads in aqueous media with different pH were investigated. The experimental results demonstrated that the presence of chitosan in the formulation caused an increase of the mechanical resistance of the bead structure and, as a consequence, a limitation of the bead swelling ability and a decrease of the drug release rate at neutral pH. Alginate-chitosan beads could be a good tool to guarantee a celecoxib colon delivery. PMID:27127680

  4. Calcium Alginate and Calcium Alginate-Chitosan Beads Containing Celecoxib Solubilized in a Self-Emulsifying Phase

    Lorena Segale


    Full Text Available In this work alginate and alginate-chitosan beads containing celecoxib solubilized into a self-emulsifying phase were developed in order to obtain a drug delivery system for oral administration, able to delay the drug release in acidic environment and to promote it in the intestinal compartment. The rationale of this work was linked to the desire to improve celecoxib therapeutic effectiveness reducing its gastric adverse effects and to favor its use in the prophylaxis of colon cancer and as adjuvant in the therapy of familial polyposis. The systems were prepared by ionotropic gelation using needles with different diameters (400 and 600 μm. Morphology, particle size, swelling behavior, and in vitro drug release performance of the beads in aqueous media with different pH were investigated. The experimental results demonstrated that the presence of chitosan in the formulation caused an increase of the mechanical resistance of the bead structure and, as a consequence, a limitation of the bead swelling ability and a decrease of the drug release rate at neutral pH. Alginate-chitosan beads could be a good tool to guarantee a celecoxib colon delivery.

  5. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung


    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

  6. Kinetic studies on toluene metabolism in ethanol- and phenobarbital-induced rat liver microsomes in vitro

    Wang Ruisheng; Nakajima, Tamie (Shinshu Univ., Matsumoto (Japan). Dept. of Hygiene)


    In vitro metabolism of toluene was investigated at substrate concentrations of 0.03-6.25 mM in liver microsomes from control and ethanol- and phenobarbital (PB)-treated rats. Three metabolites, benzylalcohol (BA), o-and p-cresol, were measured by high-performance liquid chromatograph. BA was the main metabolite of toluene, whereas o- and p-creasol contributed only 1.1-1.5% and 1.7-2.8% of total metabolites, respectively, in microsomes from control rats. Ethanol treatment showed little effect on the percentages of three metabolites, but PB increased the percentages of o- and p-cresol to as high as 5.5% and 8.0%, respectively, following the increase in toluene concentration. There were two different isozymes with different K{sub m} involved in the side-chain hydroxylation of toluene in microsomes from control and ethanol-treated rats. One had a low K{sub m} value (0.13-0.17 mM) and could be greatly induced with ethanol treatment. The other was a high K{sub m} isozyme (0.60-0.87 mM). PB-induced isozyme showed a similar K{sub m} value to that of the high K{sub m} isozyme existing in microsomes from control and ethanol-treated rats. Two isozymes were involved in the formation of p-cresol in microsomes of control rats; the low-K{sub m} type had a similar value (0.15 mM) to the low isozyme of BA formation, but the high K{sub m} isozyme had a larger value (2.04 mM) than the high isozyme of BA. Only one enzyme responsible for o-cresol formation was detected in microsomes of control rats, and had a similar K{sub m} (2.11 mM) to that of the high K{sub m} isozyme of p-cresol. The high K{sub m} and low V{sub max} values of isozymes of aromatic hydroxylation account for the minor proportion of cresols. Ethanol and PB treatments enhanced the metabolism of toluene, but they differ in that the ethanol-inducible isozyme metabolized toluene at a faster rate at low substrate concentrations, whereas PB did so at high toluene concentrations. (orig./MG).

  7. Water-solubilization of alkyloxo(methoxo)porphyrinatoantimony bromides.

    Matsumoto, Jin; Tanimura, Shin-ichiro; Shiragami, Tsutomu; Yasuda, Masahide


    In order to develop water-soluble porphyrins, alkyloxo(methoxo)porphyrinatoantimony bromides (alkyl = hexyl (1a), decyl (1b), dodecyl (1c), tetradecyl (1d), octadecyl (1e)) were prepared. 1 had more than 1 mmol dm(-3) of solubility in water. From the dependence of the half-width of the bands in the absorption spectra and surface tension on the concentration of 1, it was estimated that 1b-d were present as aggregates in concentrations higher than 10 micromol dm(-3). From the NMR analysis in D(2)O, it was deduced that the alkyloxo ligands of 1 were arranged alternately in the aggregates. The diameter of the aggregates of 1 in water was determined to be around 100 nm by the dynamic light scattering method. Since the solubilities of di(methoxo)tetraphenylporphyrinatoantimony bromide and 5-(4'-decyloxyphenyl)-10,15,20-triphenylporphyrinato(dimethoxo)antimony(v) bromide were low, it was calculated that the long alkyl axial ligands were requisite for the high solubility in water.

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

    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...

  9. In vitro metabolic clearance of pyrethroid pesticides by rat and human hepatic microsomes and cytochrome P450 isoforms

    Species differences in the intrinsic clearance (CLint) and the enzymes involved in the metabolism of pyrethroid pesticides were examined in rat and human hepatic microsomes. The pyrethroids bifenthrin, S-bioallethrin, bioresmethrin, β-cyfluthrin, cypermethrin, cis-per...

  10. Immunochemical and immunoelectron microscope studies on localization of NADPH-cytochrome c reductase on rat liver microsomes


    By the use of ferritin-conjugated antibody (conjugate) indirect immunoelectron microscopy, NADPH-cytochrome c reductase was localized on rat liver microsomes. Most microsomes in the sections had from 1 to 12 conjugates on their outer surfaces. Among the conjugates, 83% was estimated to bind to NADPH-cytochrome c reductase at a molecular ratio of 1:1, 12% at the ratio of 2:1, and 5% at the ratio of 3 or 4:1. The correlation between immunochemical and morphological data confirmed that most of the NADPH-cytochrome c reducatase reacted with the conjugates. Subsequent morphological analyses have revealed that the enzyme is distributed homogeneously on the outer surfaces of microsomes but heterogeneously within microsomes in groups of three to five enzyme molecules. PMID:812876

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

    Nicks, Diane


    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.

  12. Amide hydrolysis of a novel chemical series of microsomal prostaglandin E synthase-1 inhibitors induces kidney toxicity in the rat

    Bylund, Johan; Annas, Anita; Hellgren, Dennis; Bjurström, Sivert; Andersson, Håkan; Svanhagen, Alexander


    A novel microsomal prostaglandin E synthase 1 (mPGES-1) inhibitor induced kidney injury at exposures representing less than 4 times the anticipated efficacious exposure in man during a 7-day toxicity study in rats...

  13. Liquefaction and/or solubilization of Spanish coals by newly isolated microorganisms

    Monistrol, I.F.; Laborda, F. (Universidad de Alcala de Henares, Alcala de Henares (Spain). Dept. de Microbiologia Parasitologia)


    A screening procedure has been set up for isolating microorganisms capable of liquefying coal. Spanish coals were used in the tests, namely a brown lignite from Galicia, sub-bituminous coal from Teruel and hard coal from Minas Figaredo S.A. (Asturias). Among the isolated strains several microorganisms proved capable of liquefying untreated lignite. When lignites were pretreated a more intense and rapid liquefaction was achieved, chelating agents being among the best pretreatment. None of the isolated microorganisms could satisfactorily liquefy sub-bituminous and hard coals in solid media. On the other hand, some fungi grew specifically on the untreated coals, engulfing them and in many cases a soft slurry was obtained. Several of the isolated microorganisms were able to solubilize all three untreated Spanish coals in liquid media. Coal solubilization was measured spectrophotometrically at 300, 400 and 450 nm. 5 refs., 10 figs., 3 tabs.

  14. Influence of biochar application on potassium-solubilizing Bacillus mucilaginosus as potential biofertilizer.

    Liu, Sainan; Tang, Wenzhu; Yang, Fan; Meng, Jun; Chen, Wenfu; Li, Xianzhen


    Biochar can enhance soil fertility to increase agricultural productivity, whereas its improvement in soil microbial activity is still unclear. In this article, the influence of biochar on the cell growth and the potassium-solubilizing activity of Bacillus mucilaginosus AS1153 was examined. The impact on cell growth is related to the biochar-derived feedstocks and the particle size of biochar. Both intrinsic features and inner component fraction can promote the cell growth of B. mucilaginosus AS1153. The potassium-solubilizing activity was increased by 80% when B. mucilaginosus was incubated in conjunction with the biochar derived from corn stover. The survival time of B. mucilaginosus also was prolonged by adsorption in biochar. The experimental results suggested that the biochar containing B. mucilaginosus could be used as a potential biofertilizer to sustain crop production.

  15. Study of Acid Phosphatase in Solubilization of Inorganic Phosphates by Piriformospora indica.

    Seshagiri, Swetha; Tallapragada, Padmavathi


    Phosphorus is an essential plant macronutrient present in the soil. Only a small portion of phosphorus in soil is taken up by plants and the rest of it becomes unavailable to plants as it is immobilized. Phosphate solubilizing microorganisms play a vital role in converting the insoluble form of phosphates to the soluble form. The present paper reports the solubilization of tricalcium phosphate, rock phosphate, single super phosphate, zinc phosphate and aluminum phosphate by Piriformospora indica with the production of organic acids as well as acid phosphatase. The amount of phosphate released (4.73 mg ml(-1)) and titratable acidity (0.12%) was found to be the highest in the case of single super phosphate as compared to other phosphate sources. High performance liquid chromatography (HPLC) revealed the presence of oxalic acid, lactic acid, citric acid and succinic acid in the media. Highest phosphatase activity was observed with the cell membrane extract of the organism in the presence of zinc phosphate.

