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Incubation Mixture (incubation + mixture)
Selected AbstractsSmall heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actinFEBS JOURNAL, Issue 22 2007Anastasia V. Pivovarova Previously, we have shown that the small heat shock protein with apparent molecular mass 27 kDa (Hsp27) does not affect the thermal unfolding of F-actin, but effectively prevents aggregation of thermally denatured F-actin [Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI & Gusev NB (2005) Biochem Biophys Res Commun331, 1548,1553], and supposed that Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. In the present work, we applied dynamic light scattering, analytical ultracentrifugation and size exclusion chromatography to examine the properties of complexes formed by denatured actin with a recombinant human Hsp27 mutant (Hsp27,3D) mimicking the naturally occurring phosphorylation of this protein at Ser15, Ser78, and Ser82. Our results show that formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. All the methods show that the size of actin,Hsp27-3D complexes decreases with increasing Hsp27-3D concentration in the incubation mixture and that saturation occurs at approximately equimolar concentrations of Hsp27-3D and actin. Under these conditions, the complexes exhibit a hydrodynamic radius of ,,16 nm, a sedimentation coefficient of 17,20 S, and a molecular mass of about 2 MDa. It is supposed that Hsp27-3D binds to denatured actin monomers or short oligomers dissociated from actin filaments upon heating and protects them from aggregation by forming relatively small and highly soluble complexes. This mechanism might explain how small heat shock proteins prevent aggregation of denatured actin and by this means protect the cytoskeleton and the whole cell from damage caused by accumulation of large insoluble aggregates under heat shock conditions. [source] 2,-Deoxyadenosine causes apoptotic cell death in a human colon carcinoma cell lineJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2003Michela Giannecchini Abstract The combination of 2,-deoxyadenosine and 2,-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2,-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2,-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2,-deoxyadenosine and 2,-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:329,337, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10095 [source] Simultaneous purification and immobilization of bitter gourd (Momordica charantia) peroxidases on bioaffinity supportJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 2 2005Suhail Akhtar Abstract This paper demonstrates the construction of an inexpensive bioaffinity adsorbent by simply incubating Sephadex G 50 matrix with jack bean meal extract at room temperature. Sephadex G 50 adsorbed 17 mg Con A (concanavalin A) per g of the matrix. Con A-adsorbed Sephadex was employed for the immobilization of glycoenzymes directly from ammonium sulfate-fractionated proteins of bitter gourd. The obtained bioaffinity support was very efficient for high yield immobilization of peroxidases from bitter gourd and it bound nearly 425 enzyme units per g of the matrix. Bitter gourd peroxidase immobilized on lectin,Sephadex support showed a very high effectiveness factor, ,,,' of 1.25. Immobilized BGP preparation was quite stable against the denaturation induced by pH, heat, urea, Triton X 100, Tween 20, SDS, Surf Excel and water-miscible organic solvents: dimethyl sulfoxide and dimethyl formamide. Low concentration of detergents like SDS, Tween 20, and Triton X 100 enhanced the activity of soluble and immobilized bitter gourd peroxidase. Peroxidase bound to the bioaffinity support exhibited very high resistance to proteolysis caused by the trypsin treatment. Con A,Sephadex-bound bitter gourd peroxidase retained 85% of its initial activity after treatment with 2.5 mg trypsin per cm3 of incubation mixture for 1 h at 37 °C while the soluble enzyme lost nearly 40% of the initial activity under similar incubation conditions. Immobilized bitter gourd peroxidase preparation appeared to be more rigid to proteolysis mediated by trypsin compared with soluble bitter gourd peroxidase. Copyright © 2004 Society of Chemical Industry [source] Chylomicron accelerates C3 tick-over by regulating the role of Factor H, leading to overproduction of acylation stimulating proteinJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2007Takayuki Fujita Abstract Acylation stimulating protein (ASP) is a fragment of the third component of complement (C3) that is generated in the presence of chylomicron, and plays a role in the synthesis of triacylglycerol by transporting free fatty acids into adipocytes. However, the precise mechanism of ASP generation, especially the role of chylomicron in ASP generation, is unknown. We examined the mechanism through which chylomicron induces ASP generation. Ultracentrifugationally separated chylomicron was incubated with normal human serum (NHS) under various conditions, and the amounts of complement activation products and ASP in the incubation mixture were determined by enzyme-linked immunosorbent assay (ELISA). Upon incubation of NHS with various amounts of chylomicron for 120,min, ASP was generated in a dose-dependent manner. The time course of the production of ASP was similar to the time course of the C3 tick-over