Elisabetta Capuozzo

High sensitivity of plasma membrane ion transport ATPases from human neutrophils towards 4-hydroxy-2,3-trans-nonenal.

Lipid peroxidation results in release of 4-hydroxy-2,3-trans-nonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. The effect of HNE on the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, and calmodulin-stimulated Ca(2+)-ATPase has been studied both in erythrocyte ghosts and in neutrophil membrane preparations. Neutrophil Ca(2+)-ATPase was strongly inhibited by micromolar concentrations of HNE (IC(50) = 12 microM), that means in the range of pathophysiologically relevant HNE levels. The IC(50) value for neutrophil Na(+)/K(+)-ATPase was about 40 microM. HNE was considerably less effective against neutrophil Mg(2+)-ATPase and the erythrocyte ghost enzymes (IC(50) values range from 91 to 240 microM). The data suggest that HNE may play a specific role in the regulation of neutrophil calcium homeostasis in response to oxidative stress.

Inhibition of NADPH Oxidase-Mediated Superoxide Radical Formation in PMA-Stimulated Human Neutrophils by 4-Hydroxynonenal- Binding to -SH and -NH2 Groups'

4-Hydroxynonenal (HNE), a major lipid peroxidation product, effectively inhibits the superoxide radical formation by NADPH oxidase of phorbol myristate acetate (PMA)--stimulated human PMNL. The I50 value for the inhibition of NADPH oxidase-mediated superoxide radical formation by 4-hydroxynonenal was found to be 19 microM. The HNE inhibition involves the reaction with both -SH and -NH2 groups. Superoxide formation as final result of the NADPH oxidase cascade was almost completely restored by addition of dithiothreitol. In presence of hydroxylamine only a minor restoration of superoxide radical formation was found. A combination of dithiothreitol and hydroxylamine yielded the greatest recovery. Two other aldehydes with the same chain length as HNE but different binding to lysine, histidine and cysteine residues, trans-2,3-nonenal and nonanal, gave I50 values for the inhibition of NADPH oxidase-mediated superoxide formation rate of 110 microM or > 300 microM, respectively.

Carotenoid cleavage products modify respiratory burst and induce apoptosis of human neutrophils.

Carotenoid supplementation in the treatment of diseases associated with oxidative stress has been recently questioned because of the cell damage and the increased risk of lung cancer in male smokers. Because of the complex role of neutrophils in lung diseases, we investigated whether carotenoid derivatives could affect respiratory burst and apoptosis of human neutrophils purified from peripheral blood. Stimulation of superoxide production was induced by nanomolar and micromolar concentrations of carotenoid cleavage products with aliphatic chains of different length, but not by carotenoids lacking the carbonyl moiety. The stimulatory effect of carotenoid cleavage products was observed in cells activated by phorbol myristate acetate (PMA), while a slight inhibition of superoxide production was noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid cleavage products inhibited superoxide production in the presence of both PMA and f-MLP. In the presence of 20 microM carotenoid cleavage products, inhibition of superoxide production was accompanied by DNA fragmentation and increased level of intracellular caspase-3 activity.

Metabolism of 4-hydroxy-2-nonenal in human polymorphonuclear leukocytes.

Intracellular metabolism of 4-hydroxy-2-nonenal (HNE), a major product and mediator of oxidative stress and inflammation, is analyzed in resting and fMLP-stimulated human polymorphonuclear leukocytes (PMNL), where this compound is generated during activation of the respiratory burst. HNE consumption rate in PMNL is very low, if compared to other cell types (rat hepatocytes, rabbit fibroblasts), where HNE metabolism is always an important part of secondary antioxidative defense mechanisms. More than 98% of HNE metabolites are identified. The pattern of HNE intermediates is quite similar in stimulated and resting PMNL - except for higher water formation in resting PMNL - while the initial velocity of HNE degradation is somewhat higher in resting cells, 0.44 instead of 0.28 nmol/(min×10(6) cells). The main products of HNE metabolism are 4-hydroxynonenoic acid (HNA), 1,4-dihydroxynonene (DHN) and the glutathione adducts with HNE, HNA, and DHN. Protein-bound HNE and water account for about 3-4% of the total HNE derivatives in stimulated cells, while in resting cells protein-bound HNE and water are 4% and 20%, respectively. Cysteinyl-glycine-HNE adduct and mercapturic acids contribute to about 5%.

