Vesna Niketić

EXPOSURE OF MN AND FESODS, BUT NOT CU/ZNSOD, TO NO LEADS TO NITROSONIUM AND NITROXYL IONS GENERATION WHICH CAUSE ENZYME MODIFICATION AND INACTIVATION : AN IN VITRO STUDY

The effect of NO treatment in vitro on structural and functional alterations of Cu/Zn, Mn, and Fe type of SODs was studied. Significant difference in response to NO of Cu/ZnSOD compared to the Mn and Fe types was demonstrated. Cu/ZnSOD was shown to be stable with respect to NO: even on prolonged exposure, NO produced negligible effect on its structure and activity. In contrast, both Mn and Fe types were found to be NO-sensitive: exposure to NO led to their fast and extensive inactivation, which was accompanied by extensive structural alterations, including (in some of the samples tested) the cleavage of enzyme polypeptide chains, presumably at His residues of the enzyme metal binding sites. The generation of nitrosonium (NO+) and nitroxyl (NO-) ions in NO treated Mn and FeSODs, which produce enzyme modifications and inactivation, was demonstrated. The physiological and biomedical significance of described findings is briefly discussed.

Consequences of MnSOD interactions with nitric oxide : Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed √NO disproportionation (dismutation) into nitrosonium (NO+) and nitroxyl (NO− ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to √NO, MnSOD-derived NO− species initiate the formation of peroxynitrite (ONOO− ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO− decomposition and ONOO− -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO− is accompanied by the formation of substantial amounts of H2O2. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of √NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of √NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of √NO.

Lipid status, anti-oxidant enzyme defence and haemoglobin content in the blood of long-term clozapine-treated schizophrenic patients.

OBJECTIVE Despite clozapines unique effectiveness in patients with schizophrenia, a number of adverse effects have been recognised including abnormalities in lipid and glucose metabolisms. A high clozapine level in red blood cells (RBCs) and disturbed anti-oxidant enzyme activities in blood from schizophrenic patients prompted us to investigate lipid status and anti-oxidant enzyme defence in the blood of chronic schizophrenic patients on long-term clozapine therapy. METHODS Plasma lipids, RBC anti-oxidant enzyme activities and haemoglobin (Hb) content were measured using established procedures in a group of eighteen chronically-medicated (average 630 days of therapy) schizophrenic patients receiving clozapine (average dose of 295 mg/day) and data were compared with those from a group of eighteen well-matched normal controls. RESULTS Significantly higher levels of plasma triglycerides (by 47%, p<0.01) and total cholesterol and phospholipids (by 8% and 11%, respectively p<0.05) in patients were found. CuZn-superoxide dismutase (SOD1) activity was markedly higher (by 35%, p<0.001) while selenium-dependent glutathione peroxidase (GSH-Px1) activity was markedly lower (by 41%, p<0.001) in patients. In addition, metHb and HbA1c levels in patients were significantly higher (by 58% and 25%, respectively p<0.001). SOD1 activity was negatively correlated (p<0.001) to GSH-Px1 activity in patients. CONCLUSIONS The findings support the view that ongoing oxidative stress may be a mechanism by which clozapine induces some adverse effects that increase the risk of diabetes and metabolic syndrome. If valid, this would indicate that in parallel with long-term clozapine treatment, schizophrenic patients could be encouraged to make some lifestyle changes to limit the detrimental effects of the medication.

NO Dismutase Activity of Seven‐Coordinate Manganese(II) Pentaazamacrocyclic Complexes

Seven-coordinate Mn pentaazamacrocyclic complexes represent the most potent synthetic mimics of native superoxide dismutase (SOD), which catalyze dismutation of superoxide (O2C ) into O2 andH2O2 with efficiency that can exceed that of the mitochondrial MnSOD. A number of studies demonstrated the ability of these SOD mimics to protect cells and tissues from oxidative damage caused by superoxide (and/or the product of its reaction with nitric oxide, peroxynitrite), for example in inflammation and oxidation reperfusion injury. The members of this class of SODmimics have entered Phase II clinical trials in the USA. It is emphasized that the major advantage of Mn pentaazamacrocycles over other SOD mimics is their high selectivity for O2C and lack of reactivity with NO, a key molecule in biological processes. However, direct studies of the reaction between NO and this class of complexes, which would support such claims, have not been reported. Various metal complexes, including manganese complexes, react readily with NO, either to yield metal nitrosyls, or to produce N2O and metal nitrite complexes by NO disproportionation. Coordinated NO in metal nitrosyls can exist in one of its three formal redox states NO, NO, and NO . For a number of NO and NO complexes reactivity towards selected nucleophiles and electrophiles, respectively, has been documented. We demonstrated recently that natural MnSOD enzyme reacts with NO according to distinct catalytic NO disproportionation (dismutation) mechanism which yields both reactive species NO and NO . Collectively these results prompted us to (re)examine the reaction of these complexes with NO. The chosen approach has been 1) to establish the reactivity of complexes with NO, 2) to establish the mechanistic details of the reaction, and 3) to demonstrate the potential validity of the complex reaction with NO in a biological setting. In the present study we used [Mn(pyane)Cl2] (1), [8] as a general representative of this class of SOD mimics, and its

