Reactivity of Hg(II) with superoxide: evidence for the catalytic dismutation of superoxide by Hg(II).

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O2-) and hydrogen peroxide (H2O2) production by rat kidney mitochondria, Hg(II) (1-6 microM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H2O2 production but an apparent decrease in mitochondrial O2- production. Hg(II) also inhibited O(2-)-dependent cytochrome c reduction (IC50 approximately 2-3 microM) when O2- was produced from xanthine oxidase. In contrast, Hg(I) did not react with O2- in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O2- by Hg(II). Hg(II) also inhibited the reactions of KO2 (i.e., O2-) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO2 in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO2. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O2- dismutation, leading to increased production of H2O2 by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

Evidence for two sites of superoxide production by mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).     

Evidence for catalytic dismutation of superoxide by cobalt(II) derivatives of bovine superoxide dismutase in aqueous solution as studied by pulse radiolysis.

By using the technique of pulse radiolysis to generate O2-., it is demonstrated that Co(II) derivatives of bovine superoxide dismutase in which the copper alone and both the copper and zinc of the enzyme have been substituted by Co(II), resulting in (Co,Zn)- and (Co,Co)-proteins, are capable of catalytically dismutating O2-. with 'turnover' rate constants of 4.8 X 10(6) dm3.s-1.mol-1 and 3.1 X 10(6) dm3.s-1.mol-1 respectively. The activities of the proteins are independent of the pH (7.4-9.4) and are about three orders of magnitude less than that of the native (Cu,Zn)-protein. The rate constants for the initial interaction of O2-. with the Co-proteins were determined to be (1.5-1.6) X 10(9) dm3.s-1.mol-1; however, in the presence of phosphate, partial inhibition is apparent [k approximately (1.9-2.3) X 10(8) dm3.s-1.mol-1]. To account for the experimental observations, two reaction schemes are presented, involving initially either complex-formation or redox reactions between O2-. and Co(II). This is the first demonstration that substitution of a metal into the vacant copper site of (Cu,Zn)-protein results in proteins that retain superoxide dismutase activity.     

Reduction of Hg(II) to Hg(0) by Biogenic Magnetite from two Magnetotactic Bacteria

Understanding the biogeochemical cycle of the highly toxic element mercury (Hg) is necessary to predict its fate and transport. In this study, we determined that biogenic magnetite isolated from Magnetospirillum gryphiswaldense MSR-1 and Magnetospirillum magnetotacticum MS-1 was capable of reducing inorganic mercury [Hg(II)] to elemental mercury [Hg(0)]. These two magnetotactic bacteria (MTB) lacked mercuric resistance operons in the genomes. However, they revealed high resistance to Hg(II) under atmospheric conditions and an even higher resistance under microaerobic conditions (1% O₂ and 99% N₂). Neither strain reduced Hg(II) to Hg(0) under atmospheric conditions. However, a slow rate (0.05–0.21 µM·d^?1) of Hg(II) loss occurred from late log phase to stationary phase in two MTBs' culture media under microaerobic conditions. Increased Hg(II) entered both cells under microaerobic conditions relative to atmospheric conditions. The majority of Hg(II) was still blocked by the cell membrane. Hg(II) reduction was more effective when biogenic magnetite was extracted out, with or without the magnetosome membrane envelope. When magnetosome membrane was present, 8.55–13.53% of 250 nM Hg(II) was reduced to Hg(0) by 250 mg/L biogenic magnetite suspension within 2 hours. This ratio increased to 55.07–64.70% while magnetosome membrane was removed. We concluded that two MTBs contributed to the reduction of Hg(II) to Hg(0) at a slow rate in vivo. Such reduction was more favorable to occur when biogenic magnetite is released from dead cells. It proposed a new biotic pathway for the formation of Hg(0) in aquatic systems.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

The mechanism of superoxide production by the antimycin-inhibited mitochondrial Q-cycle

