Profiling the Linear Peptides of Venom from the Brazilian Scorpion Tityus serrulatus: Structural and Functional Characterization.

Scorpion venoms are a rich source of bioactive peptides, most of which are neurotoxic, with 30 to 70 amino acid residues in their sequences. There are a scarcity of reports in the literature concerning the short linear peptides found in scorpion venoms. This type of peptide toxin may be selectively extracted from the venom using 50% (v/v) acetonitrile. The use of LC-MS and MS/MS enabled the detection of 12 bioactive short linear peptides, of which six were identified as cryptides. These peptides were shown to be multifunctional, causing hemolysis, mast cell degranulation and lysis, edema, pain, and anxiety, increasing the complexity of the envenomation mechanism. Apparently, the natural functions of these peptide toxins are to induce inflammation and discomfort in the victims of scorpion stings.

Profiling the linear peptides of venom from the Brazilian scorpion Tityus serrulatus: structural and functional characterization

Scorpion venoms are a rich source of bioactive peptides, most of which are neurotoxic, with 30 to 70 amino acid residues in their sequences. There are a scarcity of reports in the literature concerning the short linear peptides found in scorpion venoms. This type of peptide toxin may be selectively extracted from the venom using 50% (v/v) acetonitrile. The use of LC-MS and MS/MS enabled the detection of 12 bioactive short linear peptides, of which six were identified as cryptides. These peptides were shown to be multifunctional, causing hemolysis, mast cell degranulation and lysis, edema, pain, and anxiety, increasing the complexity of the envenomation mechanism. Apparently, the natural functions of these peptide toxins are to induce inflammation and discomfort in the victims of scorpion stings.

Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells

Systemic scorpion envenomation is characterized by massive neurotransmitter release from peripheral nerves mediated primarily by scorpion venoms neurotoxins. Tityus bahiensis is one of the medically most important species in Brazil, but its venom pharmacology, especially regarding to peripheral nervous system, is poorly understood. Here, we evaluated the T. bahiensis venom activity on autonomic (sympathetic) neurotransmission by using a variety of approaches, including vas deferens twitch-tension recordings, electrophysiological measurements (resting membrane potentials, spontaneous excitatory junctional potentials and whole-cell patch-clamp), calcium imaging and histomorphological analysis. Low concentrations of venom (=?3 µg/mL) facilitated the electrically stimulated vas deferens contractions without affecting postsynaptic receptors or damaging the smooth muscle cells. Transient TTX-sensitive sustained contractions and resting membrane depolarization were mediated mainly by massive spontaneous ATP release. High venom concentrations (=?10 µg/mL) blocked the muscle contractions and induced membrane depolarization. In neuronal cells (ND7-23wt), the venom increased the peak sodium current, modified the current-voltage relationship by left-shifting the Nav-channel activation curve, thereby facilitating the opening of these channels. The venom also caused a time-dependent increase in neuronal calcium influx. These results indicate that the sympathetic hyperstimulation observed in systemic envenomation is presynaptically driven, probably through the interaction of a- and ß-toxins with neuronal sodium channels.

Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells

Systemic scorpion envenomation is characterized by massive neurotransmitter release from peripheral nerves mediated primarily by scorpion venoms neurotoxins. Tityus bahiensis is one of the medically most important species in Brazil, but its venom pharmacology, especially regarding to peripheral nervous system, is poorly understood. Here, we evaluated the T. bahiensis venom activity on autonomic (sympathetic) neurotransmission by using a variety of approaches, including vas deferens twitch-tension recordings, electrophysiological measurements (resting membrane potentials, spontaneous excitatory junctional potentials and whole-cell patch-clamp), calcium imaging and histomorphological analysis. Low concentrations of venom (= 3 µg/mL) facilitated the electrically stimulated vas deferens contractions without affecting postsynaptic receptors or damaging the smooth muscle cells. Transient TTX-sensitive sustained contractions and resting membrane depolarization were mediated mainly by massive spontaneous ATP release. High venom concentrations (= 10 µg/mL) blocked the muscle contractions and induced membrane depolarization. In neuronal cells (ND7-23wt), the venom increased the peak sodium current, modified the current-voltage relationship by left-shifting the Nav-channel activation curve, thereby facilitating the opening of these channels. The venom also caused a time-dependent increase in neuronal calcium influx. These results indicate that the sympathetic hyperstimulation observed in systemic envenomation is presynaptically driven, probably through the interaction of a- and ß-toxins with neuronal sodium channels.

