Lymphoid/nonlymphoid compartmentalization of donor leukocyte chimerism in rat recipients of heart allografts, with or without adjunct bone marrow

A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes. When suspensions of the intact bacteriophage Q beta were exposed to methylene blue plus light (MB + L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8-dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks. Formation of carbonyl groups associated with protein in the Q beta phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal. Strand breaks in the Q beta genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Q beta RNA was observable well beyond the 99% inactivation point in MB dosage. It was shown that the number of 8-oxoGua lesions were unlikely to be sufficient to account for the number of lethal events. Following exposure to MB + L, crosslink formation between Q beta RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions. A significant increase over background of RNA-protein complexes (including full-length Q beta RNA) was observed at the lowest concentration of MB tested (0.5 microM), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival). Due to its close correlation with Q beta inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Q beta following MB + L exposure.     

Formation of 8-hydroxydeoxyguanosine from 2'-deoxyguanosine and its decomposition by active oxygen

8-hydroxydeoxyguanosine (8-OHdG) was formed from dG and isolated DNA by photosensitization with rose bengal (RB) and methylene blue (MB). 8-OHdG formed from dG was decomposed by the photosensitization with these dyes. Singlet oxygen was concerned with the formation and decomposition of 8-OHdG by photosensitization. Fe++ oxidized dG to 8-OHdG, which was decomposed by the addition of H2O2. 8-OHdG was formed and decomposed by the treatment of dG with Fe++, EDTA and ascorbic acid. Hydroxy radical (.OH) participated in the formation and decomposition of 8-OHdG by Fe++.     

Pre-steady-state kinetics of nucleotide insertion following 8-oxo-7,8-dihydroguanine base pair mismatches by bacteriophage T7 DNA polymerase exo-

8-Oxo-7,8-dihydroguanine (8-oxoGua) can base pair with either cytosine (C) or adenine (A) when replicated by DNA polymerases. The 8-oxoGua.A mismatch is extended in preference to the 8-oxoGua.C pair. Using a model 25-mer/36-mer DNA duplex containing either guanine (Gua).C, 8-oxoGua.C, or 8-oxoGua.A base pairs at the primer terminus and A at the standing start position, we found that the pre-steady-state addition of dTTP opposite A following all three base pairs by bacteriophage T7 DNA polymerase exo- showed burst kinetics, suggesting that extension of all three base pairs is controlled by the rate of a step at or before phosphodiester bond formation. Substitution of dTTP alpha S for dTTP yielded modest thio effects of 1-6, suggesting that extension of all three pairs is limited by the rate of the conformational change prior to phosphodiester bond formation. Pre-steady-state values for kpol (maximum polymerization rate) were 120, 12, and 28 s-1, and Kd values were 2, 75, and 22 microM for insertion of dTTP following Gua.C, 8-oxoGua.C, and 8-oxoGua.A base pairs, respectively. Additional analysis of extension was provided by substitution of A in the standing start position by 2-aminopurine (2-AP), a fluorescent base analogue. Comparison of rapid-quench gel-based assays with stopped-flow fluorescence quenching assays suggested that during addition of dTTP opposite 2-AP phosphodiester bond formation was rate-limiting when 8-oxoGua.C or 8-oxoGua.A were the preceding base pairs, while conformational change was rate-limiting when Gua.C was the preceding base pair. Furthermore, the difference in apparent conformational change rates for addition of dTTP opposite 2-AP following the 8-oxoGua base pairs was greater than the differences in their phosphodiester bond formation rates, suggesting that discrimination in extension may be influenced more by conformational change rates than the rates of phosphodiester bond formation in this mispaired system.     

