Enzymatic properties of cytochrome P450 catalyzing 3′-hydroxylation of naringenin from the white-rot fungus Phanerochaete chrysosporium

We cloned full-length cDNAs of more than 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium, and successfully expressed 70 isoforms using a co-expression system of P. chrysosporium P450 and yeast NADPH-P450 reductase in Saccharomyces cerevisiae. Of these P450s, a microsomal P450 designated as PcCYP65a2 consists of 626 amino acid residues with a molecular mass of 68.3 kDa. Sequence alignment of PcCYP65a2 and human CYP1A2 revealed a unique structure of PcCYP65a2. Functional analysis of PcCYP65a2 using the recombinant S. cerevisiae cells demonstrated that this P450 catalyzes 3′-hydroxylation of naringenin to yield eriodictyol, which has various biological and pharmacological properties. In addition, the recombinant S. cerevisiae cells expressing PcCYP65a2 metabolized such polyaromatic compounds as dibenzo-p-dioxin (DD), 2-monochloroDD, biphenyl, and naphthalene. These results suggest that PcCYP65a2 is practically useful for both bioconversion and bioremediation.     

Comparative excretion and tissue distribution of selenium in mice and rats following treatment with the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate

In a previous preliminary investigation, we reported on the excretion, tissue disposition and metabolism of the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) in the rat, but similar studies in the mouse have not been explored. Following the oral administration of p-XSC (50 micromol/kg body weight), selenium excretion in feces was comparable to that in urine in mice, but in rats, feces was the major route of excretion. Tetraselenocyclophane (TSC) was the major metabolite detected in mouse and rat feces. In both species, levels of selenium in exhaled air were negligible. At termination, in the mouse, the stomach had the highest selenium content followed by liver and blood, but lung and kidney contained negligible levels of selenium; in the rat, the selenium level in liver was the highest followed by kidney, stomach, blood and lung. The identification of TSC as a fecal metabolite in both species let us to postulate the following metabolic pathway: p-XSC-->glutathione conjugate (p-XSeSG)-->a selenol (p-XSeH)-->TSC. Since the glutathione conjugate appears to be the proximal precursor for the selenol metabolite that may be an important intermediate in cancer chemoprevention, we report for the first time the synthesis of p-XSeSG and its other potential metabolites, namely the cysteine- and N-acetylcysteine-conjugates of p-XSC. HPLC analysis of the urine and bile showed a few metabolites of p-XSC; none of which eluted with the synthetic standards described above. When we examined the conversion of p-XSC and p-XSeSG in vitro using rat cecal microflora, TSC was formed from p-XSeSG but not from p-XSC. The formation of TSC from p-XSC in vivo but not in vitro suggests that p-XSC needs to be metabolized to p-XSeSG or an intermediate derived from its further metabolism. Thus, p-XSeSG was given orally to rats and the results showed that the pattern of selenium excretion after p-XSeSG treatment was similar to that of p-XSC; TSC was also identified as a fecal metabolite of p-XSeSG. It may be that the conversion of p-XSeSG to TSC is too facile, or the mere conjugation of p-XSC with glutathione does not occur in rats and mice.     

Effects of the aqueous extract of white tea (Camellia sinensis) in a streptozotocin-induced diabetes model of rats

White tea (WT) is very similar to green tea (GT) but it is exceptionally prepared only from the buds and young tea leaves of Camelia sinensis plant while GT is prepared from the matured tea leaves. The present study was investigated to examine the effects of a 0.5% aqueous extract of WT in a streptozotocin-induced diabetes model of rats. Six-week-old male Sprague-Dawley rats were divided into 3 groups of 6 animals in each group namely: normal control (NC), diabetic control (DBC) and diabetic white tea (DWT). Diabetes was induced by an intraperitoneal injection of streptozotocin (65 mg/kg BW) in DBC and DWT groups except the NC group. After 4 weeks feeding of 0.5% aqueous extracts of WT, the drink intake was significantly (P < 0.05) increased in the DWT group compared to the DBC and NC groups. Blood glucose concentrations were significantly decreased and glucose tolerance ability was significantly improved in the DWT group compared to the DBC group. Liver weight and liver glycogen were significantly increased and serum total cholesterol and LDL-cholesterol were significantly decreased in the DWT group compared to the DBC group. The food intake, body weight gain, serum insulin and fructosamine concentrations were not influenced by the consumption of WT. Data of this study suggest that the 0.5% aqueous extract of WT is effective to reduce most of the diabetes associated abnormalities in a steptozotocin-induced diabetes model of rats.     