Scorpion venom increases acetylcholine release by prolonging the duration of somatic nerve action potentials

Scorpionism is frequently accompanied by a massive release of catecholamines and acetylcholine from peripheral nerves caused by neurotoxic peptides present in these venoms, which have high specificity and affinity for ion channels. Tityus bahiensis is the second most medically important scorpion species in Brazil but, despite this, its venom remains scarcely studied, especially with regard to its pharmacology on the peripheral (somatic and autonomic) nervous system. Here, we evaluated the activity of T. bahiensis venom on somatic neurotransmission using myographic (chick and mouse neuromuscular preparations), electrophysiological (MEPP, EPP, resting membrane potentials, perineural waveforms, compound action potentials) and calcium imaging (on DRG neurons and muscle fibres) techniques. Our results show that the major toxic effects of T. bahiensis venom on neuromuscular function are presynaptically driven by the increase in evoked and spontaneous neurotransmitter release. Low venom concentrations prolong the axonal action potential, leading to a longer depolarization of the nerve terminals that enhances neurotransmitter release and facilitates nerve-evoked muscle contraction. The venom also stimulates the spontaneous release of neurotransmitters, probably through partial neuronal depolarization that allows calcium influx. Higher venom concentrations block the generation of action potentials and resulting muscle twitches. These effects of the venom were reversed by low concentrations of TTX, indicating voltage-gated sodium channels as the primary target of the venom toxins. These results suggest that the major neuromuscular toxicity of T. bahiensis venom is probably mediated mainly by a- and ß-toxins interacting with presynaptic TTX-sensitive ion channels on both axons and nerve terminals.

Scorpion venom increases acetylcholine release by prolonging the duration of somatic nerve action potentials

Scorpionism is frequently accompanied by a massive release of catecholamines and acetylcholine from peripheral nerves caused by neurotoxic peptides present in these venoms, which have high specificity and affinity for ion channels. Tityus bahiensis is the second most medically important scorpion species in Brazil but, despite this, its venom remains scarcely studied, especially with regard to its pharmacology on the peripheral (somatic and autonomic) nervous system. Here, we evaluated the activity of T. bahiensis venom on somatic neurotransmission using myographic (chick and mouse neuromuscular preparations), electrophysiological (MEPP, EPP, resting membrane potentials, perineural waveforms, compound action potentials) and calcium imaging (on DRG neurons and muscle fibres) techniques. Our results show that the major toxic effects of T. bahiensis venom on neuromuscular function are presynaptically driven by the increase in evoked and spontaneous neurotransmitter release. Low venom concentrations prolong the axonal action potential, leading to a longer depolarization of the nerve terminals that enhances neurotransmitter release and facilitates nerve-evoked muscle contraction. The venom also stimulates the spontaneous release of neurotransmitters, probably through partial neuronal depolarization that allows calcium influx. Higher venom concentrations block the generation of action potentials and resulting muscle twitches. These effects of the venom were reversed by low concentrations of TTX, indicating voltage-gated sodium channels as the primary target of the venom toxins. These results suggest that the major neuromuscular toxicity of T. bahiensis venom is probably mediated mainly by a- and ß-toxins interacting with presynaptic TTX-sensitive ion channels on both axons and nerve terminals.

Influence of different processing techniques on the toxicity and biochemical characteristics of Tityus serrulatus scorpion venom.