Adjuvant radiation therapy for adenocarcinoma of the rectum

The potential for electron transfer quenching of rose bengal triplet (3RB2-) to compete with energy transfer quenching by oxygen was evaluated. Rate constants for oxidative and reductive quenching were measured in buffered aqueous solution, acetonitrile and in small unilamellar liposomes using laser flash photolysis. Biologically relevant quenchers were used that varied widely in structure, reduction potential and charge. Radical ion yields (phi i) were measured by monitoring the absorption of the rose bengal semireduced (RB.3-) and semioxidized (RB.-) radicals. The results in solution were analyzed as a function of the free energy for electron transfer (delta G) calculated using the Weller equation including electrostatic terms. Exothermic oxidative quenching was about 10-fold faster than exothermic reductive quenching in aqueous solution. The quenching rate constants decreased as delta G approached zero in both aqueous and acetonitrile solution. Exceptions to these generalizations were observed that could be rationalized by specific steric or electrostatic effects or by a change in mechanism. The results suggest that electron transfer reactions with some potential quenchers in cells could compete with formation of singlet oxygen [O2(1 delta g)]. Values of phi i were generally greater for reductive quenching and, for oxidative quenching, greater in acetonitrile than in buffer. Electron transfer quenching of 3RB2- in liposomes, below the phase transition temperature was slower than in solution for both lipid-soluble and water-soluble quenchers indicating that these reactions may not compete with formation of O2(1 delta g) during cell photosensitization.     

Dependence of nucleic acid degradation on in situ free-radical production by adriamycin

Adriamycin (Adr) is one of the most powerful antitumor drugs. Its therapeutic effect may be due to its cyclic reduction-oxidation and, thus, generation of oxygen radicals. Using the spin-trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and EPR we have demonstrated that in an enzymatic system consisting of NADPH, NADPH-cytochrome P-450 reductase, and Fe(EDTA)2 Adr stimulates formation of .OH radicals in the presence of DNA or RNA with equal efficiency. Incubation of nucleic acids in the Adr-dependent reaction generating .OH radicals resulted in extensive degradation of double- and single-stranded DNA, but did not effect RNA. In contrast, both DNA and RNA were effectively destroyed in a footprinting system, ascorbate-Fe(EDTA)2-H2O2, which generates .OH radicals in massive quantities. Fluorescence assays indicated that Adr forms stable complexes with ds- and ss-DNA but reacts only slightly with RNA. We conclude that the formation of Adr-nucleic acid complex is necessary for .OH radical-mediated cleavage of the latter, and thus, Adr may be regarded as a chemical nuclease acting in situ.     

Oxidative inactivation of gastric peroxidase by site-specific generation of hydroxyl radical and its role in stress-induced gastric ulceration

We have shown earlier that restraint-cold stress-induced gastric ulceration in rats is caused by metal ion-dependent generation of hydroxyl radical (OH.) and oxidative inactivation of the gastric peroxidase (GPO), an important H2O2 scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH. generating system containing Cu2+, ascorbate, and H2O2. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process showing saturation with respect to Cu2+ concentration. The enzyme specifically requires Cu2+ and is not inactivated by the same concentration of Fe2+, Mn2+, or Zn2+. Sensitivity to catalase indicates the critical role of H2O2 in the inactivation. Inactivation is insensitive to superoxide dismutase, suggesting no role of superoxide. The rate of inactivation is not increased in D2O excluding the involvement of singlet oxygen in the process. However, OH. scavengers such as benzoate or mannitol cannot prevent inactivation. The results indicate a plausible generation of OH. within the enzyme molecule as the cause of inactivation. Fragmentation of peptide linkage or intramolecular crosslinking, gross change of tertiary structure, or change in intrinsic tryptophan fluorescence which occurs in "global" oxidation are not evident. Inactivation is dependent on pH and from a plot of K(obs) of inactivation against pH, the controlling role of an ionizable group of the enzyme having a pka of 7.8 could be suggested, deprotonation of which favors inactivation. Amino acid analysis shows a specific loss of two lysine residues in the inactivated enzyme. Competitive kinetic studies indicate that pyridoxal phosphate, a specific modifier of the lysine residue, prevents inactivation by competing with Cu2+ for binding at the GPO. A Cu2+ binding motif consisting at least of two lysine residues exists in GPO, which specifically binds Cu2+ and generates OH.. The radical oxidizes the lysine residues and perturbs the heme environment to cause inactivation. We suggest that oxidative damage of GPO is mediated by site-specific generation of OH. and not by the OH. generated in the bulk phase.     

