Sergio Marangoni

Toxins and genes isolated from scorpions of the genus Tityus

Scorpion venoms contain a variety of low mol. wt peptides toxic to different organisms. These peptides have been intensively studied because they represent excellent models for investigating structure-function relationships and they are also fine probes for studying ionic channel functions. This review deals with the biological and chemical aspects of toxic peptides that affect Na+ or K+ channels and the cloning of the cDNAs and genes encoding the main alpha and beta neurotoxins present in the venom of the three most dangerous species of Brazilian scorpion, Tityus bahiensis, Tityus stigmurus and Tityus serrulatus, and the Venezuelan scorpion Tityus discrepans. At least 16 different peptides specific for Na+ channels and five affecting K+ channels were isolated and characterized from the venom of these scorpions. The isolation of cDNAs and genes encoding four distinct toxins has permitted the elucidation of their nucleotide sequences as well as their genomic organization. Venoms and isolated toxins from scorpions of the genus Tityus were shown to enhance the secretory activity of the pancreas. Antisera obtained against venom of T. serrulatus show cross-reactivity with other species of the Brazilian scorpions.

Bothropstoxin-I : amino acid sequence and function

The complete amino acid sequence of bothropstoxin-I (BthTX-I), a myotoxin isolated fromBothrops jararacussu snake venom, is reported. The results show that BthTX-I is a Lys49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 121 amino acid residues (Mr=13,720), containing one methionine and 14 half-cystines. Although deprived of any detectable PLA2 activity, BthTX-I reveals a high degree of sequence homology with Asp49-PLA2s and with other Lys49-myotoxins. Critical mutations—such as Leu5 for Phe5; Gln11 for X11; Asn28 for Tyr28; Leu32 for Gly32; Lys49 for Asp49; and Asp71 for Asn71—which are apparently involved with the decreasing or elimination of PLA2 activity, have been detected. The same mutations occurred in myotoxin II fromBothrops asper venom, but five extra changes—namely, Pro90 for Ser90; Gly111 for Asn111; His120 for Tyr120; Phe124 for Leu124; and Pro132 for Ala132—have been found relative to myotoxin II.

Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis

Global proteomic analysis of post-mortem anterior temporal lobe samples from schizophrenia patients and non-schizophrenia individuals was performed using stable isotope labeling and shotgun proteomics. Our analysis resulted in the identification of 479 proteins, 37 of which showed statistically significant differential expression. Pathways affected by differential protein expression include transport, signal transduction, energy pathways, cell growth and maintenance and protein metabolism. The collection of protein alterations identified here reinforces the importance of myelin/oligodendrocyte and calcium homeostasis in schizophrenia, and reveals a number of new potential markers that may contribute to the understanding of the pathogenesis of this complex disease.

Mast cell degranulation induced by two phospholipase A2 homologues : Dissociation between enzymatic and biological activities

Bothropstoxin-I and bothropstoxin-II are phospholipase A2 homologues isolated from Bothrops jararacussu snake venom. The former is devoid of phospholipase A2 activity whereas the latter has very low enzymatic activity. In this study, we have investigated the in vivo (rat paw and skin oedema) and in vitro (mast cell degranulation) inflammatory effects caused by bothropstoxin-I and bothropstoxin-II. Bothropstoxin-I (25-100 microg/paw) and bothropstoxin-II (12.5-50 microg/paw) caused dose-dependent rat paw oedema. The intradermal injection of bothropstoxin-I (0.125-5 microg/site) and bothropstoxin-II (0.125-5 microg/site) into rat skin also resulted in dose-dependent oedema formation. These oedematogenic activities were largely reduced in animals pretreated with the histamine/5-hydroxytryptamine (5-HT) receptor antagonist cyproheptadine (2 mg/kg, i.p. 0.5 h before). Similarly, p-bromophenacyl bromide, a compound known to inhibit phospholipase A2 activity, significantly inhibited rat paw and skin oedema induced by both phospholipase A2 homologues. The polyanion heparin (5 IU/site) significantly reduced the rat skin oedema induced by either bothropstoxin-I or bothropstoxin-II as well as the paw oedema (50 IU/site) induced by the former. When assayed in the rat peritoneal mast cells in vitro, both bothropstoxin-I (10 and 100 microg/ml) and bothropstoxin-II (3 and 10 microg/ml) significantly caused [14C]5-HT release. The [14C]5-HT release caused by these phospholipase A2 homologues were reduced by p-bromophenacyl bromide and heparin (50 IU/ml). Our results indicate that oedema formation induced by bothropstoxin-I and bothropstoxin-II is mostly dependent on in vivo mast cell degranulation. Since heparin greatly reduced the oedematogenic activity of these phospholipase A2 homologues, it is likely that the cationic charge of these substances plays a major role in the mast cell activation. Our results also indicate that p-bromophenacyl bromide may not be a suitable pharmacological tool to investigate the correlation between enzymatic activity and the inflammatory effects of phospholipases A2.