Superoxide production from antimycin-inhibited complex III in isolated mitochondria first increased to a maximum then decreased as substrate supply was modulated in three different ways. In each case, superoxide production had a similar bell-shaped relationship to the reduction state of cytochrome b(566), suggesting that superoxide production peaks at intermediate Q-reduction state because it comes from a semiquinone in the outer quinone-binding site in complex III (Q(o)). Imposition of a membrane potential changed the relationships between superoxide production and b(566) reduction and between b(562) and b(566) redox states, suggesting that b(562) reduction also affects semiquinone concentration and superoxide production. To assess whether this behavior was consistent with the Q-cycle mechanism of complex III, we generated a kinetic model of the antimycin-inhibited Q(o) site. Using published rate constants (determined without antimycin), with unknown rate constants allowed to vary, the model failed to fit the data. However, when we allowed the rate constant for quinol oxidation to decrease 1000-fold and the rate constant for semiquinone oxidation by b(566) to depend on the b(562) redox state, the model fit the energized and de-energized data well. In such fits, quinol oxidation was much slower than literature values and slowed further when b(566) was reduced, and reduction of b(562) stabilized the semiquinone when b(566) was oxidized. Thus, superoxide production at Q(o) depends on the reduction states of b(566) and b(562) and fits the Q-cycle only if particular rate constants are altered when b oxidation is prevented by antimycin. These mechanisms limit superoxide production and short circuiting of the Q-cycle when electron transfer slows.     

Antioxidant action of sodium diethyldithiocarbamate: Reaction with hydrogen peroxide and superoxide radical

The oxidation of sodium diethyldithiocarbamate (DDC) by hydrogen peroxide or superoxide radicals has been investigated. Hydrogen peroxide oxidizes DDC, leading to the formation of a hydrated form of disulfiram, a dimer of DDC having a disulfide group. In equimolar conditions, the overall process appears as a first-order reaction (k = 0.025±0.005 s⁻¹), the first step being a second-order reaction (k = 5.0±0.1mol⁻¹.1. s⁻¹). No radical intermediate was observed in this process. In the presence of an excess of any of the reagents, the hydrated form of disulfiram transforms into different products corresponding to the fixation of oxygen by sulfur atoms or replacement of C = S group by ketone function, in the presence of an excess of hydrogen peroxide. Superoxide anions (produced by steady-state ⁶⁰Co γ-radiolysis) oxidize DDC, yielding similar products to those obtained with hydrogen peroxide with a maximum oxidation G-value of 0.3 μmol.J⁻¹. The rate constant k(O₂^·− + DDC) is equal to 900 mol⁻¹. 1. s⁻¹.     

Novel mechanisms for superoxide-scavenging activity of human manganese superoxide dismutase determined by the K68 key acetylation site

Superoxide is the primary reactive oxygen species generated in the mitochondria. Manganese superoxide dismutase (SOD2) is the major enzymatic superoxide scavenger present in the mitochondrial matrix and one of the most crucial reactive oxygen species-scavenging enzymes in the cell. SOD2 is activated by sirtuin 3 (SIRT3) through NAD⁺-dependent deacetylation. However, the exact acetylation sites of SOD2 are ambiguous and the mechanisms underlying the deacetylation-mediated SOD2 activation largely remain unknown. We are the first to characterize SOD2 mutants of the acetylation sites by investigating the relative enzymatic activity, structures, and electrostatic potential of SOD2 in this study. These SOD2 mutations affected the superoxide-scavenging activity in vitro and in HEK293T cells. The lysine 68 (K68) site is the most important acetylation site contributing to SOD2 activation and plays a role in cell survival after paraquat treatment. The molecular basis underlying the regulation of SOD2 activity by K68 was investigated in detail. Molecular dynamics simulations revealed that K68 mutations induced a conformational shift of residues located in the active center of SOD2 and altered the charge distribution on the SOD2 surface. Thus, the entry of the superoxide anion into the coordinated core of SOD2 was inhibited. Our results provide a novel mechanistic insight, whereby SOD2 acetylation affects the structure and charge distribution of SOD2, its tetramerization, and p53–SOD2 interactions of SOD2 in the mitochondria, which may play a role in nuclear–mitochondrial communication during aging.     

