research in bloodstain pattern evidence pdf
Tài liệu Báo cáo khoa học: Pathways and products for the metabolism of vitamin D3 by cytochrome P450scc docx
... identified as monohydroxyvitamin D3 Because cyclodextrin provides an artificial means of holding vitamin D3 in solution, we also examined the metabolism of vitamin D3 incorporated into the membrane of ... antiproliferative and differentiationinducing agent in different cell types, including epidermal keratinocytes [7–10] To determine whether the major products of P450scc action on vitamin D3 have similar biological ... vitamin D3 in cyclodextrin Vitamin D3 (50 lM) dissolved in cyclodextrin to a final concentration of 0.45% was incubated with 1.0 lM P450scc for the indicated times (A) Consumption of vitamin D3 and...
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Tài liệu Báo cáo khoa học: Cytochrome P450 Cyp4x1 is a major P450 protein in mouse brain doc
... determine if this represented specific binding, the antisera were preincubated with purified recombinant Cyp4x1 protein, resulting in the ablation of detection of the 55-kDa protein in brain (Fig ... and initial side chain coordinates were assigned using the rules of Summers and Karplus [39] Main chain coordinates for residues in loops were obtained using the search method implemented in sybyl ... predicted CYP4x1 protein has characteristics of a functional P450, including conservation of the haembinding cysteine in the RNCIG motif, and so we undertook molecular modelling to examine its structure...
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Tài liệu Báo cáo khóa học: Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism doc
... protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 41 Omura, T & Sato, R (1964) Tha carbon monoxyde binding pigment of liver microsomes I Evidence for its hemoprotein ... Papilio polyxenes is involved in furanocoumarin metabolism It has been demonstrated that three amino acids are involved in protein structure stability (F116, H117 and F484) and two in substrate specificity ... diametrically to the pole carrying the amino acids involved in substrate binding As far as we know, there has been no report about structure activity relationships in this area of the cytochrome...
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Tài liệu Báo cáo khoa học: Models and mechanisms of O-O bond activation by cytochrome P450 A critical assessment of the potential role of multiple active intermediates in oxidative catalysis doc
... constitutive or inducible hemoprotein isoforms [281], exhibiting insignicant sequence identity with P450s in the heme-binding region [282], but bearing a C-terminal avoprotein fragment in the single polypeptide ... Mayer, B (1994) The pteridine binding site of brain nitric oxide synthase Tetrahydrobiopterin binding kinetics, specicity, and allosteric interaction with the substrate domain J Biol Chem 269, 1386113866 ... aromatic amine involving H2O2, formed through dismutation of autoxidatively generated superoxide, to produce the active intermediate, Hb(III)-OOH [149] In line with this, alkaline hemin (ferriprotoporphyrin...
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Báo cáo khoa học: Arabidopsis thaliana CYP77A4 is the first cytochrome P450 able to catalyze the epoxidation of free fatty acids in plants potx
... monomer into the cutin In this context, it is interesting to note that inhibition studies allowed LeQueu et al [49] to demonstrate the involvement of a peroxygenase in the formation of cutin of ... generated in incubations of linoleic acid with microsomes from yeast expressing CYP77A4 Microsomes were incubated with 100 lM [1-14C]linoleic acid in the absence (A) or presence (B) of NADPH Incubations ... encoding a fatty acid x-hydroxylase, a putative epoxide hydrolase, several genes encoding enzymes involved in the synthesis of fatty acids in plastids, including the stearoyl acyl carrier protein...
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Báo cáo khoa học: Inter-flavin electron transfer in cytochrome P450 reductase – effects of solvent and pH identify hidden complexity in mechanism potx
... data have been obtained by anaerobically mixing oxidized CPR with excess NADPH in a stopped-ow instrument and following either the decrease in absorbance at 450 nm indicative of avin reduction or ... chemical and pH gating mechanisms We were principally interested in the inter-avin ET reactions, so the pH dependence of the kinetic behaviour at 600 nm was analysed, reporting on the formation ... dotted lines being intermediate spectra (Inset) Extinction coefcient changes at 600 nm versus solution potential (for clarity not all data points are shown) errors in the estimation of the midpoint...
