Biochemical analysis of the molecular mechanism of action of natural integrin inhibitors.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
M A Belisario
Publications and source records attributed to M A Belisario.
Site-specific atherosclerotic plaques in the carotid arteries of middle-aged women from southern Italy: associations with traditional risk factors and oxidation markers.
BACKGROUND AND PURPOSE: Studies on cardiovascular disease have preferentially involved men because of the lower frequency of the disease in preelderly women. The aim of this analysis was to examine, with the use of a standardized ultrasound protocol, a cohort of women to differentiate early atherosclerotic lesions in different carotid segments in relation to traditional (lipoprotein abnormalities, high blood pressure, cigarette smoking) and nontraditional (oxidation markers) cardiovascular risk factors. METHODS: More than 5000 clinically healthy, middle-aged women (n=5062; age range, 30 to 69 years) living in the area of Naples in southern Italy participated in the Progetto Atena, a population-based study on the etiology of cardiovascular disease and cancer in the female population. A subsample of 310 participants underwent high-resolution B-mode ultrasound to assess intima-media thickness of common carotid artery and carotid bifurcation. RESULTS: Early atherosclerotic plaques (intima-media thickness >1.2 mm) were detected within the common carotid arteries in 37 women, in the carotid bifurcations in 77 women, and in both sites in 91 women. After age adjustment, common carotid plaques were found to be associated with higher systolic blood pressure (143 versus 138 mm Hg; P<0.05) and higher body mass index (29 versus 27 kg/m(2); P<0.01), while lesions at the carotid bifurcations were associated with higher LDL cholesterol (4.3 versus 3.8 mmol/L; P<0.01) and with smoking habit. Multivariate odds ratios for the presence of common carotid plaques were related to antibodies against oxidized LDL (odds ratio, 2.72; 95% CI, 1.46 to 5.07), and those for plaques at the bifurcation were related to lipid peroxides (odds ratio, 1.90; 95% CI, 1.04 to 3.47), and both relationships were independent of age, LDL cholesterol concentrations, body mass index, smoking habit, and systolic blood pressure. CONCLUSIONS: In a cohort of clinically healthy, middle-aged women, we found a site-specific association of traditional risk factors and oxidation markers with early atherosclerotic lesions in arterial segments differing in geometry, shear stress, extracellular matrix composition, and cell type populations.
Adult↗
Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation.
Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet alpha(IIb)beta(3) binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of alpha(IIb)beta(3) conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADP-activated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72(syk) and pp125(FAK). Inhibitors of platelet signalling, such as apyrase, prostaglandin E(1), cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125(FAK) phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125(FAK) phosphorylation.
Adenosine Diphosphate↗
H(2)O(2) activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation.
Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H(2)O(2) concentrations on platelet adhesion to immobilized fibrinogen and on pp72(syk) and pp125(FAK) tyrosine phosphorylation. Our results demonstrate that: (1) H(2)O(2) does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H(2)O(2) does not affect pp72(syk) phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H(2)O(2) reduces, in a dose-dependent fashion, pp125(FAK) phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H(2)O(2) near to physiological values (10-12 microM) are able to strengthen the subthreshold activation of pp125(FAK) induced by epinephrine in apyrase-treated platelets; (5) H(2)O(2) doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H(2)O(2) to modulate pp125(FAK) phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation.
Apyrase↗
Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK-paxillin interaction in fibronectin-adherent melanoma cells.
Echistatin, a snake-venom RGD-containing protein, was previously shown to disrupt cell-matrix adhesion by a mechanism that involves the reduction of pp125FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125FAK dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskeleton and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125FAK. One hour of cell exposure to echistatin caused a 39% decrease of pp125FAK Tyr397 phosphorylation and a 31% reduction of pp125FAK autophosphorylation activity as measured by immune-complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125FAK. Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo.
Animals↗
The mycotoxin fumonisin B1 inhibits integrin-mediated cell-matrix adhesion.
Fumonisin B1 (FB1), a mycotoxin produced by the corn fungus Fusarium moniliforme, causes a variety of animal diseases and is a suspected human carcinogen. The FB1 molecule bears remarkable structural resemblance to the long-chain sphingoid base backbones of sphingolipids. The toxicity and carcinogenicity of FB1 has been ascribed to its ability to inhibit ceramide synthase, a key enzyme in the metabolism of complex sphingolipids. In this study we have investigated whether the exposure of B16-BL6 mouse melanoma cells to FB1 affects cell growth and integrin-mediated cell matrix adhesion. Cell treatment with the highest tested dose (75 microM) of FB1 for 72 h induced an about 20% inhibition of cell growth. FB1 strongly affected B16-BL6 cell adhesion to immobilized fibronectin, by causing a dose-dependent inhibition of cell attachment to this substrate. FB1 also inhibited in a dose-dependent manner the adhesion of B16-BL6 cells to the immobilized anti-fibronectin receptor antibody, whereas it affected only to a low extent cell attachment to concanavalin A. Our results demonstrate that FB1 treatment alters integrin adhesive activity, thus affecting all cellular integrin-dependent functions.