Identification of Direct and Indirect Effectors of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable cation channel involved in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca²⁺ and ADP-ribose (ADPR) but also modulated in intact cells by several additional factors. Superfusion of TRPM2-expressing cells with H₂O₂ or intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) activates, whereas dialysis of AMP inhibits, TRPM2 whole-cell currents. Additionally, H₂O₂, cADPR, and NAADP enhance ADPR sensitivity of TRPM2 currents in intact cells. Because in whole-cell recordings the entire cellular machinery for nucleotide and Ca²⁺ homeostasis is intact, modulators might affect TRPM2 activity either directly, by binding to TRPM2, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca²⁺. To identify direct modulators of TRPM2, we have studied the effects of H₂O₂, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide in inside-out patches from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H₂O₂ (1 mm) and enzymatically purified cADPR (10 μm) failed to activate, whereas AMP (200 μm) failed to inhibit TRPM2 currents. NAADP was a partial agonist (maximal efficacy, ∼50%), and nicotinic acid adenine dinucleotide was a full agonist, but both had very low affinities (K_0.5 = 104 and 35 μm). H₂O₂, cADPR, and NAADP did not enhance activation by ADPR. Considering intracellular concentrations of these compounds, none of them are likely to directly affect the TRPM2 channel protein in a physiological context.     

Intramolecular oxidation of the ligand 4-methyl-2-N-(2-pyridylmethyl)aminophenol (Hpyramol) upon coordination with iron(II) chloride and manganese(II) perchlorate

The ligand Hpyramol (Hpyramol=4-methyl-2-N-(2-pyridylmethyl)aminophenol) is found to undergo an oxidative dehydrogenation of its amine function to an imine group upon coordination with iron(II) chloride and manganese(II) perchlorate. X-ray diffraction analyses for both complexes shows differences in the coordination geometry of the complexes most likely because of the two different counter-ions namely the strong coordinating chloride anions and the weak coordinating perchlorate anions. The coordination sphere of the iron(III) complex in [FeCl₂(pyrimol)(MeOH)](MeOH) is best described as a distorted octahedral FeN₂O₂Cl₂ chromophore, while the manganese(II) ions in [Mn(ClO₄)(pyrimol)(Hpyrimol)]₂ are in a distorted octahedral MnN₄O₂ environment with a 2:1 ligand to metal ratio instead of 1:1.     

Silver(I), mercury(II) and palladium(II) complexes of functionalized N-heterocyclic carbenes: Synthesis, structural studies and catalytic activity

A series of NHC silver(I), mercury(II) and palladium(II) complexes, [(1,3-diethylbimy)₆Ag₄I₃]I (2), [(1-benzyl-3-picolylbimy)Ag₂Br₂]_n (3), [(1-benzyl-3-picolylbimy)HgI(CH₂CN)]₂ (4), {[(1-picolyl-3-npropylbimy)₂Hg][Hg₂I₆]}_n (5) and [(1,3-dipicolylbimy)PdCl]Cl (6), as well as one anionic complex [1,3-diethylbimidazolium]₂[HgI₄] (1) (bimy = benzimidazol-2-ylidene), have been prepared and characterized. Interestingly, a wind wheel-like Ag₄I₃ arrangement in 2 is formed, 1D polymeric chain containing 12-membered macrometallocycles and quadrangle Ag₂Br₂ units in 3 is generated, and the α-carbon atom of deprotonated acetonitrile ([CH₂CN]⁻) in 4 participates in coordination with mercury(II) atom. In the crystal packings of complexes 1-6, 2D supramolecular layers or 3D supramolecular architectures are formed via intermolecular weak interactions, including π-π interactions, hydrogen bonds, C-H···π contacts, weak Hg···I bonds and I···I bonds. Additionally, the catalytic activity of the NHC palladium(II) complex 6 in Suzuki-Miyaura cross-coupling reaction was studied.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Copper(II) complexes as superoxide dismutase mimics: Synthesis, characterization, crystal structure and bioactivity of copper(II) complexes