Studies of scorpion venoms have used different venom drying methods: lyophilization, desiccation, lyophilization after mixing with 0.9% saline or purified water and centrifugation. The aim of this study was to see if these different approaches cause some alteration in the composition of the venom or interfere with its biological effects. Mice were injected (i.p.) with T. serrulatus scorpion venom in the liquid form (G-liq) or dried by different methods (lyophilized - G-lyo; centrifuged and the supernatant lyophilized - G-cen; desiccated - G-des), and observed regarding the occurrence of the symptoms respiratory difficulty, convulsion and death. The occurrence of seizures, although occurring in all groups and with the various doses used, did not prove to be effective to determine differences between the different handling techniques. Respiratory distress appeared to be useful in analyzing differences between groups, where this effect was less pronounced in the G-liq and G-des groups. In general, death occurred in a certain proportion with increasing dose for all groups. G-liq and G-des seemed to be more "active" at lower doses and G-cen and G-lyo at higher doses. The electrophoretic and chromatographic profile demonstrated main differences between G-liq and the dried groups. In the electrophoretic profile, the liquid venom showed bands of proteins of higher concentration and greater number of major bands and the three dried venom had the lowest number of protein bands. The HPLC profile and densitometry of the electrophoretic profiles showed some differences that may be associated with different protein conformation/aggregation. Our data indicated that lyophilization is the most suitable method for processing T. serrulatus scorpion venom after extraction.

Proteomic endorsed transcriptomic profiles of venom glands from Tityus obscurus and T. serrulatus scorpions.

BACKGROUND: Except for the northern region, where the Amazonian black scorpion, T. obscurus, represents the predominant and most medically relevant scorpion species, Tityus serrulatus, the Brazilian yellow scorpion, is widely distributed throughout Brazil, causing most envenoming and fatalities due to scorpion sting. In order to evaluate and compare the diversity of venom components of Tityus obscurus and T. serrulatus, we performed a transcriptomic investigation of the telsons (venom glands) corroborated by a shotgun proteomic analysis of the venom from the two species. RESULTS: The putative venom components represented 11.4% and 16.7% of the total gene expression for T. obscurus and T. serrulatus, respectively. Transcriptome and proteome data revealed high abundance of metalloproteinases sequences followed by sodium and potassium channel toxins, making the toxin core of the venom. The phylogenetic analysis of metalloproteinases from T. obscurus and T. serrulatus suggested an intraspecific gene expansion, as we previously observed for T. bahiensis, indicating that this enzyme may be under evolutionary pressure for diversification. We also identified several putative venom components such as anionic peptides, antimicrobial peptides, bradykinin-potentiating peptide, cysteine rich protein, serine proteinases, cathepsins, angiotensin-converting enzyme, endothelin-converting enzyme and chymotrypsin like protein, proteinases inhibitors, phospholipases and hyaluronidases. CONCLUSION: The present work shows that the venom composition of these two allopatric species of Tityus are considerably similar in terms of the major classes of proteins produced and secreted, although their individual toxin sequences are considerably divergent. These differences at amino acid level may reflect in different epitopes for the same protein classes in each species, explaining the basis for the poor recognition of T. obscurus venom by the antiserum raised against other species.

Effects of Brazilian scorpion venoms on the central nervous system.

In Brazil, the scorpion species responsible for most severe incidents belong to the Tityus genus and, among this group, T. serrulatus, T. bahiensis, T. stigmurus and T. obscurus are the most dangerous ones. Other species such as T. metuendus, T. silvestres, T. brazilae, T. confluens, T. costatus, T. fasciolatus and T. neglectus are also found in the country, but the incidence and severity of accidents caused by them are lower. The main effects caused by scorpion venoms - such as myocardial damage, cardiac arrhythmias, pulmonary edema and shock - are mainly due to the release of mediators from the autonomic nervous system. On the other hand, some evidence show the participation of the central nervous system and inflammatory response in the process. The participation of the central nervous system in envenoming has always been questioned. Some authors claim that the central effects would be a consequence of peripheral stimulation and would be the result, not the cause, of the envenoming process. Because, they say, at least in adult individuals, the venom would be unable to cross the blood-brain barrier. In contrast, there is some evidence showing the direct participation of the central nervous system in the envenoming process. This review summarizes the major findings on the effects of Brazilian scorpion venoms on the central nervous system, both clinically and experimentally. Most of the studies have been performed with T. serrulatus and T. bahiensis. Little information is available regarding the other Brazilian Tityus species.

Profiling the short, linear, non-disulfide bond-containing peptidome from the venom of the scorpion Tityus obscurus.

