Spectral properties of the higher oxidation states of prostaglandin H synthase

Prostaglandin H (PGH) synthase reacts with organic hydroperoxides and fatty acid hydroperoxides on a millisecond time scale to generate an intermediate that is spectrally similar to compound I of horseradish peroxidase. Compound I of PGH synthase is converted to compound II within 170 ms. Compound II decays to resting enzyme in a few seconds. Thus, the peroxidase reaction of PGH synthase appears to involve a cycle of native enzyme, compound I, and compound II, typical of heme-containing peroxidases. The Soret absorption maximum of compound I appears to occur at 412 nm but a small amount of compound II may be present. Soret maxima occur at 420, 433, and 419 for compound II, the ferrous enzyme, and the oxyferrous enzyme (compound III), respectively. Rapid scan analysis of the reaction of PGH synthase with arachidonic acid reveals the absorbance of compound II but no evidence for ferrous or oxyferrous enzyme.     

Coupling of dihydroriboflavin oxidation to the formation of the higher valence states of hemeproteins.

The reactions between hydrogen peroxide and hemeproteins have been coupled to the oxidation of dihydroriboflavin so as to provide a simple method for measuring the rate constant of hemeprotein peroxidation. Dihydroriboflavin rapidly reduces the higher oxidation states of iron and the hydroxy radicals which are the products of the hemeprotein/hydrogen peroxide reaction. The rapid reduction of these highly reactive compounds prevents the hemeproteins from undergoing irreversible chemical modifications and thus allows the kinetics of peroxidation to be studied. The rate constants at pH 7.2 and 23 degrees C for the peroxidation of horseradish peroxidase, myoglobin, and ferrocytochrome c are found to be 6.2 x 10(6), 7.5 x 10(4), and 8 x 10(3)M-1s-1, respectively. These studies suggest that reduced riboflavin might efficiently protect cells from oxidative damage such as that occurring in inflammation and reperfusion injury.     

Myeloperoxidase oxidation states involved in myeloperoxidase-oxidase oxidation of thiols.

The human red-blood-cell glyoxalase system was modified by incubation with high concentrations of glucose in vitro. Red-blood-cell suspensions (50%, v/v) were incubated with 5 mM- and 25 mM-glucose to model normal and hyperglycaemic glucose metabolism. There was an increase in the flux of methylglyoxal metabolized to D-lactic acid via the glyoxalase pathway with high glucose concentration. The increase was approximately proportional to initial glucose concentration over the range studied (5-100 mM). The activities of glyoxalase I and glyoxalase II were not significantly changed, but the concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, and the percentage of glucotriose metabolized via the glyoxalase pathway, were significantly increased. The increase in the flux of intermediates metabolized via the glyoxalase pathway during periodic hyperglycaemia may be a biochemical factor involved in the development of chronic clinical complications associated with diabetes mellitus.     

Reactions of the NAD radical with higher oxidation states of horseradish peroxidase

The reactions of the NAD radical (NAD.) with ferric horseradish peroxidase and with compounds I and II were investigated by pulse radiolysis. NAD. reacted with the ferric enzyme and with compound I to form the ferrous enzyme and compound II with second-order rate constants of 8 X 10(8) and 1.5 X 10(8) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of NAD. with native compound II at pH 10.0 nor with diacetyldeutero-compound II at pH 5.0-8.0 could be detected. Other reducing species generated by pulse radiolysis, such as hydrated electron (eaq-), superoxide anion (O2-), and benzoate anion radical, could not reduce compound II of the enzyme to the ferric state, although the methylviologen radical reduced it. The results are discussed in relation to the mechanism of catalysis of the one-electron oxidation of substrates by peroxidase.     

A novel DC therapy with manipulation of MKK6 gene on nickel allergy in mice

BACKGROUND: Although the activation of dermal dendritic cells (DCs) or Langerhans cells (LCs) via p38 mitogen-activated protein kinase (MAPK) plays a crucial role in the pathogenesis of metal allergy, the in vivo molecular mechanisms have not been identified and a possible therapeutic strategy using the control of dermal DCs or LCs has not been established. In this study, we focused on dermal DCs to define the in vivo mechanisms of metal allergy pathogenesis in a mouse nickel (Ni) allergy model. The effects of DC therapy on Ni allergic responses were also investigated. METHODS AND FINDING: The activation of dermal DCs via p38 MAPK triggered a T cell-mediated allergic immune response in this model. In the MAPK signaling cascade in DCs, Ni potently phosphorylated MAP kinase kinase 6 (MKK6) following increased DC activation. Ni-stimulated DCs could prime T cell activation to induce Ni allergy. Interestingly, when MKK6 gene-transfected DCs were transferred into the model mice, a more pronounced allergic reaction was observed. In addition, injection of short interfering (si) RNA targeting the MKK6 gene protected against a hypersensitivity reaction after Ni immunization. The cooperative action between T cell activation and MKK6-mediated DC activation by Ni played an important role in the development of Ni allergy. CONCLUSIONS: DC activation by Ni played an important role in the development of Ni allergy. Manipulating the MKK6 gene in DCs may be a good therapeutic strategy for dermal Ni allergy.     