The interaction of a new anti‐tumour drug, KAR‐2 with calmodulin

KAR‐2 (3′′‐(β‐chloroethyl)‐2′′,4′′‐dioxo‐3,5′′‐spiro‐oxazolidino‐4‐deacetoxy‐vinblastine) is a semisynthetic bis‐indol derivative, with high anti‐microtubular and anti‐tumour activities but with low toxicity. KAR‐2, in contrast to other biologically active bis‐indols (e.g. vinblastine), did not show anti‐calmodulin activity in vitro (enzyme kinetic, fluorescence anisotropy and immunological tests). Direct binding studies (fluorescence resonance energy transfer, circular dichroism) provided evidence for the binding of KAR‐2 to calmodulin. The binding affinity of KAR‐2 to calmodulin (dissociation constant was about 5 μM) in the presence of Ca2+ was comparable to that of vinblastine. KAR‐2 was able to interact with apo‐calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature. The effect of drugs on Ca2+ homeostasis in human neutrophil cells was investigated by means of a specific fluorescent probe. Trifluoperazine extensively inhibited the elevation of intracellular Ca2+ level, vinblastine did not appreciably affect it, KAR‐2 stimulated the Ca2+ influx and after a transient enhancement the Ca2+ concentration reached a new steady‐state level. Comparison of the data obtained with KAR‐2 and bis‐indols used in chemotherapy suggests that the lack of anti‐calmodulin potency resides on the spiro‐oxazolidino portion of KAR‐2. This character of KAR‐2 manifested itself in various systems and might result in its low in vivo toxicity, established in an anti‐tumour test.

Effects of calmodulin antagonists on calcium pump and cytosolic calcium level in human neutrophils.

The concentration of cytosolic free calcium was monitored in suspensions of intact human neutrophils in phosphate-buffered saline by means of the fluorescent indicator Indo 1 trapped in the cytosol. Trifluoperazine and n-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide markedly reduced the amplitude of the transient increase in cytosolic Ca2+ triggered by CaCl2 as well as by N-formyl-methionyl-leucyl-phenylalanine. The effect of the calmodulin antagonists on the calcium burst observed upon cell activation was much more pronounced in the presence of extracellular free calcium than in EGTA-containing media; it was not inhibited by wortmannin or thapsigargin. Nevertheless, trifluoperazine and n-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited the plasma-membrane Ca2+ ATPase if added to plasma membrane-enriched fractions of neutrophils. These results suggest that calmodulin antagonists affect calcium ion influx even if they inhibit plasma membrane Ca2+ ATPase.

Sheep ceruloplasmin: isolation and characterization.

SummaryCeruloplasmin has been isolated from sheep plasma by a procedure involving two chromatographic steps and (NH4)2SO4 fractionation.The ovine protein is similar to ceruloplasmins from other species previously described (human, bovine), having a single chain of about 125 Kdal with a very high degree of homology in the amino acid composition. It differs, however, from human and bovine ceruloplasmin because of its lower copper content and its higher specific enzyme activity.The oxidase activity as well as the spectroscopic properties were found to be pH sensitive in the pH range 5–8 with a pH optimum for activity of 6.3.

