Biochemical characterization and insecticidal activity of isolated peptides from the venom of the scorpion Centruroides tecomanus.

The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 µg/100 mg of cricket weight, but low amounts of peptides (0.8 µg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant ß-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.

Biochemical characterization of the venom from the Mexican scorpion Centruroides ornatus, a dangerous species to humans.

Every year in Mexico, around 300,000 people suffer from accidents related to scorpion stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose venom characterization is described here. From this venom, a total of 114 components were found using chromatographic separation and mass spectrometry analysis. The most abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da respectively, similar to other known K+ and Na+-channel specific scorpion peptides. Using intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these peptides using heterologously expressed human Na+-channels revealed a typical ß-toxin effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3 among other ß-toxins from Centruroides scorpions. Peptide Co52 segregates among peptides of unknown defined functions.

Molecular composition of the paralyzing venom of three solitary wasps (Hymenoptera: Pompilidae) collected in southeast Mexico.

The chemical and biological characterization of peptide and protein components of the paralyzing venom from three Pompilidae solitary spider wasps (Pepsis mexicana, Pepsis terminata, and Anoplius nigritus) is described for the first time. The molecular masses of the most abundant peptides were determined. The N-terminal sequences of two cysteine-rich peptides were obtained from Pepsis. Metalloproteinase and hyaluronidase activities were identified in the venom of P. mexicana. A novel non-lethal method to collect venom is described.

Venom characterization of the Amazonian scorpion Tityus metuendus.

The soluble venom from the scorpion Tityus metuendus was characterized by various methods. In vivo experiments with mice showed that it is lethal. Extended electrophysiological recordings using seven sub-types of human voltage gated sodium channels (hNav1.1 to 1.7) showed that it contains both α- and ß-scorpion toxin types. Fingerprint analysis by mass spectrometry identified over 200 distinct molecular mass components. At least 60 sub-fractions were recovered from HPLC separation. Five purified peptides were sequenced by Edman degradation, and their complete primary structures were determined. Additionally, three other peptides have had their N-terminal amino acid sequences determined by Edman degradation and reported. Mass spectrometry analysis of tryptic digestion of the soluble venom permitted the identification of the amino acid sequence of 111 different peptides. Search for similarities of the sequences found indicated that they probably are: sodium and potassium channel toxins, metalloproteinases, hyaluronidases, endothelin and angiotensin-converting enzymes, bradykinin-potentiating peptide, hypothetical proteins, allergens, other enzymes, other proteins and peptides.

General characterization of the venoms from two species of rattlesnakes and an intergrade population (C. lepidus x aquilus) from Aguascalientes and Zacatecas, Mexico.

The venoms from two species of rock rattlesnakes and an intergrade population were studied. Differences were noted in SDS-PAGE and RP-HPLC profiles and only the venom from the intergrade population showed low procoagulant activity. Also, a Crotoxin-like neurotoxic PLA2 was identified in the venom of C. l. klauberi, the most toxic of the analyzed venoms. This is the first report of such a toxin in C. l. klauberi from Aguascalientes, Mexico.

Comprehensive analysis of venom from the scorpion Centruroides tecomanus reveals compounds with antimicrobial, cytotoxic, and insecticidal activities.

Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10-100 µg/mL concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 µg/mL concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 µg/mL.

Turkish scorpion Buthacus macrocentrus: general characterization of the venom and description of Bu1, a potent mammalian Na⁺-channel α-toxin.

The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200-4500 Da, and 6000-7500 Da. They very likely correspond to K⁺-channel and Na⁺-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na⁺ currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country.

GP35 ANOAL, an abundant acidic glycoprotein of female Anopheles albimanus saliva.