Two new copper(II) complexes of the type [Cu(L)X₂), where L = (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide X = Cl/Br have been synthesized and characterized by elemental analyses, FAB (fast atomic bombardment) magnetic measurements, electronic absorption, conductivity measurements cyclic voltammetry (CV) and Electron paramagnetic resonance (epr) spectroscopy. The structures of these complexes determined by single crystal X-ray crystallography show a distorted square based pyramidal (DSBP) geometry around copper(II) metal center. The distorted CuN₂OX (X = Cl/Br) basal plane in them is comprised of two nitrogen and one oxygen atoms of the meridionally coordinated ligand and a chloride or bromide ion and axial position is occupied by other halide ion. The epr spectra of these complexes in frozen solutions of DMSO showed a signal at g ca. 2. The trend in g-value (g_|| > g_⊥ > 2.00) suggest that the unpaired electron on copper(II) has d_x²-y² character. Biological activities in terms of superoxide dismutase (SOD) and antimicrobial properties of copper(II) complexes have also been measured. The superoxide dismutase activity reveals that these two complexes catalyze the fast disproportionation of superoxide in DMSO solution.     

Reaction of superoxide with trityl radical: implications for the determination of superoxide by spectrophotometry

Superoxide radicals can be measured by redox methods which utilize the oxidation/reduction reactions of specific compounds. The redox methods, however, suffer from various interferences, which limit their use in the assay of superoxide. Electron paramagnetic resonance (EPR) spectroscopy using spin traps has been widely used as an alternative and direct technique to measure superoxide radicals. In our recent study, we have demonstrated the detection of superoxide in cellular system by EPR spectroscopy with triarylmethyl (trityl) free radical, TAM Ox063. TAM is highly water-soluble and stable in the presence of many biological oxidizing and reducing agents such as hydrogen peroxide, ascorbate, and glutathione. TAM reacts with superoxide with an apparent second order rate constant of 3.1x10(3)M(-1)s(-1). In the present work, we investigated the feasibility of a spectrophotometric assay of superoxide by taking advantage of the newly formed distinct absorption peak corresponding to the product formed from the reaction between TAM and superoxide. The effects of different fluxes of superoxide and concentrations of TAM on the efficiency and sensitivity of quantification of superoxide were investigated and compared with the widely used cytochrome c method of superoxide determination. The results demonstrated that the TAM method is comparable to the cytochrome c method for the assay of superoxide and further revealed that the assay is not affected by the presence of hydrogen peroxide. In summary, the TAM spectrophotometric assay of superoxide provides a suitable alternative method to the cytochrome c assay to measure superoxide and further complements our earlier reported TAM-EPR assay of superoxide.     

Understanding the influence of the protein environment on the Mn(II) centers in Superoxide Dismutases using High-Field Electron Paramagnetic Resonance

One of the most puzzling questions of manganese and iron superoxide dismutases (SODs) is what is the basis for their metal-specificity. This review summarizes our findings on the Mn(II) electronic structure of SODs and related synthetic models using high-field high-frequency electron paramagnetic resonance (HFEPR), a technique that is able to achieve a very detailed and quantitative information about the electronic structure of the Mn(II) ions. We have used HFEPR to compare eight different SODs, including iron, manganese and cambialistic proteins. This comparative approach has shown that in spite of their high structural homology each of these groups have specific spectroscopic and biochemical characteristics. This has allowed us to develop a model about how protein and metal interactions influence protein pK, inhibitor binding and the electronic structure of the manganese center. To better appreciate the thermodynamic prerequisites required for metal discriminatory SOD activity and their relationship to HFEPR spectroscopy, we review the work on synthetic model systems that functionally mimic Mn-and FeSOD. Using a single ligand framework, it was possible to obtain metal-discriminatory “activity” as well as variations in the HFEPR spectra that parallel those found in the proteins. Our results give new insights into protein-metal interactions from the perspective of the Mn(II) and new steps towards solving the puzzle of metal-specificity in SODs.     

Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line

Previous studies have reported a neuroprotective role for cellular prion protein (PrP(C)) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrP(C) prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrP(C) or PrP(C) lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrP(C) enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrP(C) protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrP(C) plays an essential role in regulating apoptosis and SOD activity.     

Complexation between Hg(II) and dissolved organic matter in stream waters: an application of fluorescence spectroscopy

Complexation between Hg(II) and dissolved organic matter (DOM) collected from streams in Ontario, Canada, was studied using three-dimensional excitation emission matrix (3DEEM) fluorescence spectroscopy. The results show that DOM reacted with Hg(II) rapidly, and the complexation reached pseudo-equilibrium within 20 s. Maximum excitation/emission (Ex/Em) wavelengths shifted towards the longer wavelengths, indicating that DOM structure changed during its interaction with Hg(II). Using fluorescence quenching titrations, complexing parameters, conditional stability constants and the percentage of fluorophores participating in the complexation, were estimated by the modified Stern–Volmer equation. The experimental and field survey results suggest that the Hg–DOM complexation in various streams was related to water quality parameters, e.g. DOC, Cl–, and cation concentrations, and was strongly affected by UV irradiation.     

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.     

The mitochondrial benzodiazepine receptor: Evidence for association with the voltage-dependent anion channel (VDAC)

Specific, high-affinity receptors for numerous drugs have recently been localized to mitochondrial membrane proteins. This review discusses the association of the mitochondrial receptor for benzodiazepines (mBzR) with the voltage-dependent anion channel (VDAC), indicating a possible auxiliary role for VDAC as a putative drug binding protein. The proposed subunit composition of the purified mBzR complex isolated from rat kidney mitochondria includes VDAC, which functions as a recognition site for benzodiazepines (e.g., flunitrazepam), the adenine nucleotide carrier (ADC), and an 18 kDa outer membrane protein identified by covalent labelling with the mBzR antagonists isoquinoline carboxamides (e.g., PK 14105).Abbreviations and chemical names: Ro5-4864: 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepin-2-one; Ro15-1788: ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-]-[1,4]benzodiazepine-3-carboxylate; AHN-086: (1-(2-isothiocyanatoethyl-7-chloro-1,3-dihydro-5-(4-chlorophenyl)-2H-1,4-benzodiazepin-2-one hydrochloride;) PK11195: 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-isoquinoline-3-carboxamide; PK14105: 1-(2-fluoro-5-nitrophenyl)-3-isoquinoline-carboxylic acid.     

Hg(II)-coordination by sugar-acids: role of the hydroxy groups

A solution study on the ability of some derivatised sugars [glucuronic acid (GluA), galacturonic acid (GalA) and glucosaminic acid (GlNA)] to complex the Hg(II) ion is reported. The stability constants of the complex species were determined by potentiometric measurements while (1)H NMR experiments allow to define the coordination sites of sugar molecules. GluA coordinates the metal ion through the carboxylic oxygen and the O-4 hydroxyl group and is found to form more stable complexes with respect to GalA in which metal ligation is from the carboxylic oxygen and the O-5 ring oxygen. GlNA forms stable complexes chelating Hg(II) ion through carboxylic oxygen and the alpha-amino group. The ternary 2,2'-bipyridine containing systems were also investigated by means of potentiometric studies. The ML(2) complexes were also isolated in the solid state and characterised by IR spectroscopy.