Animals↗
Species-dependent specificity of platelet aggregation inhibitors from snake venom.
Explore the source record for details and available documents.
Adenosine Diphosphate↗
Croton ruizianus: platelet proaggregating activity of two new pregnane glycosides.
The MeOH extract of the aerial parts of Croton ruizianus afforded two new pregnane glycosides 1 and 2, together with the morphinandienone alkaloids flavinantine (3) and O-methylflavinantine (4). Their structures were elucidated by NMR experiments including 1H-1H (1D TOCSY and 2D DQF-COSY) and 1H-13C (HSQC, HMBC) spectroscopy. The proaggregating activity of the MeOH extract and the isolates were evaluated. Although the MeOH extract and pregnane glycosides (at different doses) were found to promote platelet aggregation, flavinantine (3) and O-methylflavinantine (4) showed only slight activity. The ability of the MeOH extract and the four compounds to act synergistically with thrombin was also evaluated. All the tested compounds were successful in augmenting the aggregating effect of thrombin, although to different degrees.
Blood Platelets↗
Effect of N-acetyl-cysteine on lymphomonocyte glutathione and response to interferon treatment in C-virus chronic hepatitis.
BACKGROUND/AIM: Much controversy exists concerning effect of N-acetyl-cysteine, a precursor of glutathione, on the response to interferon treatment in patients with C-virus chronic hepatitis. The aim of this study was to evaluate the efficacy of interferon therapy with and without oral N-acetyl-cysteine. We also measured glutathione concentrations in lymphomonocytes of 25 patients with chronic C-virus hepatitis before and after interferon treatment and correlated the results with treatment response. METHODS: Glutathione was extracted from lymphomonocytes and measured with a modified high performance liquid chromatographic method in the 25 hepatitis patients and 12 healthy controls. RESULTS/CONCLUSIONS: 1) Hepatitis patients and controls had similar basal concentrations of lymphomonocytic glutathione; 2) neither interferon nor N-acetyl-cysteine significantly affected glutathione concentrations in patients; and 3) N-acetyl-cysteine did not affect response to interferon.
Acetylcysteine↗
Metal-ion catalyzed oxidation affects fibrinogen activity on platelet aggregation and adhesion.
Exposure of fibrinogen to the Fe3+/ascorbate oxidative system resulted in structural modifications and altered functionality of the glycoprotein. The overnight treatment of fibrinogen by oxidants caused a 20-fold increase of carbonyl content with respect to the native protein. Formation of dityrosines as well as loss of tryptophan following fibrinogen oxidation were observed. The occurrence of conformational changes of the fibrinogen molecule as a consequence of the oxidative treatment was also established. Oxidized fibrinogen showed a distinct capability from the native molecule to mediate platelet aggregation and adhesion. The percentage of ADP-induced platelet aggregation decreased as a function of fibrinogen oxidative damage. Further, both unstimulated platelets and ADP-activated platelets showed a reduced ability to adhere to oxidized fibrinogen than to the native protein. These results suggest that oxidative treatment alters fibrinogen domains involved in the recognition and the binding of this molecule by the platelet receptor GP IIb/IIIa.
Ascorbic Acid↗
Echistatin inhibits pp72syk and pp125FAK phosphorylation in fibrinogen-adherent platelets.
The adhesion of ADP-stimulated platelets to immobilized fibrinogen induces the tyrosine phosphorylation of multiple proteins which include pp72syk and pp125FAK. The phosphorylation of these two proteins increases as function of time of platelet adhesion to fibrinogen; however, pp72syk results strongly phosphorylated already after 15 min, whereas pp125FAK reaches high levels of phosphorylation after 1 h of platelet adhesion. Phosphorylation of both proteins is only slightly detectable when platelets are held in suspension or when platelets are allowed to adhere to bovine serum albumin, a non-specific substrate. Echistatin, an Arg-Gly-Asp (RGD)-containing snake-venom protein, affects protein tyrosine phosphorylation promoted by platelet adhesion to fibrinogen, by causing an approximately 44% and 39% decrease of pp72syk and pp125FAK phosphorylation, respectively. The interaction of echistatin with fibrinogen receptor glycoprotein IIb-IIIa on platelet surface might be responsible for the block of integrin-mediated signaling cascade, including pp72syk and pp125FAK inactivation.