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Báo cáo khoa học: D88A mutant of cytochrome P450nor provides kinetic evidence for direct complex formation with electron donor NADH ppt
... by replacing the BssHII–PstIfragment containing P450nor cDNA [15] in pT7-nor with the corresponding portion of the mutant cDNA The introduced mutations were again confirmed by sequencing of the ... The resulting PCR product was inserted into pGEM-T (Promega), and then the mutation was confirmed by sequencing of the inserted nucleotide fragment All plasmids expressing mutant proteins were ... ferric resting (solid line), dithionite-reduced (dotted line), and CO-bound (broken line) forms, respectively, of P450nor (5.0 lM) in 10 mM potassium phosphate buffer pH 7.2 indicating that each...
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Báo cáo khoa học: Testosterone 1b-hydroxylation by human cytochrome P450 3A4 pdf
... containing pmol of P450 3A4 (Varying amounts of P450 were used because of differences in rates of the systems, in order to maintain linearity of product formation vs time.) A typical system contained ... H2O; initial conditions 5% A/95% B (v/v), followed by a linear gradient to 95% A/5% B (v/v) over 30 min; 10-min linear gradient to 100% A and held for min; back to initial conditions in 0.1 min; ... to dryness The resulting solid was dissolved in 300 lL of CD3OD and filtered prior to injection HPLC (including UV) The HPLC system consisted of an Agilent 1100 System including a vacuum degasser,...
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Báo cáo khoa học: Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacteriumsp. strain HE5 ppt
... with saturating concentrations of ferredoxin However, kinetic plots did not show a typical Michaelis–Menten curve Instead of reaching a plateau, velocities continued to increase in a linear dependence ... terpineol [26] or cineole [21] In streptomycetes, P450s are often involved in the biosynthesis of macrolide antibiotics such as pikromycin [1], oleandomycin [2], rapamycin [3] or nikkomycin [4], ... compared with the binding of camphor [43] One should also note that binding of obtusifoliol to CYP51 resulted in only a minor change in the absorption spectra [28] The binding of deoxycorticosterone...
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Báo cáo khoa học: The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism docx
... and bovine P450scc after introduction of a 25-hydroxyl group into the cholesterol side chain [20,24] Since 25-hydroxylation of vitamin D3 is a limiting step in its activation, the latter finding ... of 20(S)-hydroxyvitamin D3 (Fig 1A) indicates easy release from the active site of the enzyme with only a minor portion remaining (or rebinding) for further hydroxylation In fact, the yield of ... arising from contaminating cholesterol by showing that it is not generated when FEBS Journal 272 (2005) 4080–4090 ª 2005 FEBS P450scc hydroxylates vitamin D3 vitamin D3 is incubated with recombinant...
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Báo cáo Y học: Baboon cytochrome P450 17a-hydroxylase/17,20-lyase (CYP17) Characterization of the adrenal microsomal and cloned enzymes docx
... substrate docking and binding, the active site including the heme-binding domain and redox partner binding domain [10] Baboon and human CYP17 are excellent candidates for identifying regions in the ... prediction of domains involved in substrate binding and redox partner interaction [10,17] Although site-directed mutageneses and naturally occurring CYP17 mutants have pinpointed specific amino acid residues ... predicted 508 amino acid protein The 28 amino acid differences between baboon and human CYP17 are predominantly conservative with some differences resulting in a change in side chain size and polarity...
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Báo cáo Y học: Substrates modulate the rate-determining step for CO binding in cytochrome P450cam (CYP101) A high-pressure stopped-flow study pdf
... and slow CO binding kinetics (1R-camphor and camphane) (b) There are two complexes which show two-phase CO binding kinetics (substrate-free, camphane) In the following both these findings will be ... for P450s that the observed rate constant for the CO binding is linearly related to the CO concentration indicating bimolecular binding kinetics [11,16] To get the kon rate constants the time ... that the entry of CO into the protein is the rate-limiting step of CO binding In contrast, a negative activation volume points to the Fe-CO bond formation as the rate-limiting step However, the...
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Báo cáo khoa học: Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation pdf
... secondary kinetic isotope effects [19,27–29] The existence of a gem secondary kinetic isotope effect has the effect of attenuating the rate of C-H bond-breaking, so in principle the intrinsic kinetic ... effects are interpreted to mean that the step(s) involving the chemistry of C-H bond cleavage are ratelimiting in the reaction even in liver microsomes, as opposed to steps involving generation ... values of the intramolecular values in Table are reasonable but should be considered lower estimates of the intrinsic kinetic deuterium isotope effects Intermolecular competitive kinetic isotope...