phenomenon that occurred by depletion of factor H from the serum. The complement activation induced by chylomicron was different from the usual complement activation that occurs under the regulation of factor H and factor I with respect to the time course and the amount of ASP produced. Our results indicate that chylomicron accelerates C3 tick-over by regulating the role of factor H, leading to the overproduction of ASP. J. Clin. Lab. Anal. 21:14,23, 2007. © 2007 Wiley-Liss, Inc. [source] In vitro Metabolism of Genistein and Tangeretin by Human and Murine Cytochrome P450sBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2003Vibeke M. Breinholt Analysis of the metabolic profile from incubations with genistein and human liver microsomes revealed the production of five different metabolites, of which three were obtained in sufficient amounts to allow a more detailed elucidation of the structure. One of these metabolites was identified as orobol, the 3,-hydroxylated metabolite of genistein. The remaining two metabolites were also hydroxylated metabolites as evidenced by LC/MS. Orobol was the only metabolite formed after incubation with CYP1A2. The two major product peaks after incubation of tangeretin with human microsomes were identical with 4,-hydroxy-5,6,7,8-tetramethoxyflavone and 5,6-dihydroxy-4,,7,8-trimethoxyflavone, previously identified in rat urine in our laboratory. By comparison with UV spectra and LC/MS fragmentation patterns of previously obtained standards, the remaining metabolites eluting after 14, 17 and 20 min. were found to be demethylated at the 4,,7-, 4,,6-positions or hydroxylated at the 3,- and demethylated at the 4,-positions, respectively. Metabolism of tangeretin by recombinant CYP1A2, 3A4, 2D6 and 2C9 resulted in metabolic profiles that qualitatively were identical to those observed in the human microsomes. Inclusion of the CYP1A2 inhibitor fluvoxamine in the incubation mixture with human liver microsomes resulted in potent inhibition of tangeretin and genistein metabolism. Other isozymes-selective CYP inhibitors had only minor effects on tangeretin or genistein metabolism. Overall the presented observations suggest major involvement of CYP1A2 in the hepatic metabolism of these two flavonoids. [source] Simultaneous detection of monohydroxybenzo[a]pyrene positional isomers by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 7 2002Hideki Sasaki A liquid chromatographic (LC) method has been developed for the separation of 11 monohydroxybenzo[a]pyrenes (OH BaPs) positional isomers, and for their detection using electrospray ionization mass spectrometry (ESI-MS). All OH BaPs isomers were separated on an octadecylsilyl (C18)-bonded amorphous organosilica column utilizing gradient elution with acetonitrile,water and triethylamine (TEA) at pH 11.0 and determined by MS, except 2- and 8-OH BaPs which were coeluted. The lower detection limits were in the range from 1.6,µg/L for 12-OH BaP to 12,µg/L for 5-OH BaP without any sample enrichment. The relative standard deviations of area response were in the range from 1.8% (9-OH BaP) to 4.9% (12-OH BaP) except for 9.4% (7-OH BaP). The developed method was successfully applied to incubation mixtures of BaP and CYP1A1/epoxide hydrolase. This method identified 1-, 3- and 9-OH BaPs as the major metabolites, and 2- (and/or 8-) and 12-OH BaPs as the minor metabolites in the incubation mixture. Copyright © 2002 John Wiley & Sons, Ltd. [source] A rapid assay method for catechol- O -methyltransferase activity by flow injection analysisBIOMEDICAL CHROMATOGRAPHY, Issue 4 2002Nozomi Aoyama A rapid assay employing flow injection analysis (FIA) to determine the activity of purified catechol- O -methyltransferase (COMT) from porcine liver is described. The method was based on the determination of normetanephrine, the 3- O -methyl metabolite of the substrate norepinephrine. Excess norepinephrine was removed from the incubation mixture by alumina extraction twice to allow normetanephrine to be subjected to flow injection analysis, coulometrical oxidation, fluorogenic reaction with ethylenediamine and fluorescence detection. Km and Vmax values for COMT obtained with the system were 503,µM and 4.51 nmol/min/mg protein, respectively. The method is suitable for screening of COMT inhibitors or activators, as a large number of samples, up to 200, can be processed in one working day. Copyright © 2002 John Wiley & Sons, Ltd. [source] New method for the simultaneous estimation of intrinsic hepatic clearance and protein binding by matrix inhibitionBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 1 2008Takahide Uchimura Abstract The purpose of this study was to develop a method for estimating the hepatic clearance (CLh) without using a protein binding test. This method allows the simultaneous evaluation of the intrinsic hepatic clearance (CLint) with a correction for microsomal binding, and the free fraction in the serum (fu). It uses the decrease in metabolic velocity achieved by decreasing the free fraction of a compound in the incubation mixture (fuinc) by the addition of serum, and by changing the microsomal protein concentration. This method is denoted as the ,matrix inhibition method', because it uses the inhibition of the metabolic velocity by the incubation matrix. The metabolic rates of eight compounds (diazepam, imipramine, warfarin, and compounds A,E) were