Salivary glands of female mosquitoes produce proteins, not completely described yet, that participate in carbohydrate and blood feeding. Here, we report an acidic glycoprotein of 35 kDa (GP35 ANOAL) secreted in the saliva of the malaria vector mosquito Anopheles albimanus. GP35 ANOAL is produced exclusively in the distal lateral lobes of adult female salivary glands, it has a pI of 4.45 and is negatively stained by regular silver stain. An 888 bp cDNA clone encoding a predicted product of 240 amino acids has a signal peptide, potential post-translational modification sites, and a disintegrin signature RGD. The GP35 ANOAL sequence depicts high similarities with the 30 kDa saliva allergen of Aedes aegypti, 30 kDa allergen-like hypothetical proteins, and GE-rich proteins present in several Anopheles species, as well as in Ae. albopictus and Culex pipiens quinquefasciatus. The function of this protein family is still unknown.

Proteomic analysis of the venom from the scorpion Tityus stigmurus: biochemical and physiological comparison with other Tityus species.

The venom from the Brazilian scorpion Tityus stigmurus was fractionated by high performance liquid chromatography (HPLC) and the corresponding components were used for molecular mass determination using electrospray ion trap mass spectrometry. One hundred distinct components were clearly assigned showing molecular masses from 216.5 to 44,800.0 Da. Fifteen new components were isolated and sequenced, four of them to completion: Tst-3 (similar to Na(+) channel specific scorpion toxins), Tst-17 (a K(+) channel blocking peptide similar to Tc1), Tst beta KTx (a peptide with identical sequence as that of TsTX-K beta toxin earlier described to exist in T. serrulatus venom) and finally a novel proline-rich peptide of unknown function. Among the eleven components partially sequenced were two enzymes: hyaluronidase and lysozyme. The first enzyme has a molecular mass of 44,800.0 Da. This enzyme showed high activity against the substrate hyaluronan in vitro. Amino acid sequence of the second enzyme showed that it is similar to other known lysozymes, with similar molecular mass and sequence to that of bona fide lysozymes reported in public protein data banks. Finally, this communication reports a correlation among HPLC retention times and molecular masses of folded scorpion toxins as well as a comparative structural and physiological analysis of components from the venom of several species of the genus Tityus.

Functional characterization of brain mitochondrial nitric oxide synthase during hypertension and aging.

Nitric oxide (NO*) plays an important role in various physiological processes. The aim of the present study was to investigate if brain mitochondrial nitric oxide synthase (mtNOS) is active and functional during hypertension. L-citrulline production, an indicator of nitric oxide synthesis, was concentration-dependent on L-arginine in all strains and all ages tested, and was inhibited by 7-Nitroindazole (7-NI). Brain mitochondria of 1 month-old (prehypertensive) spontaneously hypertensive rats (SHR) exhibited a significantly (p < 0.05) low basal L-citrulline content as compared to age-matched Wistar (W) and Wistar-Kyoto (WKY) rats. L-citrulline synthesis in SHR rats showed a significant (p < 0.01) low response to L-arginine in 3 and 7 months-old rats. Respiratory rates in states 3 and 4 increased with low L-arginine concentration in all strains and all ages. The results suggest that in rat brain mitochondria, L-citrulline synthesis is constant once age-related hypertension is installed and NO* does not regulate oxidative phosphorylation.

Textural assessment in digital mammograms.

This work focuses on testing textural and morphological parameters to assess characteristic features of digital mammograms. The selected images were radiological studies from the Institute Nacional de Cancerologia in Mexico City, evaluated as BI-RADS 4 or 5, meaning "probably malign" or "malign" findings, respectively. All patients were subjected to a biopsy procedure after the image was taken. The study group consisted of patients diagnosed with cancer, while the control group included those without cancer. We propose to analyze textural roughness by the mean height-width ratio of extrema (MHWRE) and morphological features by circularity. Results show good differentiation (correct diagnosis) for 46% of the images, bad differentiation (wrong diagnosis) for 25%, and undetermined diagnosis for 29% of the cases.