Spectroscopic studies of the oxidation of ferric CYP153A6 by peracids: Insights into P450 higher oxidation states

Our previous rapid-scanning stopped-flow studies of the reaction of substrate-free cytochrome P450cam with peracids [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-20309; J. Inorg. Biochem. 100 (2006) 2034-2044; J. Biol. Inorg. Chem. 13 (2008) 599-611] spectrally characterized compound I (ferryl iron plus a porphyrin π-cation radical (Fe^IVO/Por⁺)), Cpd ES, and Cpd II (Fe^IVO/Tyr or Fe^IVO). We now report that reactions of CYP153A6 with peracids yield all these intermediates, with kinetic profiles allowing better resolution of all forms at pH 8.0 compared to similar reactions with WT P450cam. Properties of the reactions of these higher oxidation state intermediates were determined in double-mixing experiments in which intermediates are pre-formed and ascorbate is then added. Reactions of heptane-bound CYP153A6 (pH 7.4) with mCPBA resulted in conversion of P450 to the low-spin ferric form, presumably as heptanol was formed, suggesting that CYP 153A6 is a potential biocatalyst that can use peracids with no added NAD(P)H or reducing systems for bioremediation and other industrial applications.     

Coordination chemistry of higher oxidation states. Part 15. Cobalt(III) complexes of bi- and multi-dentate phenylphosphines

Six-coordinate cobalt(III) complex trans-[Co{o-C₆H₄(PPh₂)₂}₂X₂]ClO₄, fac-[Co{PhP(CH₂CH₂PPh₂)₂}X₃],cis-[Co{P(CH₂CH₂PPh₂)₃}X₂]ClO₄ and cis-β-[Co{-CH₂P(Ph)CH₂CH₂PPh₂}₂X₂]PF₆ (X = Cl, Br) have been prepared by halogen oxidation of the Co(II) analogues, and characterised by IR, electronic and ³¹P NMR spectroscopy. The failure to obtain complexes with X = I, and with some related ligands is discussed, and the rather low stability of the above complexes is rationalised in terms of steric crowding at the metal centre.     

Green tea extract improves endurance capacity and increases muscle lipid oxidation in mice

Green tea contains a high level of polyphenolic compounds known as catechins. We investigated the effects of green tea extract (GTE), which is rich in catechins, on endurance capacity, energy metabolism, and fat oxidation in BALB/c mice over a 10-wk period. Swimming times to exhaustion for mice fed 0.2-0.5% (wt/wt) GTE were prolonged by 8-24%. The effects were dose dependent and accompanied by lower respiratory quotients and higher rates of fat oxidation as determined by indirect calorimetry. In addition, feeding with GTE increased the level of beta-oxidation activity in skeletal muscle. Plasma lactate concentrations in mice fed GTE were significantly decreased after exercise, concomitant with increases in free fatty acid concentrations in plasma, suggesting an increased lipid use as an energy source in GTE-fed mice. Epigallocatechin gallate (EGCG), a major component of tea catechins, also enhanced endurance capacity, suggesting that the endurance-improving effects of GTE were mediated, at least in part, by EGCG. The beta-oxidation activity and the level of fatty acid translocase/CD36 mRNA in the muscle was higher in GTE-fed mice compared with control mice. These results indicate that GTE are beneficial for improving endurance capacity and support the hypothesis that the stimulation of fatty acid use is a promising strategy for improving endurance capacity.     

Nickel in higher plants: further evidence for an essential role

Soybeans (Glycine max [L.] Merr.) grown in Ni-deficient nutrient solutions accumulated toxic urea concentrations which resulted in necrosis of their leaflet tips, a characteristic of Ni deficiency. Estimates of the Ni requirement of a plant were made by using seeds produced with different initial Ni contents. When compared to soybeans grown from seeds containing 2.5 nanograms Ni, plants grown from seeds containing 13 nanograms Ni had a significantly reduced incidence of leaflet tip necrosis. Plants grown from seeds containing 160 nanograms Ni produced leaves with almost no leaflet tip necrosis symptoms. Neither Al, Cd, Sn, nor V were able to substitute for Ni.

