Optimization of reaction variables for sucrose monoester production using lipase in a solvent free system by Taguchi's method

Taguchi's method was attempted successfully to optimize the reaction variables and their ranges for sucrose monoester production. Optimal conditions determined by Taguchi's method were 35°C, 200rpm, capric acid (40mmol) and sucrose (3mmol), 24h, 0.5g lipase, 0.72g Ba(OH)2 ·8H2O, 0.72g Ba(OH)2·1H2O. A product yield of 21% was obtained. © Rapid Science Ltd. 1998     

Banding pattern observed in human chromosomes by the modified BSG technique

Summary A successful modification of the BSG technique to reveal C and R bands simultaneously in human chromosomes is described. Conventional air dried preparations were treated first with 0.1 N HCl for 30 min at room temperature, then denatured in freshly prepared 3% aqueous solution of Ba(OH)₂8H₂O for 10 min at 50°C. After rinsing, the slides were incubated for 1 h at 60°C in 2xSSC, and stained with Giemsa. The striking intense staining pattern could be observed in chromosome No. 19.The factors involved in the present technique were analyzed changing the concentrations of the reagents and the treatment time. It was evident that R, T and C bands correspond to a progressive destruction of the chromosome sructure mainly by the Ba(OH)₂8H₂O solution.     

The biological activity of hydrogen peroxide VII. L-Histidine increases incorporation of H(2)O(2) into cells and enhances formation of 8-oxodeoxyguanosine by UV-C plus H(2)O(2) but not by H(2)O(2) alone

L-Histidine (L-His) enhances the clastogenic effects of hydrogen peroxide (H(2)O(2)). We previously suggested the involvement of active transport in the efficient influx of an L-His--H(2)O(2) adduct into cells (Oya-Ohta et al. [1]). In this study, we detected intracellular H(2)O(2) by monitoring formation of 2',7'-dichlorofluorescein (DCF) from its precursor. More fluoroproduct accumulated dose-dependently in cells treated with a mixture of L-His and H(2)O(2) (mixture) than with H(2)O(2) alone. This observation supports our hypothesis that active transport is involved in the enhanced incorporation of H(2)O(2) into cells. Moreover, both mixture and the L-His--H(2)O(2) adduct were less active in the generation of hydroxyl radicals (*OH) upon addition of FeCl(2) than was H(2)O(2) alone in a cell-free system. This result suggests that the Fenton reaction might occur more effectively around the nucleus in cells. An immunohistochemical assay using 8-oxodG-specific monoclonal antibodies did not reveal whether the accumulation of H(2)O(2) generates 8-oxodeoxyguanosine (8-oxodG). No 8-oxodG was evident in cells treated with mixture or with H(2)O(2) alone, or even in cells treated with H(2)O(2) at high doses up to 20 mM and, in some cases, pre-treated with catalase inhibitors. It appears, therefore, that *OH and, specifically, *OH derived from intracellular Fenton reactions, might not play a role in the formation of 8-oxodG. However, exposure to UV-C of cells treated with H(2)O(2) yielded more 8-oxodG in the presence of L-His than in the absence of L-His. Thus, the previously observed enhancing effects of L-His were also noted during the induction of formation of 8-oxodG by UV-C plus H(2)O(2). The formation of 8-oxodG in response to UV-C alone was very limited and, hence, H(2)O(2) seemed to be an effective source of *OH only in the presence of UV-C. It is suggested that the *OH that induces formation of 8-oxodG is not *OH formed via intracellular Fenton reactions but is *OH formed via the dissociation of H(2)O(2) under UV-C.     