Identification and characterization of gp65, a salivary-gland-specific molecule expressed in the malaria vector Anopheles albimanus.

A group of salivary-gland-specific proteins, designated gp65, were identified in the mosquito Anopheles albimanus. Two-dimensional gel electrophoresis resolved this group into at least four molecules with pI 6.4-6.5. The N-terminal amino acid sequence was determined for the major species, gp65-1, and degenerate oligonucleotide primers were used to amplify a specific probe for library screening. A 1312 bp cDNA clone encoding a predicted translation product of 386 amino acids was recovered. gp65-1 is expressed abundantly in the medial and distal-lateral lobes of the adult female glands, and is secreted in the saliva. The amino acid sequence has potential sites for N-glycosylation, phosphorylation and myristylation, and is similar to a number of proteins of unknown function from other mosquito species.

Ardiscretin a novel arthropod-selective toxin from Tityus discrepans scorpion venom.

A new arthropod selective toxin was purified from the venom of the Venezuelan scorpion Tityus discrepans, and its amino acid sequence, cDNA clone and biological activity are reported here. The amino acid sequence of this peptide, named ardiscretin (from arthropod toxin of T. discrepans) was completed by Edman degradation and mass spectrometry. It is a single polypeptide composed by 61 amino acids with an amidated cysteine residue at the C-terminal end, closely packed by four disulfide bridges. The atomic mass unit (a.m.u.) experimentally determined was 7103.8 a.m.u. This peptide was shown to be specific for invertebrates (crickets, triatomides, crabs and squids), but non-toxic to mice, at the dose assayed. Ardiscretin inhibits the Na(+)-currents of squid giant axons in an apparent irreversible manner, whose inhibitory effect is reached at 30 microM toxin concentration. Sequence comparison showed that it is phylogenetically closely related to insect-specific scorpion toxins. Ardiscretin produced a small depolarization and induced repetitive firing in squid axons resembling those of DDT [1,1'(p-chlorobenzyl)2-tricloretane] in its ability to slow down action potential, to induce repetitive firing, and in that the concentration required for any effect in squid axon is rather high.

Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation.

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60-65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices alpha1 and alpha2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the alpha1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.

Slotoxin, alphaKTx1.11, a new scorpion peptide blocker of MaxiK channels that differentiates between alpha and alpha+beta (beta1 or beta4) complexes.

A novel peptide from Centruroides noxius Hoffmann scorpion venom was isolated and sequenced. The 37 amino acid peptide belongs to the charybdotoxin sub-family (alphaKTx1) and was numbered member 11. alphaKTx1.11 has 75% sequence identity with iberiotoxin and 54% with charybdotoxin. alphaKTx1.11 revealed specificity for mammalian MaxiK channels (hSlo), thus, was named slotoxin. Slotoxin blocks the MaxiK pore-forming alpha subunit reversibly (K(d)=1.5 nM). Slotoxin association with alpha+beta (beta1 or beta4) channels was approximately 10 times slower than iberiotoxin and charybdotoxin, leading to a lack of effect on alpha+beta4 when tested at 100 nM for 5 min. Thus, slotoxin is a better tool to distinguish MaxiK alpha+beta complexes.

Time-resolved sperm responses to an egg peptide measured by stopped-flow fluorometry.

Speract, a decapeptide from sea urchin egg jelly, induces various sperm responses. Stopped-flow fluorometry was used to examine the binding of labeled speract and the intracellular changes in pH (pH(i)) and Ca2+ ([Ca2+]i) it induces in sperm. We observed significant time delays for the increase in pH(i) and [Ca2+]i induced by 200 nM speract (69 and 190 ms, respectively). Also, we found that the receptor undergoes a pH(i)-dependent affinity change at around 129 ms. These time delays probably reflect biochemical processes underlying each sperm response to speract that circumscribe the time sequence of the signaling events.

Hadrurin, a new antimicrobial peptide from the venom of the scorpion Hadrurus aztecus.

