Biomedical subjects

H Karoui

Publications and source records attributed to H Karoui.

At least 19 recordsLinked to original sources

PubMed2003

Molecular cloning and nucleotide sequence analysis of encoded anti-insect toxin BotIT2 from the scorpion Buthus occitanus tunetanus venom.

Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.

Amino Acid Sequence↗

PubMed2002

[In vivo and in vitro protection against lethal activity of Buthus occitanus tunetanus venom with a recombinant protein].

We report the use of recombinant scorpion toxin in the form of fusion protein as antigen for mice immunisation. The aim is to produce protective antisera against lethal activity of the venom from Tunisian scorpion Buthus occitanus tunetanus, responsible for several annually reported human cases of scorpion stings. The gene encoding Bot III (the most toxic alpha toxin of Buthus occitanus tunetanus) was fused to the sequence encoding synthetic ZZ domains of staphylococcal protein A. The construct ZZ-Bot III was expressed in the periplasm of E. coli as a fusion protein and purified by affinity chromatography. The recombinant fusion protein was characterized and used as antigen to generate antibodies in mice. The antibodies against the recombinant protein neutralize the toxic venom (10 LD50/ml) and also confer protection for immunized mice against antigenically related mammal toxins.

Animals↗

PubMed2001

Synthesis and biochemical applications of a solid cyclic nitrone spin trap: a relatively superior trap for detecting superoxide anions and glutathiyl radicals.

A novel cyclic nitrone spin trap, 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) as a pure white solid has been synthesized for the first time. BMPO offers several advantages over the existing spin traps in the detection and characterization of thiyl radicals, hydroxyl radicals, and superoxide anions in biological systems. The corresponding BMPO adducts exhibit distinct and characteristic electron spin resonance (ESR) spectral patterns. Unlike the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-derived superoxide adduct, the BMPO superoxide adduct does not non-enzymatically decompose to the BMPO hydroxyl adduct. This feature is clearly perceived as a definite advantage of BMPO in its biological applications. In addition, the ESR spectrum of the BMPO glutathionyl adduct (BMPO/*SG) does not fully overlap with the spectrum of its hydroxyl adduct. This spectral feature is again distinctly different from that of DMPO because the ESR spectral lines of DMPO glutathionyl and hydroxyl radical adducts largely overlap. Finally, the ESR spectra of BMPO-derived adducts exhibit a much higher signal-to-noise ratio in biological systems. These favorable chemical and spectroscopic features make BMPO ideal for the detection of superoxide anions, hydroxyl and thiyl radicals in biochemical oxidation and reduction.

Cyclic N-Oxides↗

PubMed2001

Amino acid structure and characterization of a heterodimeric disintegrin from Vipera lebetina venom.

A heterodimeric disintegrin designed as lebein was isolated from crude Vipera lebetina venom using gel filtration, anion and cation exchange chromatographies on FPLC. The amino acid sequence of each subunit determined by Edman degradation contains 64 residues with ten half-cystines and an RGD site at the C-terminal part of the molecule. The molecular mass of native lebein determined by mass spectrometry was found to be 14083.4 Da and those of alpha and beta subunits were 6992.05 and 7117.62, respectively. These value are in good agreement with those calculated from the sequences. This protein strongly inhibits ADP induced platelet aggregation on human platelet rich plasma with IC(50)=160 nM. Sequences of this protein subunits displayed significant sequence similarities with many other monomeric and dimeric disintegrins reported from snake venoms. We identified an amino acid residue (N) in the hairpin loop of both subunits (CNRARGDDMNDYC) which is different from all other reported motifs of disintegrins and this subtle difference may contribute to the distinct affinities and selectivities of this class of proteins.

Amino Acid Sequence↗

PubMed2000

Amino acid sequence of VlF: identification in the C-terminal domain of residues common to non-hemorrhagic metalloproteinases from snake venoms.

The complete amino acid sequence of a non-hemorrhagic fibrino(geno)lytic enzyme (VlF) isolated from Vipera lebetina venom has been determined. VlF was subjected to separate enzymatic and chemical digestions. Resulting fragments were purified by RP-HPLC and subjected for sequencing by automated Edman degradation. The amino terminus of VlF was determined by mass spectrometry. VlF was shown to be composed of 202 residues having a relative molecular mass of 22,826 Da and containing a zinc-binding site and a catalytically active residue. It displayed significant sequence similarities with many other mature metalloproteinases reported from snake venoms. Sequence comparison of hemorrhagic and non-hemorrhagic mature metalloproteinases revealed the presence at the C-terminal part of the enzymes of two residues common to only hemorrhagic metalloproteinases and two others shared by only non-hemorrhagic ones.

Amino Acid Sequence↗

PubMed2000

Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications.