Expression of the retinoblastoma (RB) tumor suppressor gene inhibits tumor cell invasion in vitro

To determine if replacement of the retinoblastoma (RB) tumor suppressor gene could inhibit invasion of RB-defective tumor cells, the capacity of tumor cells to migrate or invade was quantitated by the Boyden chamber assay. The studies were done in a diverse group of stable RB-reconstituted human tumor cell lines, including those derived from the osteosarcoma and carcinomas of the lung, breast and bladder. The expression of the exogenous wild-type RB protein in these tumor cell lines was driven by either a constitutively active promoter or an inducible promoter. It was found that significantly more tumor cells from the parental RB-defective cell lines and the RB revertants than from the RB-reconstituted RB+ cell lines penetrated through the Matrigel (P<0.001, two-tailed t-test), although both RB+ and RB- cells migrated at approximately the same rate on uncoated polycarbonate filters in the Boyden chambers. Of note, the inhibition of invasiveness of various RB-defective tumor cells by RB replacement was apparently well correlated with suppression of their tumorigenicity in vivo. In contrast, although either functional RB or p53 re-expression effectively suppressed tumor formation in nude mice of the RB-/p53null osteosarcoma cell line, Saos-2, replacement of the wild-type p53 gene had much less impact on their invasiveness as compared to the RB gene. These studies provided an insight into the broader biological basis of the RB-mediated tumor suppression in RB-defective tumor cells.     

Investigation of the mechanism of non-turnover-dependent inactivation of purified human 5-lipoxygenase. Inactivation by H2O2 and inhibition by metal ions

Human 5-lipoxygenase is a non-heme iron protein which is reported to be highly unstable in the presence of oxygen. The results of this investigation demonstrate that H2O2 generated during air oxidation of thiols is the main factor in non-turnover-dependent inactivation of purified recombinant human 5-lipoxygenase for the following reasons: catalase protects against oxygen-dependent inactivation of the enzyme in the presence of dithiothreitol; the active, stable enzyme can be prepared under aerobic conditions with the exclusion of dithiothreitol and contaminating metal ions; 10 microM H2O2 causes the rapid inactivation of the enzyme. The native (ferrous) enzyme is approximately seven times more sensitive to inactivation by H2O2 than the ferric enzyme, suggesting that the mechanism of inactivation involves a Fenton-type reaction of the ferrous enzyme with H2O2, resulting in the formation of an activated oxygen species. Purification of 5-lipoxygenase under aerobic conditions (no dithiothreitol) results in an increase in both the specific activity of the purified protein [up to 70 mumol 5(S)-hydroperoxy-6-trans-8, 11, 14-cis-icosatetraenoic acid (5-HPETE)/mg protein] and in the ratio of specific activity to enzyme iron content compared to enzyme purified under anaerobic conditions in the presence of dithiothreitol. The reaction of the highly active 5-lipoxygenase enzyme shows a dependence on physiological intracellular calcium concentrations, half-maximal product formation being obtained at 0.9 microM free Ca2+. The maximal enzyme activity is also dependent on EDTA and dithiothreitol and low amounts of carrier protein, as well as the known activators PtdCho and ATP. Ca2+ can be substituted by Mn2+, Ba2+ and Sr2+, although lower levels of stimulation are obtained. 5-Lipoxygenase is strongly inhibited by low concentrations (< or = 10 microM) of Zn2+ and Cu2+. The inhibition by Cu2+ is apparently irreversible, whereas that by Zn2+ is slowly reversed (t1/2 = 2 min) in the presence of excess EDTA. These observations on the mechanism of non-turnover-dependent inactivation of 5-lipoxygenase, and the optimisation of assay conditions, have facilitated the purification of large quantities of relatively stable enzyme that will be useful for further kinetic and physical studies.     

The measurement of bioreductive capacity of tumor cells using methylene blue

PURPOSE: Methylene blue (MB) can be used as an intracellular electron acceptor. The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell suspensions. METHODS AND MATERIALS: We measured oxygen consumption by Clark electrode and pentose cycle activity by release of 14CO2 from 1-14C-glucose. RESULTS: Methylene blue catalyzes the reaction of intracellular reductants NADPH, NADH, and reduced glutathione (GSH) with oxygen, causing the production of hydrogen peroxide. The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction. In a cellular system MB causes the electron flow from cellular endogenous substrates to oxygen. It is activated by the disruption of the NADP+/NADPH ratio due to several processes. These are direct oxidation of NADPH and GSH, the GSH peroxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidation by oxidized glutathione (GSSG). This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cycle (PC) in the presence of MB. The cellular effect of MB differs from other electron accepting drugs. Diamide and tert-butylhydroperoxide act as direct oxidants, while MB is an electron carrier to oxygen. Accordingly, MB shows the highest effect on PC activation and oxygen consumption. CONCLUSIONS: Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measured by oxygen consumption and PC activation.     