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Báo cáo khoa học: The first cytochrome P450 in ferns Evidence for its involvement in phytoecdysteroid biosynthesis in Polypodium vulgare doc
... balance in plant tissues, as depicted in Fig In vivo E bioconversion measured in callus culture was a linear function of the P450 microsomal protein content, and was inhibited by P450 inhibitors ... protein was determined using the Bradford procedure with bovine serum albumin as a standard [42] The protein concentration of partially or total purified P450 protein was estimated from silver-stained ... of microgram quantities of protein utilizing the principle of protein–dye binding Anal Biochem 72, 248–254 43 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of...
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Báo cáo khoa học: Two CCAAT/enhancer binding protein sites in the cytochrome P4503A1 locus Potencial role in the glucocorticoid response ppt
... heat inactivated fetal bovine serum and maintained at 37 °C in humidified 5% (v/v) CO2 Transactivation assays To minimize variations in transfection efficiency, replicates were transfected in single ... 2003 interactions of the different C/EBP proteins with other factors binding to neighbouring regulatory sites The in vivo observations of the induction of C/EBPa mRNA and protein preceding the ... transferred to poly(vinylidene difluoride) membranes, and the membranes incubated with a specific anti-C/EBPa Ig Protein loading control using Amido Black staining of the membrane is shown in the bottom...
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Báo cáo khoa học: Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1 ppt
... with increased side chain flexibility or reduced bulkiness in the CA ring region are expected to have increased binding efficiencies compared to CA This is actually the case, with a gain in binding ... results in close to 50% loss in C4H activity, does not affect I2C hydroxylation As initial cinnamate binding is not in uenced by this mutation (Table 2) and binding kinetics are first-order (indicating ... residue in the initial binding and correct positioning of CA during catalysis A role of N302 in anchoring the side-chain carboxylate of CA is further supported by the enhanced binding of amine substituted...
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Báo cáo khoa học: Metabolism of N-methyl-amide by cytochrome P450s Formation and characterization of highly stable carbinol-amide intermediate pdf
... free in carbinol-amines and partially delocalized in the peptide bond in carbinol-amides or in the vicinal aromatic rings in N-hydroxymethylcarbazole Finally, cyclic constraints have marginal ... characterization of stable carbinol-amide L Perrin et al intermediates remains scarce and frequently speculative, in both in vivo and in vitro studies Interestingly, N-hydroxymethyl derivatives ... Computational study In order to determine a plausible mechanism connecting M1 to M2 and to assess the factors that govern the stability of carbinol-amide and carbinol-amine, we initially computed...
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Báo cáo khoa học: Cytochrome P450 metabolizing fatty acids in plants: characterization and physiological roles pptx
... to help attract insect pollinators, giving CYP77A6 an indirect role in reproduction It is possible that the introduction of in- chain hydroxyl allows cross-linking of cutin, leading to reticulation ... x-hydroxylation x-hydroxylation in- chain hydroxylation in- chain hydroxylation x-hydroxylation x-hydroxylation in- chain hydroxylation Epoxidation Epoxidation in- chain hydroxylation in- chain hydroxylation x-hydroxylation ... key step in exine formation during plant evolution The apparent absence of the CYP77 family in the moss genome [58] might indicate that cuticles based on cutin polyesters containing in- chain hydroxylated...
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Báo cáo khoa học: The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism pdf
... acid-producing strain Just like C maltosa, C tropicalis mutant strains are used in industrial dicarboxylic acid production, the strains are among others blocked in b-oxidation by inactivation ... found in some bacterial and fungal cell membranes Interestingly, bacterial sphingolipid synthesis predominantly occurs in anaerobic species One such anaerobe is Sphingomonas paucimobilis Its sphingolipids ... carcinogenic The 17 fumonisin biosynthetic genes are located in a gene cluster and three of them are P450 enzymes The FUM6 protein (CYP505B1) intervenes in one of the first steps in fumonisin synthesis...
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Báo cáo khoa học: An alternative pathway of vitamin D2metabolism Cytochrome P450scc (CYP11A1)-mediated conversion to 20-hydroxyvitamin D2and 17,20-dihydroxyvitamin D2 pot
... position and coupling patterns in vitamin D2 and metabolite 2, indicating that the B-ring stays intact Therefore, the second hydroxylation must occur in the C-ring The 14-CH in this metabolite ... for 24 h in Ham’s F10 medium containing serum and vitamin D2 or its metabolites, and [3H]-thymidine (B) HaCaT keratinocytes were transfected with a construct containing the involucrin promoter ... P450scc, which include brain [33], gastrointestinal tract [34], kidney [35], and skin [18], could alternatively produce and use the same metabolites in local paracrine, autocrine or intracrine roles...
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