Kinetics and mechanism of epoxidation of olefins by a novel ruthenium(IV)-oxo complex

[Ru^IV(tpy)(pic)(O)]⁺ (1) was synthesized by chemical oxidation of the corresponding aqua-complex [Ru^II(tpy)(pic)(H₂O)]⁺ (2) and characterized by analytical, spectroscopic (UV-vis and IR) and magnetic moment studies. Complex 1 effected epoxidation of styrene and substituted styrenes, cis- and trans-stilbenes and cyclohexene, in CH₃CN at room temperature. Epoxides were found to be the major product for styrenes and stilbenes, whereas, the oxidation of cyclohexene yielded allylic oxidation product. Detailed kinetic studies were performed under pseudo-first order conditions of excess alkene concentrations. A working mechanism in agreement with the rate and activation parameters is presented, and the results are discussed in reference to the data reported for the alkene oxidation by relevant Ru^IVO system in CH₃CN.     

On the isolation of polychlorinated dibenzo-p-dioxins and furans from serum samples using immunoaffinity chromatography prior to high-resolution gas chromatography–mass spectrometry

Immunoaffinity chromatography (IAC) for the purification of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) from biological samples was explored as a means to simplify the cleanup procedure and thereby decrease the time and cost of dioxin analysis. A monoclonal antibody (DD3) was used to produce IAC columns and to isolate the PCDD/Fs from serum. Native and ¹³C-labeled PCDD/Fs were spiked at the ppq to ppt range into serum. Quantitation of the PCDD/Fs was performed by a standard dioxin analytical method, i.e. high-resolution gas chromatography–mass spectrometry (GC–MS), which was easily compatible with IAC. Five of the most toxic PCDD/Fs consistently showed acceptable recoveries (>25%) and were reliably quantitated. The congeners specifically recovered by this method represent almost 80% of the toxic equivalency of dioxins and furans present in the serum samples. Dioxin-like polychlorinated biphenyls (PCBs) were not recognized by this antibody column. Compared to conventional dioxin cleanup methods, IAC decreased solvent usage by 1.5 l/sample and took only 2 h to process a sample for analysis.     

Serotonin synthesis inhibition by pre-treatment of p-CPA alters sleep-electrophysiology in an animal model of acute and chronic heat stress

The effects of pre-treatment of para-chlorophenylalanine (p-CPA) on sleep–wake electroencephalograms (EEG) have been demonstrated in three age groups of rats subjected to heat stress. Each age group for both p-CPA pre-treated and untreated subjects was sub-divided into three groups: (i) acute heat stress—subjected to a single heat exposure for 4 h at 38 °C; (ii) chronic heat stress—exposed for 21 days daily for 1 h in the incubator at 38 °C; and (iii) handling control groups. Digital polygraphic sleep recordings were performed just after the heat exposure from acute stressed rats and on the 22nd day from chronic stressed rats. The analyses of results demonstrated that many changes associated with sleep-EEG (either in sleep–wake parameter or in EEG frequencies) due to acute and chronic heat stress were reversed (changes were analyzed; P<0.05 or better) in p-CPA pre-treated groups of rats. However, differential observations between acute and chronic heat stress groups of subjects were recorded, which are thought to have happened due to acclimatization of subjects to the hot environment. The results of present study supported the previous hypothesis about the significant involvement of serotonin in sleep–wake parameters and also demonstrated its participation in brain electrophysiological alterations in stressed conditions.     

HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester,and linkage of pHBA into cell walls

As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.     

Interactions of tertiary phosphines with p-benzoquinones; X-ray structures of [HO(CH2)3]3PC6H2(O)(OH)(MeO) and Ph3PC6H3(O)(OH)

Phosphonium zwitterions of a known type were obtained in high yield via a 1:1 reaction of p-benzoquinone or methoxy-p-benzoquinone with the tertiary phosphines R₃P [R = (CH₂)₃OH, Ph, Et, Me] and Ph₂MeP, in acetone or benzene at room temperature. In all cases, attack of the P-atom occurs at a C-atom rather than at an O-atom. The products were characterized to various degrees by elemental analysis, ³¹P{¹H}, ¹H and ¹³C NMR spectroscopies, and mass spectrometry, and two of the zwitterions, the new [HO(CH₂)₃]₃P⁺C₆H₂(O⁻)(OH)(MeO) and the known Ph₃P⁺C₆H₃(O⁻)(OH), were structurally characterized by X-ray analysis. The PEt₃ reaction also produces small amounts of the ‘dimeric’, μ-oxo co-product Et₃P⁺C₆H₂(O⁻)(OH)-O-C₆H₃(O⁻)P⁺Et₃ that is tentatively characterized by 1D- and 2D-NMR data. 2,5-Di-tert-butyl- and 2,3,5,6-tetramethyl-p-benzoquinone do not react with [HO(CH₂)₃]₃P under the conditions noted above. Heating D₂O solutions of the water-soluble zwitterions R₃P⁺C₆H₃(O⁻)(OH) [R = (CH₂)₃OH, Et] at 90 °C for 72 h leads to complete H/D exchange of the H-atom in the position ortho to the phosphonium center.     

Chitosan-graft poly(p-dioxanone) copolymers: preparation, characterization, and properties

A new biodegradable copolymer of chitosan and poly(p-dioxanone) (PPDO) was prepared through a protection-graft-deprotection procedure using N-phthaloyl-chitosan as an intermediate. PPDO terminated with the isocyanate group was allowed to react with hydroxyl groups of the N-phthaloyl-protected chitosan, and then the phthaloyl group was cleaved to give the free amino groups. The length of PPDO graft chains can be controlled easily by using the prepolymers of PPDO with different molecular weights. The resulting products were thoroughly characterized with FT-IR, ¹H NMR, TG, DSC, SEM, and WAXD. The copolymers were used as drug carriers for sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methyl-9α,13α,14α-morphinan-6-one) and these exhibited a significant controlled drug-releasing behavior whether in artificial gastric juice or in neutral phosphate buffer solution.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Kinetics and mechanism of large rate enhancement in an acidic aqueous cleavage of the tertiary amide bond of N-(2-methoxyphenyl)-N'-morpholinophthalamide (1)

The rate of conversion of 1 to N-(2-methoxyphenyl)phthalimide (2) within [HCl] range 5.0 × 10⁻³-1.0 M at 1.0 M ionic strength (by NaCl) reveals the presence of both uncatalyzed and specific acid-catalyzed kinetic terms in the rate law. Intramolecular carboxamide group-assisted cleavage of amide bond of 1 reveals rate enhancement of much larger than 10⁶-fold compared to the expected rate of analogous intermolecular reaction.     

Recrudescence of Toxoplasma gondii infection in chronically infected rats (Rattus norvegicus)

The kinetics of Toxoplasma gondii infection reactivation in the brain and muscles was analyzed in this study to determine the preferred tissue by the parasite during immunosuppression. Two groups of Wistar rats (G1 and G2) were inoculated with 10⁴ bradyzoites of BTU10 strain (genotype I), p.o., and other two groups (G3 and G4) were inoculated with 0.9% saline solution. G2 and G4 were immunosuppressed with dexamethasone (DXM) and hydrocortisone sodium succinate (HSS). The presence of antibodies was researched in all groups through modified agglutination test (MAT) on days 0 and 21 p.i., and brain and muscle tissues of the rats were bioassayed in mice. G2 rats died at approximately 19.2 days after drug treatment, while G1 rats survived. The reactivation was initially observed in G1 brain and G2 muscles. Thus, the initial reactivation in muscles after immunosuppression allows doctors to save precious time to control the evolution of reactivated infection, preventing brain damage to the host.     