  16. Molecular size and amino acid composition of H-2d antigen solubilized in Nonidet P-40.

    Rossowski, W; Kloczewiak, M; Radzikowski, C; Strzadala, L


    H-2d antigenic material solubilized by the detergent Nonidet P-40 from L-1210 mouse leukemia cells was isolated by gel filtration on Bio-Gel P-100. A single peak eluted in the void volume consisted of about 90% protein, 8% hexose and traces of sialic acids. In sedimentation velocity runs, the antigen sedimented as a single peak of 3-1 S. Molecular weight determined by sedimentation equilibrium as well as calculated from amino acid composition was found to be in the range of 53,000 daltons and approx. 45,000-51,000 when calculated from sodium dodecyl sulfate polyacrylamide gel electrophoresis. Secondary structure of H-2d glycoprotein was predicted from the amino acid composition. For NP-40-solubilized H-2d antigen, about 34% of helix, 13% beta sheet and 41% turns was found.

  17. Solubilization of benzene, toluene, and xylene (BTX) in aqueous micellar solutions of amphiphilic imidazolium ionic liquids.

    Łuczak, Justyna; Jungnickel, Christian; Markiewicz, Marta; Hupka, Jan


    Water-soluble ionic liquids may be considered analogues to cationic surfactants with a corresponding surface activity and ability to create organized structures in aqueous solutions. For the first time, the enhanced solubility of the aromatic hydrocarbons, benzene, toluene, and xylene, in aqueous micellar systems of 1-alkyl-3-methylimidazolium chlorides was investigated. Above a critical micelle concentration, a gradual increase in the concentration of aromatic hydrocarbons in the miceller solution was observed. This phenomenon was followed by means of the molar solubilization ratio, the micellar/water partition coefficient, and the number of solubilizate molecules per IL micelle. The molar solubilization ratio for ionic liquid micelles was found to be significantly higher when compared to that of ionic surfactants of similar chain length. The incorporation of the hydrocarbon into the micelle affects also an increase of the aggregation number.

  18. Novel application of hydrotropic solubilizing additives in the estimation of aspirin in tablets

    Maheshwari R


    Full Text Available Highly concentrated aqueous solutions of various hydrotropic agents like sodium benzoate, sodium salicylate, sodium acetate, sodium citrate, nicotinamide and sodium ascorbate have been observed to enhance aqueous solubilities of a large number of poorly water-soluble drugs. In the present investigation hydrotropic solubilization technique has been employed to solubilize poorly water-soluble aspirin (analgesic and antipyretic drug by 0.5 M ibuprofen sodium solution to carry out titrimetric analysis of aspirin in tablet dosage form. Results of analysis by proposed method and Phamacopoeial method are very comparable. Proposed method is new, rapid, simple, accurate, and reproducible. Statistical data proved the accuracy, reproducibility and the precision of proposed method.

  19. Effective solubilization of chalcones in micellar phase: Conductivity and voltammetric study

    Ahmed, Safeer; Khan, Gul Tiaz; Shah, Syed Sakhawat


    The solubilization of four chalcones, between aqueous and micellar phases of ionic surfactants (SDS and CTAB), was investigated by conductivity and cyclic voltammetry (CV) techniques. From conductivity data, a decrease in the critical micellar concentration (CMC) of the surfactants, in presence of the chalcones was ascribed to the decreased charge density over the surfactants. The results were seconded by thermodynamic parameters including degree of ionization (α), counter ion binding (β), and standard Gibbs free energy of micellization (Δ G {m/○}). The added surfactant decreased the peak current of the oxidized chalcone and shifted the peak potential either positively (in presence of SDS) or negatively (in presence of CTAB). The effect is rationalized as chalcone-surfactant interaction and quantitated as binding constant ( K b) assorting values from 8.78 to 552.97 M-1. The preferred solubilization of the chalcones in the micellar phase has been inferred.

  20. Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: identification of intact signal peptide.

    Mollay, C; Vilas, U; Kreil, G


    It has previously been shown that rat liver microsomes contain a proteolytic enzyme that cleaves honeybee prepromelittin to yield promelittin. This enzyme has now been further purified by centrifugation on a sucrose-deoxycholate gradient and then reconstituted into phospholipid vesicles. Incubation of prepromelittin with vesicles in the presence of melittin yields, in addition to promelittin, a hydrophobic peptide. The latter could be isolated by extraction with l-butanol and paper electrophoresis in 30% formic acid and was shown to be intact signal peptide by analysis of peptic fragments and automated Edman degradation. The microsomal enzyme is thus an endoprotease that hydrolyzes prepromelittin exclusively at the pre-pro junction. The precision of this cleavage of an insect preprotein by a rat liver enzyme indicates that we are dealing with the ubiquitous eukaryotic signal peptidase. PMID:7048315

  1. Formation of carcinogenic and inactive chrysene metabolites by rat liver microsomes of various monooxygenase activities

    Jacob, J.; Grimmer, G.; Schmoldt, A.


    Microsomal oxidation of chrysene in rat liver occurs at various positions (1,2-; 3,4-; 5,6-). This has been verified by means of gas chromatography/mass spectrometry (GC/MS) and comparison with synthetic reference substances. After various rat pretreatments with inducers of the monooxygenase system the oxidation at the 3,4-position predominated in isolated microsomes. The formation of the ultimate carcinogen of chrysene - 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene - was not detectable in untreated rats. However, it was observed as 1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene-TMS-ether formed under workup and derivatisation conditions after pretreating the rats with phenobarbital, polychlorinated biphenyl, benzoflavone, or various polycyclic aromatic hydrocarbons. Polychlorinated biphenyls and benzoflavone were the most potent inducers for the formation of this metabolite.

  2. Effects of calmodulin antagonists on radiation-induced lipid peroxidation in microsomes

    Varshney, R.; Kale, R.K. (Jawaharlal Nehru Univ., New Delhi (India). School of Life Sciences)


    Rat liver microsomes were irradiated with {gamma}-rays at a dose of 1.31 Gy s{sup -1}. The extent of lipid peroxidation, measured in terms of malondialdehyde (MDA) formed, increased with radiation dose. The presence of calmodulin antagonists during irradiation decreased lipid peroxidation. The order of their protective efficiency was: chlorpromazine (CPZ)>promethazine (PMZ)>trimeprazine (TMZ). Their protective effect was diminished in the presence of ferrous (Fe{sup 2+}) ions and was restored on addition of EDTA. However, calmodulin antagonists considerably inhibited radiation-induced lipid peroxidation in the presence of ferric (Fe{sup 3+}) ions. Calmodulin antagonists also decreased the cytochrome P-450 content of microsomes. These results are discussed with respect to their applicability to radiotherapy. A possible mechanism for the inhibition of radiation-induced lipid peroxidation is suggested. (author).

  3. High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes.

    Tuttle, R H; Hall, J E; Tidwell, R R


    Fisher rat liver microsomes metabolized the antimicrobial drug pentamidine to four new compounds detected by gradient elution reversed-phase high-performance liquid chromatography with variable wavelength detection. Coelution experiments with pentamidine metabolite standards determined the new peaks to be previously identified hydroxylated metabolites of pentamidine, with 1,5-bis(4'-amidinophenoxy)-3-pentanol and 1,5-di-(4'-amidinophenoxy)-2-pentanol formed in the greatest amount. The data contradict a previous report that Fisher rat liver homogenates do not metabolize pentamidine. Pentamidine and its known primary metabolites have almost identical absorption spectra; thus, pentamidine metabolism must be evaluated using gradient elution HPLC to resolve pentamidine from its metabolites. The current assay has now been used to demonstrate that Fisher and Sprague-Dawley rat, mouse, rabbit and human liver microsomes all metabolize pentamidine in vitro.

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

    Zielinski, Jana; Mevissen, Meike


    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.

  5. Identification of metabolites of fosinopril produced by human and rat liver microsomes with liquid chromatography-mass spectrometry.

    Uutela, Päivi; Monto, Matti; Iso-Mustajärvi, Ilona; Madetoja, Mari; Yliperttula, Marjo; Ketola, Raimo A


    Metabolic profiles of prodrug fosinopril and pharmacologically active metabolite fosinoprilat were studied using human or rat liver microsomes and S9 fractions. Metabolites were identified by ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) using electrospray ionization in the positive and negative ion mode. They were characterized by accurate MS and MS/MS spectra and based on their different fragmentation pathways. With human liver microsomes fosinopril was metabolized via hydroxylation, glucuronidation, and hydrolysis to fosinoprilat. As expected the main metabolite was fosinoprilat and it was further hydroxylated and glucuronidated. However, these metabolites were not detected after incubation of fosinoprilat with human liver microsomes, indicating that metabolic reactions occur in sequence and fosinopril is hydrolyzed after glucuronidation or hydroxylation. With the developed UHPLC/Q-TOF-MS method once or twice hydroxylated fosinopril metabolites were detected for the first time and different regioisomers were separated. It was observed that the hydrolysis of fosinopril to fosinoprilat was more efficient with rat than with human liver microsomes, and therefore more hydroxylated fosinoprilat metabolites were detected when rat liver microsomes were used. Glucuronidation of fosinopril was not observed with rat liver microsomes.

  6. Binding properties of solubilized gonadotropin-releasing hormone receptor: role of carboxylic groups

    Hazum, E.


    The interaction of /sup 125/I-buserelin, a superactive agonist of gonadotropin-releasing hormone (GnRH), with solubilized GnRH receptor was studied. The highest specific binding of /sup 125/I-buserelin to solubilized GnRH receptor is evident at 4/sup 0/C, and equilibrium is reached after 2 h of incubation. The soluble receptor retained 100% of the original binding activity when kept at 4 or 22/sup 0/C for 60 min. Mono- and divalent cations inhibited, in a concentration-dependent manner, the binding of /sup 125/I-buserelin to solubilized GnRH receptor. Monovalent cations require higher concentrations than divalent cations to inhibit the binding. Since the order of potency with the divalent cations was identical with that of their association constants to dicarboxylic compounds, it is suggested that there are at least two carboxylic groups of the receptor that participate in the binding of the hormone. The carboxyl groups of sialic acid residues are not absolutely required for GnRH binding since the binding of /sup 125/I-buserelin to solubilized GnRH receptor was only slightly affected by pretreatment with neuraminidase and wheat germ agglutinin. The finding that polylysines stimulate luteinizing hormone (LH) release from pituitary cell cultures with the same efficacy as GnRH suggest that simple charge interactions can induce LH release. According to these results, the authors propose that the driving force for the formation of the hormone-receptor complex is an ionic interaction between the positively charged amino acid arginine in position 8 and the carboxyl groups in the binding site.