A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae)

A novel trypsin inhibitor was purified from the seeds of Peltophorum dubium (Spreng.). SDS-PAGE under reducing conditions showed that the inhibitor consisted of a single polypeptide chain (ca. 20 kDa). The dissociation constants of 4 x 10(-10) and 1.6 x 10(-10) M were obtained with bovine and porcine trypsin, respectively. This constant was lower (2.6 x 10(-7) M) for chymotrypsin. The inhibitory activity was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the P. dubium inhibitor showed a high degree of homology with other Kunitz-type inhibitors. When fed to the insect Anagasta kuehniella, in an artificial diet (inhibitor concentration 1.6%), the inhibitor produced approximately 56% and delayed the development of this lepidopteran. The concentration of inhibitor in the diet necessary to cause a 50% reduction in the weight (ED50) of fourth instar larvae was approximately 1%. The action of the P. dubium trypsin inhibitor (PDTI) on A. kuehniella may involve inhibition of the trypsin-like activity present in the larval midgut, resistance of the inhibitor to digestion by midgut enzymes and bovine trypsin, and association of the inhibitor with a chitin column and chitinous structures in the peritrophic membrane and/or midgut of the insect.

Inhibition of carrageenin‐induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2

1 The effect of purified crotapotin, a non‐toxic non‐enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 μg/paw) and 5‐hydroxytryptamine (5‐HT) (3 μg/paw) in the rat hind‐paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea‐pig isolated lung were also investigated. 2 Subplantar co‐injection of crotapotin (1 and 10 μg/paw) with carrageenin or injection of crotapotin (10 μg/paw) into the contralateral paw significantly inhibited the carrageenin‐induced oedema. This inhibition was also observed when crotapotin (10–30 μg/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60°C) failed to inhibit carrageenin‐induced oedema. Subplantar injection of crotapotin (10 μg/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5‐HT‐induced oedema. 3 In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5‐HT stores. 4 Crotapotin (30 μg/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 mm) and platelet activating factor (1 μm) in human platelet‐rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml−1) in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 μg/paw) did not affect the release of 6‐oxo‐prostaglandin F1α and TXB2 induced by ovalbumin in sensitized guinea‐pig isolated lungs. 5 Our results indicate that the anti‐inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo‐oxygenase activity. It is possible that crotapotin may interact with extracellular PLA2 generated during the inflammatory process thereby reducing its hydrolytic activity.

Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties.

A trypsin inhibitor from Dimorphandra mollis seeds was isolated to apparent homogeneity by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange and affinity chromatographic techniques. SDS-PAGE analysis gave an apparent molecular weight of 20 kDa, and isoelectric focusing analysis demonstrated the presence of three isoforms. The partial N-terminal amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz family of inhibitors. This inhibitor, which inhibited trypsin activity with a Ki of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site.

Structural, enzymatic and biological properties of new PLA2 isoform from Crotalus durissus terrificus venom

We isolated a new PLA(2) from the Crotalus durissus terrificus venom that designated F15, which showed allosteric behavior with a V(max) of 8.5nmol/min/mg and a K(m) of 38.5 mM. The incubated heparin salt of this isolated F15 act a positive allosteric effector by increasing the V(max) to 10.2 nmol/min/mg, with decreasing the V(max) value to 20.5 mM. The crotapotin, on the other hand acts as a negative allosteric effector by increasing the V(max) values to 58.4 mM. F15 also showed high calcium dependence for its catalysis similar to that found for other PLA(2) enzymes isolated from these snake venoms. The replacement of calcium by other divalent ions such Mg(2+), Mn(2+), Cd(2+), Sn(2+) and Cu(2+) resulted in lower enzymatic activity. The optimum pH and temperature for the enzyme was 8.5 and 18 degrees C, respectively. F15 alone showed moderate neurotoxic activity in isolated mouse phrenic nerve diaphragm in comparison to other strong myotoxic PLA(2) such as bothropstoxin-I (BThtx-I), but this activity was highly neurotoxic in a chick biventrer cervis preparation, whereas BthTx-I did not reveal this high neurotoxicity. This new protein showed a high bactericidal effect against both Gram-negative and Gram-positive bacterial strains. F15 contained 122 amino acid residues, with a primary structure of: HLLQFNKMIKFETRKNAVPFYAFYGCYCGWGGQRRPKDATDRCCFVHDCCYGKLTKCNTKWDIYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPKSRCRRPSETC. Its molecular mass and isoeletric point were 14.5 kDa and 8.85, both estimated by two dimensional electrophoresis. The amino acid sequence of the F15 revealed high sequence homology with F16 and F17. F15 and the other PLA(2)s revealed highly conserved amino acid sequences principally for calcium binding loop and active site helix. F15 also showed a high homology with the lysine-rich region of myotoxic PLA(2).

Purification and Characterization of a New Trypsin Inhibitor from Dimorphandra mollis Seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10−9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.

Isolation and partial characterization of a novel lectin from Talisia esculenta seeds that interferes with fungal growth

A novel plant lectin has been isolated from the seeds of Talisia esculenta and partially characterized. The purified lectin showed two protein bands in SDS-PAGE (20,000 and 40,000 kDa) and agglutinated human and animal erythrocytes. Of the various sugars tested, the lectin was best inhibited by mannose. A search of sequence databases showed that the N-terminal sequence had no homology to any known protein. The lectin inhibited the growth of the fungi Fusarium oxysporum, Colletotrichum lindemuthianum and Saccharomyces cerevisiae.