Susceptibility of Eimeria bovis and Toxoplasma gondii to oxygen intermediates and a new mathematical model for parasite killing

Eimeria bovis and Toxoplasma gondii differ in their susceptibility to macrophages activated by lymphokines. Interferon-gamma can activate macrophages to totally inhibit E. bovis sporozoite development, whereas growth of T. gondii tachyzoites in macrophages is not totally affected. The susceptibility of these parasites to oxygen intermediates and their ability to evade the oxidative burst by macrophages were investigated in cell-free systems. Using a logistic model to assess growth inhibition, T. gondii growth was impaired by 50% at 10(-4.25) M (56 microM) H2O2, with 30 min as the optimum time for measuring inhibition. Preliminary results indicate that T. gondii follows mode-one and mode-two killing with relation to time after exposure to H2O2, implying a role for OH. and the induction of a DNA repair mechanism. The same model was used to assess inhibition of E. bovis growth that was more susceptible, being inhibited to 50% by 10(-5) M (10 microM) H2O2. Both parasites were susceptible to the effects of xanthine-xanthine oxidase that releases a full complement of oxygen intermediates (H2O2, OH., (1)O2, and O2-). Adding quenchers or scavengers to the system confirmed that T. gondii was susceptible to products of the interaction of O2- and H2O2 (OH. and (1)O2), and that E. bovis sporozoites were at least partially susceptible to H2O2 and O2-, but extremely susceptible to OH.. These data were supported by studies on scavenging enzymes present in the parasites. Toxoplasma gondii was rich in superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPO), and E. bovis had less catalase and SOD.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the radiation chemistry of thymine in aqueous solution

The reactions of thymine in aqueous solution with radiation-induced radicals OH, H, and e-aq were studied under various conditions. Competition studies using scavengers of OH radicals (methanol, ethanol, iodide) or of e-aq and/or H atoms (N2O, H+, O2) led to the conclusion that OH and H radicals destroy the chromophoric group of thymine, but e-aq does not. A trace of O2 proved to be necessary to obtain maximal destruction. Removal of the last traces of O2 resulted in a decrease of the destruction yield, possibly through restitution reactions. It was found that (1) alcohol radicals destroy thymine, even in the presence of O2; (2) the rate constant, k(OH + thymine) = 4.3 X 10(9) M(-1) sec(-1) (from competition with iodide); and (3) k(H + thymine) = 8 X 10(8) M(-1) sec(-1) (from competition with O2 in acid solution).     

Reactive oxygen species induced structural alterations of substance P

Substance P (SP(1-11)) was exposed to a continuous flux of superoxide (O2-) or hydroxyl radicals ((.)OH) in a hypoxanthine (HX)/xanthine oxidase (86 mU) system in the presence of 1 mM deferoxamine and 40 mM D-mannitol or 50 muM FeCI(3). 6H(2)O and 50 muM EDTA, respectively. O2- caused fragmentation between the Phe(7) and Phe(8), whereas (.)OH induced cleavage also between the Phe(8) and Gly(9). Reactive oxygen species H(2)O(2) and HCIO did not cause fragmentation, but modification of the amino acid side chains and/or aggregation with altered hydrophobicity in reverse phase high performance liquid chromatography compared to native SP(1-11). Furthermore, exposure of SP(1-11) to phorbol myristate acetate preactivated neutrophils resuited in products similar to those observed upon exposure to superoxide or hydroxyl radicals in a cell-free HX/xanthine oxidase system. This study suggests that, in contrast to rigid proteins, fragmentation is relatively easily induced in a small peptide like SP(1-11), perhaps due to strain on the peptide and t-carbon bonds caused by the movable, random coil configuration acquired by SP(1-11) in an aqueous solution. Oxidative modification might modulate paracrine actions of SP(1-11) at site of inflammation.     