We describe the synthesis and biological applications of a novel nitrogen-15-labeled nitrone spin trap, 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide ([(15)N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min(-1)) and NADPH/calcium-calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5'-dimethyl-1-pyrroline N-oxide (DMPO). Unlike DMPO-superoxide adduct (DMPO-OOH), the superoxide adduct of EMPO (EMPO-OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide is more persistent than EMPO-OOH, the electron spin resonance spectra of [(14)N]EMPO-OOH and [(15)N]EMPO-OOH are less complex and easier to interpret. Potential uses of [(15)N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.

Calmodulin↗

PubMed2000

Purification and characterization of a growth factor-like which increases capillary permeability from Vipera lebetina venom.

We have investigated the effect of Vipera lebetina venom on capillary permeability and isolated an increasing capillary permeability protein (ICPP) which is devoid of arginine ester hydrolase and phospholipase A2 activities. This protein was purified with a yield of about 0.2% by fast protein liquid chromatography (FPLC) using successively Superose 12, Mono Q, and Mono S columns and by high-pressure liquid chromatography (HPLC) on a C8 reverse-phase column. The purified protein migrated on SDS-PAGE as a band of about 27 kDa under nonreducing conditions and as a band of about 16 kDa under reducing conditions. Chromatography on a C8 column of reduced and alkylated protein yielded a single peak suggesting that this protein is homodimeric. This protein was refractory to Edman degradation chemistry. We used successfully a chemical unblocking involving the incubation of the protein with HCl in anhydrous methanol. The N-terminal amino acid sequence clearly shows considerable similarity to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).

Amino Acid Sequence↗

PubMed2000

Purification, characterization and molecular modelling of two toxin-like proteins from the Androctonus australis Hector venom.

Two toxin-like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross-reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins. In order to study structure-function relationships, their complete amino-acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three-dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion alpha-toxins specific to voltage dependent Na+ channels. The models showed also that amino-acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.

Amino Acid Sequence↗

PubMed1999

Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase.

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.

Animals↗

PubMed1999

Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase.

NOS is a ubiquitous enzyme that has an oxygenase and reductase activity. NOS reduces electron acceptors, at the reductase domain, by a one-electron mechanism that is not inhibited by SOD. One example of this activity is the direct reduction of ferricytochrome c by nNOS. Redox cycling electron acceptors (EA in Scheme 1), such as lucigenin and NBT, are reduced by NOS to generate an intermediate radical (EAred). This radical can then be reoxidized to the parent compound by oxygen, and in the process generate superoxide. Consequently, both NBT and lucigenin will enhance NADPH-dependent superoxide generation in the presence of flavoprotein reductases such as NOS. The artificial generation of superoxide from lucigenin and NBT is a major pitfall in the use of these compounds as superoxide probes. We conclude that the use of ESR spin-trapping techniques, although not free of problems, is a viable technique for the detection and quantification of superoxide in systems containing nNOS.

Acridines↗

PubMed1999

Effect of redox-active drugs on superoxide generation from nitric oxide synthases: biological and toxicological implications.

In this article, we address the mechanism of superoxide formation from constitutive nitric oxide synthases (NOS). Merits and drawbacks of the various superoxide detection assays are reviewed. One of the most viable techniques for measuring superoxide from NOS is electron spin resonance (ESR) spin-trapping using a novel phosphorylated spin trap. Implications of superoxide and peroxynitrite formation from NOS enzymes in cardiovascular and cerebrovascular disorders are discussed.

Animals↗

PubMed1998

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors.

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

Animals↗

PubMed1998

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2.

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5, 5'-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/.OH formation from the SOD and SOD mutants. The relative ratios of DMPO/.17OH and DMPO/.16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/.17OH and 37% DMPO/.16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/.17OH (64%); DMPO/.16OH (36%) from A4V and DMPO/.17OH (62%); and DMPO/.16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/.OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/.OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/.OH or DEPMPO/.OH formed nor in the relative incorporation of oxygen from H2O2 or water.

Amyotrophic Lateral Sclerosis↗

PubMed1997

Cerastotin, a serine protease from Cerastes cerastes venom, with platelet-aggregating and agglutinating properties.

Cerastotin, a thrombin-like enzyme from the venom of the desert viper Cerastes cerastes, has been purified by gel filtration on Sephadex G-75 and two ion-exchange chromatographies on Mono S columns. It is a neutral glycoprotein (pI = 6.6), present as a single polypeptide chain of 40 kDa. Its N-terminal sequence shows strong similarity with those of other thrombin-like enzymes from snake venoms. Cerastotin possesses esterase and amidolytic activities measured with N(alpha)-tosyl-L-arginine methyl ester and the thrombin chromogenic substrate D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide, respectively. The amidolytic activity is inhibited by phenylmethylsulfonyl fluoride, N(alpha)-tosyl-L-lysine chloromethane, N(alpha)-tosyl-L-phenylalanyl chloromethane, D-phenylalanyl-L-prolyl-L-arginyl chloromethane and benzamidine, suggesting that cerastotin is a serine protease. Cerastotin efficiently clots human plasma and cleaves preferentially the alpha chain of fibrinogen. Cerastotin did not induce aggregation of washed normal platelets, but did aggregate platelets in the presence of exogenous fibrinogen. A monoclonal antibody directed against glycoprotein (GPIb), which specifically inhibits induced agglutination by ristocetin also completely blocks platelet aggregation induced by cerastotin. However, another anti-GPIb monoclonal antibody, which specifically inhibits alpha-thrombin binding to GPIb, did not prevent this aggregation. Furthermore, platelets which were desensitised by alpha-thrombin still aggregate in the presence of cerastotin, but not alpha-thrombin. Similarly a monoclonal antibody, anti-GPIIb-IIIa, which blocks fibrinogen binding, did not inhibit cerastotin-induced platelet aggregation. This activity is abolished in the presence of 1 mM phenylmethylsulfonyl fluoride and/or 10 mM EDTA. Cerastotin also agglutinates formalin-fixed and washed platelets, only in the simultaneous presence of fibrinogen and of Von Willebrand factor.

