Habib Karoui

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.

Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes.

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.

Molecular cloning and functional expression of the alpha-scorpion toxin BotIII: pivotal role of the C-terminal region for its interaction with voltage-dependent sodium channels

Alpha scorpion toxins bind to receptor site 3 on voltage-dependent sodium channels and inhibit their inactivation. The alpha-scorpion toxin BotIII is the most toxic protein of Buthus occitanus tunetanus. Its sequence differs only by three amino acid residues from that of AahII, the most active alpha-toxin. Due to their high affinity and selectivity for mammalian sodium channels, BotIII and AahII represent powerful tools for studying the molecular determinants of specificity for voltage-dependent sodium channels. Sequence analysis of BotIII gene has revealed two exons separated by a 381-bp intron and a signal peptide of 19 amino acids. We succeeded in expressing BotIII in significantly higher amounts than AahII the only expressed strict alpha anti-mammalian scorpion toxin reported in the literature. We have also modified specific amino acid residues of BotIII. The recombinant and the natural toxins differ by the amidation of the C-terminal residue. Toxicity and binding experiments indicated: (a) the affinity of rBotIII-OH and rAahII-OH (rBotIII-OH with the 3 mutations R10V, V51L, N64H) for the voltage-dependent sodium channels is reduced compared to the natural toxins. This data revealed the important role of the C-terminal amidation for the biological activity of BotIII and AahII; (b) the single mutation N64H is responsible for the difference of toxicity and affinity between rBotIII-OH and rAahII-OH; (c) the addition of the sequence GR to rBotIII-OH leads to the loss of biological activity. This study is in agreement with the important role attributed to the C-terminal sequence of alpha-toxins in their interaction with sodium channels receptors.

Quince (Cydonia oblonga Miller) peel polyphenols modulate LPS-induced inflammation in human THP-1-derived macrophages through NF-κB, p38MAPK and Akt inhibition.

Chronic inflammation is a hallmark of several pathologies, such as rheumatoid arthritis, gastritis, inflammatory bowel disease, atherosclerosis and cancer. A wide range of anti-inflammatory chemicals have been used to treat such diseases while presenting high toxicity and numerous side effects. Here, we report the anti-inflammatory effect of a non-toxic, cost-effective natural agent, polyphenolic extract from the Tunisian quince Cydonia oblonga Miller. Lipopolysaccharide (LPS) treatment of human THP-1-derived macrophages induced the secretion of high levels of the pro-inflammatory cytokine TNF-α and the chemokine IL-8, which was inhibited by quince peel polyphenolic extract in a dose-dependent manner. Concomitantly, quince polyphenols enhanced the level of the anti-inflammatory cytokine IL-10 secreted by LPS-treated macrophages. We further demonstrated that the unexpected increase in IL-6 secretion that occurred when quince polyphenols were associated with LPS treatment was partially responsible for the polyphenols-mediated inhibition of TNF-α secretion. Biochemical analysis showed that quince polyphenols extract inhibited the LPS-mediated activation of three major cellular pro-inflammatory effectors, nuclear factor-kappa B (NF-κB), p38MAPK and Akt. Overall, our data indicate that quince peel polyphenolic extract induces a potent anti-inflammatory effect that may prove useful for the treatment of inflammatory diseases and that a quince-rich regimen may help to prevent and improve the treatment of such diseases.

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.

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.

PURIFICATION AND CHARACTERIZATION OF A FIBRINOGENASE FROM VIPERA LEBETINA (DESERT ADDER) VENOM

A fibrinogenase from Vipera lebetina venom was isolated by gel filtration in a Superose 12 column prep grade HR 16/50 and by ion-exchange in a Mono Q HR 5/5 column. The purified enzyme, which was obtained with a yield of 8 mg from 60 mg of crude venom, is a glycoprotein having an isoelectric point of 5.9 +/- 0.1 and a mol. wt of 26,000 +/- 1000 as estimated by SDS-PAGE. The biochemical characterization of the enzyme revealed that it hydrolyzes readily the B beta chain of fibrinogen and the A alpha chain as well as fibrin and casein. Over a pH range from 4 to 11 the enzyme was not inactivated by a 20 min treatment at 90 degrees C. The isolated fibrinogenase is inhibited by ethylenediamine tetraacetic acid, dithiothreitol and L-cysteine but not by phenylmethylsulfonyl fluoride. On the other hand, it is activated by Ca2+ and Mg2+. Purified fibrinogenase up to a dose of 100 micrograms/mouse shows no toxicity and has no hemorrhagic activity.

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 organs. 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. VlFs 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.

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.

Quince peel polyphenolic extract blocks human colon adenocarcinoma LS174 cell growth and potentiates 5-fluorouracil efficacy

BackgroundDevelopment of alternative cancer-specific drugs would be of paramount importance to overcome toxicity toward normal tissues and tumor resistance. Here, we investigated the potential anti-tumoral effect of peel (Peph) and pulp polyphenolic extracts from the Tunisian quince Cydonia oblonga Miller on both no-tumorigenic cells NIH 3T3 Fibroblasts and HEK 293 cells and human colon adenocarcinoma LS174 cells.MethodsCell proliferation and cytotoxicity were measured with MTT and LDH assays respectively. Cell cycle distribution and the apoptosis levels were assessed by flow cytometry. Intracellular reactive oxygen species (ROS) levels were determined using the fluorescent probe CM-H2DCFDA. Western blot was used to further characterize cell death and analyze the signaling pathways affected by Peph treatment. The expression level of VEGF-A was evaluated by real time quantitative PCR and further verified by quantifying the secreted cytokines by enzyme-linked immunosorbent assay.ResultsWe found that Peph extract displayed the highest anti-proliferative effect specifically on LS174 cells. However, each Peph phenolic compound alone did not exhibit any anti-proliferative activity, suggesting a synergistic effect of phenolic molecules. Such effect was associated with a cell cycle arrest in the G1/S phase, a caspase-independent apoptosis and an increase of the ROS production. Peph extract inhibited the pro-survival signaling pathway NFκB and suppressed the expression of various cellular markers known to be involved in cell cycling (cyclin D1) and angiogenesis (Vascular Endothelial Growth Factor, VEGF). Interestingly, the combination Peph extract and 5-FU exerted synergistic inhibitory effect on cell viability.ConclusionThese data propose the quince Peph extract as a promising cost effective non toxic drug to employ alone or in combination with conventional anti-colorectal cancer. Moreover, quince rich regimen may prevent the development and the progress of colon cancer.