  7. [Filamentous and phosphate solubilizing fungi relationships with some edaphic parameters and coffee plantations management].

    Posada, Raúl Hernando; Sánchez de Prager, Marina; Sieverding, Ewald; Aguilar Dorantes, Karla; Heredia-Abarca, Gabriela Patricia


    Soil properties and the environment have multiple outcomes on fungal communities. Although, the interaction effects between management intensity, pH, available phosphorus, organic carbon, soil texture and different fractions of water stable macro-aggregates on the communities of microscopic filamentous fungi (MFF), iron phosphate solubilizing fungi (PSF-Fe), and iron and calcium phosphate solubilizing fungi (PSF-(Fe+Ca)), have been previously evaluated in field conditions, this has never been performed in terms of their combined effects, neither with phosphate solubilizing fungi. To assess this, we collected 40 composite soil samples from eight Mexican and Colombian coffee plantations, with different management intensities and physico-chemical edaphic parameters, during 2008-2009. We isolated different communities of MFF, PSF-Fe and PSF-(Fe+Ca), by wet sieving and soil particles culture in Potato-Dextrose-Agar from soil samples, and we classified isolates in terms of their phosphate solubilizing ability. Following the principal component analysis results, we decided to analyze fungal communities and abiotic factors interactions for each country separately. Structural Equation Models revealed that organic carbon was positively associated to MFF richness and number of isolates (lambda>0.58), but its relationship with PSF-Fe and PSF-(Fe+Ca) were variable; while the available phosphorus, pH and water stable macro-aggregate fractions did not show a clear pattern. Management intensity was negatively related to PSF-Fe (lambda fungi (PSF). This study highlights the need to take into account edaphic and geographic context in order to reach a better understanding of the intensity management effects on MFF and PSF function in agroecosystems.

  8. Initial pH of medium affects organic acids production but do not affect phosphate solubilization

    Leandro M. Marra


    Full Text Available The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP. The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici, UFLA03-09 (Acinetobacter sp., UFLA03-10 (Paenibacillus kribbensis, UFLA03-106 (Paenibacillus kribbensis and UFLA03-116 (Paenibacillus sp.. The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO42 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO42 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization.

  9. Effects of Phosphate-solubilizing Microbial Inoculums on Soil Nutrients and Corn Growth

    QIAN Lin-zhao


    Full Text Available Phosphate-solubilizing microorganisms(PSMs, which are commonly found in soil, can solubilize insoluble phosphate(P, reduce phosphorus fertilizer fixation, increase the utilization ratio of P fertilizers, and promote crop growth. In order to analyse the effects of different phosphate-solubilizing microbial inoculums in field, the experiment was conducted to study the effects of soil nutrients and output of corn af-ter three phosphate-solubilizing microbial inoculums in plot experiment. The results showed that available phosphate in soil was increased remarkably after Penicillium oxalate I1(I1, Aspergillus niger H1(H1and Bacillus Megaterium(BMwere inoculated. When the concer-tration of P2O5 were 0, 46, 75 kg·hm -2 and 92 kg·hm -2, the content of soil available phosphate was increased 6.47%-37.13%. At prophase of corn growth, when P2O5 was 92 kg·hm -2, the soil pH was decreased, and the soil exchangeable Ca, Mg and available Fe, Cu and Zn were in-creased significantly, and the activity of SOD, POD and CAT enzyme was also increased. However, at the same condition, the content of available Mn was decreased higher than CK. As a whole, the ability of increasing exchangeable Ca, Mg and available Zn was H1>I1>BM, the ability of increasing available Fe was H1>BM>I1, and the ability of increasing available Cu was BM>H1>I1. The output of corn increased remarkable after three inoculums were inoculated, and the output were increased 8.2%-12.1% higher than CK at 92 kg·hm -2 of P2O5.

  10. Use of Phosphate Solubilizing Bacteria to Leach Rare Earth Elements from Monazite-Bearing Ore

    Doyun Shin


    Full Text Available In the present study, the feasibility to use phosphate solubilizing bacteria (PSB to develop a biological leaching process of rare earth elements (REE from monazite-bearing ore was determined. To predict the REE leaching capacity of bacteria, the phosphate solubilizing abilities of 10 species of PSB were determined by halo zone formation on Reyes minimal agar media supplemented with bromo cresol green together with a phosphate solubilization test in Reyes minimal liquid media as the screening studies. Calcium phosphate was used as a model mineral phosphate. Among the test PSB strains, Pseudomonas fluorescens, P. putida, P. rhizosphaerae, Mesorhizobium ciceri, Bacillus megaterium, and Acetobacter aceti formed halo zones, with the zone of A. aceti being the widest. In the phosphate solubilization test in liquid media, Azospirillum lipoferum, P. rhizosphaerae, B. megaterium, and A. aceti caused the leaching of 6.4%, 6.9%, 7.5%, and 32.5% of calcium, respectively. When PSB were used to leach REE from monazite-bearing ore, ~5.7 mg/L of cerium (0.13% of leaching efficiency and ~2.8 mg/L of lanthanum (0.11% were leached by A. aceti, and Azospirillum brasilense, A. lipoferum, P. rhizosphaerae and M. ciceri leached 0.5–1 mg/L of both cerium and lanthanum (0.005%–0.01%, as measured by concentrations in the leaching liquor. These results indicate that determination of halo zone formation was found as a useful method to select high-capacity bacteria in REE leaching. However, as the leaching efficiency determined in our experiments was low, even in the presence of A. aceti, further studies are now underway to enhance leaching efficiency by selecting other microorganisms based on halo zone formation.

  11. Nanolipoprotein particles and related methods and systems for protein capture, solubilization, and/or purification

    Chromy, Brett A.; Henderson, Paul; Hoeprich, Jr, Paul D.


    Provided herein are methods and systems for assembling, solubilizing and/or purifying a membrane associated protein in a nanolipoprotein particle, which comprise a temperature transition cycle performed in presence of a detergent, wherein during the temperature transition cycle the nanolipoprotein components are brought to a temperature above and below the gel to liquid crystalling transition temperature of the membrane forming lipid of the nanolipoprotein particle.

  12. Molecular identification of phosphate-solubilizing native bacteria isolated from the rhizosphere of Prosopis glandulosa in Mexicali valley.

    Moreno-Ramírez, L; González-Mendoza, D; Cecena-Duran, C; Grimaldo-Juarez, O


    One of the main limitations in intensive crop production in Northwestern Mexico is the dependence on the use of phosphate fertilizer. In this study, we isolated indigenous microorganisms with phosphate solubilization capacities from mesquite (Prosopis glandulosa) present in the Mexicali valley. In total, 4 bacteria were isolated from the rhizosphere of mesquite, including ICA01, ICA02Ba, ICA03Bs, and ICA04Ma. The bacterial isolates were identified based on their phenotypic and 16S rRNA gene sequencing data to be Acinetobacter calcoaceticus. The results showed that ICA01 was the most efficient in solubilizing phosphate, followed by ICA02Ba and ICA03Bs, while ICA04Ma showed the lowest phosphate-solubilizing activity. The pH value of the culture medium decreased with bacterial growth, suggesting that these strains produce organic acids that solubilize phosphorus. These results will be useful for biotechnological studies and A. calcoaceticus may be employed for biofertilization programs in northwest Mexico.

  13. Different strategies of fungi to solubilize coal: a comparison of the deutermycetes Trichoderma atroviride and Fusarium oxysporum

    Hoelker, U.; Ludwig, S.; Moenkemann, H.; Scheel, T.; Hoefer, M. [Bonn Univ. (Germany). Botanisches Inst.


    Four different mechanisms can be envisaged which are used by microorganisms to solubilize coal: the production of alkaline substances, the extrusion of chelators, the action of biotensides, and of special interest in terms of biotechnology, the action of enzymes. Whether these mechanisms are operating seperately or in varying combinations has not yet been settled. The two deuteromycetes Fusarium oxysporum and Trichoderma altroviride solubilize coal by synergistic effects of different mechanisms depending on the cell metabolism. F. oxysporum seems to solubilize coal by increasing the pH of the mycelial surroundings and by the action of chelators induced during growth in glutamate containing media (without involvement of enzymes). T. atroviride, on the other hand, appears to use, in addition to an alkaline pH and a high chelator activity, at least two classes of enzymes to attack coal: hydrolytic activity for coal solubilization and ligninolytic activity for degradation of humic acids. (orig.)

  14. Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes.

    Weng, Zuquan; Greenhaw, James; Salminen, William F; Shi, Qiang


    Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1-100μM for 30min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1μM EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1μM EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults.

  15. Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes.

    Rodriguez, R J; Proteau, P J; Marquez, B L; Hetherington, C L; Buckholz, C J; O'Connell, K L


    Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation.

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

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


    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.

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

    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.


    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 fr

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

    Marker, E K; Kulkarni, A P


    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.

  19. Effect of pretreatment with hepatic microsomal enzyme inducers on the toxicity of diazinon in calves.

    Abdelsalam, E B; Ford, E J


    The pretreatment of calves with a single dose of 10 mg kg-1 dieldrin or 21 daily doses of 10 mg kg-1 phenobarbitone increased the toxicity of diazinon as reflected by the development of more severe clinical signs and greater depression in whole blood cholinesterase activity in the pretreated calves. Induction by dieldrin or phenobarbitone of the hepatic microsomal enzyme amidopyrine-N-demethylase was also accompanied by a concurrent rise in the liver carboxylesterase activity.