Thiotaurine Prevents Apoptosis of Human Neutrophils: A Putative Role in Inflammation

Thiotaurine, a metabolic product of cystine, contains a sulfane sulfur atom that can be released as H(2)S, a gaseous molecule with a regulatory activity on inflammatory responses. The influence of thiotaurine on human leukocyte spontaneous apoptosis has been evaluated by measuring caspase-3 activity in human neutrophils. Addition of 100 μM thiotaurine induced a 55% inhibition of caspase-3 activity similar to that exerted by 100 μM H(2)S. Interestingly, in the presence of 1 mM GSH, an increase of the inhibition of apoptosis by thiotaurine has been observed. These results indicate that the bioactivity of thiotaurine can be modulated by GSH, which promotes the reductive breakdown of the thiosulfonate generating H(2)S and hypotaurine. As thiotaurine is able to incorporate reversibly reduced sulfur, it is suggested that the biosynthesis of this thiosulfonate could be a means to transport and store H(2)S.

Formation and ways of detecting advanced glycation end-products in isolated human glomerular basement membrane and human serum albumin nonenzymatically glycated in vitro

The formation of advanced glycation end-products (AGE) was investigated in samples of isolated human glomerular basement membrane (hGBM) and human serum albumin (hSA) which had been nonenzymatically glycated in vitro. In order to measure AGE, two methods which differ in principle--the standard spectrofluorescence technique and the spectrophotometric diazonium salt reaction--have been used and compared. Samples of finely homogenized hGBM and hSA were incubated for 10 days in buffer containing 500 mmol/L (9 x 10(3) mg%) and 100 mmol/L (1.8 X 10(3) mg%) D-glucose respectively. At the end of the incubation period, the ambient glucose was removed and the samples were incubated for a further 10 days in glucose-free buffer. During this time, loosely bound sugar was released into the buffer; at the end of the incubation, the emission fluorescence at 440 nm (following continuous excitation at 370 nm) and the absorbance at 492 nm of the glycated hGBM and hSA samples were measured and found to be significantly increased by comparison with native samples (1-way ANOVA: p less than 0.05 with both techniques). Comparison of the two techniques used for AGE detection showed a positive linear correlation (Pearsons correlation coefficient r = 0.714; n = 8; p = 0.02). The released glucose probably originates from reversal of the Schiff base (the first and reversible step of the nonenzymatic glycation reaction), whereas fluorescence and photometric findings prove the presence of stable AGE on both hGBM and hSA. It is concluded that AGE can indeed be formed and detected by two different methods in hGBM (and hSA) subjected to nonenzymatic glycation in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation of Hypotaurine and Cysteine Sulfinic Acid by Peroxidase-generated Reactive Species

Hypotaurine (HTAU) and cysteine sulfinic acid (CSA) are the metabolic intermediates in the mammalian pathway leading from cysteine to taurine. Strong evidence has been presented that the formation of taurine (TAU) and cysteic acid (CA) is the result of the interaction of both sulfinates with various oxidizing agents that may be present in biological systems. The purpose of the present study is to investigate the oxidation of sulfinates, HTAU and CSA, by peroxidase-generated reactive species. Reactive nitrogen and oxygen species can be produced during the process of nitrite oxidation catalyzed by heme peroxidases, such as horseradish peroxidase (HRP) or myeloperoxidase, in the presence of hydrogen peroxide (H2O2). Nitrite is the major end product of nitric oxide (NO) metabolism. Oxidation of nitrite by such mechanisms could be important at sites of inflammatory processes. The formation of reactive nitrogen species (RNS) via peroxidase-catalyzed oxidation of nitrite could represent an additional mechanism of the formation of taurine. The oxidation of the sulfinates, HTAU and CSA, by HRP/H2O2/nitrite system has been evaluated by monitoring the oxygen consumption and the production of the corresponding sulfonates, TAU and CA. Moreover, the effect of HTAU and CSA, on nitrotyrosine formation by HRP/H2O2/nitrite system has been studied. During the HRP/H2O2-dependent oxidation of tyrosine, tyrosyl radicals are formed. The results provide evidence that sulfinates inhibit nitrotyrosine formation not only by scavenging RNS but also by reducing the peroxidase-generated tyrosyl radical.