Many scorpion accidents occur in the Brazilian Amazonian region and are frequently caused by Tityus obscurus. Approximately 5% of the crude venom of this species is composed of short linear, non-disulfide-bridged peptides, which have not been intensively investigated. As a consequence, only a few of these peptides have been structurally and functionally characterized to date. In the present paper, the peptide fraction of the venom was subjected to peptide profiling using an LCMS-IT-TOF/MS and MSn system. The analysis detected 320 non-disulfide bond-containing peptides (NDBPs), of which twenty-seven had their sequences assigned; among them, thirteen peptides were characterized, constituting novel toxins in T. obscurus venom. Some of the novel peptides showed similarities to hypotensin-like toxins, while other peptides appear to be natural fragments of neurotoxins. The novel peptides were submitted to a series of bioassays, revealing that many are multifunctional toxins that cause, for example, pain, edema formation and hemolysis to potentiate strong inflammatory processes and alterations in the locomotion and lifting activities in the victims of stinging. Knowledge of the complex matrix of peptides composing the venom of T. obscurus will contribute to better understanding of the complex mechanism of envenoming caused by stinging accidents. SIGNIFICANCE: The scorpion Tityus obscurus causes many envenoming accidents of medical importance in Brazilian Amazon region; despite to this, very few is known about the toxinology of this animal. The knowledge about the venom composition and mechanisms of action is very important to understand the physiopathology processes related to the envenoming caused by this animal. The proteopeptidomic investigations of scorpion venoms in general have focused mainly the neurotoxins (which are disulfide bonds containing peptides) and large proteins. The short, linear, non-disulfide bonds containing peptides (NDBPs) represent up to 5% of scorpion venom compositions; however, they have been few investigated in comparison with the neurotoxins. The present study used a mass spectrometric approach to detect 320 NDBPs and to sequence 27 of them; pharmacological assays permitted to characterize 13 NDBPs as novel toxins involved with inflammation, pain and edema formation.

Effects of Brazilian scorpion venoms on the central nervous system

In Brazil, the scorpion species responsible for most severe incidents belong to the Tityus genus and, among this group, T. serrulatus, T. bahiensis, T. stigmurus and T. obscurus are the most dangerous ones. Other species such as T. metuendus, T. silvestres, T. brazilae, T. confluens, T. costatus, T. fasciolatus and T. neglectus are also found in the country, but the incidence and severity of accidents caused by them are lower. The main effects caused by scorpion venoms - such as myocardial damage, cardiac arrhythmias, pulmonary edema and shock - are mainly due to the release of mediators from the autonomic nervous system. On the other hand, some evidence show the participation of the central nervous system and inflammatory response in the process. The participation of the central nervous system in envenoming has always been questioned. Some authors claim that the central effects would be a consequence of peripheral stimulation and would be the result, not the cause, of the envenoming process. Because, they say, at least in adult individuals, the venom would be unable to cross the blood-brain barrier. In contrast, there is some evidence showing the direct participation of the central nervous system in the envenoming process. This review summarizes the major findings on the effects of Brazilian scorpion venoms on the central nervous system, both clinically and experimentally. Most of the studies have been performed with T. serrulatus and T. bahiensis. Little information is available regarding the other Brazilian Tityus species.

Influence of different processing techniques on the toxicity and biochemical characteristics of Tityus serrulatus scorpion venom

Studies of scorpion venoms have used different venom drying methods: lyophilization, desiccation, lyophilization after mixing with 0.9% saline or purified water and centrifugation. The aim of this study was to see if these different approaches cause some alteration in the composition of the venom or interfere with its biological effects. Mice were injected (i.p.) with T. serrulatus scorpion venom in the liquid form (G-liq) or dried by different methods (lyophilized – G-lyo; centrifuged and the supernatant lyophilized – G-cen; desiccated – G-des), and observed regarding the occurrence of the symptoms respiratory difficulty, convulsion and death. The occurrence of seizures, although occurring in all groups and with the various doses used, did not prove to be effective to determine differences between the different handling techniques. Respiratory distress appeared to be useful in analyzing differences between groups, where this effect was less pronounced in the G-liq and G-des groups. In general, death occurred in a certain proportion with increasing dose for all groups. G-liq and G-des seemed to be more "active" at lower doses and G-cen and G-lyo at higher doses. The electrophoretic and chromatographic profile demonstrated main differences between G-liq and the dried groups. In the electrophoretic profile, the liquid venom showed bands of proteins of higher concentration and greater number of major bands and the three dried venom had the lowest number of protein bands. The HPLC profile and densitometry of the electrophoretic profiles showed some differences that may be associated with different protein conformation/aggregation. Our data indicated that lyophilization is the most suitable method for processing T. serrulatus scorpion venom after extraction.

