Hyaluronidases in Loxosceles intermedia (Brown spider) venom are endo-beta-N-acetyl-d-hexosaminidases hydrolases.

In studying Loxosceles venom, we detected degradation of purified hyaluronic acid (HA) and hydrolysis of purified chondroitin sulphate (CS) while neither dermatan sulphate, heparin or heparan sulphate were affected. In addition, with HA-degrading kinetic assays, we show that a hydrolase enzyme was involved in the HA cleavage. By use of the Reissig colorimetric reaction, we found that venom hyaluronidase is an endo-beta-N-acetyl-d-hexosaminidase that generates terminal N-acetylglucosamine residues upon cleavage of HA. Zymogram analysis of L. intermedia venom showed HA lytic activities at 41 and 43kDa, and, when CS was used as a substrate, zymograph experiments resulted in 41 and 43kDa lytic zones. Thus, these results support the hypothesis that the same molecules are involved in cleaving HA and CS residues. Experiments to compare L. intermedia electrostimulated venom and venom gland extract also demonstrated very similar HA lytic activity, suggesting again that hyaluronidases are self-components of Loxosceles spider venom instead of oral egesta contamination. HA degradation as a function of pH in these hydrolase enzymes showed no apparent activities at low or high pH, with optimal activity at 6.0-8.0 pH. Finally, we confirmed the cleaving action of the venom hyaluronidases on HA in the extracellular matrix of the dermis of rabbit by fluorescence reaction to HA and confocal microscope analysis. Thus, hyaluronidases type hydrolases endo-beta-N-acetyl-d-hexosaminidase are implicated as self-components of Loxosceles spider venom and can be involved in venom effects as spreading factors.     

A potent vasoactive cytolysin isolated from Scorpaena plumieri scorpionfish venom

A new vasoactive cytolytic toxin, referred to as Sp-CTx, has been purified from the venom of the scorpionfish Scorpaena plumieri by a combination of gel filtration and anion exchange chromatographies. An estimation of Sp-CTx native molecular mass, performed by size exclusion chromatography, demonstrated that it is a 121 kDa protein. Further physicochemical studies revealed its glycoproteic nature and dimeric constitution, comprising subunits of approximately 65 kDa (MALDI-TOF-MS). Such protein has proved to possess a potent hemolytic activity on washed rabbit erythrocytes (EC₅₀ 0.46 nM), whose effect was strongly reduced after treatment with antivenom raised against stonefish venom - Synanceja trachynis (SFAV). This cross-reactivity has been confirmed by western blotting. Like S. plumieri whole venom (100 μg/mL), Sp-CTx (1-50 nM) caused a biphasic response on phenylephrine pre-contracted rat aortic rings, characterized by an endothelium- and dose-dependent relaxation phase followed by a contractile phase. The vasorelaxant activity has been abolished by l-NAME, demonstrating the involvement of nitric oxide on the response. We report here the first isolation of a cytolytic/vasoactive protein from scorpionfish venom and the data provided suggest structural and functional similarities between Sp-CTx and previously published stonefish hemolytic toxins.     

Cloning and molecular characterization of BmHYA1, a novel hyaluronidase from the venom of Chinese red scorpion Buthus martensi Karsch

In this communication, the full protein sequence of a novel venom hyaluronidase BmHYA1 was reported. It is the first full hyaluronidase amino acid sequence from scorpion venom. It was deduced from nucleotide sequence by 3′ Rapid Amplification of cDNA Ends (RACE) PCR cloning, followed by alignment with the N-terminal amino acid sequence, which was obtained by Edman degradation. BmHYA1 has 385 amino acid residues containing five potential N-glycosylation sites. The phylogenetic analysis indicates early divergence and independent evolution of BmHYA1 from other hyaluronidases.     

Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels

The β-toxins purified from the New World scorpion venoms of the Centruroides species affect several voltage-gated sodium channels (VGSCs) and thus are essential tools not only for the discrimination of different channel sub-types but also for studying the structure-function relationship between channels and toxins. This communication reports the results obtained with four different peptides purified from three species of Centruroides scorpions and assayed on seven distinct isoforms of VGSC (Na_v1.1-Na_v1.7) by specific functional analysis conducted through single cell electrophysiology. The toxins studied were CssII from Centruroides suffusus suffusus, Cll1 and Cll2 from Centruroides limpidus limpidus and a novel toxin from Centruroides noxius, which was characterized for the first time here. It has 67 amino acid residues and four disulfide bridges with a molecular mass of 7626 Da. Three different functional features were identified: current reduction of macroscopic conductance, left shift of the voltage-dependent activation and induction of resurgent currents at negative voltages following brief, strong depolarizations. The isoforms which revealed to be more affected resulted to be Na_v1.6 > 1.1 > 1.2 and, for the first time, a β-toxin is here shown to induce resurgent current also in isoforms different from Na_v1.6. Additionally, these results were analyzed with molecular modelling. In conclusion, although the four toxins have a high degree of identity, they display tri-modal function, each of which shows selectivity among the different sub-types of Na⁺-channels. Thus, they are invaluable as tools for structure-function studies of β-toxins and offer a basis for the design of novel ion channel-specific drugs.     

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

Stonefish (Synanceia trachynis) Antivenom: In Vitro Efficacy and Clinical Use

Certain species of fish have long been recognised as venomous. Although venomous fish do not represent a substantial source of human mortality, they are responsible for a number of envenomations each year that are serious enough to warrant clinical treatment. To the author's knowledge, the only fish antivenom still commercially available is the stonefish antivenom produced by CSL Ltd. in Australia. This antivenom consists of the purified F(ab)₂ fragment of equine IgG antibodies raised against the venom of Synanceia trachynis. The antivenom is cheap, and effective in neutralising all known clinical effects of serious S. trachynis envenomation. In addition, there is experimental evidence that stonefish antivenom neutralises the pharmacological effects of other fish venoms, particularly those of the lionfish (Pterois volitans) and the soldierfish (Gymnapistes marmoratus), as well as displaying cross‐reactivity with them in Western immuno‐blotting. The potential therefore exists for the use of stonefish antivenom in the treatment of severe envenomations by species of fish other than Synanceia spp.     

JZTX-XIII, a Kv channel gating modifier toxin from Chinese tarantula Chilobrachys jingzhao

Jingzhaotoxin-XIII (JZTX-XIII), a 35 residue polypeptide, with the ability to inhibit voltage-dependent potassium channels in the shab (Kv2) and shal (Kv4) subfamilies, was purified from the venom of the Chinese tarantula Chilobrachys jingzhao. Electrophysiological recordings carried out in Xenopus laevis oocytes showed that JZTX-XIII acted as gating modifier of voltage-dependent K⁺ channels which inhibited the Kv2.1 channel and Kv4.1 channel, with the IC₅₀ value of 0.47 μM and 1.17 μM, respectively. JZTX-XIII shares high sequence similarity with gating modifier toxins inhibiting a wide variety of ion channels including Nav1.5 subtype, but it showed no Nav1.5 channel activity. Structure-function analysis indicates that the acidic residues of Glu10 and Glu17 in JZTX-XIII might be responsible for the loss of the Nav1.5 channel inhibitory potency for JZTX-XIII.     

Electrophysiological characterization of a novel small peptide from the venom of Conus californicus that targets voltage-gated neuronal Ca channels

Conus californicus belongs to a genus of marine gastropods with more than 700 extant species. C. californicus has been shown to be distantly related to all Conus species, but showing unusual biological features. We report a novel peptide isolated from C. californicus with a significant inhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by 13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) is strikingly different from regular ω-conotoxins. In the HPLC purification procedure, the venom fraction eluted in the first 10-15 min produced a significant decrease (54% ± 3%) of the Ca²⁺ current in Xenopus laevis oocytes transfected with purified rat-brain mRNA. A specific peptide obtained from the elution at 13 min decreased the Ca²⁺ current in the adult rat dorsal-root ganglion neurons in a primary culture by 34% ± 2%. The cysteine pattern of this peptide corresponds to the framework XVI described for the M-superfamily of conopeptides and is unprecedented among Conus peptides acting on Ca²⁺ channels.     

Identification, cDNA cloning and heterologous expression of a hyaluronidase from the tarantula Brachypelma vagans venom

Hyaluronidases (Hyal) present in the venom of poisonous animals have been considered as “spreading factors” that facilitate a fast penetration of the venom in the prey. We have found that hyaluronidase from the tarantula Brachypelma vagans venom (BvHyal) displays a substrate-specific Hyal activity against hyaluronan. By using a combined strategy based on peptide sequencing and RT-PCR, we have cloned a BvHyal cDNA. Active recombinant BvHyal was efficiently expressed in a baculovirus system in insect cell.     