  20. Solubilization of Phenanthrene and Fluorene in Equimolar Binary Mixtures of Gemini/Conventional Surfactants

    Huma Siddiqui; Mohammad Kamil; Manorama Panda; Kabir-ud-Din


    abstract This study deals with the enhanced solubilization of polycyclic aromatic hydrocarbons (PAHs) such as phenan-threne (PHE) and fluorene (FLR) in a pure cationic gemini (G6) and three conventional surfactants [polyethylene glycol dodecyl ether (Brij35), cetyltrimethyl ammonium bromide (CTAB) and sodium lauryl sulfate (SDS)] as well as in their equimolar binary combinations (G6-Brij35, G6-CTAB and G6-SDS). Their solubilization efficiency toward PHE and FLR has been quantified in terms of the molar solubilization ratio (MSR) and the micelle-water partition coefficient (Km). The ideality/nonideality of the mixed micelles is discussed with the help of Clint, Rubingh and Rosen's approaches. These theories determine the deviation of experimental critical micelle concen-tration (CMC) values from ideal critical micelle concentration, which was measured by evaluating the interaction parameters (βm andβσ). Negative values ofβm were observed in all the equimolar binary systems, which show synergism in the mixed micelles. Whereas at air/liquid interface synergism was observed in the systems G6-CTAB and G6-Brij35; G6-SDS exhibited an antagonistic effect. The order of MSR and Km was G6-CTAB N G6-Brij35 N G6-SDS for phenanthrene as well as for fluorene.

  1. Host-derived Loss of Dentin Matrix Stiffness Associated with Solubilization of Collagen

    Carrilho, Marcela R.; Tay, Franklin R.; Donnelly, Adam M.; Agee, Kelli A.; Tjäderhane, Leo; Mazzoni, Annalisa; Breschi, Lorenzo; Foulger, Stephen; Pashley, David H.


    Matrix metalloproteinases (MMPs) bound to dentin matrices are activated during adhesive bonding procedures and are thought to contribute to the progressive degradation of resin-dentin bonds over time. The purpose of this study was to evaluate the changes in mechanical, biochemical and structural properties of demineralized dentin treated with or without chlorhexidine (CHX), a known MMP-inhibitor. After demineralizing dentin beams in EDTA or phosphoric acid (PA), the baseline modulus of elasticity (E) of each beam was measured by 3-point flexure. Specimens were pretreated with water (control) or with 2% CHX (experimental) and then incubated in artificial saliva (AS) at 37°C for 4 weeks. The E of each specimen was remeasured weekly and, the media was analyzed for solubilized dentin collagen at first and fourth week of incubation. Some specimens were processed for electron microscopy (TEM) immediately after demineralization and after 4 weeks of incubation. In EDTA and PA-demineralized specimens, the E of the control specimens fell (p<0.05) after incubation in AS, while there were no changes in E in the CHX-pretreated specimens over time. More collagen was solubilized from PA-demineralized controls (p<0.05) than from EDTA-demineralized matrices after 1 or 4 weeks. Less collagen (p<0.05) was solubilized from CHX-pretreated specimens demineralized in EDTA compared to PA. TEM examination of control beams revealed that prolonged demineralization of dentin in 10% PA (12 h) did not denature the collagen fibrils. PMID:19090493

  2. Rates of solubilization and biodegradation of PAH compounds in porous media. [Quarterly report

    Luthy, R.G.


    Microbial degradation of hydrophobic organic compounds in soils and aquifer media is dependent on rates of desorption of these compounds from solids and rates of solubilization from residual nonaqueous phase liquids (NAPLs). The couples processes involving microbial degradation and hydrophobic compound availability are not well understood. The proposed research effort explores certain physicochemical phenomena that may have a significant affect on the rate of microbial degradation of hydrophobic organic compounds in porous media. The investigation will examine rates of biomineralization of polycyclic aromatic hydrocarbon (PAH) compounds that are leached from a residual saturation of coal tar. Batch and continuously-stirred reactor studies will be used to measure solute equilibrium concentrations and rates of solubilization of PAH compounds from coal tar imbided into microporous silica media. These rates will be compared with rates of mineralization of {sup 14}C-labeled compounds in similar systems inoculated with a culture of PAH degrading microorganisms. Column experiments will also be conducted to assess the rates of solubilization and mass transfer coefficients from coal tar entrapped in a sandy aquifer material by capillary forces.

  3. Rates of solubilization and biodegradation of PAH compounds in porous media

    Luthy, R.G.


    Microbial degradation of hydrophobic organic compounds in soils and aquifer media is dependent on rates of desorption of these compounds from solids and rates of solubilization from residual nonaqueous phase liquids (NAPLs). The couples processes involving microbial degradation and hydrophobic compound availability are not well understood. The proposed research effort explores certain physicochemical phenomena that may have a significant affect on the rate of microbial degradation of hydrophobic organic compounds in porous media. The investigation will examine rates of biomineralization of polycyclic aromatic hydrocarbon (PAH) compounds that are leached from a residual saturation of coal tar. Batch and continuously-stirred reactor studies will be used to measure solute equilibrium concentrations and rates of solubilization of PAH compounds from coal tar imbided into microporous silica media. These rates will be compared with rates of mineralization of {sup 14}C-labeled compounds in similar systems inoculated with a culture of PAH degrading microorganisms. Column experiments will also be conducted to assess the rates of solubilization and mass transfer coefficients from coal tar entrapped in a sandy aquifer material by capillary forces.

  4. Host-derived loss of dentin matrix stiffness associated with solubilization of collagen.

    Carrilho, Marcela R; Tay, Franklin R; Donnelly, Adam M; Agee, Kelli A; Tjäderhane, Leo; Mazzoni, Annalisa; Breschi, Lorenzo; Foulger, Stephen; Pashley, David H


    Matrix metalloproteinases (MMPs) bound to dentin matrices are activated during adhesive bonding procedures and are thought to contribute to the progressive degradation of resin-dentin bonds over time. The purpose of this study was to evaluate the changes in mechanical, biochemical, and structural properties of demineralized dentin treated with or without chlorhexidine (CHX), a known MMP-inhibitor. After demineralizing dentin beams in EDTA or phosphoric acid (PA), the baseline modulus of elasticity (E) of each beam was measured by three-point flexure. Specimens were pretreated with water (control) or with 2% CHX (experimental) and then incubated in artificial saliva (AS) at 37 degrees C for 4 weeks. The E of each specimen was remeasured weekly and, the media was analyzed for solubilized dentin collagen at first and fourth week of incubation. Some specimens were processed for electron microscopy (TEM) immediately after demineralization and after 4 weeks of incubation. In EDTA and PA-demineralized specimens, the E of the control specimens fell (p < 0.05) after incubation in AS, whereas there were no changes in E of the CHX-pretreated specimens over time. More collagen was solubilized from PA-demineralized controls (p < 0.05) than from EDTA-demineralized matrices after 1 or 4 weeks. Less collagen (p < 0.05) was solubilized from CHX-pretreated specimens demineralized in EDTA compared with PA. TEM examination of control beams revealed that prolonged demineralization of dentin in 10% PA (12 h) did not denature the collagen fibrils.

  5. Solubilization, Activation and Partial Purification of a Sialidase from Horse Liver



    Full Text Available Using sialyl-methylumbelliferyl -glycoside as substrate, sialidase in horse liver was detected as a membrane-bound enzyme. A yield of about 50% of sialidase activity was found in supernatant when solubilized in 0.1 M sodium-phosphate buffer pH 5.5, containing 0.15 M NaCl, 0.25 M sucrose, and 0.5% Triton X-100. Sialidase in the solubilisate could be activated by incubating in acidic pH at 37 oC. Incubation of this solubilized enzyme at 37 oC for 1.5 h at pH 5.0 led to 10% increase of activity and to the precipitation of about 50% of contaminating protein. Using cation-exchange chromatography on S-Sepharose FF and affinity chromatography on p-aminophenyl oxamic acid-agarose following solubilization and activation, about 6% of total sialidase activity was recovered with the purification factor of about 500. The pH and temperature optimum were measured at pH 4.3 and between 37-45 oC, respectively. Neu5Ac2en was a strong inhibitor, while p-aminophenyl oxamic acid had only a weak inhibitory effect.

  6. Pressure cell assisted solubilization of xyloglucans: tamarind seed polysaccharide and detarium gum.

    Picout, David R; Ross-Murphy, Simon B; Errington, Neil; Harding, Stephen E


    To improve the solubilization of two water-soluble xyloglucans, tamarind seed polysaccharide and detarium gum, by reducing substantially molecular aggregation, a "pressure cell" heating method was used. Conditions allowing solubilization and chain depolymerization were produced by varying appropriately the pressure, time, and temperature applied. The various MW fractions of solubilized xyloglucans were characterized by capillary viscometry and light scattering techniques in order to extract, with reliability, fundamental macromolecular parameters. Mark-Houwink and Flory exponents were found to be 0.67 +/- 0.04 and 0.51 +/- 0.06, respectively for both xyloglucan data combined, consistent with linear random coil behavior. A detailed analysis of the data seems to suggest that tamarind gum solutions are slightly perturbed by the effect of excluded volume, whereas detarium gum samples are close to the theta state. Chain flexibility parameters such characteristic ratio, C( proportional, variant ), and persistence length, L(p), were calculated for tamarind and detarium using the Burchard-Stockmayer-Fixman (BSF) geometric method. L(p) values of 6-8 nm were estimated for xyloglucans. The seemingly linear structure of tamarind and detarium, as suggested by the value of the Mark-Houwink and Flory exponents obtained, follows from analysis of the data by the classical Zimm method but not when employing the square root or Berry method which suggests a more branched chain profile. This was the approach adopted in our previous work on the characterization of detarium samples.

  7. Screening for phosphate solubilizing bacteria inhabiting the rhizoplane of rice grown in acidic soil in Bangladesh.

    Sarkar, Animesh; Islam, Tofazzal; Biswas, Gokul Chandra; Alam, Shohidul; Hossain, Mikail; Talukder, Nur Mohammad


    The objectives of the research were to isolate phosphate solubilizing bacteria (PSB) from the rhizoplane of rice (Oryza sativa L.) cv. BRRIdhan 29 cultivated in acidic soils of Tangail in Bangladesh and evaluate their performances in phosphate solubilization in both in vitro and in vivo conditions. A total of 10 bacterial strains were isolated and purified by repeated streak culture on nutrient agar medium. Upon screening, five isolates (OS01, OS03, OS07, OS08 and OS10) showed varying levels of phosphate solubilizing activity in agar plate and broth assays. Among them, the strain OS07 (B1) and two previously isolated PSB strains B2 and B3 were selected for evaluation for their performances in rice alone or in combination of TSP (triple super phosphate: P1) and rock phosphate (P2). Plant height and the number of tillers per plant were significantly increased by all PSB isolates when used in combination with TSP but PSB alone did not influence much on plant height and the number of tillers except B1. The levels of mineral nutrients content in rice plant tissues were generally increased by the application of the PSB in combination with TSP, while the performances of B1 isolate was superior in all aspects to B2 and B3 isolates.