Tb II-I, a fraction isolated from Tityus bahiensis scorpion venom, alters cytokines': level and induces seizures when intrahippocampally injected in rats

Scorpion venoms are composed of several substances with different pharmacological activities. Neurotoxins exert their effects by targeting ion channels resulting in toxic effects to mammals, insects and crustaceans. Tb II-I, a fraction isolated from Tityus bahiensis scorpion venom, was investigated for its ability to induce neurological and immune-inflammatory effects. Two putative -sodium channel toxins were identified in this fraction, Tb2 II and Tb 4, the latter having been completely sequenced by mass spectrometry. Male Wistar rats, stereotaxically implanted with intrahippocampal cannulas and electrodes, were injected with Tb II-I (2 mu g/2 mu L) via the intrahippocampal route. The behavior, electrographic activity and cellular integrity of the animals were analyzed and the intracerebral level of cytokines determined. Tb II-I injection induced seizures and damage in the hippocampus. These alterations were correlated with the changes in the level of the cytokines tumoral necrosis factor- (TNF-) and interleukin-6 (IL-6). Therefore, the binding of Tb II-I to its target in the central nervous system may induce inflammation resulting in neuropathological and behavioral alterations.

Proteomic endorsed transcriptomic profiles of venom glands from Tityus obscurus and T. serrulatus scorpions

Background Except for the northern region, where the Amazonian black scorpion, T. obscurus, represents the predominant and most medically relevant scorpion species, Tityus serrulatus, the Brazilian yellow scorpion, is widely distributed throughout Brazil, causing most envenoming and fatalities due to scorpion sting. In order to evaluate and compare the diversity of venom components of Tityus obscurus and T. serrulatus, we performed a transcriptomic investigation of the telsons (venom glands) corroborated by a shotgun proteomic analysis of the venom from the two species. Results The putative venom components represented 11.4% and 16.7% of the total gene expression for T. obscurus and T. serrulatus, respectively. Transcriptome and proteome data revealed high abundance of metalloproteinases sequences followed by sodium and potassium channel toxins, making the toxin core of the venom. The phylogenetic analysis of metalloproteinases from T. obscurus and T. serrulatus suggested an intraspecific gene expansion, as we previously observed for T. bahiensis, indicating that this enzyme may be under evolutionary pressure for diversification. We also identified several putative venom components such as anionic peptides, antimicrobial peptides, bradykinin-potentiating peptide, cysteine rich protein, serine proteinases, cathepsins, angiotensin-converting enzyme, endothelin-converting enzyme and chymotrypsin like protein, proteinases inhibitors, phospholipases and hyaluronidases. Conclusion The present work shows that the venom composition of these two allopatric species of Tityus are considerably similar in terms of the major classes of proteins produced and secreted, although their individual toxin sequences are considerably divergent. These differences at amino acid level may reflect in different epitopes for the same protein classes in each species, explaining the basis for the poor recognition of T. obscurus venom by the antiserum raised against other species.

Profiling the short, linear, non-disulfide bond-containing peptidome from the venom of the scorpion Tityus obscurus