Stonefish (Synanceia spp.) antivenom neutralises the in vitro and in vivo cardiovascular activity of soldierfish (Gymnapistes marmoratus) venom.

The soldierfish (Gymnapistes marmoratus), which is related to the stonefish (Synanceia spp.), inhabits the western, southern and lower eastern coastlines of Australia. We have previously found that G. marmoratus venom possesses pharmacological activity similar to Synanceia trachynis venom (Hopkins, B.J., Hodgson, W.C., 1998. Cardiovascular studies on venom from the soldierfish (Gymnapistes marmoratus). Toxicon 36, 973-872; Church, J.E., Hodgson, W.C., 2000. Dose-dependent cardiovascular and neuromuscular effects of stonefish (Synanceja trachynis) venom. Toxicon 38, 391-407), namely an action at muscarinic receptors and adrenoceptors. The aim of this study was to determine the effectiveness of Synanceia antivenom in neutralising the in vitro and in vivo cardiovascular activity of G. marmoratus venom. Venom extract (0.4-12 microg protein/ml) caused dose- and endothelium-dependent relaxation in porcine U46619-precontracted coronary arteries. This relaxation was abolished by 10 min prior exposure of the tissue to Synanceia antivenom (3 units/ml). In rat paced (5 ms, 2 Hz, 7-12 V) left atria, G. marmoratus venom extract (40 microg protein/ml) produced a transient negative, followed by a sustained positive inotropic response. In spontaneously beating right atria, venom extract (40 microg protein/ml) produced similar changes in rate. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the inotropic response, and abolished the positive chronotropic response. In the anaesthetised rat, venom extract (400 microg protein/kg, i.v.) produced a transient depressor response, followed by a more sustained pressor response. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the in vivo response. As Synanceia antivenom neutralised the cardiovascular activity of G. marmoratus venom both in vitro and in vivo, we suggest that the venoms of the two species may share a similar component(s).     

Stonefish antivenom neutralises the inflammatory and cardiovascular effects induced by scorpionfish Scorpaena plumieri venom

Venomous fish are often involved in human accidents and symptoms of envenomation include local (intense pain and swelling) and systemic effects (cardiovascular and neurological disorders). However the only commercially available antivenom is against the Indo-Pacific stonefish Synanceja trachynis Stonefish Antivenom (SFAV). The aim of the present study was to evaluate the potential of SFAV in neutralising the in vivo effects of some toxic activities of scorpionfish Scorpaena plumieri venom (SpV), and the in vitro immuno cross-reactivity. The SpV (7.5–100 μg/animal) caused nociceptive and dose-dependent edematogenic responses in the mice footpad. In rats SpV (300 μg/kg, i.v.) produced immediate and transient increase in arterial blood pressure and decrease in heart rate. Prior incubation of SpV with SFAV (1 μg SpV/1 U SFAV) abolished the inflammatory response, and significantly attenuated the cardiovascular effects induced by SPV. Western blotting analysis on two-dimensional SDS-PAGE of S plumieri venom proteins using SFAV proved that the epitopes recognized by SFAV are shared with the ～98 kDa proteins. This is the first report of venom similarities between Indo-Pacific and Atlantic venomous fish, suggesting that the SpV compound responsible for inflammatory and cardiovascular effects possesses similar biochemical and antigenic properties to those found in stonefish venom.     

Green synthesis of silver nanoparticles using aqueous extracts of three Sideritis species from Turkey and evaluations bioactivity potentials