Interactions between metal ions and carbohydrates: the coordination behavior of neutral erythritol to transition metal ions

The single crystals of coordinated complexes of neutral erythritol (C4H10O4) with various transition metal ions were synthesized and studied using FT-IR and single crystal X-ray diffraction analysis. Two CuCl2-erythritol complexes (denoted as CuE(I) and CuE(II)) were obtained. In CuE(I), Cu2+ coordinates with two chloride ions and four OH groups from two erythritol molecules. Two copper centers are linked by one erythritol molecule to form a zigzag chain. For CuE(II), each Cu2+ coordinates with two OH groups from an erythritol molecule and two chloride ions. The crystal of CuE(II) contains complexed and free erythritol, the dimers of [Cu2Cl4(C4H10O4)] further form a [Cu2Cl4(C4H10O4)]infinity chain via secondary Cu...Cl bonds, both the dimer unit of [Cu2Cl4.(C4H10O4)] and non-coordinated C4H10O4 unit exist side by side in the crystal. MnCl2-erythritol complex whose structure is similar to CuE(I) is also acquired. The OH groups of erythritol act as ligand to coordinate to metal ions on one hand, one the other hand, OH groups form hydrogen bonds network that link chain and layer together to build three-dimensional structures.     

An alternative method for the rapid synthesis of partially O-methylated alditol acetate standards for GC-MS analysis of carbohydrates

Mixtures of partially O-methylated alditol acetate standards (PMAAs) of Glc, Gal, and Man were synthesized rapidly. Methylation of methyl glycosides was carried out in the presence of BaO/Ba(OH)(2) x 8H(2)O giving rise to mixtures of partially methylated glycosides (PMGs), whose degree of methylation was monitored by TLC. The batch containing the largest mixture of methyl ethers was converted into partially O-methylated alditol acetate derivatives (PMAAs), via successive hydrolysis, reduction, and acetylation, and then subjected to GC and GC-MS analysis. Detailed data on retention times, TIC, and EIMS are now provided.     

Paired Bacillus anthracis Dps (mini-ferritin) have different reactivities with peroxide

Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe(2+)/H(2)O(2)/O(2) reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe(2+)/O(2) kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe(2+) were novel in several ways: 1) Ba Dps2 reactions (Fe(2+)/H(2)O(2)) proceeded via an A(650 nm) intermediate, with similar rates to maxi-ferritins (Fe(2+)/O(2)), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe(2+)/O(2) versus Fe(2+)/O(2) + H(2)O(2)) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe(2+)/H(2)O(2) catalysis, inhibited protein-independent Fe(2+)/H(2)O(2) reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H(2)O(2) (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H(2)O(2) destruction, important in host/pathogen interactions. Destruction of H(2)O(2) by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A(650 nm)) and kinetically to the maxi-ferritin diferric peroxo complex.     

1,25-Dihydroxyvitamin D3 activates secretion of hydrogen peroxide by human monocytes

Human blood monocytes cultured in the presence of 1,25(OH)2D3 developed enhanced competence for secretion of H2O2 relative to cells suspended in media. This effect was maximal at a concentration of 10(-8) M 1,25(OH)2D3. After 3 days of incubation, monocyte-derived macrophages (MDM) exposed to 1,25(OH)2D3 demonstrated competence for secretion of H2O2 equivalent to cells exposed to recombinant IFN-gamma. Both IFN-gamma and 1,25(OH)2D3 offset decay of this function among cells in culture after 7 days. Simultaneous exposure of cells to 1,25(OH)2D3 and IFN-gamma did not activate competence for H2O2 secretion more than either agent alone. 24,25(OH)2D3 and 25(OH)2D3 activated MDM but at higher concentration than required for 1,25(OH)2D3. Progesterone did not affect H2O2 production. Incubation of MDM with a monoclonal antibody directed against IFN-gamma inhibited activation induced by lymphokine, and to a lesser extent by cells activated with IFN-gamma; this antibody had an insignificant effect on cells treated with 1,25(OH)2D3. These results suggest that 1,25(OH)2D3 exerts a receptor-mediated effect on monocyte function that results in cellular activation as manifested by enhanced competence for secretion of H2O2. It is possible that smaller concentrations of 1,25(OH)2D3 present in serum are permissive for macrophage activation, or that monocytic phagocytes are exposed to high concentrations of vitamin D metabolites under some clinical circumstances.     