Many scorpion accidents occur in the Brazilian Amazonian region and are frequently caused by Tityus obscurus. Approximately 5% of the crude venom of this species is composed of short linear, non-disulfide-bridged peptides, which have not been intensively investigated. As a consequence, only a few of these peptides have been structurally and functionally characterized to date. In the present paper, the peptide fraction of the venom was subjected to peptide profiling using an LCMS-IT-TOF/MS and MSn system. The analysis detected 320 non-disulfide bond-containing peptides (NDBPs), of which twenty-seven had their sequences assigned; among them, thirteen peptides were characterized, constituting novel toxins in T. obscurus venom. Some of the novel peptides showed similarities to hypotensin-like toxins, while other peptides appear to be natural fragments of neurotoxins. The novel peptides were submitted to a series of bioassays, revealing that many are multifunctional toxins that cause, for example, pain, edema formation and hemolysis to potentiate strong inflammatory processes and alterations in the locomotion and lifting activities in the victims of stinging. Knowledge of the complex matrix of peptides composing the venom of T. obscurus will contribute to better understanding of the complex mechanism of envenoming caused by stinging accidents. Significance: The scorpion Tityus obscurus causes many envenoming accidents of medical importance in Brazilian Amazon region; despite to this, very few is known about the toxinology of this animal. The knowledge about the venom composition and mechanisms of action is very important to understand the physiopathology processes related to the envenoming caused by this animal. The proteopeptidomic investigations of scorpion venoms in general have focused mainly the neurotoxins (which are disulfide bonds containing peptides) and large proteins. The short, linear, non-disulfide bonds containing peptides (NDBPs) represent up to 5% of scorpion venom compositions; however, they have been few investigated in comparison with the neurotoxins. The present study used a mass spectrometric approach to detect 320 NDBPs and to sequence 27 of them; pharmacological assays permitted to characterize 13 NDBPs as novel toxins involved with inflammation, pain and edema formation.

Effects of Brazilian scorpion venoms on the central nervous system

In Brazil, the scorpion species responsible for most severe incidents belong to the Tityus genus and, among this group, T. serrulatus, T. bahiensis, T. stigmurus and T. obscurus are the most dangerous ones. Other species such as T. metuendus, T. silvestres, T. brazilae, T. confluens, T. costatus, T. fasciolatus and T. neglectus are also found in the country, but the incidence and severity of accidents caused by them are lower. The main effects caused by scorpion venoms - such as myocardial damage, cardiac arrhythmias, pulmonary edema and shock - are mainly due to the release of mediators from the autonomic nervous system. On the other hand, some evidence show the participation of the central nervous system and inflammatory response in the process. The participation of the central nervous system in envenoming has always been questioned. Some authors claim that the central effects would be a consequence of peripheral stimulation and would be the result, not the cause, of the envenoming process. Because, they say, at least in adult individuals, the venom would be unable to cross the blood-brain barrier. In contrast, there is some evidence showing the direct participation of the central nervous system in the envenoming process. This review summarizes the major findings on the effects of Brazilian scorpion venoms on the central nervous system, both clinically and experimentally. Most of the studies have been performed with T. serrulatus and T. bahiensis. Little information is available regarding the other Brazilian Tityus species.

Effects of Brazilian scorpion venoms on the central nervous system

In Brazil, the scorpion species responsible for most severe incidents belong to the Tityus genus and, among this group, T. serrulatus, T. bahiensis, T. stigmurus and T. obscurus are the most dangerous ones. Other species such as T. metuendus, T. silvestres, T. brazilae, T. confluens, T. costatus, T. fasciolatus and T. neglectus are also found in the country, but the incidence and severity of accidents caused by them are lower. The main effects caused by scorpion venoms - such as myocardial damage, cardiac arrhythmias, pulmonary edema and shock - are mainly due to the release of mediators from the autonomic nervous system. On the other hand, some evidence show the participation of the central nervous system and inflammatory response in the process. The participation of the central nervous system in envenoming has always been questioned. Some authors claim that the central effects would be a consequence of peripheral stimulation and would be the result, not the cause, of the envenoming process. Because, they say, at least in adult individuals, the venom would be unable to cross the blood-brain barrier. In contrast, there is some evidence showing the direct participation of the central nervous system in the envenoming process. This review summarizes the major findings on the effects of Brazilian scorpion venoms on the central nervous system, both clinically and experimentally. Most of the studies have been performed with T. serrulatus and T. bahiensis. Little information is available regarding the other Brazilian Tityus species.(AU)

Influence of different processing techniques on the toxicity and biochemical characteristics of Tityus serrulatus scorpion venom