The members of the genus Sideritis have a wide variety of phytochemicals and thus the genus are gaining interest to fabricate nanoparticles (via green synthesis) as sources reducing or stabilizer agents. In the present study, silver nanoparticles (AgNPs) were synthesized by an easy and eco-friendly method using aqueous extracts of three Sideritis species (Sideritis argyrea (SA), S. brevidens (SB), and S. lycia (SL)). These AgNPs were investigated in terms of cholinesterase (AChE: acetylcholinesterase and BChE: butyrylcholinesterase) and tyrosinase activities. The presence of (111), (200), (220) and (311) planes in Bragg's reflections verified the fcc (face-cantered-cubic) crystalline AgNPs. Sideritis species-directed AgNPs were characterized by surface plasmon resonance at 428–440 nm. Transmission electron microscopy (TEM) characterizations were showed the spherical and monodispersed of the AgNPs with an average particle size of 22–26 nm. Fourier transform infrared (FTIR) spectra were revealed functional groups responsible for the reduction of silver ions. Also, for the AgNPs were confirmed by the characterizations of Zeta Potential and Dynamic Light Scattering (DLS). Chlorogenic acid (CGA) was found as major component for all three species. We demonstrated that Sideritis-directed AgNPs showed excellent inhibitory activity against BChE, while Sideritis extracts have no effective inhibitory activity against AChE. Among AgNPs, SA AgNPs exhibited the greatest tyrosinase inhibitory activity with the value of 33.02 mg kojic acid equivalents (KAE)/g, following CGA AgNPs (18.31 mg KAE/g) and SB AgNPs (5.46 mg KAE/g). Regarding the extracts, they had similar tyrosinase inhibition activity (33.61–36.34 mg KAE/g). Our findings suggest that the green synthesis by using Sideritis extracts could be open a new horizon in the biotechnological applications such as bioactivity and drug delivery.     

Retention and tissue damage of PSP and NSP toxins in shrimp: Is cultured shrimp a potential vector of toxins to human population?

Toxic microalgae outbreaks have caused significant economic losses in the Mexican aquaculture industry. Blooms that involve PSP and NSP phycotoxins are two of the most dangerous, causing harmful effects to the environment, economy and public health. The exact metabolic mechanism of these toxins in shrimp still remains unknown. Because shrimp consume microalgae their edible tissues are clearly possible vectors for human toxic syndrome. This study examined and verified the toxicological effects for white leg shrimp (Litopenaeus vannamei) exposed to different cell densities of Gymnodinium catenatum and Karenia brevis. Acute assays demonstrated good survival rates of shrimp at low densities of dinoflagellates (10³ cell/L), while mortality and abnormal behavior were observed with higher densities (>10⁴ cell/L). Chronic assays showed significant differences in survival rates, percentage of feed and weight gain of organisms exposed to the dinoflagellates with respect to controls. Furthermore, PSP and NSP toxins were detected in all the edible tissues. Gastric glands and muscle retained toxins for a longer period of time compared to other tissues, even after a depuration period. Histology damages were observed in the heart, gastric gland and brain. This study strongly supports that shrimp represent a potential risk for humans as unconventional vectors of phycotoxins.     

Milking and partial characterization of venom from the Brazilian spider Vitalius dubius (Theraphosidae)

The theraphosid spider genus Vitalius contains several species found in southeastern Brazil. In this work, we used electrostimulation to obtain venom from Vitalius dubius and examined its general composition. Male spiders yielded significantly less (p < 0.05) venom (12.5 ± 0.7 mg of liquid/spider, n = 16; mean ± S.E.M.) than female spiders (25.5 ± 2.0 mg of liquid/spider, n = 11). However, when corrected for spider weight, males yielded slightly more venom (2.89 ± 0.16 mg/g vs. 2.45 ± 0.76 mg/g for males and females, respectively, p < 0.05). Venom yield correlated linearly with spider weight for spiders weighing up to ∼12-13 g, but decreased in very heavy females. There was a marked decrease in venom yield after the first milking. The protein concentration of pooled venom was 18.3 ± 2.4 mg/ml (n = 4) and accounted for 16.6 ± 4.7% of the dry venom weight. The venom contained high hyaluronidase activity (275 ± 24 TRU/mg of protein, n = 4), with a molecular mass of ∼45 kDa estimated by zymography. SDS-PAGE revealed a few proteins with molecular masses >14 kDa but showed two staining bands of peptides <14 kDa. The venom reacted in ELISA with affinity-purified IgG from commercial arachnidic antivenom. Immunoblotting with this IgG detected proteins of 30-140 kDa only. Fractionation of the venom by reverse-phase chromatography resulted in five major and eight minor peaks.     

Bioactive proteins from stonefish venom

1. Of all the venomous fish known, the stonefish is one of the most commonly encountered by man. Studies on its venom started in the 1950s, but little work was performed after that until several groups revived interest in the venom in the 1980s after easier accessibility to the fish. 2. Stonefish venom is a mixture of proteins, containing several enzymes, including hyaluronidase of high specific activity. A purified stonefish hyaluronidase has been characterized. 3. Several of the effects of the crude venom have been isolated to a protein lethal factor that has cytolytic, neurotoxic and hypotensive activity. This protein is stonustoxin from Synanceja horrida, trachynilysin from Synanceja trachynis and verrucotoxin from Synanceja verrucosa. 4. The biochemical properties and activities of these protein lethal factors are reviewed.     