  8. Mineral Phosphate Solubilizing Bacteria Isolated from Various Plant Rhizosphere under Different Aluminum Content

    Dolly Iriani Damarjaya


    Full Text Available The objectives of this study was to isolate and characterize the mineral phosphate solubilizing bacteriafrom rhizosphere and evaluate their potential as plant growth promoting bacteria in Al-toxic soils. The halozone formation method was used to isolate PSB using the media containing insoluble phosphates (Ca-P or Al-Pas a source of phosphate. Eight of acid and Al-tolerant PSB isolates that were able to solubilize Ca-P wereobtained from rhizosphere of clover, wheat, corn, and sunflower grown in Al-toxic soil. Identification of theisolates based on the 16S rRNA gene sequence analysis demonstrated that the isolates were strains of Burkholderia(5 strains, Pseudomonas (1 strain, Ralstonia (1 strain, and unidentified bacterium (1 strains. All PSB isolatesshowed the capability to dissolve Ca-P, and only 1 strain (Ralstonia strain was able to dissolve Al-P in agar platemedium. The P-solubilization by these isolates was correlated with pH of medium. Inoculation of the bacterialstrains on clover on Al-toxic medium showed that all isolates increased the plant dry weight compared withuninoculated treatment. Our results showed that those PSB isolates have potential to be developed as a biofertilizerto increase the efficiency of P-inorganic fertilizer used in Al-toxic soils.

  9. Arsenic-hyperaccumulator Pteris vittata efficiently solubilized phosphate rock to sustain plant growth and As uptake.

    Fu, Jing-Wei; Liu, Xue; Han, Yong-He; Mei, Hanyi; Cao, Yue; de Oliveira, Letuzia M; Liu, Yungen; Rathinasabapathi, Bala; Chen, Yanshan; Ma, Lena Q


    Phosphorus (P) is one of the most important nutrients for phytoremediation of arsenic (As)-contaminated soils. In this study, we demonstrated that As-hyperaccumulator Pteris vittata was efficient in acquiring P from insoluble phosphate rock (PR). When supplemented with PR as the sole P source in hydroponic systems, P. vittata accumulated 49% and 28% higher P in the roots and fronds than the -P treatment. In contrast, non-hyperaccumulator Pteris ensiformis was unable to solubilize P from PR. To gain insights into PR solubilization by plants, organic acids in plant root exudates were analyzed by HPLC. The results showed that phytic acid was the predominant (>90%) organic acid in P. vittata root exudates whereas only oxalic acid was detected in P. ensiformis. Moreover, P. vittata secreted more phytic acid in -P and PR treatments. Compared to oxalic acid, phytic acid was more effective in solubilizing PR, suggesting that phytic acid was critical for PR utilization. Besides, secretion of phytic acid by P. vittata was not inhibited by arsenate. Our data indicated that phytic acid played an important role in efficient use of insoluble PR by P. vittata, shedding light on using insoluble PR to enhance phytoremediation of As-contaminated soils.

  10. Solubilization of tea seed oil in a food-grade water-dilutable microemulsion.

    Lingli Deng

    Full Text Available Food-grade microemulsions containing oleic acid, ethanol, Tween 20, and water were formulated as a carrier system for tea seed oil (Camellia oleifera Abel.. The effect of ethanol on the phase behavior of the microemulsion system was clearly reflected in pseudo-ternary diagrams. The solubilization capacity and solubilization efficiency of tea seed oil dispersions were measured along the dilution line at a 70/30 surfactant/oil mass ratio with Tween 20 as the surfactant and oleic acid and ethanol (1:3, w/w as the oil phase. The dispersed phase of the microemulsion (1.5% weight ratio of tea seed oil to the total amount of oil, surfactant, and tea seed oil could be fully diluted with water without phase separation. Differential scanning calorimetry and viscosity measurements indicated that both the carrier and solubilized systems underwent a similar microstructure transition upon dilution. The dispersion phases gradually inverted from the water-in-oil phase ( 45% water along the dilution line.

  11. Atomistic Simulation of Solubilization of Polycyclic Aromatic Hydrocarbons in a Sodium Dodecyl Sulfate Micelle.

    Liang, Xujun; Marchi, Massimo; Guo, Chuling; Dang, Zhi; Abel, Stéphane


    Solubilization of two polycyclic aromatic hydrocarbons (PAHs), naphthalene (NAP, 2-benzene-ring PAH) and pyrene (PYR, 4-benzene-ring PAH), into a sodium dodecyl sulfate (SDS) micelle was studied through all-atom molecular dynamics (MD) simulations. We find that NAP as well as PYR could move between the micelle shell and core regions, contributing to their distribution in both regions of the micelle at any PAH concentration. Moreover, both NAP and PYR prefer to stay in the micelle shell region, which may arise from the greater volume of the micelle shell, the formation of hydrogen bonds between NAP and water, and the larger molecular volume of PYR. The PAHs are able to form occasional clusters (from dimer to octamer) inside the micelle during the simulation time depending on the PAH concentration in the solubilization systems. Furthermore, the micelle properties (i.e., size, shape, micelle internal structure, alkyl chain conformation and orientation, and micelle internal dynamics) are found to be nearly unaffected by the solubilized PAHs, which is irrespective of the properties and concentrations of PAHs.

  12. Biosurfactant-producing strains in enhancing solubilization and biodegradation of petroleum hydrocarbons in groundwater.

    Liu, Hong; Wang, Hang; Chen, Xuehua; Liu, Na; Bao, Suriguge


    Three biosurfactant-producing strains designated as BS-1, BS-3, and BS-4 were screened out from crude oil-contaminated soil using a combination of surface tension measurement and oil spreading method. Thin layer chromatography and infrared analysis indicated that the biosurfactants produced by the three strains were lipopeptide, glycolipid, and phospholipid. The enhancement of solubilization and biodegradation of petroleum hydrocarbons in groundwater employing biosurfactant-producing strains was investigated. The three strain mixtures led to more solubilization of petroleum hydrocarbons in groundwater, and the solubilization rate was 10.5 mg l−1. The combination of biosurfactant-producing strains and petroleum-degrading strains exhibited a higher biodegradation efficiency of 85.4 % than the petroleum-degrading strains (71.2 %). Biodegradation was enhanced the greatest with biosurfactant-producing strains and petroleum-degrading strains in a ratio of 1:1. Fluorescence microscopy images illustrate that the oil dispersed into smaller droplets and emulsified in the presence of biosurfactant-producing strains, which attached to the oil. Thus, the biodegradation of petroleum hydrocarbons in groundwater was enhanced.

  13. Gluconic acid production and phosphate solubilization by the plant growth-promoting bacterium Azospirillum spp.

    Rodriguez, Hilda; Gonzalez, Tania; Goire, Isabel; Bashan, Yoav


    In vitro gluconic acid formation and phosphate solubilization from sparingly soluble phosphorus sources by two strains of the plant growth-promoting bacteria A. brasilense (Cd and 8-I) and one strain of A. lipoferum JA4 were studied. Strains of A. brasilense were capable of producing gluconic acid when grown in sparingly soluble calcium phosphate medium when their usual fructose carbon source is amended with glucose. At the same time, there is a reduction in pH of the medium and release of soluble phosphate. To a greater extent, gluconic acid production and pH reduction were observed for A. lipoferum JA4. For the three strains, clearing halos were detected on solid medium plates with calcium phosphate. This is the first report of in vitro gluconic acid production and direct phosphate solubilization by A. brasilense and the first report of P solubilization by A. lipoferum. This adds to the very broad spectrum of plant growth-promoting abilities of this genus.

  14. Water solubilization capacity of pharmaceutical microemulsions based on Peceol®, lecithin and ethanol.

    Mouri, Abdelkader; Diat, Olivier; Lerner, Dan Alain; El Ghzaoui, Abdeslam; Ajovalasit, Alessia; Dorandeu, Christophe; Maurel, Jean-Claude; Devoisselle, Jean-Marie; Legrand, Philippe


    Biocompatible microemulsions composed of Peceol(®), lecithin, ethanol and water developed for encapsulation of hydrophilic drugs were investigated. The binary mixture Peceol(®)/ethanol was studied first. It was shown that the addition of ethanol to pure Peceol(®) has a significant fluidifying and disordering effect on the Peceol(®) supramolecular structure with an enhancement in water solubilization. The water solubilization capacity was improved by adding lecithin as a third component. It was then demonstrated that the ethanol/lecithin weight ratio played an important role in determining the optimal composition in term of water solubilization efficiency, a necessary property for a nutraceutical or pharmaceutical application. The optimal ethanol/lecithin weight ratio in the Peceol(®) rich region was found to be 40/60. Combination different techniques such as SAXS, fluorimetry, rheology and conductivity, we analyzed the water uptake within the microemulsion taking into account the partitioning of ethanol between polar and apolar domains. This ethanol distribution quantified along a water dilution line has a major effect on microemulsion properties. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Reversibly enhanced aqueous solubilization of volatile organic compounds using a redox-reversible surfactant

    Yingjie Li; Senlin Tian; Hong Mo; Ping Ning


    Surfactant-enhanced remediation (SER) is an effective method for the removal of volatile organic compounds (VOCs) from contaminated soils and groundwater.To reuse the surfactant the VOCs must be separated from the surfactant solutions.The water solubility of VOCs can be enhanced using reversible surfactants with a redox-acive group,(ferrocenylmethyl)dodecyldimethylammonium bromide (Fc12) and (ferrocenylmethyl)tetradecanedimethylammonium bromide (Fc14),above and below their critical micelle concentrations (CMC) under reducing (I+) and oxidative (I2+) conditions.The CMC values of Fc12 and Fc14 in I+ are 0.94 and 0.56 mmol/L and the solubilization of toluene by Fc12 and Fc14 in I+ for toluene is higher than the solubilization achieved with sodium dodecyl sulfate,cetyltrimethylammonium bromide and Trition X-114.The solubilization capacity of the ferrocenyl surfactants for each tested VOCs ranked as follows:ethylbenzene > toluene > benzene.The solubilities of VOCs by reversible surfactant in I+ were 30% higher than those in 12+ at comparable surfactant concentrations.The effects of Fc14 concentrations on VOCs removal efficiency were as follows:benzene > toluene > ethylbenzene.However,an improved removal efficiency was achieved at low ferrocenyl surfactant concentrations.Furthermore,the reversible surfactant could be recycled through chemical approaches to remove organic pollutants,which could significantly reduce the operating costs of SER technology.

