alpha-Tocopherol increases caspase-3 up-regulation and apoptosis by beta-carotene cleavage products in human neutrophils.

It has been found that beta-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as alpha-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that alpha-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by alpha-tocopherol. Both alpha-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of alpha-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of alpha-tocopherol and beta-ionone, a cyclohexenyl degradation product of beta-carotene with shorter aliphatic chain.

Interaction of the polyene antibiotic etruscomycin with large unilamellar lipid vesicles: binding and proton permeability inducement.

The effect of the polyene antibiotic etruscomycin on the permeability of large unilamellar lipid vesicles was investigated. Proton leakage was induced in egg-yolk phosphatidylcholine (EPC) vesicles only when sterol was present in the membrane; the extent of leakage was limited. High etruscomycin/lipid ratios (R) were necessary (R greater than 0.1). Higher percentages of sterol increased the permeability, slightly more strongly for ergosterol than for cholesterol. Dipalmitoylphosphatidylcholine (DPPC) vesicles were more sensitive to permeability inducement, even in the absence of sterol in the bilayer (inducement for R greater than 0.06). The interactions of etruscomycin with the vesicles were examined by circular dichroism, fluorescence and 31P-NMR. In the range of antibiotic concentration where permeability was induced, R greater than 0.1 for EPC vesicles, R greater than 0.06 for DPPC vesicles, etruscomycin exhibited characteristic circular dichroism spectra independent of the presence of sterol. Under the same conditions, 31P-NMR and fluorescence studies indicated a destruction or a fusion of the vesicle bilayer. At lower etruscomycin concentrations (R less than 0.03), the etruscomycin circular dichroism spectra were different, indicating that the interaction with membranes containing ergosterol differed from that with membranes containing cholesterol. From correlating the increase in fluorescence intensity with this interaction, as well as from exchange experiments, it was inferred that etruscomycin at a low antibiotic/lipid ratio is more strongly bound to ergosterol-containing vesicles than to cholesterol-containing vesicles. These results and their comparison with the results obtained with other polyene antibiotics indicate that at low R etruscomycin resembles amphotericin rather than filipin in its preferential binding to ergosterol-containing vesicles. At higher R, that is in conditions where permeability is induced, the selectivity is different. The corresponding mechanism seems not to involve the formation of an etruscomycin-sterol channel, since the hydrophobic chain of the complex would be too short to form a channel.

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.

Inhibition of ion transport ATPases by HNE.

4-Hydroxy-2,3-trans-nonenal (HNE), a major lipid peroxidation product, has been shown to react with specific amino acid residues of proteins and alter their function. In vitro exposure of erythrocyte ghosts and neutrophil membranes to HNE results in the inhibition of ion transport ATPases. Neutrophil membrane Ca2+-ATPase is strongly inhibited by micromolar concentrations of HNE, while HNE is considerably less effective against neutrophil Mg2+-ATPase and the erythrocyte ghost enzymes.

Effect of carotenoid oxidation products on neutrophil viability and function.

Human neutrophils are short-lived cells that play important roles in host defense and acute inflammation by releasing hydrolytic and cytotoxic proteins and reactive oxygen derivatives. Apoptosis, a physiological mechanism for cell death, regulates both production and survival of neutrophils, representing a basic biological mechanism for this type of cells. Carotenoids may react with toxic oxygen metabolites released by neutrophils to form a multitude of carotenoid cleavage products that exert, in turn, relevant prooxidative biological effects. Recent data suggest that carotenoid oxidation products may affect neutrophil viability and function by exerting proapoptotic activity and interfering with superoxide production by activated cells. The prooxidant and proapoptotic activities of carotenoid oxidation products could account, at least in some cases, for the procancerogenic properties of carotenoid rich diet.

Combined effects exerted by amphotericin B and lucensomycin on cation permeability in a unilamellar vesicle system.

The permeability of artificial unilamellar vesicles and of plasma membrane vesicles from homogenized yeast in aqueous solutions of polyene antibiotics (amphotericin B and lucensomycin) was studied by measuring proton leakage by a pH-stat method. Micromolar concentrations of amphotericin B induced a remarkable proton efflux from the vesicles. Lucensomycin exerted similar effects only at 100 times higher concentrations. The latter antibiotic, at concentrations one order of magnitude lower than those necessary to induce a detectable proton efflux, seemed to protect the vesicles from the subsequent permeabilizing action of amphotericin B.

Interaction of lucensomycin with membranes: the role of non-steroidal components.

The binding of lucensomycin to unilamellar phospholipid/cholesterol vesicles and to colloidal emulsions of cholesterol in aqueous solutions was studied by monitoring the changes in the electronic absorption spectra of the polyene antibiotic. The total extent of the absorption variations was a direct function of cholesterol concentration and quite independent of the nature of the emulsion. The rate of binding, relatively slow in the colloidal systems, was greatly enhanced when cholesterol was included in phospholipid-containing membranes. The rate of lucensomycin binding to colloidal cholesterol increased with increasing cholesterol concentrations and/or stirring the heterogeneous suspension. The time course of lucensomycin binding to vesicles appeared to be independent of the concentrations of phospholipids and cholesterol.

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

1. KAR-2 (3"-(beta-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). 2. 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 microM) in the presence of Ca2+ was comparable to that of vinblastine. 3. KAR-2 was able to interact with apo-calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature. 4. 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. 5. 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.

Polyene antibiotics inhibit superoxide-producing NADPH oxidase in a polymorphonuclear cell-free system.

We studied the effect of polyene macrolide antibiotics on the NADPH-dependent superoxide production induced by arachidonic acid in a cell-free system consisting of the membrane and cytosolic fractions obtained from bovine polymorphonuclear leukocytes. Preincubation of the membrane fraction with polyenes before addition of the soluble components of the reaction mixture resulted in a dose-dependent inhibition of superoxide production.

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.

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.

Interaction of lucensomycin with cholesterol in membranes: kinetic and structural studies.

The variations of optical density and fluorescence of lucensomycin are good indices of the binding of this polyenic antibiotic to membranes. The former parameter reflects more generally the binding to any site present in the membrane, while the latter is more specific for binding to cholesterol. Equilibrium titration experiments performed in the presence of an excess of membrane-bound cholesterol suggest that lucensomycin self-associates and that the binding and polymerization sites of the antibiotic are identical or quasi-identical; hence polymerization leads to a loss of binding sites and vice versa. The non-steroidal components of the membrane (such as proteins and lipids) seem to affect the rate of the individual reaction leading to the formation of the cholesterol-lucensomycin complexes, rather than the ratio among these heterologous aggregates at equilibrium. Polyene concentrations, which induce detectable proton release from unilamellar vesicles, are at least two orders of magnitude higher than those necessary to perform a spectroscopic titration of membrane cholesterol.

Inhibition of erythrocyte ghost ATPase by polyene antibiotics.

The effect of micromolar concentrations of polyene antibiotics on erythrocyte ghost ATPase activities has been studied. (Mg2+)-ATPase is inhibited by amphotericin B and amphotericin B methyl ester, whereas (Na+ + K+ + Mg2+)-ATPase is inhibited by amphotericin B and lucensomycin. (Ca2+ + Mg2+)-ATPase is only slightly affected by polyene antibiotics.

Sheep ceruloplasmin: isolation and characterization.

Ceruloplasmin 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 range 5-8 with a pH optimum for activity of 6.3.