The oxidative inactivation of mitochondrial electron transport chain components and ATPase

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elevation of hydrogen peroxide after spinal cord injury detected by using the Fenton reaction.

To reveal whether reactive oxygen species (ROS) play a role after spinal cord injury, we developed a unique method for assaying hydrogen peroxide (H2O2) and determined the time course of its concentration changes following impact injury to the rat spinal cord. Microdialysis was used to sample H2O2 in the extracellular space and the dialysates were collected into a vial containing salicylate and ferrous chloride (FeCl2). H2O2 collected in the vial was converted to hydroxyl radicals (*OH) by FeCl2 catalysis. 2,3- and 2,5-dihydroxybenzoic acid produced by reaction of *OH with salicylate in the collecting vial were measured by HPLC and calibrated to H2O2 concentrations. The postinjury levels of H2O2 were significantly increased (p = 0.02) for over 11 h. FeCl2 administered through the dialysis fiber catalyzes H2O2 conversion in the cord to *OH. This *OH does not reach the collecting vial due to its extremely short lifetime (nanoseconds). The reduced H2O2 levels in the vials validate the measurement of H2O2. The relatively long-lasting formation of H2O2 and superoxide reported herein and previously suggests that ROS may be important in secondary spinal cord damage and that removal of ROS may be a realistic treatment strategy for reducing injury caused by free radicals.     

Protein damage and degradation by oxygen radicals. II. Modification of amino acids

Exposure of proteins to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical (.OH + O2-) causes gross structural modification. Such modified proteins can undergo spontaneous fragmentation or can exhibit substantial increases in proteolytic susceptibility. In the present study, with the representative protein bovine serum albumin (BSA), we report that alterations to primary structure underlie such gross structural modifications. All amino acids in BSA were susceptible to modification by both .OH and .OH + O2- +O2), although tryptophan, tyrosine, histidine, and cysteine were particularly sensitive. At a radical/BSA molar ratio (nmol of radicals/nmol of BSA) of 10, we observed an average 9-10% destruction of amino acids; whereas at a ratio of 100, the average loss was 45%. Decreasing tryptophan fluorescence provided a useful index of amino acid loss and exhibited a clear dose dependence with .OH or with .OH + O2- (+O2). Linear production of the biphenol bityrosine was observed with .OH treatment. In contrast, .OH + O2- (+O2) induced only a limited bityrosine production rate which reached an early plateau. Studies with various chemical scavengers (t-butyl alcohol, isopropyl alcohol, mannitol, urate) and gasses (N2O, N2, O2, air) revealed that .OH is the primary radical responsible for all amino acid modifications, but that O2- and O2 can further transform the products of .OH reactions. Thus, O2-/O2 can potentiate .OH-dependent destruction of many amino acids (e.g. tryptophan) while inhibiting production of bityrosine by reacting with tyrosyl (phenoxyl) radicals. No amino acid loss or bityrosine production occurred with exposure to O2- (+O2) alone. Amino acid modifications caused both by .OH alone and by .OH + O2- (+O2) progressively affected the overall electrical charge of BSA. In a pH range of 3.7-6.2, some 16 new isoelectric focusing bands were induced by .OH, and some eight new bands were induced by .OH + O2- (+O2). The alterations to primary structure observed provide the key to an understanding of the link between oxidative modification and increased proteolytic susceptibility.     