Quantification of singlet oxygen from hematoporphyrin derivative by electron spin resonance

The mechanism of the generation and the quantitative analysis of singlet oxygen (1O2) formed by the exposure of a hematoporphyrin derivative (HpD) to light was re-evaluated by electron spin resonance (ESR) combined with 2,2,6,6,-tetramethyl-4-piperidine (TMPD). The change from TMPD to 2,2,6,6,-tetramethyl-4-piperidine-N-oxide (TAN) has been reported to depend on singlet oxygen. However, we confirmed that this reagent also react with superoxide anion (O2-) and hydroxyl radicals (OH). Therefore, the reactions between TMPD and 1O2, O2- and OH were re-examined using a kinetic approach. We found that the generation of TAN was proportional to the concentration of TMPD and HpD, as well as to the duration and strength of the illumination. The generation of TAN was not inhibited by dimethyl-sulfoxide (DMSO) or superoxide dismutase (SOD). The reaction rate between TMPD and 1O2 was determined to be 5.0 x 10(-7) M min-1. The generation of 1O2 from HpD was 2.7 x 10(-7) M min-1 under our conditions. The competitive reaction observed between 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and TMPD for O2- or OH shows that TMPD reacts with both forms of active oxygen, but gave no ESR signal. The second-order reaction rate constant of TMPD between O2- and OH was calculated as 73 M-1 s-1 and 1.5 x 10(9) M-1 s-1, respectively. The photochemical generation of 1O2 from methylene blue, another sensitizer, was also demonstrated by this method. These results show that ESR signal of TAN can be used for the highly selective monitoring of 1O2.     

Direct effects of reactive oxygen species on cochlear outer hair cell shape in vitro

Reactive oxygen species (ROS) have been implicated in the ototoxicity of various agents. This study examines the effects of superoxide anion (O2), hydroxyl radical (OH.) and hydrogen peroxide (H2O2), on isolated cochlear outer hair cell (OHC) morphology. OHCs were superfused with artificial perilymph (AP) or AP containing a specific ROS scavenger, and then with AP, ROS system or scavenger plus ROS system for 90 min. The generation of ROS as well as the scavenging properties of other agents were confirmed by specific biochemical assays. Control cells decreased 4.8% in mean length, and showed no obvious membrane damage. Generation of O2. or OH. resulted in high rates (85.7 and 42.9%, respectively) of bleb formation at the synaptic pole, and decreased (O2., 15.2%; OH., 17.3%) mean cell length. Length change and bleb formation rate were H2O2 concentration-dependent. 20 mM H2O2 led to 33.3% decreased mean cell length, and only 20% bleb formation; 0.1 mM H2O2 led to 83.3% bleb formation, with no length decrease. Superoxide dismutase, deferoxamine and catalase protected against O2., OH. and H2O2 effects, respectively. Bleb formation and diminished cell length likely represent differential lipid peroxidative outcomes at supra- and infranuclear membranes, and are consistent with effects of certain ototoxicants.     

Determination of stability of Brucella abortus RB51 by use of genomic fingerprint, oxidative metabolism, and colonial morphology and differentiation of strain RB51 from B. abortus isolates from bison and elk

Brucella abortus RB51 and isolates from cattle, bison, and elk were characterized by pulsed-field gel electrophoresis and standard techniques for biotyping Brucella species, which included biochemical, morphological, and antigenic techniques, phage susceptibility, and antibiotic resistance. The objectives were to ascertain the stability of RB51 and to differentiate RB51 from other brucellae. Genomic restriction endonuclease patterns produced by pulsed-field gel electrophoresis demonstrated a unique fingerprint for RB51 relative to other brucellae. Comparisons of the oxidative metabolic profiles of RB51 after time in vivo (14 weeks) and in vitro (75 passages) showed no change in characteristic patterns of oxygen uptake on selected amino acid and carbohydrate substrates. Strain RB51 was biotyped as a typical rough B. abortus biovar 1 (not strain 19) after animal passage or a high number of passages in vitro and remained resistant to rifampin or penicillin and susceptible to tetracycline. No reactions with A or M antiserum or with a monoclonal antibody to the O antigen of Brucella lipopolysaccharides were detected; however, RB51 agglutinated with R antiserum. The results indicate that the genomic fingerprint and rough colonial morphology of RB51 are stable characteristics and can be used to differentiate this vaccine strain from Brucella isolates from cattle, bison, and elk.     