Heminecrolysin, the first hemolytic dermonecrotic toxin purified from scorpion venom

Envenomation caused by Hemiscorpius (H.) lepturus from Liochlidae family presents clinical features that have not been previously described for the Buthidae family scorpions. The most significant manifestations of H. lepturus envenomation are hemolysis and dermonecrosis which could lead in severe cases to renal, cardio-respiratory failure, and death. In this study, we aimed to identify and characterize the protein(s) causing these effects. We have purified a 33 kDa protein from the venom of H. lepturus and named it Heminecrolysin. Tryptic digestion and MS/MS analysis of obtained peptides showed homology with previously described brown spider sphingomyelinases D. Functional characterization of Heminecrolysin indicated a sphingomyelinase D, a complement-dependent hemolysis properties and a dermonecrosis activity. Heminecrolysin displayed higher hemolytic activity to human erythrocytes (ED50 of 0.025 μg/ml), a stronger inflammatory and dermonecrotic effects when injected intra-dermally to rabbit skins, while its efficiency to hydrolyze sphingomyelin seems weaker than other known spider dermonecrotic SMasesD (149 ± 32.5 nmol/mg). Step of sensitization of human erythrocytes by Heminecrolysin was shown to be Mg²⁺ and Ca²⁺-independent while hemolysis step in the presence of complement required both bivalent ions. Heminecrolysin is the first hemolytic dermonecrotic toxin identified in venom other than spiders. Except in spider Loxosceles genus and some pathogenic strains of Corynebacteria, sphingomyelinase D activity is unknown in the animal kingdom.     

Cytotoxic proteins of Amanita virosa Secr. mushroom: Purification, characteristics and action towards mammalian cells

A new hemolytic lectin was purified from the fruit bodies of Amanita virosa Secr. mushroom by the affinity chromatography on the cross-linked ovomucin. This lectin destroyed erythrocytes of human and animals of various species, and its hemolytic activity decreased in the row: rabbit > rat > human > dog. The erythrocytes of sheep, cow and carp were resistant to such hemolytic action of the lectin (1 mg/mL). The lectin-mediated hemolysis was blocked by the polyethylene glycol with molecular mass over 1350. A. virosa lectin, unlike Amanita phalloides lectin, did not interact with tested monosaccharides. However, the 4-nitrophenyl derivates of the monosaccharides inhibited the action of A. virosa lectin which did not prefer targeting O-type glycoproteins over the N-type glycoproteins. Murine leukemia cells of L1210 line and human leukemia T-cells of CEM T4 and Jurkat lines were shown to be sensitive to toxic effect of the lectin and another protein toxovirin isolated from A. virosa fruit bodies It was found that toxovirin possessed an enzymatic activity of l-amino acid oxidase. Since both toxic proteins - the lectin and toxovirin - are sensitive to an elevated temperature, it is suggested that they play a significant role in human poisoning only when the unbaked mushroom is eaten.     

First report of saxitoxin production by a species of the freshwater benthic cyanobacterium, Scytonema Agardh

Saxitoxins or paralytic shellfish poisons (PSP) are neurotoxins produced by some species of freshwater cyanobacteria and marine dinoflagellates. Samples collected from the metaphyton of a drinking-water supply’s pre-treatment reservoir and a small eutrophic lake in New Zealand returned positive results when screened using a Jellett PSP Rapid Test Kit. The dominant species in the sample was identified as Scytonema cf. crispum. A non-axenic clonal culture (UCFS10) was isolated from the lake. The partial 16S rRNA gene sequence shared only a 91% or less sequence similarity with other Scytonema species, indicating that it is unlikely that this genus is monophyletic and that further in-depth phylogenetic re-evaluation is required. The sxtA gene, which is known to be involved in saxitoxin production, was detected in UCFS10. Saxitoxin concentrations were determined from the lake samples and from UCFS10 using pre-column oxidation high performance liquid chromatography with fluorescence detection. Saxitoxin was the only variant detected and this was found at concentrations of 65.6 μg g⁻¹ dry weight in the lake sample and 119.4 μg g⁻¹ dry weight or 1.3 pg cell⁻¹ in UCFS10. This is the first confirmation of a saxitoxin-producing species in New Zealand and the first report of saxitoxin production by a species of Scytonema.