Studies of scorpion venoms have used different venom drying methods: lyophilization, desiccation, lyophilization after mixing with 0.9% saline or purified water and centrifugation. The aim of this study was to see if these different approaches cause some alteration in the composition of the venom or interfere with its biological effects. Mice were injected (i.p.) with T. serrulatus scorpion venom in the liquid form (G-liq) or dried by different methods (lyophilized – G-lyo; centrifuged and the supernatant lyophilized – G-cen; desiccated – G-des), and observed regarding the occurrence of the symptoms respiratory difficulty, convulsion and death. The occurrence of seizures, although occurring in all groups and with the various doses used, did not prove to be effective to determine differences between the different handling techniques. Respiratory distress appeared to be useful in analyzing differences between groups, where this effect was less pronounced in the G-liq and G-des groups. In general, death occurred in a certain proportion with increasing dose for all groups. G-liq and G-des seemed to be more "active" at lower doses and G-cen and G-lyo at higher doses. The electrophoretic and chromatographic profile demonstrated main differences between G-liq and the dried groups. In the electrophoretic profile, the liquid venom showed bands of proteins of higher concentration and greater number of major bands and the three dried venom had the lowest number of protein bands. The HPLC profile and densitometry of the electrophoretic profiles showed some differences that may be associated with different protein conformation/aggregation. Our data indicated that lyophilization is the most suitable method for processing T. serrulatus scorpion venom after extraction.

Tb II-I, a fraction isolated from Tityus bahiensis scorpion venom, alters cytokines': level and induces seizures when intrahippocampally injected in rats

Scorpion venoms are composed of several substances with different pharmacological activities. Neurotoxins exert their effects by targeting ion channels resulting in toxic effects to mammals, insects and crustaceans. Tb II-I, a fraction isolated from Tityus bahiensis scorpion venom, was investigated for its ability to induce neurological and immune-inflammatory effects. Two putative -sodium channel toxins were identified in this fraction, Tb2 II and Tb 4, the latter having been completely sequenced by mass spectrometry. Male Wistar rats, stereotaxically implanted with intrahippocampal cannulas and electrodes, were injected with Tb II-I (2 mu g/2 mu L) via the intrahippocampal route. The behavior, electrographic activity and cellular integrity of the animals were analyzed and the intracerebral level of cytokines determined. Tb II-I injection induced seizures and damage in the hippocampus. These alterations were correlated with the changes in the level of the cytokines tumoral necrosis factor- (TNF-) and interleukin-6 (IL-6). Therefore, the binding of Tb II-I to its target in the central nervous system may induce inflammation resulting in neuropathological and behavioral alterations.

Proteomic endorsed transcriptomic profiles of venom glands from Tityus obscurus and T. serrulatus scorpions

Background Except for the northern region, where the Amazonian black scorpion, T. obscurus, represents the predominant and most medically relevant scorpion species, Tityus serrulatus, the Brazilian yellow scorpion, is widely distributed throughout Brazil, causing most envenoming and fatalities due to scorpion sting. In order to evaluate and compare the diversity of venom components of Tityus obscurus and T. serrulatus, we performed a transcriptomic investigation of the telsons (venom glands) corroborated by a shotgun proteomic analysis of the venom from the two species. Results The putative venom components represented 11.4% and 16.7% of the total gene expression for T. obscurus and T. serrulatus, respectively. Transcriptome and proteome data revealed high abundance of metalloproteinases sequences followed by sodium and potassium channel toxins, making the toxin core of the venom. The phylogenetic analysis of metalloproteinases from T. obscurus and T. serrulatus suggested an intraspecific gene expansion, as we previously observed for T. bahiensis, indicating that this enzyme may be under evolutionary pressure for diversification. We also identified several putative venom components such as anionic peptides, antimicrobial peptides, bradykinin-potentiating peptide, cysteine rich protein, serine proteinases, cathepsins, angiotensin-converting enzyme, endothelin-converting enzyme and chymotrypsin like protein, proteinases inhibitors, phospholipases and hyaluronidases. Conclusion The present work shows that the venom composition of these two allopatric species of Tityus are considerably similar in terms of the major classes of proteins produced and secreted, although their individual toxin sequences are considerably divergent. These differences at amino acid level may reflect in different epitopes for the same protein classes in each species, explaining the basis for the poor recognition of T. obscurus venom by the antiserum raised against other species.