Oxidation of low-density lipoproteins by OH. and OH./O2-. free radicals produced by gamma radiolysis

The aim of this study was to analyze quantitatively the oxidative modification of low-density lipoproteins (LDLs) induced by OH. free radicals produced by gamma radiolysis, in the absence or in the presence of oxygen (action of OH. free radicals, or simultaneous action of OH. and O2-. free radicals, respectively). The effects of increasing radiation doses on aqueous LDL solutions have been monitored by several parameters: a decrease in endogenous vitamin E, the formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes, the appearance of a differential fluorescence (excitation wavelength = 360 nm), and an increase in the relative electrophoretic mobility. Initial radiation yields (decrease in vitamin E, formation of TBARS) have been determined at pH 7 as a function of LDL concentration (from 0.75 to 9 g liter-1). From the comparison of these yields with those of OH. and O2-. free radicals produced by water radiolysis, we have deduced reaction mechanisms for the initiation of oxidation of LDLs by OH. and OH./O2-. free radicals.     

Differential response to UV stress and DNA damage during the yeast replicative life span

The intensity dependence of the rose bengal (RB)-photosensitized inhibition of red blood cell acetylcholinesterase has been studied experimentally and the results compared to a quantitative excitation/deactivation model of RB photochemistry. Red blood cell membrane suspensions containing 5 microM RB were irradiated with 532 nm, 8 ns laser pulses with energies between 1 and 98.5 mJ. A constant dose (7 J) was delivered to all samples by varying the total number of pulses. At incident energies greater than approximately 4.5 mJ/pulse, the efficiency for photosensitized enzyme inhibition decreased as the energy/pulse increased. The generation of RB triplet state was monitored as a function of laser energy and the triplet-triplet absorption coefficient was determined to be 1.9 x 10(4) M-1 cm-1 at 530 nm. The number of singlet oxygen molecules produced at each intensity was calculated from both the physico-mathematical model and from laser flash photolysis results. The results indicated that the photosensitized inhibition of acetylcholinesterase was exclusively mediated by singlet oxygen, even at the highest laser intensities employed.     

Singlet oxygen involvement in ultraviolet (254 nm) radiation-induced formation of 8-hydroxy-deoxyguanosine in DNA

In the present article, we report that ultraviolet (UV 254 nm) radiation substantially induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in purified DNA. The formation of 8-OHdG, a hallmarker of oxidative DNA damage, increased linearly up to 25 kJ/m2 and was dependent on the presence of oxygen in the solution. Deoxygenation by nitrogen significantly reduced the yield of 8-OHdG by UV radiation, whereas oxygenation with 100% oxygen substantially enhanced the yield. The hydroxyl radical (HO.) scavenger dimethysulfoxide (DMSO) dramatically quenched the formation of 8-OHdG by the ionizing radiation and Fenton reaction, but enhanced the formation of UV-induced 8-OHdG. Further studies showed that DMSO and mannitol, two predominant HO. scavengers, enhanced the levels of UV-induced 8-OHdG in a dose-dependent fashion, suggesting that UV-induced 8-OHdG is independent of the generation of HO.. The use of deuterium oxide (D2O), which prolongs the half life of singlet oxygen (1O2), substantially enhanced the yield of 8-OHdG by UV radiation, but not that by Fenton reaction. In contrast, sodium azide, a more and less specific 1O2 quencher, substantially reduced the levels of 8-OHdG by both UV radiation and Fenton reaction, indicating that sodium azide lacks the quenching specificity of 1O2 and HO.. It is proposed that UV induced 8-OHdG proceeds through a singlet oxygen involvement mechanism, rather than the generation of hydroxyl radicals.     