Biotransformation of nitric oxide in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Abstract Recent findings indicate that nitric oxide (NO•) over-production might be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). We measured significantly higher concentrations of uric acid and thiol group-containing molecules (R–SH groups) in the cerebrospinal fluid (CSF) from SALS patients compared to controls. The above factors, together with a slightly increased free iron concentration found in the CSF, favour conditions necessary for the formation of the dinitrosyl iron complex, capable of NO• bio-transformation. Thus, we performed ex vivo saturation of CSF (from both SALS patients and controls) with NO•. A decrease in the level of R–SH was found. This was more pronounced in the CSF from SALS patients. In the CSF from SALS patients the production of nitrite and hydroxylamine was greater than that observed in the CSF from controls. Moreover, we also found increased Cu,Zn-SOD activity in the CSF from SALS patients (when compared to control subjects) but no activity corresponding to Mn-SOD in any CSF samples. As Cu,Zn-SOD can react with nitroxyl forming NO•, the conditions for a closed, but continuous, loop of NO• biotransformation are present in the CSF of ALS patients.

The Radiolysis of Aqueous Ammonium Cyanide: Compounds of Interest to Chemical Evolution Studies

SummaryOxygen-free aqueous solutions of NH4CN (0.1 M, pH 9) were exposed to gamma rays from a60Co source, the mixture of nonvolatile products was fractionated, and the fractions were analyzed. The procedures were chosen to make effective investigations of radiolytic products, and to minimize the contributions of chemical changes which are known to occur in aqueous solution in the absence of ionizing radiation. It has been found that the main constituents are: urea, 25.9%; an oligomer, very likely oligoimine (18.4%); and several fractions (about 50%) which release amino acids on hydrolysis. These fractions differ considerably, as shown by amino acid assay, enzymatic digestion, IR spectra, and biuret reaction. All these tests were found to be positive for two fractions; in two further fractions the enzymatic cleavage was absent, but other tests were positive. Negative enzymatic and biuret tests, and no bands characteristic of amide or peptide, were found for a fraction whose hydrolysate consisted of 55% glycine. Although most of the isolated materials were found to be composite, the results of the analyses were sufficient for getting a reliable over-all picture of the chemical action of the ionizing radiation. The role of free radicals in reactions leading to the formations of radiolytic products was considered.

Identification, purification and characterization of a novel collagenolytic serine protease from fig (Ficus carica var. Brown Turkey) latex.

A novel collagenolytic serine protease was identified and then purified (along with ficin) to apparent homogeneity from the latex of fig (Ficus carica, var. Brown Turkey) by two step chromatographic procedure using gel and covalent chromatography. The enzyme is a monomeric protein of molecular mass of 41 ± 9 kDa as estimated by analytical gel filtration chromatography. It is an acidic protein with a pI value of approximately 5 and optimal activity at pH 8.0-8.5 and temperature 60°C. The enzymatic activity was strongly inhibited by PMSF and Pefabloc SC, indicating that the enzyme is a serine protease. The enzyme showed specificity towards gelatin and collagen (215 GDU/mg and 24.8 CDU/mg, respectively) and non-specific protease activity (0.18 U/mg against casein). The enzyme was stable and retained full activity over a broad range of pH and temperature. The fig latex collagenolytic protease is potentially useful as a non-microbial enzyme with collagenolytic activity for various applications in the fields of biochemistry, biotechnology and medicine.

Effect of atypical antipsychotics on antioxidant enzyme activities in human erythrocytes (in vitro study)

This study was set out to examine the impact of atypical antipsychotic drugs: aripiprazole, clozapine, ziprasidone, olanzapine, quetiapine, sertindole and amisulpride on the activity of antioxidant defence enzymes in human erythrocytes in vitro.

Radiolysis of aqueous solutions of hydrogen cyanide (pH∼6): Compounds of interest in chemical evolution studies

SummaryOxygen-free aqueous solutions of hydrogen cyanide, 0.1 M and pH∼6, were exposed to gamma rays from a60Co source, the mixture of nonvolatile products was fractionated and the fractions were analysed. It has been found that the complex mixture contains oligomers and polymers with molecular weights up to 20,000 daltons, mainly polyamides with urea and peptidic fragments. Among the constituents are carbamyl glycinonitrile and carbamyl glycinamide that represent 6.4% and 3.1% of the total of unfractionated material respectively. Urea content is 2.6%, but the derivatives of urea are more abundant. Acid hydrolysis releases several amino acids. Glycine is the most abundant (75% or more of total amino acid content), and its concentration considerably increases in some fractions when the hydrolysis is carried out at 130°C. The role of free radicals in reactions leading to the formations of radiolytic products is considered. Some comparisons are made between findings in the present work, at initial pH∼6, and an earlier study of ammonium cyanide at pH 9.

Enzymatic characterization of peptidic materials isolated from aqueous solutions of ammonium cyanide (pH 9) and hydrocyanic acid (pH 6) exposed to ionizing radiation

The enzymatic digestion of some radiolytically produced peptidic materials was examined. The substrates were compounds isolated from 0.1 molar solutions of NH4CN (pH 9) and HCN (pH 6), after their exposure toγ rays from a60Co source (15–20 Mrad doses). Commercial proteolytic enzymes pronase and aminopeptidase M were used. The examined materials were of composite nature and proteolytic action was systematically observed after their subsequent purification. In some fractions the effect was found to be positive with up to 30% of peptide bonds cleaved with respect to the amino acid content. These findings support our previous conclusions on the free radical induced formation of peptidic backbones without the intervention of amino acids. Some side effects were also noted which might be of interest in observations on enzymatic cleavage of other composite peptidic materials of abiotic origin.