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.

Antioxidative defense in human myocardial reperfusion injury.

The activity of glutathione peroxidase (GSH-Px) as well as the activities of other antioxidative enzymes such as CuZn superoxide dismutase (CuZn SOD), catalase (CAT), glutathione reductase (GR) in erythrocytes, the plasma activity of glutathione-S-transferase (GST), and the plasma levels of vitamin E and vitamin C were evaluated in nine patients with acute myocardial infarction (AMI). Blood samples were taken before and 1, 3, 6, and 24 hours after the institution of thrombolytic therapy. The results were compared with those in 30 healthy volunteers. A significant decrease in catalase (CAT) activity and vitamin E content in patients before and after thrombolytic therapy as compared with controls was recorded. Our results confirmed that a disturbed oxidative/antioxidative balance is present after AMI and after thrombolytic therapy.

Covalent glycoinositolphospholipid binding to hemoglobin: a new post-translational modification of Hb occurring in hyperinsulinism with concomitant hypoglycemia.

In this work a novel hitherto unrecognised minor hemoglobin (Hb) fraction, which we detected previously in hemolysates of erythrocytes exposed to a high concentration of insulin under hypoglycemic conditions, both in vivo and in vitro, is analysed. The modification of Hb in HbA1x was shown to be due the addition of glycoinositolphospholipid (GPI) to the C termini of both beta polypeptide chains. A structurally related minor Hb fraction was identified in erythrocytes exposed in vitro to insulin-mimetic agent, trypsin. To our knowledge this is the first demonstration of such a modification of Hb, as well as the first demonstration of post-translational GPI binding to proteins in response to insulin. The mechanism proposed for GPI-Hb formation is briefly described.

Expression of the vascular endothelial growth factor receptor, KDR, in human placenta.

Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor known to act directly on vascular endothelial cells by promoting cell proliferation and permeability. To date, 3 structurally related cell surface receptors for VEGF, Flt-1, Flt-4 and KDR, have been identified and shown to be human type III receptor tyrosine kinases. The establishment of a vascular network is crucial to the development of the placenta and occurs through both angiogenesis and vasculogenesis. The signals controlling these processes are unclear. Immunohistochemical and in situ hybridisation techniques have localised VEGF in the trophoblast layers and VEGF binding to placental vascular endothelial cells and haemangioblasts has been shown, suggesting a role for VEGF and its receptors in development of the vascular network. In this study we have used specific antibodies to localise KDR and endothelial cells in 1st and 3rd trimester human placenta. The staining showed a colocalisation of KDR with endothelial cells and haemangioblasts. No staining of trophoblast cells was observed, but strong staining of the endothelial cells was seen in the villous stroma adjacent to areas of trophoblast proliferation.

Activities of antioxidant enzymes and formation of the glutathione adduct of hemoglobin (Hb ASSG) in epileptic patients with long-term antiepileptic therapy.

The activities of antioxidant enzymes (AOE) and amounts of glutathione adduct of Hb (Hb ASSG) in erythrocytes of chronic epileptic patients treated with mono or mixed therapy of phenobarbital (PB), carbamazepin (CBZ) and valproic acid (VPA), were evaluated and the results were compared with those for normal individuals. The activity of superoxide dismutase (SOD) was lowered for 40% to 60% in epileptic patients relative to normal controls. The lowest activity of SOD was found in patients treated with CBZ and PB. Activity of glutathione peroxidase (GSH-Px) was lowered comparing to the control from cca 15% in patients treated with PB to 50% in those treated with either CBZ alone or with CBZ in combination with PB. For the later two sub-groups of patients significantly lowered activities of catalase and glutathione reductase (cca 20% and 30-40%, respectively) were also characteristic. The highest amounts of Hb ASSG (cca 4% of the tota lHb) were found in hemolysates of patients with the lowest AOE levels i.e., in those treated with CBZ alone or with CBZ in combination with PB. In hemolysates of patients treated with PB, Hb ASSG represented 2.4% of the total Hb, while it was totally absent in those treated with VPA, although SOD and GSH-Px levels in erythrocytes of both of these sub-groups of patients were lowered to the same extent.

The effect of phenobarbital on the nonenzymatic glycation of hemoglobin.

The effect of phenobarbital (PB) on nonenzymatic glycation of hemoglobin in vivo and in vitro was studied. In epileptic patients on long-term therapy with PB, the yield of glycated hemoglobin fraction HbA1c was found to be significantly higher than in normal control. In samples of Hb exposed to glucose in vitro in the presence of PB, HbA1c was also found to be significantly higher than in those exposed to glucose in the absence of PB. Enhanced reactivity of beta-NH2 terminus of Hb with glucose, was partly explained by observing that its pKa was decreased in the presence of PB. It was shown that the effect of PB on pKa of beta-NH2 terminus is one of the consequences of the Hb-PB interactions which were studied in this work by electrophoretic and NMR techniques.