Fenton and Electro-Fenton Methods for Oxidation of H-Acid and Reactive Black 5

Oxidative treatment of H-acid (HA) and Reactive Black 5 (RB5) using Fenton reagent (Fe2+/H2O2) and the electro-Fenton (EF) method is reported. Optimization of doses of ferrous iron and hydrogen peroxide was carried out in each case using HA; and the oxidation of RB5 was also carried out under the optimized conditions. Approximately 71% chemical oxygen demand (COD) was removed in 2 h using the conventional Fenton method at optimized doses: Fe2+ = 0.3?g/L (5.37 mM), H2O2 = 6?mL/L (53.0 mM), H2O2/Fe2+ = 10. In contrast, more than 92% COD was removed in 15 min using the EF method with an optimized Fe2+ dose of 0.130?g/L (2.34 mM) and 8?ml/L (70.6 mM) of H2O2. The pseudo-first-order rate constants (k) for the Fenton reagent and EF method were 0.054 and 0.38?min?1. The COD removal through the EF method was seven times faster. The calculated energy requirement of the EF method was 0.82?kg?COD/kW?h at the minimum applied current (0.25 A) when approximately 92.5% COD was removed. In the case of RB5, about 67 and 87% COD was removed under optimized Fenton and electro-Fenton conditions, respectively. The higher efficiency of the EF method was attributed to incremental addition of Fe2+ and accompanying higher H2O2/Fe2+ molar ratio. The results are discussed in the light of the mechanism for Fenton’s oxidation.     

Mechanism-based inactivation of cytochrome P450 2B1 by 8-methoxypsoralen and several other furanocoumarins

Of several furanocoumarins [5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 5-hydroxypsoralen (5-OH-P), 8-hydroxypsoralen (8-OH-P), 4',5'-dihydro-8-MOP (DH-8-MOP), and psoralen (P)] tested as mechanism-based inactivators (MBIs) of purified reconstituted cytochrome P450 (P450) 2B1, 8-MOP was found to be the most potent (KI, kinact, and partition ratio of 2.9 microM, 0.34 min-1, and 1.3, respectively). The inactivation was not prevented by reactive oxygen species scavengers or nucleophilic trapping agents and proceeded with a decrease in P450 spectral content. Liquid chromatography (LC) separation of the reconstituted enzyme mixture, followed by liquid scintillation counting, indicated that [14C]-8-MOP binding was specific to the apoprotein of P450 2B1 with a binding stoichiometry of 0.7:1. The major metabolites formed by P450 2B1 from the furanocoumarins that were MBIs were characterized by LC electrospray ionization tandem mass spectrometry (ESI-MS/MS) as dihydro diols. Results from H218O incorporation experiments supported initial oxidation of 8-MOP and P to an epoxide which can react with some nucleophilic active site residue and inactivate the enzyme or partition to a dihydro diol metabolite by hydrolytic ring opening. On the other hand, 5-MOP was converted to an epoxide or gamma-keto enal intermediate prior to inactivation or dihydro diol formation. Comparison of the ESI mass spectra of P450 2B1 and furanocoumarin exposed P450 2B1, indicated a mass difference consistent with the covalent addition of a furanoepoxide to P450 2B1.     

Hydroxyl groups in nonmetamict chevkinite-(Ce):a crystal chemical discussion

The minerals of chevkinite group were commonly considered to be anhydrous minerals. The infrared absorption spectrum of natural nonmetamict chevkinite-(Ce) from the aegirine-alkali granite, Mianning, Sichuan Province, China, exhibited two broad peaks in the 3600-2800 cm-1 region owing to the OH stretching. The corresponding H2O content required for the charge balance in formula was 1.27%. The O-H ··· O bond lengths maight cover from 0.2658 to 0.2794 nm by the correlated OH stretching energies. An electrostatic charge balance for chevkinite-(Ce) based on the assigned site-population from chemical data was calculated without the hydrogen contribution. The resulting empirical bond-valence sum on O6, O8, O2, O3, O5, and O4 ranged from 1.73 to 1.95 vu. The partial substitution of O by OH may occur in four atom sites: O6, O2, O4, and O5. The small differences in the bond-valence sums between the supposed donors and acceptors may mean a mixed donor/acceptor role of the involved oxygen atoms. The IR spectral features between 3394 and 3035 cm-1 consisted of various hydrous species at different structural sites and orientations. The OH groups in the chevkinite-(Ce) appeared to be involved in local charge imbalance in the structure and to be present when the mineral crystallized hydrothermally.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-1-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (.OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the .OH generation. UV and O2 consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H2O2 did not generate any significant amount of .OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H2O2-->Co(II) + .OH + OH-] seems responsible for .OH generation. H2O2 is produced from O2.- via dismutation, O2.- is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or beta-ananyl-3-methyl-L-histidine alters, its oxidation-reduction potential and makes Co(II) capable of generating .OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H2O2-->Co(III) + .OH + OH-]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and beta-ananyl-3-methyl-L-histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.