  16. Metabolism of dictamnine in liver microsomes from mouse, rat, dog, monkey, and human.

    Wang, Pei; Zhao, Yunli; Zhu, Yingdong; Sun, Jianbo; Yerke, Aaron; Sang, Shengmin; Yu, Zhiguo


    Dictamnine, a furoquinoline alkaloid isolated from the root bark of Dictamnus dasycarpus Turcz. (Rutaceae), is reported to have a wide range of pharmacological activities. In this study, the in vitro metabolic profiles of dictamnine in mouse, rat, dog, monkey, and human liver microsomes were investigated and compared. Dictamnine was incubated with liver microsomes in the presence of an NADPH-regenerating system, resulting in the formation of eight metabolites (M1-M8). M1 is an O-desmethyl metabolite. M5 and M6 are formed by a mono-hydroxylation of the benzene ring of dictamnine. M8 was tentatively identified as an N-oxide metabolite. The predominant metabolic pathway of dictamnine occurs through the epoxidation of the 2,3-olefinic to yield a 2,3-epoxide metabolite (M7), followed by the ring of the epoxide opening to give M4. Likewise, cleavage of the furan ring forms M2 and M3. Slight differences were observed in the in vitro metabolic profiles of dictamnine among the five species tested. A chemical inhibition study with a broad and five specific CYP450 inhibitors revealed that most of the dictamnine metabolites in liver microsomes are mediated by CYP450, with CYP3A4 as the predominant enzyme involved in the formation of M7, the major metabolite. These findings provide vital information to better understand the metabolic processes of dictamnine among various species.

  17. Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes.

    Tan, Bo; Cai, Weimin; Zhang, Jinlian; Zhou, Ning; Ma, Guo; Yang, Ping; Zhu, Qing; Zhu, Yizhun


    Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far. Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one. Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CLint) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities. Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.

  18. Formation of 4-hydroxyochratoxin A from ochratoxin A by rat liver microsomes.

    Størmer, F C; Pedersen, J I


    Hydroxyochratoxin A was isolated and identified from the urine of rats after injection with ochratoxin A. By incubating ochratoxin A with rat liver microsomes and reduced nicotinamide adenine dinucleotide phosphate, one major (90%) and two minor metabolites, more polar than ochratoxin A, were formed. Thin-layer chromatography revealed that the major metabolite had Rf values identical to those of hydroxyochratoxin A in six different solvent systems. Formation of the metabolites in vitro was inhibited by carbon monoxide and by metyrapone, and the rate of formation increased after pretreatment of the rats with phenobarbital. A type I spectrum appeared upon binding of ochratoxin A to microsomes with a spectral dissociation constant (Ks) of 37.6 microM. These findings strongly suggest the involvement of a cytochrome P-450 in the hydroxylation of ochratoxin A by rat liver microsomes. Apparent Km and Vmax values for the formation of hydroxyochratoxin A were determined to 50 microM and 5.5 nmol/mg of protein per h, respectively. PMID:7396488

  19. Enantioselective Degradation of (2RS, 3RS)-Paclobutrazol in Rat Liver Microsomes.

    Wu, Shuchun; Yu, Miao; Zhang, Hu; Han, Jianzhong; Qian, Mingrong


    Paclobutrazol, with two stereogenic centers, but gives only (2R, 3R) and (2S, 3S)-enantiomers because of steric-hindrance effects, is an important plant growth regulator in agriculture and horticulture. Enantioselective degradation of paclobutrazol was investigated in rat liver microsomes in vitro. The degradation kinetics and the enantiomer fraction were determined using a Lux Cellulose-1 chiral column on a reverse-phase liquid chromatography-tandem mass spectrometry system. The t1/2 of (2R, 3R)-paclobutrazol is 18.60 min, while the t1/2 of (2S, 3S)-paclobutrazol is 10.93 min. Such consequences clearly indicated that the degradation of paclobutrazol in rat liver microsomes was stereoselective and the degradation rate of (2S, 3S)-paclobutrazol was much faster than (2R, 3R)-paclobutrazol. In addition, significant differences between the two enantiomers were also observed in enzyme kinetic parameters. The Vmax of (2S, 3S)-paclobutrazol was more than 2-fold of (2R, 3R)-paclobutrazol and the Clint of (2S, 3S)-paclobutrazol was higher than that of (2R, 3R)-paclobutrazol after incubation in rat liver microsomes. These results may have potential implications for better environmental and ecological risk assessment for paclobutrazol.

  20. Toxic dark effects of protoporphyrin on the cytochrome P-450 system in rat liver microsomes.

    Williams, M; Van der Zee, J; Van Steveninck, J


    In erythropoietic protoporphyria, accumulation of protoporphyrin has been found in various tissues and liver cirrhosis occurs frequently in this disease, probably due to toxic dark effects of protoporphyrin. We have studied the effect of porphyrins on various enzymic functions in rat liver microsomes. Incubation of microsomes with protoporphyrin resulted in a concentration-dependent inhibition of the oxidation of 7-ethoxycoumarin and aminopyrine by the cytochrome P-450 system. Kinetic analysis showed a decrease in Vmax., whereas the Km was not affected (non-competitive inhibition). Furthermore, reduction of cytochrome c by the NADPH-cytochrome P-450 reductase and by the NADH-cytochrome b5 reductase was inhibited. However, the activity of the reductases was only affected when the microsomes were pre-incubated with protoporphyrin, and it was found that the inhibition was dependent on the duration of the pre-incubation. Kinetic analysis again revealed non-competitive inhibition. When these experiments were repeated with uroporphyrin, no inhibition could be observed. With Stern-Volmer plots it was demonstrated that this was most likely caused by the localization of the porphyrins: protoporphyrin is localized in the membrane, whereas uroporphyrin remains in solution. From these results it is concluded that accumulation of protoporphyrin in the liver may markedly affect the cytochrome P-450 system and thus its detoxification function. PMID:1332695

  1. Fluoroaluminate treatment of rat liver microsomes inhibits GTP-dependent vesicle fusion.

    Comerford, J G; Dawson, A P


    1. Inhibition of GTP-dependent membrane fusion of rat liver microsomes requires preincubation of the membranes with GDP (17 microM) and relatively high Mg2+ concentration (0.5 mM) as well as AlCl3 (30 microM) and KF (5 mM). Preincubation is required for maximal inhibition (75%). 2. Vesicle fusion in rat liver microsomes has been demonstrated in the absence of polyethylene glycol (PEG). Further, inhibition by AlF4- of GTP-dependent vesicle fusion in the absence of PEG has been demonstrated. 3. Under similar preincubation conditions AlF4- can bring about inhibition (80%) of the high-affinity PEG-stimulated GTPase activity in rat liver microsomes, previously described by Nicchitta, Joseph & Williamson [(1986) FEBS Lett. 209, 243-248]. 4. Preincubation of small-Mr GTP-binding proteins (Gn proteins) on nitrocellulose strips with GDP (20 pM), AlCl3 (30 microM) and KF (5 mM) results in inhibition of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gn proteins. The extent of inhibition of this binding differs for different Gn proteins. PMID:1747106

  2. Effect of GTP on the dolichol pathway for protein glycosylation in rat liver microsomes.

    Bossuyt, X; Blanckaert, N


    Incubation of native rat liver microsomes with GTP resulted in enhanced incorporation of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc into lipid acceptors. The stimulation of GlcNAc transfer by GTP was specific for GTP; ATP exerted no effect. The GTP effect was blocked by a non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate, indicating that GTP hydrolysis was crucial. Though dolichyl pyrophosphate NN'-diacetylchitobiose [Dol-PP-(GlcNAc)2] was the main radiolabelled product formed upon incubation of GTP-treated microsomes with UDP-GlcNAc, GTP selectively stimulated UDP-GlcNAc:dolichyl phosphate (Dol-P) N-acetylglucosaminyl 1-phosphotransferase (N-acetylglucosaminyl 1-phosphotransferase). This conclusion was reached on the basis of experiments in which tunicamycin was used to selectively inhibit N-acetylglucosaminyl 1-phosphotransferase. The enhanced transformation of Dol-P to dolichyl pyrophosphate N-acetylglucosamine (Dol-PP-GlcNAc) by GTP ultimately led to enhanced protein glycosylation. GTP-induced stimulation of GlcNAc incorporation in lipid and protein by GTP was observed also in microsomes fully permeabilized with Staph. aureus alpha-toxin. These findings refute the previous proposal [Godelaine, Beaufay, Wibo and Ravoet (1983) J. Cell Biol. 97, 340-350] that increased membrane permeability constitutes the mechanism whereby GTP activates the reactions of the dolichol pathway. PMID:8280061

  3. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.


    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  4. The stimulatory effects of asbestos on NADPH-dependent lipid peroxidation in rat liver microsomes.