In other experiments, a small excess of EDTA was included in the nutrient solution in addition to that needed to chelate micronutrient metals. Under these conditions, nodulated nitrogen-fixing soybeans had a high incidence of leaflet tip necrosis, even when 1 micromolar NiEDTA was supplied. However, in nutrient solutions containing inorganic sources of N, 1 micromolar NiEDTA almost completely prevented leaflet tip necrosis, although no significant increase in leaf urease activity was observed. Cowpeas (Vigna unguiculata [L.] Walp) grown in Ni-deficient nutrient solutions containing NO₃ and NH₄ also developed leaflet tip necrosis, which was analogous to that produced in soybeans, and 1 micromolar NiEDTA additions prevented these symptoms.

These findings further support our contention that Ni is an essential element for higher plants.

     

Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract.

Duchenne muscular dystrophy is characterized by the absence of dystrophin from muscle cells. Dystrophic muscle cells are susceptible to oxidative stress. We tested the hypothesis that 3 wk of endurance exercise starting at age 21 days in young male mdx mice would blunt oxidative stress and improve dystrophic skeletal muscle function, and these effects would be enhanced by the antioxidant green tea extract (GTE). In mice fed normal diet, average daily running distance increased 300% from week 1 to week 3, and total distance over 3 wk was improved by 128% in mice fed GTE. Running, independent of diet, increased serum antioxidant capacity, extensor digitorum longus tetanic stress, and total contractile protein content, heart citrate synthase, and heart and quadriceps beta-hydroxyacyl-CoA dehydrogenase activities. GTE, independent of running, decreased serum creatine kinase and heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate synthase activity. These data suggest that both endurance exercise and GTE may be beneficial as therapeutic strategies to improve muscle function in mdx mice.     

Lymphocyte subpopulations in sensitization and specific immunotherapy in an experiment. Lymphocyte subpopulations in the specific immunotherapy of microbial allergy and subsequent heterologous pollen sensitization

The influence exerted by the specific immunotherapy (SIT) of delayed hypersensitivity (DH) to staphylococci and subsequent sensitization with tarragon pollen on the level of immunocompetent cells in the blood and lymphoid organs of guinea pigs was studied. On the whole, SIT normalized the characteristics of T- and B-lymphocytes, altered as the result of experimentally induced DH: the content of T-cells in the peripheral blood and the lymph nodes increased, while the number of B-cells in the blood and T gamma-suppressors increased. The subsequent heterologous sensitization with pollen abolished the effect of SIT, inducing the general decrease of the level of T gamma-lymphocytes and enhancing the number of T-lymphocytes in the lymph nodes.     

Radiation damage in DNA: possible role of higher triplet states

Triplet states of deoxyribose are expected to dissociate efficiently into radicals, leading to strand breaks. Such states could be excited by slow secondary electrons (A) or result from ion recombination in spurs containing two or more ion pairs (B). Estimates of the efficiencies of these processes are presented and the mechanisms discussed in the light of recent work with electrons, vacuum ultraviolet (VUV) photons, and X rays. Route B could play a significant role in producing double-strand breaks, while route A may be a better approach to characterizing the process experimentally.     

Costimulation blockade inhibits allergic sensitization but does not affect established allergy in a murine model of grass pollen allergy

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.     

Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase.

Addition of arachidonic acid or 5-phenyl-4-pentenylhydroperoxide to manganese-prostaglandin endoperoxide synthase (Mn-PGH synthase) produced a species with an absorbance maximum at 418 nm. This maximum is distinct from those of resting enzyme (372 and 468 nm) or reduced enzyme (434 nm). The formation of the 418 nm-absorbing species was observed immediately after the addition of hydroperoxide to enzyme but only after a 10-s lag period following addition of arachidonate. Mn-PGH synthase exhibited a peroxidase activity that was 0.8% that of Fe-PGH synthase. Addition of peroxidase reducing substrates to the oxidized form of Mn-PGH synthase diminished the absorbance at 418 nm. In the case of N,N,N',N'-tetramethylphenylenediamine, reduction of the 418 nm-absorbing species was accompanied by an increase in absorbance at 610 nm due to the oxidized form of the amine. Thus, the spectral and chemical properties of the 418 nm-absorbing species are consistent with its existence as a higher oxidation state of Mn-PGH synthase. Kinetic analysis indicated that formation of the higher oxidation state preceded or was coincident with oxygenation of the fatty acid substrate, eicosa-11,14-dienoic acid. The cyclooxygenase activity of Mn-PGH synthase was inhibited by the combination of glutathione and human plasma glutathione peroxidase at a glutathione peroxidase concentration 227-fold lower than the concentration that inhibited Fe-PGH synthase. The results suggest that Mn-PGH synthase forms a higher oxidation state following reaction with hydroperoxides added exogenously or generated endogenously from polyunsaturated fatty acid substrates. This higher oxidation state functions in the peroxidase catalytic cycle of Mn-PGH synthase, and its formation appears to be essential for activation of the cyclooxygenase catalytic cycle.