The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent Annexin A5

N-[4-(3)H]Benzoylglycylglycylglycine ([(3)H]BzG(3)) was tested as a probe for detecting hydroxyl radicals (*OH). Aerated solutions of l-ascorbate generated *OH, which oxidized [(3)H]BzG(3), yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. The (3)H(2)O was separated from organic products and remaining [(3)H]BzG(3) on Dowex-1. (3)H(2)O production was much greater with *OH than with other reactive oxygen species (ROS) (e.g., H(2)O(2), superoxide). The slight (3)H(2)O production in the presence of H(2)O(2) or superoxide was blocked by *OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H(2)O(2) or superoxide. This indicates that (3)H(2)O production was caused by *OH and that other ROS only generated any (3)H(2)O by forming traces of *OH. Doses of *OH that caused detectable nonenzymic polysaccharide scission also caused (3)H(2)O production, indicating that [(3)H]BzG(3) is a sensitive *OH probe in studies of polymer scission. The ability of scavengers and chelators to protect against ascorbate-mediated polysaccharide scission paralleled their ability to inhibit concurrent (3)H(2)O production, indicating that both processes were due to *OH. Thus, [(3)H]BzG(3) is a simple, specific, sensitive, and robust probe for detecting *OH production in vitro. It may have applications for in vivo detection of extracellular *OH in arthritic joints and of apoplastic *OH in plant cell walls.     

Effects of metal ion chelators on DNA strand breaks and inactivation produced by hydrogen peroxide in Escherichia coli: detection of iron-independent lesions.

In order to study the role of metallic ions in the H2O2 inactivation of Escherichia coli cells, H2O2-sensitive mutants were treated with metal ion chelators and then submitted to H2O2 treatment. o-Phenanthroline, dipyridyl, desferrioxamine, and neocuproine were used as metal chelators. Cell sensitivity to H2O2 treatment was not modified by neocuproine, suggesting that copper has a minor role in OH production in E. coli. On the other hand, prior treatment with iron chelators protected the cells against the H2O2 lethal effect, indicating that iron participates in the production of OH. However, analysis of DNA sedimentation profiles and DNA degradation studies indicated that these chelators did not completely block the formation of DNA single-strand breaks by H2O2 treatment. Thiourea, a scavenger of OH, caused a reduction in both H2O2 sensitivity and DNA single-strand break production. The breaks observed after treatment with metal chelators and H2O2 were repaired 60 min after H2O2 elimination in xthA but not polA mutant cells. Therefore, we propose that there are at least two pathways for H2O2-induced DNA lesions: one produced by H2O2 through iron oxidation and OH production, in which lesions are repaired by the products of the xthA and polA genes, and the other produced by an iron-independent pathway in which DNA repair requires polA gene products but not those of the xthA gene.     

Dinuclear macrocyclic polyamine zinc(II) complexes: syntheses, characterization and their interaction with plasmid DNA

The syntheses, characteristics of dinuclear macrocyclic polyamine zinc complexes and their interaction with plasmid DNA are reported. The two cyclen (1,4,7,10-tetraazacyclododecane) moieties are bridged by rigid and flexible linkages. The crystal structures of Zn2C27H43N8O15Cl4 [5c.(ClO4)3.2H2O] and Zn2C30H43N10O13Cl3 [5e.(ClO4)3.H2O] have been determined. The complexes crystallize in the monoclinic space group C2/c and P2(1)/c with the following unit cell parameters: 5c.(ClO4)3.2H2O: a=32.568(4)A, b=14.8593(17)A, c=19.443(2)A, alpha=90.00 degrees , beta=119.435(4) degrees , gamma=90.00 degrees , Dc=1.551 mg/m3, FW=956.71, F(000)=3932; 5e.(ClO4)3.H2O: a=15.807(2)A, b=16.756(2)A, c=16.161(2)A, alpha=90.00 degrees , beta=97.062(4) degrees , gamma=90.00 degrees , Dc=1.546 mg/m3, FW=988.83, F(000)=2032. The distance between the two Zn(II) ions is about 4.0 A. The structures show that two zinc ions can synergistically interact with the substrate DNA. With this novel structural characteristics, the dinuclear macrocyclic polyamine Zn(II) complexes via the synergetic effect between the two zinc ions can catalyze the cleavage of plasmid DNA (pUC18) with unprecedented speed at physiological conditions.     