    Fontecave, M; Mansuy, D; Jaouen, M; Pezerat, H


    Lipid peroxidation in rat liver microsomes induced by asbestos fibres, crocidolite and chrysotile, is greatly increased in the presence of NADPH, leading to malondialdehyde levels comparable with those induced by CCl4, a very strong inducer of lipid peroxidation. This synergic effect only occurs during the first minutes and could be explained by an increase or a regeneration of the ferrous active sites of asbestos by NADPH, which in turn could rapidly be prevented by the adsorption of microsomal proteins on the surface of the fibres. It is not inhibited by superoxide dismutase, catalase and mannitol, indicating that oxygen radicals are not involved in the reaction. It is also not inhibited by desferrioxamine, indicating that it is not due to a release of free iron ions in solution from the fibres. Lipid peroxidation in NADPH-supplemented microsomes is also greatly increased upon addition of magnetite. This could be linked to the presence of ferrous ions in this solid iron oxide, since the ferric oxides haematite and goethite are completely inactive. PMID:3036068

  5. CYP3A catalyses schizandrin biotransformation in human, minipig and rat liver microsomes.

    Cao, Y-F; Zhang, Y-Y; Li, J; Ge, G-B; Hu, D; Liu, H-X; Huang, T; Wang, Y-C; Fang, Z-Z; Sun, D-X; Huo, H; Yin, J; Yang, L


    Schizandrin is recognized as the major absorbed effective constituent of Fructus schisandrae, which is extensively applied in Chinese medicinal formula. The present study aimed to profile the phase I metabolites of schizandrin and identify the cytochrome P450 (CYP) isoforms involved. After schizandrin was incubated with human liver microsomes, three metabolites were isolated by high-performance liquid chromatography (HPLC) and their structures were identified to be 8(R)-hydroxyl-schizandrin, 2-demethyl-8(R)-hydroxyl-schizandrin, 3-demethyl-8(R)-hydroxyl-schizandrin, by liquid chromatography-mass spectrometry (LC-MS), (1)H-nuclear magnetic resonance (NMR), and (13)C-NMR, respectively. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that CYP3A4 was the main hepatic isoform that cleared schizandrin. Rat and minipig liver microsomes were included when evaluating species differences, and the results showed little difference among the species. In conclusion, CYP3A4 plays a major role in the biotransformation of schizandrin in human liver microsomes. Minipig and rat could be surrogate models for man in schizandrin pharmacokinetic studies. Better knowledge of schizandrin's metabolic pathway could provide the vital information for understanding the pharmacokinetic behaviours of schizandrin contained in Chinese medicinal formula.

  6. In Vitro Metabolism Studies of Polybrominated Diphenyl Ethers Using Rat and Human Liver Microsomes

    Shun W. Cheng


    Full Text Available A number of studies have recently reported the bioaccumulation of the commonly used fire retardants, Polybrominated Diphenyl Ethers (PBDEs, in humans and wildlife. Exposure of animals to PBDEs has been shown to result in developmental neurological, reproductive abnormalities and the disruption of endocrine function. Thyroid hormone equilibria was also shown to be altered by PBDE exposure. There is evidence that hydroxylated metabolites of PBDEs are directly involved in some of these adverse effects. Although metabolites of PBDEs have been isolated and characterized during in vivo studies, the identification of metabolites from an in vitro system has been problematic. We investigated the in vitro metabolism of four PBDEs, with varying numbers of bromine atoms, in rat and human liver microsomes. The addition of small amounts of a nonionic surfactant to the reaction mixture was necessary to obtain measurable amounts of metabolites due to the low aqueous solubility of the PBDEs. Using gas chromatography/mass spectroscopy, mono and/or dihydroxylated metabolites were identified from three of the four PBDEs with phenobarbitol- and β-naphthoflavone-induced rat liver microsomes. When using uninduced rat or human liver microsomes, metabolites were found with only one of the PBDEs. The ease of PBDE metabolism appears to be inversely related to the number of bromine atoms on the parent compound.

  7. Formation of Schiff base adduct between acetaldehyde and rat liver microsomal phosphatidylethanolamine.

    Kenney, W C


    Recent studies have established the formation of acetaldehyde adducts of proteins even at low concentrations of acetaldehyde expected to occur in vivo under conditions of ethanol metabolism. Although formation of acetaldehyde adducts with phospholipids has been obtained at high pH values and at high concentrations of acetaldehyde, the occurrence of such adducts under more physiological conditions had yet to be demonstrated. Rat liver microsomes were incubated with 0.2 mM [14C]acetaldehyde at pH 7.4 and 37 degrees C. After treatment with sodium borohydride to reduce any Schiff bases formed, the phospholipids were isolated. The major radioactive component within the phospholipid fraction had chromatographic properties identical to N-ethylphosphatidylethanolamine. In addition, the nitrogenous base derived therefrom by acid hydrolysis was identical to N-ethylethanolamine. These results indicate that a Schiff base adduct between acetaldehyde and microsomal phosphatidylethanolamine had been formed during incubation of low concentrations of acetaldehyde with rat liver microsomes.

  8. Inhibition of rat brain microsomal cytochrome P450-dependent dealkylation activities by an oxidative stress.

    Lagrange, P; El-Bachá, R D; Netter, P; Minn, A


    There is increasing evidence that an oxidative stress not only alters cellular lipids and nucleic acids, but also numerous proteins. This oxidation results in alterations of some cellular functions, either by reversible modifications allowing a post-transcriptional regulation of enzyme activities or receptor affinities, or by irreversible modifications of the protein, triggering its inactivation and destruction. In the present work, we examined the effects of an experimental oxidative stress on rat brain microsomal cytochrome P450-dependent dealkylation activities. For that purpose, superoxide anions were produced either by the NADPH-dependent redox cycling of a quinine, menadione, or by the addition of apomorphine, which produces by autoxidation both superoxide anions and apomorphine-derived quinones. The inhibition of brain cytochrome P450-dependent alkoxyresorufin O-dealkylase activities was dependent on both menadione or apomorphine concentrations. Simultaneously, an increase of microsomal carbonyl groups was recorded. Immunoblotting characterization of brain microsomal oxidized protein was carried out, using antibodies raised against 2,4-dinitrophenylhydrazine as a reagent of protein carbonyl groups, and a revelation by a chemiluminescence method. We observed an increase in cerebral CYP1A protein oxidation, related to menadione concentration, suggesting that oxidation of cytochrome P450 protein may result in its catalytic inactivation.

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

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


    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.

  10. Effect of succinate on phosphate solubilization in nitrogen fixing bacteria harbouring chick pea and their effect on plant growth.

    Iyer, Bhagya; Rajput, Mahendrapal Singh; Rajkumar, Shalini


    Diverse nitrogen fixing bacteria harbouring chick pea rhizosphere and root nodules were tested for multiple plant growth promoting traits like tricalcium phosphate (TCP) and rock phosphate (RP) solubilization, production of ammonia, indole 3-acetic acid, chitinase, phytase and alkaline phosphatase. Isolates belonged to diverse genus like Enterobacter, Acinetobacter, Erwinia, Pseudomonas, Rhizobium, Sinorhizobium, Ensifer, Klebsiella, etc. Most isolates solubilized TCP and RP along with the lowering of media pH, indicating acidification to be the chief mechanism behind this solubilization. However, lowering of media pH and P release decreased by 32-100% when media was supplemented with succinate, a major component of plant root exudates indicating succinate mediated repression of P solubilization. Maximum TCP and RP solubilization with P release of 850μg/mL and 2088μg/mL was obtained with lowering of media pH up to 2.8 and 3.3 for isolate E43 and PSB1 respectively. This pH drop changed to 4.4 and 4.8 with 80% and 87% decrease in P solubilization in the presence of succinate. Maximum 246μg/mL indole 3-acetic acid production in Lh3, 44.8U/mL chitinase activity in MB3, 11.3U/mL phytase activity in I91 and 9.4U/mL alkaline phosphatase activity in SM1 were also obtained. Most isolates showed multiple PGP traits which resulted in significant plant growth promotion of chick pea plants. Present study shows repression of P solubilization by succinate for various bacterial groups which might be one of the reasons why phosphate solubilizing bacteria which perform well in vitro often fail in vivo. Studying this repression mechanism might be critical in understanding the in vivo efficacy. Copyright © 2017. Published by Elsevier GmbH.

  11. Large scale solubilization of coal and bioconversion to utilizable energy. Quarterly report, October 1, 1995--December 31, 1995

    Mishra, N.C.


    The ability of Neurospora to solubilize and bioconvert coal was investigated. The coal solubilizing activity (CSA) was fractionated to isolate the enzyme responsible for this activity. The enzyme was purified in order to obtain the amino acid sequence. From that sequence potential oligonucleotide probes were synthesized and used to screen genomic library of Neurospora. The gene so identified was isolated. CSA appears to be an phenol oxidase or is tyrosinase.

  12. The removal characteristics of natural organic matter in the recycling of drinking water treatment sludge: Role of solubilized organics.

    Zhou, Zhiwei; Yang, Yanling; Li, Xing; Ji, Siyang; Zhang, Hao; Wang, Shuai; Zeng, Qingping; Han, Xinghang


    To clarify the role of solubilized organics derived from drinking water treatment sludge (DWTS) in the elimination of natural organic matter (NOM) in the DWTS recycling process, a probe sonoreactor at a frequency of 25 kHz was used to solubilize the organics at varied specific energies. The coagulation behavior related to NOM removal in recycling the sonicated DWTS with and without solubilized organics was evaluated, and the effect on organic fractionations in coagulated water was determined. The study results could provide useful implications in designing DWTS recycling processes that avoid the enrichment of organic matter. Our results indicate that DWTS was disrupted through a low release of soluble chemical oxygen demand (SCOD) and proteins, which could deteriorate the coagulated water quality under the specific energy of 37.87-1212.1 kW h/kg TS. The optimal coagulation behavior for NOM removal was achieved by recycling the sonicated DWTS without solubilized organics at 151.5 kW h/kg TS specific energy. Recycling the sonicated DWTS could increase the enrichment potential of weakly hydrophobic acid, hydrophilic matter, and <3 kDa fractions; the enrichment risks could be reduced by discharging the solubilized organics. Fluorescent characteristic analysis indicated that when recycling the sonicated DWTS without solubilized organics, the removal of humic-like substances was limited, whereas removal of protein-like substances was enhanced, lowering the enrichment potential of protein-like substances.