Blood Platelets↗
Vitamin E nutritional status and serum lipid pattern in normal weanling rabbits.
In a 6-wk postweaning trial (Trial 1), 30 male New Zealand White rabbits were fed a starter diet with three different levels of all-rac-alpha-tocopheryl acetate (60, 160, or 260 mg/kg diet). Plasma concentrations of alpha-tocopherol and serum concentrations of total cholesterol, high density lipoprotein (HDL) cholesterol, and triglycerides were measured at 2, 4, and 6 wk. In a 20-wk postweaning trial (Trial 2), 48 male New Zealand White rabbits were divided into two groups; one group was fed a vitamin E-free diet, and the other was fed the same diet supplemented with 60 mg/kg of all-rac-alpha-tocopheryl acetate. Plasma concentrations of alpha-tocopherol and serum total cholesterol, HDL cholesterol, and triglycerides were measured at 2, 4, 6, and 20 wk. In Trial 1, split-plot ANOVA showed that dietary all-rac-alpha-tocopheryl acetate increased plasma alpha-tocopherol (P < .01); in addition, serum total cholesterol and triglycerides declined more rapidly (P < .01) and HDL cholesterol increased more rapidly (P < .005) in rabbits fed supplemental all-rac-alpha-tocopheryl acetate than in controls. The all-rac-alpha-tocopheryl acetate intake was significantly correlated with serum total cholesterol reduction and HDL cholesterol increase only when plasma alpha-tocopherol was relatively low (< 23.5 mumol/ L). In trial 2, the vitamin E-free diet caused an increase (P < .05) in total serum cholesterol and a reduction in HDL cholesterol (P < .05 at 6 wk and P < .01 at 20 wk). The overall results of this study suggest that vitamin E plays an important role in the regulation of serum concentrations of cholesterol and lipoproteins in weanling rabbits. Consequently, the maintenance of an adequate nutritional status of vitamin E in the postweaning period is important to avoid alterations of serum lipid pattern.
Aging↗
Echistatin induces decrease of pp125FAK phosphorylation, disassembly of actin cytoskeleton and focal adhesions, and detachment of fibronectin-adherent melanoma cells.
B16-BL6 mouse melanoma cells cultured on fibronectin-coated dishes were detached by treatment with echistatin, an RGD-containing disintegrin. Echistatin was active at micromolar concentrations and was not cytotoxic. Its effect was dose-dependent and reversible. Sequential morphological changes leading to rounding up of the cells were detected by phase-contrast microscopy and by immunofluorescence analysis. A dramatic reduction in the number and size of focal adhesions and loss of cytoplasmic actin filaments were observed well before cell detachment occurred. Echistatin treatment down-regulated the phosphorylation of pp125FAK in fibronectin-adherent cells in a dose- and time-dependent fashion. The reduction of pp125FAK phosphorylation preceded cell detachment and occurred even in the presence of orthovanadate, an inhibitor of protein tyrosine phosphatases. These results suggest that echistatin detaches cells from the fibronectin substratum by inducing a decrease of pp125FAK phosphorylation and that echistatin acts by inhibiting protein tyrosine kinase activity rather than activating protein tyrosine phosphatases.
Actin Cytoskeleton↗
Erythrocyte enzymes catalyze 1-nitropyrene and 3-nitrofluoranthene nitroreduction.
Nitroarenes are environmental contaminants produced during incomplete combustion processes. Nitroreduction, the most important pathway of nitroarene toxification, occurs mainly in the liver and intestine. In the present study, we show that human red cells may also possess the metabolic competence to reduce 1-nitropyrene (NP) and 3-nitrofluoranthene (NF), the nitroarenes chosen as model compounds, to their corresponding amino derivatives, 1-aminopyrene (AP) and 3-aminofluoranthene (AF). The requirement of the cofactor couple NADH/FMN suggests that erythrocyte nitroreductase activity occurs via one electron transfer. The presence of oxygen strongly inhibited the haemolysate-catalyzed nitroarene reduction, whether measured as amine formation or nitroarene disappearance. Intermediate reactive species, that bind covalently to haemoglobin and/or other erythrocyte proteins, are formed during nitroreduction catalyzed by human haemolysate. In fact, the reduced metabolites AP and AF were released after mild acid hydrolysis of red cell proteins exposed to NP and NF, thus suggesting that sulphinamide adducts have been formed.