Cysteine suppresses oxidative stress-induced myofibrillar proteolysis in chick myotubes

The effects of cysteine as an antioxidant nutrient on change in protein modification and myofibrillar proteolysis in chick myotubes by induction of oxidative stress by H(2)O(2) treatment were investigated. Myotubes were treated for 1 h with H(2)O(2) (1 mM). After this treatment, the H(2)O(2) was removed and the cells were cultured in cysteine (0.1 and 1 mM) containing serum-free medium for 24 h. Protein carbonyl content as an index of protein modification and N(tau)-methylhistidine release as an index of myofibrillar proteolysis were increased at 24 h after H(2)O(2) treatment, and the increment was reduced by cysteine. Calpain, proteasome and cathepsin (B+L and D) activities were increased at 24 h after H(2)O(2) treatment, and the increment was also reduced by cysteine. These results indicate that cysteine suppresses protein modification by oxidative stress, resulting in a decrease of protease acitivities, finally resulting in a decrease in myofibrillar proteolysis in chick myotubes.     

Induction of maternal behaviors in primigravid rats by ovariectomy,hysterectomy, or ovariectomy plus hysterectomy: Effect of length of gestation

The effects of terminating pregnancy by means of ovariectomy (O), hysterectomy (H), or ovariectomy plus hysterectomy (OH) at different stages of gestation upon the latency to initiation of maternal behavior were examined in primigravid rats. O, H, or OH on either Day 13 or 17 of pregnancy resulted in an accelerated onset of maternal behaviors (median range = 1.0–2.0 day latency for O, H, or OH animals vs 4.0–5.0 day latency for sham-operated intacts). However, O and H on Day 8 of pregnancy were ineffective in inducing a rapid onset of maternal behavior, while OH on Day 8 of pregnancy only slightly facilitated the onset of maternal behavior when compared to animals sham-operated on Day 8 of pregnancy. O, H, and OH on either Day 13 or 17 of gestation were also significantly more effective in rapidly inducing maternal behaviors than the corresponding surgeries performed on Day 8 of pregnancy. These data suggest that the onset of maternal behavior in the pregnant rat can be rapidly induced after mid-pregnancy by surgical procedures that presumably result in a rapid decline in serum steroids of ovarian origin.     

Inactivation of xanthine oxidase by hydrogen peroxide involves site-directed hydroxyl radical formation.

The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.     

Metal-independent production of hydroxyl radicals by halogenated quinones and hydrogen peroxide: an ESR spin trapping study

The metal-independent production of hydroxyl radicals (*OH) from H(2)O(2) and tetrachloro-1,4-benzoquinone (TCBQ), a carcinogenic metabolite of the widely used wood-preservative pentachlorophenol, was studied by electron spin resonance methods. When incubated with the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), TCBQ and H(2)O(2) produced the DMPO/*OH adduct. The formation of DMPO/*OH was markedly inhibited by the *OH scavenging agents dimethyl sulfoxide (DMSO), ethanol, formate, and azide, with the concomitant formation of the characteristic DMPO spin trapping adducts with *CH(3), *CH(CH(3))OH, *COO(-), and *N(3), respectively. The formation of DMPO/*OH and DMPO/*CH(3) from TCBQ and H(2)O(2) in the absence and presence, respectively, of DMSO was inhibited by the trihydroxamate compound desferrioxamine, accompanied by the formation of the desferrioxamine-nitroxide radical. In contrast, DMPO/*OH and DMPO/*CH(3) formation from TCBQ and H(2)O(2) was not affected by the nonhydroxamate iron chelators bathophenanthroline disulfonate, ferrozine, and ferene, as well as the copper-specific chelator bathocuproine disulfonate. A comparative study with ferrous iron and H(2)O(2), the classic Fenton system, strongly supports our conclusion that *OH is produced by TCBQ and H(2)O(2) through a metal-independent mechanism. Metal-independent production of *OH from H(2)O(2) was also observed with several other halogenated quinones.