  13. Generally recognized as safe (GRAS) Lactococcus lactis strains associated with Lippia sidoides Cham. are able to solubilize/mineralize phosphate.

    de Lacerda, Jackeline Rossetti Mateus; da Silva, Thais Freitas; Vollú, Renata Estebanez; Marques, Joana Montezano; Seldin, Lucy


    Eight strains isolated from the stems of Lippia sidoides were identified as belonging to Lactococcus lactis, a bacterial species considered as "generally recognized as safe". Their capacity to solubilize/mineralize phosphate was tested in vitro with different inorganic and organic phosphorus (P) sources. All strains were able to solubilize calcium phosphate as an inorganic P source, and the best result was observed with strain 003.41 which solubilized 31 % of this P source. Rock phosphate, a mined rock containing high amounts of phosphate bearing minerals, was solubilized by five strains. When calcium phytate was the organic P source used, the majority of the strains tested showed phosphate mineralization activity. Moreover, all strains were able to solubilize/mineralize phosphate from poultry litter, a complex P source containing inorganic and predominantly organic P. The presence of genes coding for phytase and alkaline phosphatase was searched within the strains studied. However, only gene sequences related to alkaline phosphatase (phoA and phoD) could be detected in the majority of the strains (excepting strain 006.29) with identities varying from 67 to 88 %. These results demonstrate for the first time the potential of L. lactis strains for phosphate solubilization/mineralization activity using a broad spectrum of P sources; therefore, they are of great importance for the future development of more safe bioinoculants with possible beneficial effects for agriculture.

  14. Solubilization and purification of the ATPase from the tonoplast of Hevea.

    Marin, B; Preisser, J; Komor, E


    The tonoplast-bound ATPase of Hevea brasiliensis (caoutchouc tree) was solubilized with dichloromethan and purified 100-fold with two ammonium sulfate precipitation steps and a G-200 gel filtration step. The resulting ATPase activity eluted according to a molecular mass of approximately 200 kDa and chromatographed at an isoelectric pH of 5.3. Subunits of molecular mass 110 kDa, 68 kDa, 24 kDa and 12 kDa appeared after treatment with 1% sodium dodecyl sulfate or spontaneously during storage of the solubilized ATPase. Dodecyl sulfate/polyacrylamide gel electrophoresis yielded four polypeptides of molecular mass 54 kDa, 66 kDa, 23 kDa and 13 kDa. From protein determination by ultraviolet absorption and Coomassie stain it appears that the 54-kDa and the 66-kDa polypeptides exist in multiple copies. No close resemblance to the membrane-bound ATPase of mitochondria, plastids, plasmalemma, chromaffin granules and synaptic vesicles is seen. No antibody cross-reaction to F1 of bacteria is observed. Therefore it is concluded that the vacuolar ATPase represents a novel type of ATPase. Many properties of the tonoplast-bound ATPase such as pH-dependence, substrate specificity, ion-dependence and inhibitor sensitivity did not change when the enzyme had been solubilized and purified. The phosphatase activity was lost during the purification procedure. The stimulation of ATP-hydrolysis in tonoplast vesicles by uncouplers and ionophores was absent in the solubilized ATPase, and also the stimulation by chloride was significantly reduced. Anion channel blockers, such as triphenyltin and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, which are strong inhibitors of membrane-bound ATPase, fully or partly lost their inhibiting effect after solubilization of the ATPase. These results are interpreted to indicate that ionophores do not directly affect the ATPase molecule, whereas chloride might have a small direct effect on the ATPase besides its effect as a permeating anion.

  15. Pulmonary surfactant protein A inhibits the lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria and microsomes.

    Terrasa, Ana M; Guajardo, Margarita H; de Armas Sanabria, Elizabeth; Catalá, Angel


    Reactive oxygen species play an important role in several acute lung injuries. The lung tissue contains polyunsaturated fatty acids (PUFAs) that are substrates of lipid peroxidation that may lead to loss of the functional integrity of the cell membranes. In this study, we compare the in vitro protective effect of pulmonary surfactant protein A (SP-A), purified from porcine surfactant, against ascorbate-Fe(2+) lipid peroxidation stimulated by linoleic acid hydroperoxide (LHP) of the mitochondria and microsomes isolated from rat lung; deprived organelles of ascorbate and LHP were utilized as control. The process was measured simultaneously by chemiluminescence as well as by PUFA degradation of the total lipids isolated from these organelles. The addition of LHP to rat lung mitochondria or microsomes produces a marked increase in light emission; the highest value of activation was produced in microsomes (total chemiluminescence: 20.015+/-1.735 x 10(5) cpm). The inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (2.5 to 5.0 microg) of SP-A in rat lung mitochondria and 2.5 to 7.5 microg of SP-A in rat lung microsomes. The inhibitory effect reaches the highest values in the mitochondria, thus, 5.0 microg of SP-A produces a 100% inhibition in this membranes whereas 7.5 microg of SP-A produces a 51.25+/-3.48% inhibition in microsomes. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized membranes was found in the arachidonic acid content; this decreased from 9.68+/-1.60% in the native group to 5.72+/-1.64% in peroxidized mitochondria and from 7.39+/-1.14% to 3.21+/-0.77% in microsomes. These changes were less pronounced in SP-A treated membranes; as an example, in the presence of 5.0 microg of SP-A, we observed a total protection of 20:4 n-6 (9.41+/-3.29%) in mitochondria, whereas 7.5 microg of SP-A produced a 65% protection in microsomes (5

  16. Comparative metabolism of chloroacetamide herbicides and selected metabolites in human and rat liver microsomes.

    Coleman, S; Linderman, R; Hodgson, E; Rose, R L


    Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methyl-phenyl)-acetamide], alachlor [N-(methoxymethyl)-2-chloro-N-(2, 6-diethyl-phenyl)acetamide], butachlor [N-(butoxymethyl)-2-chloro-N-(2,6-diethyl-phenyl)acetamide], and metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] are pre-emergent herbicides used in the production of agricultural crops. These herbicides are carcinogenic in rats: acetochlor and alachlor cause tumors in the nasal turbinates, butachlor causes stomach tumors, and metolachlor causes liver tumors. It has been suggested that the carcinogenicity of these compounds involves a complex metabolic activation pathway leading to a DNA-reactive dialkylbenzoquinone imine. Important intermediates in this pathway are 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) produced from alachlor and butachlor and 2-chloro-N-(2-methyl-6-ethylphenyl)acetamide (CMEPA) produced from acetochlor and metolachlor. Subsequent metabolism of CDEPA and CMEPA produces 2,6-diethylaniline (DEA) and 2-methyl-6-ethylaniline (MEA), which are bioactivated through para-hydroxylation and subsequent oxidation to the proposed carcinogenic product dialkylbenzoquinone imine. The current study extends our earlier studies with alachlor and demonstrates that rat liver microsomes metabolize acetochlor and metolachlor to CMEPA (0.065 nmol/min/mg and 0.0133 nmol/min/mg, respectively), whereas human liver microsomes can metabolize only acetochlor to CMEPA (0.023 nmol/min/mg). Butachlor is metabolized to CDEPA to a much greater extent by rat liver microsomes (0.045 nmol/min/mg) than by human liver microsomes (< 0.001 nmol/min/mg). We have determined that both rat and human livers metabolize both CMEPA to MEA (0.308 nmol/min/mg and 0.541 nmol/min/mg, respectively) and CDEPA to DEA (0.350 nmol/min/mg and 0.841 nmol/min/mg, respectively). We have shown that both rat and human liver microsomes metabolize MEA (0.035 nmol/min/mg and 0.069 nmol/min/mg, respectively

  17. Solubilization of immune complexes in complement factor deficient sera and the influence of temperature, ionic strength and divalent cations on the solubilization reaction

    Baatrup, Gunnar; Petersen, Ivan; Svehag, Svend-Erik;


    The complement-mediated solubilization (CMS) of immune complexes (IC) and the initial kinetics (IKS) of this reaction in human sera depleted of or deficient in C2, C3, C8, factors B, P and I were investigated. Sera depleted of B or P and those lacking native C3 or factor I showed virtually no CMS...... by a radioassay and kinetic data for the binding of C3b to preformed immune complexes. The CMS capacity reached maximum at 39-41 degrees C and at an ionic strength of approximately 0.20 mu. Selective chelation of Mg2+ completely abolished the CMS of IC. Maximal CMS was observed at Mg2+ concentration of about 2m...

  18. Kinetics of naphthalene metabolism in target and non-target tissues of rodents and in nasal and airway microsomes from the Rhesus monkey

    Buckpitt, Alan, E-mail: [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)


    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.

  19. Metabolism of methyl tert-butyl ether and other gasoline ethers in mouse liver microsomes lacking cytochrome P450 2E1.

    Hong, J Y; Wang, Y Y; Bondoc, F Y; Yang, C S; Gonzalez, F J; Pan, Z; Cokonis, C D; Hu, W Y; Bao, Z


    To reduce the production of pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE) and other ethers such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Metabolism of these gasoline ethers is catalyzed by cytochrome P450 (P450) enzymes. P450 2E1, which metabolizes diethyl ether, was suggested to be an enzyme involved. The present study used 2E1 knock-out mice (2E1-/-) to assess the contribution of 2E1 to the metabolism of MTBE, ETBE and TAME. Liver microsomes prepared from the 2E1 knock-out mice lacked 2E1 activity (assayed as N-nitrosodimethylamine demethylation), but were still active in metabolizing all three gasoline ethers. The levels of ether-metabolizing activity (nmol/min per mg) in the liver microsomes from 7 week old female 2E1 knock-out mice were 0.54+/-0.17 for MTBE, 0.51+/-0.24 for ETBE and 1.14+/-0.25 for TAME at a 1 mM substrate concentration. These activity levels were not significantly different from those of the sex- and age-matched C57BL/6N and 129/Sv mice, which are the parental lineage strains of the 2E1 knock-out mice and are both 2E1+/+. Our results clearly demonstrate that 2E1 plays a negligible role in the metabolism of MTBE, ETBE and TAME in mouse livers.

  20. Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L. ) microsomal and soluble protein tryptophanyl residues in vitro

    Caldwell, C.R. (Dept. of Agriculture, Beltsville, MD (United States))


    The in vitro effects of ultraviolet B (280--320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen species. The optimum wavelengths for loss of tryptophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24[degrees]C, which influenced protein structure and tryptophan photosensitivity. 29 refs., 6 figs., 1 tab.