Giardia duodenalis: Kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG₁, IgG_2a, IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10¹, 10², 10³, 10⁴, 10⁵) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.     

Kinetics of the cis-to-planar interconversion of cis-diaqua(1,4,7,11-tetraazacyclotetradecane)nickel(II) ion

In order to examine the effects of coordinated hydroxide ion and free hydroxide ion in configurational conversion of a tetraamine macrocyclic ligand complex, the kinetics of the cis-to-planar interconversion of cis-[Ni(isocyclam)(H₂O)₂]²⁺ (isocyclam, 1,4,7,11-tetraazacyclotetradecane) has been studied spectrophotometrically in basic aqueous solution. The interconversion requires the inversion of one sec-NH center of the folded cis-complex to have the planar species. Kinetic data are satisfactorily fitted by the rate law, R = k_OH[OH⁻][cis-[Ni(isocyclam)(H₂O)₂]²⁺], where k_OH = 3.84 × 10³ dm³ mol⁻¹ s⁻¹ at 25.0 ± 0.1 °C with I = 0.10 mol dm⁻³ (NaClO₄). The large ΔH^≠, 61.7 ± 3.2 kJ mol⁻¹, and the large positive ΔS^≠, 30.2 ± 10.8 J K⁻¹ mol⁻¹, strongly support a free-base-catalyzed mechanism for the reaction.     

Adsorptive removal of Pb(II) by activated carbon prepared from Spartina alterniflora: Equilibrium,kinetics and thermodynamics

Low-cost activated carbon was prepared from Spartina alterniflora by phosphoric acid activation for the removal of Pb(II) from dilute aqueous solution. The effect of experimental parameters such as pH, initial concentration, contact time and temperature on the adsorption was studied. The obtained data were fitted with the Langmuir and Freundlich equations to describe the equilibrium isotherms. The kinetic data were fitted with the Lagergren-first-order, pseudo-second-order and Elovich models. It was found that pH played a major role in the adsorption process. The maximum adsorption capacity for Pb(II) on S. alterniflora activated carbon (SAAC) calculated from Langmuir isotherm was more than 99 mg g⁻¹. The optimum pH range for the removal of Pb(II) was 4.8–5.6. The Freundlich isotherm model was found to best describe the experimental data. The kinetic rates were best fitted to the pseudo-second-order model. Thermodynamic study showed the adsorption was a spontaneous exothermic process.     

Electronic effects on the interactions of complexes [Ru(phen)2(p-L)] (L=MOPIP, HPIP, and NPIP) with DNA

In order to explore the electronic effects of Ru(II) complexes binding to DNA, a series of Ru(II) complexes [Ru(phen)₂ (p-MOPIP)]²⁺ (1), [Ru(phen)₂ (p-HPIP)]²⁺ (2), and [Ru(phen)₂(p-NPIP)]²⁺ (3) were synthesized and characterized by elementary, ¹H NMR, and ES-MS analysis. The binding properties of these complexes to CT-DNA were investigated with spectroscopic methods and viscosity experiments. Furthermore, the computations for these complexes applying the density functional theory (DFT) method have also been performed. The results show that all of these complexes can well bind to DNA in intercalation mode and DNA-binding affinity of these complexes is greatly influenced by electronic effects of intercalating ligands. The intrinsic binding constants for 1, 2, and 3 are 0.20, 0.69, and 1.56 × 10⁵ M⁻¹, respectively. This order is in accordance with that of the electron-withdrawing ability of substituent [-OR < -OH < -NO₂]. Such a trend in electronic effects of Ru(II) complexes binding to DNA can be reasonably explained by the DFT calculations.