Effect of oxidative stress on membrane phospholipid and protein organization in human erythrocytes

Membrane phospholipid and protein organization was studied in intact human erythrocytes exposed to phenylhydrazine, an oxidative agent inducer. The evaluation of the membrane phospholipid and protein organization was carried out in terms of asymmetric distribution across the membrane bilayer for the phospholipids, and in terms of accessibility of cleavable sites present on the outer membrane surface for the proteins. Treatment of phenylhydrazine-exposed erythrocytes either with bee venom phospholipase A2 or with trinitrobenzenesulfonic acid indicated that phosphatidylserine (PS), which is the only phospholipid not formally present on the outer leaflet of the membrane, was translocated to the outer surface of the cell membrane. The extent of this phenomenon was directly proportional to the concentration of the oxidant having a peak value at 0.1 mM. Phosphatidylcholine and phosphatidylethanolamine conserved their original distribution across the erythrocyte membrane throughout the study. The oxidant, at a dose which did not induce any modification of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis cytoskeleton membrane protein pattern, did not provoke any alteration of the membrane protein surface architecture, although the translocation of PS to the membrane outer leaflet in intact erythrocytes was present.     

Crystal structure of myoglobin form a synthetic gene

Crystal have been grown of myoglobin produced in Escherichia coli from a synthetic gene, and the structure has been solved to 1.9 Å resolution. The space group of the crystals is P6, which is different from previously solved myoglobin crystal forms. The synthetic myoglobin is essentially identical to myoglobin isolated from sperm whale tissue, except for the retention of the initiator methionine at the N-terminus and the substitution of asparagine for aspartic acid at position 122. Superposition of the coordinates of native and synthetic sperm whale myoglobins reveals only minor changes in the positions of main chain atoms and roeientation of some surface side chains. Crystals of variant of the “synthetic” myoglobin have also been grown for structural analysis of the role of key amino acid residues in ligand and specificity.     

Cadmium effects on growth and antioxidant enzymes activities in Miscanthus sinensis

Plants of Miscanthus sinensis (cv. Giganteus) were grown in hydroponics for three months in nutrient solution with 0, 2.2, 4.4 and 6.6 μM CdNO₃. Growth parameters, catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities were analysed in leaves and roots collected after 1-and 3-month exposure. Dry biomass of all miscanthus organs was affected by Cd concentration both after 1-and 3-month exposure. No visible symptoms of Cd toxicity were observed in shoots and rhizomes of plants grown in presence of Cd. In contrast, roots became shorter and thicker and the whole root system more dense and compact already after one month of treatment with 6.6 μM Cd. The lower Cd concentration increased the enzymes activities after 3 months in leaves and only after 1-month in roots, while a decrease in activity was observed at higher Cd concentrations.     

An acetylcholinesterase biosensor for determination of low concentrations of Paraoxon and Dichlorvos

The characterization of an economic and ease-to-use carbon paste acetylcholinesterase (AChE) based biosensor to determine the concentration of pesticides Paraoxon and Dichlorvos is discussed. AChE hydrolyses acetylthiocholine (ATCh) in thiocoline (TC) and acetic acid (AA). When AChE is immobilized into a paste carbon working electrode kept at +410 mV vs. Ag/AgCl electrode, the enzyme reaction rate using acetylthiocholine chloride (ATCl) as substrate is monitored as a current intensity. Because Paraoxon and Dichlorvos inhibit the AChE reaction, the decrease of the current intensity, at fixed ATCl concentration, is a measure of their concentration. Linear calibration curves for Paraoxon and Dichlorvos determination have been obtained. The detection limits resulted to be 0.86 ppb and 4.2 ppb for Paraoxon and Dichlorvos, respectively, while the extension of the linear range was up 23 ppb for the former pesticide and up to 33 ppb for the latter. Because the inhibited enzyme can be reactivated when immediately treated with an oxime, the biosensor reactivation has been studied when 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were used. TMB-4 resulted more effective. The comparison with the behavior of similar AChE based biosensors is also presented.     

Mechanisms of reoxygenation injury in myocardial infarction: implications of a myoglobin redox cycle

The addition of ascorbate to ischemic rat hearts prevents the myocardial damage associated with reoxygenation. H2O2 oxidizes myoglobin (Mb+2) to higher oxidation states (Mb+4 and Mb+5) which are rapidly reduced by ascorbate. It is proposed that the operation of a myoglobin redox cycle, in which H2O2 causes the two-electron oxidation of myoglobin, is a critical determinant of reperfusion injury. Conversely, the reduction of myoglobin, in one-electron steps, may represent an essential protective mechanism against such injury in the heart.     

Participation of tyrosine residues of myoglobin in the disproportionateness reaction of heme iron (II) and (IV)]

By means of the comparison of the constant oxidation reactions of both the myoglobin modified by N-acetylimidazole and the intact myoglobin in the presence of H2O2 or ferrylmyoglobin we characterized the role of the tyrosine residues (Tyr) of myoglobin in the synproportionation reaction between heme iron (II) of one molecule and heme iron (IV)--of another. It was demonstrated that Tyr derivatization resulted in the decrease of the velocity of redox interaction between deoxymyoglobin (II) and ferrylmyoglobin (IV) and led to the decrease of the efficiency of oxymyoglobin deoxygenation. The effects were shown to be independent on Tyr quantity in myoglobin molecule and to have the same character for both the sperm-whale myoglobin and the horse myoglobin.     

Enhanced potency of human Sonic hedgehog by hydrophobic modification

Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.     

Cell age-related monovalent cations content and density changes in stored human erythrocytes

Conversion of erythrocyte membrane protein 4.1b to 4.1a occurs through a non-enzymatic deamidation reaction in most mammalian erythrocytes, with an in vivo half-life of approximately 41 days, making the 4.1a/4.1b ratio a useful index of red cell age [Inaba and Maede, Biochim. Biophys. Acta 944 (1988) 256-264]. Normal human erythrocytes distribute into subpopulations of increasing cell density and cell age when centrifuged in polyarabinogalactan density gradients. We have observed that, when erythrocytes were stored at 4 degrees C under standard blood bank conditions, the deamidation was virtually undetectable, as cells maintained the 4.1a/4.1b ratio they displayed at the onset of storage. By measuring the 4.1a/4.1b values in subpopulations of cells of different density at various time points during storage, a modification of the normal 'cell age/cell density' relationship was observed, as erythrocytes were affected by changes in cell volume in an age-dependent manner. This may stem from a different impact of storage on the imbalance of monovalent cations, Na(+) and K(+), in young and old erythrocytes, related to their different complement of cation transporters.