Glutathione adduct of hemoglobin (Hb ASSG) in hemolysates of patients on long-term antiepileptic therapy.

1. A novel, hitherto unrecognized, hemoglobin fraction, HbA1x, has been detected in amounts up to 7% of the total Hb, in hemolysates from epileptic patients with long-term therapy containing phenobarbital (PB) and carbamazepine (Carb). 2. HbA1x was shown to represent glutathione adduct of Hb (Hb ASSG). 3. To our knowledge, this is the first demonstration of the occurrence of Hb ASSG in vivo.

Modified hemoglobin in hypoglycemia caused by hyperinsulinism.

Hemoglobin (Hb) was found to be modified in patients with prolonged hypoglycemia caused by hyperinsulinism. This modification was of a nonpolar nature, did not affect the overall charge of the Hb and caused its aggregation in aqueous solution. After normal concentrations of glucose and insulin were reestablished, this modification slowly disappeared. Hemoglobin from normal erythrocytes incubated in vitro with high concentrations of insulin showed similar behavior.

Glycated hemoglobin in hypoglycemia.

The effect of hypoglycemia--caused by hyperinsulinism in insulinoma patients and in diabetic patients with frequent episodes of hypoglycemia--on glycated hemoglobin was studied. The amount of sugar bound to total hemoglobin in hypoglycemia samples was found to be significantly lower than in those which were normal or hyperglycemic. The amount of total HbA1 fraction, as determined by the mini-column method, was significantly higher than expected on the basis of the corresponding values for total glycated hemoglobin. Evidence is presented to show that this is due to the formation of a hitherto unrecognized HbA1 constituent(s) denoted here as HbA1x.

Radiation chemistry of an aqueous solution of glycine: compounds of interest to chemical evolution studies.

We have examined the radiolysis of an O2-free aqueous solution of glycine at absorbed doses of 60Co gamma-radiation of up to 20 Mrad. At least 20 compounds are formed during radiolysis, among them several amino acids, an oligoamine, and the nitrogen-free polymers (Mw less than or equal to 28,000 daltons). When dicyandiamide is present in the solution of glycine, various nitrogen-containing products, including some polymers (Mw less than or equal to 12,000 daltons), are synthesized along with radiolytic products of glycine; polyglycines are not formed. We have determined the radiation-chemical yields of radiolytic-product formation and of decomposition of glycine, and have considered possible free-radical reactions leading to the radiation-induced changes observed.

Radiation synthesis of polymers in aqueous cyanides.

Structures and properties of polymers isolated from the reaction mixtures obtained upon irradiation of dilute aqueous solutions of ammonium cyanide and HCN with gamma rays of 60Co were studied. On the basis of spectroscopic and chemical data it was concluded that two principal classes of polymers occur each of them having molecular weights that range from about 5000 to over 20000. In both types of polymers peptidic, urea-formaldehyde, and complex heterocyclic fragments are identified. In one type aliphatic fragments are more pronounced, while in the other heterocyclic structures predominate. The polymers interact with nucleic acid bases and some of them show catalytic properties as demonstrated by the hydrolysis of p-nitrophenylacetate.

The radiolysis of aqueous ammonium cyanide: polymers of interest to chemical evolution studies.

Some of the polymers isolated from the mixture of products obtained upon irradiation of dilute oxygen-free solutions of NH4CN with gamma rays from a 60Co source were examined. Their molecular weights were estimated to be in the range 4000-20000 daltons; elements of their structure were inferred from chemical and spectroscopic studies; and their interactions with nucleic acid components were examined. Significance of these results for chemical evolution studies is summarized.

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 to gamma rays from a 60Co 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.

The radiolysis of aqueous ammonium cyanide: compounds of interest to chemical evolution studies.

Oxygen-free aqueous solutions of NH4CN (0.1 M, pH9) were exposed to gamma rays from a 60Co 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.

The radiolysis of aqueous acetonitrile: compounds of interest to chemical evolution studies.

Oxygen-free aqueous solutions of CH3CN (0.1 M, pH 6) were exposed to gamma rays from a 60Co source, the mixture of nonvolatile radiolytic products was fractionated and the fractions were analysed. Succinic, maleic, fumaric, malonic and pyruvic acids were identified. Glycol aldehyde, glucose and probably ribose were observed in the hydrolysate of fractionated material. It has been suggested that an oligomer is formed which has a fragment with the polyhydroxy structure and on hydrolysis releases the carbohydrates. Radiolytic products which release amino acids on hydrolysis were found in several fractions. The amino acid contents of the hydrolysates were up to about 2.8% of the fraction mass. The presence of several protein and nonprotein amino acids suggests that their origin should be in a peptidic structure, which is probably a fragment of an oligomer radiolytically produced. A direct analysis of the irradiated solution shows the presence of acetaldehyde, propionaldehyde, glyoxal and of biacetyl. Experimental findings are discussed and a free-radical mechanism is proposed to account for the chemical changes observed.