A new antimicrobial peptide, hadrurin, was isolated from the venom of the Mexican scorpion Hadrurus aztecus, by gel filtration on a Sephadex G-50 column, followed by high performance liquid chromatography. It is a basic peptide composed of 41 amino-acid residues with a molecular mass of 4436 Da, and contains no cysteines. A model of the three-dimensional folding of hadrurin is compatible with that of an amphipatic molecule with two alpha-helical segments. Hadrurin demonstrates antimicrobial activity at low micromolar concentration, inhibiting the growth of bacteria such as: Salmonella thyphi, Klebsiella pneumoniae, Enterococcus cloacae, Pseudomonas aeruginosa, Escherichia coli and Serratia marscences. It also shows cytolytic activity when tested in human erythrocytes. Hadrurin and two analogs (C-terminal amidated, and all D-enantiomer) were chemically synthesized. They were used to study the possible molecular mechanism of action by testing their ability to dissipate the diffusion potential of liposomes of different compositions. The results obtained indicate that there are no specific receptor molecules for the action of hadrurin, and the most probable mechanism is through a membrane destabilization activity. It is surmised that hadrurin is used by the scorpion as both an attack and defense element against its prey and putative invasive microorganisms. It is a unique peptide among all known antimicrobial peptides described, only partially similar to the N-terminal segment of gaegurin 4 and brevinin 2e, isolated from frog skin. It would certainly be a model molecule for studying new antibiotic activities and peptide-lipid interactions.

Scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom.

A novel peptide, scorpine, was isolated from the venom of the scorpion Pandinus imperator, with anti-bacterial activity and a potent inhibitory effect on the ookinete (ED(50) 0.7 microM) and gamete (ED(50) 10 microM) stages of Plasmodium berghei development. It has 75 amino acids, three disulfide bridges with a molecular mass of 8350 Da. Scorpine has a unique amino acid sequence, similar only to some cecropins in its N-terminal segment and to some defensins in its C-terminal region. Its gene was cloned from a cDNA library.

Phospholipin, a novel heterodimeric phospholipase A2 from Pandinus imperator scp6pion venom.

The primary structure of a phospholipase A2, with unique structural and functional characteristics, was determined. The large subunit has 108 amino acid residues, linked by a disulfide bridge to the small subunit, which contains 17 residues. Its gene was cloned from a cDNA library. The nucleotide sequence showed that the same RNA messenger encodes both subunits, separated only by a pentapeptide, that is processed during maturation.

Antibody BCF2 against scorpion toxin Cn2 from Centuroides noxius Hoffmann: primary structure and three-dimensional model as free Fv fragment and complexed with its antigen.

The antibody BCF2 generated against the mammal-specific toxin Cn2 of the scorpion Centuroides noxius Hoffmann neutralizes the effect of both the toxin and the venom. We cloned and sequenced the genes coding for the Fv fragment of BCF2. A three-dimensional (3D) model of the Fv fragment was generated using a knowledge-based approach. Furthermore, a 3D model of the complex Cn2-BCF2 was built using the nuclear magnetic resonance (NMR) structure of Cn2 and experimental results on a putative epitope region around the N and C termini. The initial complex conformations were submitted to a new refinement procedure of rigid-body energy minimization combined with flexible-side-chain molecular dynamics. The final complex, selected after an extensive evaluation, uses the loop 7-11 as the central part of the epitope. The generated complex allows the following conclusions: 1) the neutralizing capacity of BCF2 toward the venom of C. noxius might rather be caused by the high venom concentration and toxicity of Cn2 than by a broad specificity, 2) the region involved in the binding of Cn2 to the Na(+) channel, should overlap with the employed epitope region, and 3) contact residues SerL91, AsnL92, LeuH50, AspH56, TyrH95, and TyrH98 of BCF2 are candidates for mutations to broaden its specificity. Proteins 1999;37:130-143.