evaluated under several incubation conditions using rat serum and microsomes. The correlation of CLint evaluated using the method and using equilibrium dialysis after the CLint was corrected for microsomal binding was r,=,0.968. The correlation of fu,·,CLint was r,=,0.996. Although the method required a high enough fu and fumicrosomes difference among the reaction conditions for each compound, it could evaluate CLint and fu simultaneously and easily by adding additional reaction conditions to the metabolic stability tests performed in ADME screening. Copyright © 2007 John Wiley & Sons, Ltd. [source] CYP7B1-mediated metabolism of dehydroepiandrosterone and 5,-androstane-3,,17,-diol , potential role(s) for estrogen signalingFEBS JOURNAL, Issue 8 2008Hanna Pettersson CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5,-androstane-3,,17,-diol (3,-Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1-dependent metabolism involving dehydroepiandrosterone or 3,-Adiol may play an important role for estrogen receptor ,-mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1-related steroids on estrogen receptor , activation. In the present study, we investigated CYP7B1-mediated conversions of dehydroepiandrosterone and 3,-Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3,-Adiol (a CYP7B1 substrate) and 7,-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor , activation. The data obtained indicated that 3,-Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 catalysis may decrease estrogen receptor , activation. Our data on metabolism indicate that the efficiencies of CYP7B1-mediated hydroxylations of dehydroepiandrosterone and 3,-Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the Kcat/Km values were in the same order of magnitude. A high dehydroepiandrosterone/3,-Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1-mediated 3,-Adiol metabolism. As the efficiencies of CYP7B1-mediated hydroxylation of dehydroepiandrosterone and 3,-Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1-mediated metabolism of dehydroepiandrosterone or 3,-Adiol. Consequently, tissue-specific steroid concentrations may have a strong impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1-related steroids that can influence estrogen receptor , signaling. [source] Moxidectin and ivermectin metabolic stability in sheep ruminal and abomasal contentsJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 5 2005A. LIFSCHITZ The oral administration of macrocyclic lactones to sheep leads to poorer efficacy and shorter persistence of the antiparasitic activity compared to the subcutaneous treatment. Gastrointestinal biotransformation occurring after oral treatment to ruminant species has been considered as a possible cause of the differences observed between routes of administration. The current work was addressed to evaluate on a comparative basis the in vitro metabolism of moxidectin (MXD) and ivermectin (IVM) in sheep ruminal and abomasal contents. Both compounds were incubated under anaerobic conditions during 2, 6 and 24 h in ruminal and abomasal contents collected from untreated adult sheep. Drug concentrations were measured by high-performance liquid chromatography with fluorescence detection after sample clean up and solid phase extraction. Neither MXD nor IVM suffered metabolic conversion and/or chemical degradation after 24-h incubation in ruminal and abomasal contents collected from adult sheep. Unchanged MXD and IVM parent compounds represented between 95.5 and 100% of the total drug recovered in the ruminal and abomasal incubation mixtures compared with those measured in inactive control incubations. The partition of both molecules between the solid and fluid phases of both sheep digestive contents was assessed. MXD and IVM were extensively bound (>90%) to the solid material of both ruminal and abomasal contents collected from sheep fed on lucerne hay. The results reported here confirm the extensive degree of association to the solid digestive material and demonstrates a high chemical stability without evident metabolism and/or degradation for both MXD and IVM in ruminal and abomasal contents. [source] Simultaneous detection of monohydroxybenzo[a]pyrene positional isomers by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometryBIOMEDICAL CHROMATOGRAPHY, Issue 7 2002Hideki Sasaki A liquid chromatographic (LC) method has been developed for the separation of 11 monohydroxybenzo[a]pyrenes (OH BaPs) positional isomers, and for their detection using electrospray ionization mass spectrometry (ESI-MS). All OH BaPs isomers were separated on an octadecylsilyl (C18)-bonded amorphous organosilica column utilizing gradient elution with acetonitrile,water and triethylamine (TEA) at pH 11.0 and determined by MS, except 2- and 8-OH BaPs which were coeluted. The lower detection limits were in the range from 1.6,µg/L for 12-OH BaP to 12,µg/L for 5-OH BaP without any sample enrichment. The relative standard deviations of area response were in the range from 1.8% (9-OH BaP) to 4.9% (12-OH BaP) except for 9.4% (7-OH BaP). The developed method was successfully applied to incubation mixtures of BaP and CYP1A1/epoxide hydrolase. This method identified 1-, 3- and 9-OH BaPs as the major metabolites, and 2- (and/or 8-) and 12-OH BaPs as the minor metabolites in the incubation mixture. Copyright © 2002 John Wiley & Sons, Ltd. [source] | |||||||||||||