Biotransformation↗
In vitro effect of avarone and avarol, a quinone/hydroquinone couple of marine origin, on platelet aggregation.
We investigated the effect on human platelet aggregation of the naturally occurring quinone/hydroquinone couple, avarone and avarol. Avarone exerted antiplatelet activity both on platelet-rich plasma and, to a greater extent, on washed platelets. The quinone inhibited the platelet aggregatory process with all the agonists used. The highest inhibitory potency occurred with arachidonic acid or A23187 as stimulating agents. In the case of agonists such as adenosine 5' diphosphate, platelet-activating factor or U46619, the antiaggregatory effect was more pronounced on the second wave. Inhibition of the aggregatory process paralleled thromboxane B2 formation. Avarol also exerted antiplatelet activity, even though its inhibitory potency was much lower than that of avarone.
Adenosine Diphosphate↗
Arylation of sulfhydryl groups in vitro by the naturally occurring sesquiterpenoid benzoquinone avarone.
Avarone (AQ) is a naturally occurring sesquiterpenoid benzoquinone possessing antileukaemic activity. Its reactivity towards glutathione (GSH) and protein sulfhydryl (SH) groups was investigated. The stoichiometry of AQ reaction with GSH at [GSH]/[AQ] ratios lower than unity proved to be 1:2 (thiol:quinone), consistent with the formation of the corresponding hydroquinone (avarol) as well as a quinone-thioether in the reaction. Conversely, when the [GSH]/[AG] ratio was higher than unity, a hydroquinone-thioether was the only reaction product. AQ/protein interaction was also investigated by using bovine serum albumin (BSA) as model compound. As observed with GSH, arylation rather than oxidation of SH groups appeared to be the mechanism responsible for the AQ-induced depletion of protein SH groups. However, AQ proved to be less effective in depleting BSA sulfhydryls than that of GSH. AQ disappearance after BSA addition was greater than expected on the basis of the total SH groups depleted, if a stoichiometric ratio 1:2 (thiol:quinone) was assumed. It also occurred in the presence of BSA with blocked SH groups, thus suggesting that AQ may react with other nucleophilic protein residues, such as amino or imino groups. When HepG2 cells were exposed to AQ, depletion of both protein SH groups and GSH occurred. However, in contrast to the above, AQ proved to be more effective, probably because of its lipophilic nature, in depleting protein SH groups than GSH. Also, in intact cells AQ appeared to arylate both SH and other nucleophilic groups in proteins. This mechanism may play a major role in AQ-induced cytotoxicity.
Animals↗
Simultaneous evaluation of vitamins A and E in human plasma by normal phase HPLC.
The production of toxic oxidants such as H2O2; OH. and O2.- which follows aerobic metabolism is highly enhanced in aging, alcohol consumption and in a variety of degenerative diseases including neoplasias. Among the different antioxidant agents that in human organisms counterbalance the oxidative stress, which damages lipid, protein and DNA, are included beta-carotene, alpha-tocopherol, glutathione, ascorbate, ubiquinol and specific enzymes such as superoxide dismutase, catalase and glutathione peroxidase. In particular, vitamins A and E are the most important lipid-soluble antioxidants in human tissues protecting polyunsaturated fatty acid from peroxidation. In this paper we report an improved method, obtained after modification of a normal phase HPLC method, which allows the simple and rapid simultaneous evaluation of vitamins A and E in plasma. The separation of vitamins is achieved by eluting the HPLC column with a linear gradient from 1 to 5% isopropanol in n-hexane. The eluate is simultaneously monitored by U.V. and fluorescence detection to identify and quantify vitamins A and E, respectively.
Chromatography, High Pressure Liquid↗
Effect of avarol and avarone on in vitro-induced microsomal lipid peroxidation.
Lipid peroxidation was employed as an experimental model to study the antioxidant properties of avarol, a sesquiterpenoid hydroquinone and of its quinone, avarone. In the NADPH- or ascorbate-linked lipid peroxidation, avarol and avarone were shown to be more effective as inhibitors than in the t-BuOOH-dependent peroxidative process. However, in all three systems employed avarol was a more powerful inhibitor than avarone. The chemical structure of avarol, having an easily donatable hydrogen atom and its kinetics of inhibition suggested that the hydroquinone acted mainly as a radical scavenger. Conversely avarone appeared to interfere mainly with the initiation phase of lipid peroxidation. However, avarol and the semiquinone intermediate may contribute to the inhibitory action of the quinone. In fact avarone reduction to avarol has been shown to occur in the presence of reducing agents such as ascorbate or Fe(II) and to be catalyzed by NADPH-supplemented microsomes.
Animals↗