Agglutination↗

PubMed1997

Further characterization and thrombolytic activity in a rat model of a fibrinogenase from Vipera lebetina venom.

Vipera lebetina fibrinogenase (VlF) was shown to render fibrinogen incoagulable and to solubilize fibrin. The fibrinogenolytic activity of this enzyme was found to be 33 mg fibrinogen/min/mg protein. The study of the specificity of this enzyme revealed that it has no effect on purified factor X, prothrombin and protein C and on the specific chromogenic substrates of their active form. Plasminogen was not activated by VlF but slightly degraded. We have also compared the effect of VlF and plasmin on fibrinogen and shown that these two enzymes have a different sites of cleavage. This enzyme inhibited human platelet aggregation on PRP initiated by ADP and collagen but was without effect on the aggregation of washed rabbit platelets using thrombin as agonist. Administration of VlF in rat did not show any necrosis or hemorrhage in treated rats organ's. We therefore, examined the thrombolytic activity of VlF in a rat model of venous thrombosis. Thrombus was produced in the posterior vena cava by injection of human fibrinogen and thrombin. Injection of 5 mg/Kg body weight showed an evident flow restoration after one hour and measurement of the fibrinogen level a decrease of about 30% after 3 hrs. VlF's action is not dependent on plasminogen activators and may act synergistically with them, thereby providing an intriguing potential clinical application for dissolution of blood clots.

Animals↗

PubMed1997

Spin-trapping of free radicals formed during the oxidation of glutathione by tetramethylammonium peroxynitrite.

Glutathionyl radical (GS.) formed during the oxidation of glutathione by tetramethylammonium peroxynitrite ([NMe4][ONOO]) was spin-trapped with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). This radical reacted with ammonium formate to form the carbon dioxide anion radical (CO2-.). The superoxide anion formed during oxidation of GSH by peroxynitrite salt was trapped with DMPO and detected as the DMPO-hydroxyl adduct. Addition of SOD mimic completely abolished the spectrum of the hydroxyl adduct but not the spectrum of the DMPO-glutathionyl radical adduct. Addition of seleno-DL-cystine or its reduced form caused a dramatic inhibition in the formation of spin adducts, suggesting that seleno-DL-cysteine is a more effective scavenger of peroxynitrite. The oxygen uptake observed during oxidation of GSH by peroxynitrite salt was inhibited by spin traps. In the presence of catalase, approximately 50% of the oxygen consumed was restored, indicating stoichiometric conversion of O2 to H2O2 during oxidation of GSH by peroxynitrite salt. Results indicate that nitrite and glutathione disulfide are formed as the major products during oxidation of GSH by peroxynitrite.

Catalase↗

PubMed1997

Purification, structure and activity of three insect toxins from Buthus occitanus tunetanus venom.

One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.

Action Potentials↗

PubMed1997

In vivo protection against Androctonus australis hector scorpion toxin and venom by immunization with a synthetic analog of toxin II.

A synthetic peptide mimicking the North African scorpion Androctonus australis hector toxin II was designed and produced by chemical solid-phase synthesis. It contains the entire sequence of toxin II (64 amino acid residues), with each half-cystine being replaced by the isosteric residue a-aminobutyric acid, and was thus devoid of disulfide bridges. This construct was totally nontoxic in mice even if large amounts, equivalent to 1000 times the LD50 of the original toxin, were injected by the intracerebroventricular route. The synthetic peptide, either as a monomer or polymerized by means of glutaraldehyde, induced the production of antitoxin neutralizing antibodies in immunized mice and rabbits. After three injections with either the monomeric or polymerized synthetic peptide, the immunized mice were protected against several lethal doses of the corresponding native toxin or scorpion venom. Six months after immunization, the mice were completely protected against challenge with eight LD50 of the original toxin. The protection was better when the polymerized synthetic peptide was used. One month after the start of the immunization program, it showed a good correlation between antibody titer and protection. However, antibody titer decreased with time but protection remained high. This suggests that additional factors other than circulating antibodies play a role in protective activity.

Animals↗