Deteriorating insulin resistance due to WL15 peptide from cysteine and glycine-rich protein 2 in high glucose-induced rat skeletal muscle L6 cells.

This study investigates the antioxidant and antidiabetic activity of the WL15 peptide derived from Channa striatus on regulating the antioxidant property in the rat skeletal muscle cell line (L6) and enhancing glucose uptake via glucose metabolism. Increased oxidative stress plays a major role in the development of diabetes and its complications. Strategies are needed to mitigate the oxidative stress that can reduce these pathogenic processes. Our results showed that with treatment with WL15 peptide, the reactive oxygen species significantly decreased in L6 myotubes in a dose-dependent manner, and increased antioxidant enzymes help to prevent the formation of lipid peroxidation in L6 myotubes. The cytotoxicity of WL15 is evaluated in the L6 cells and found to be non-cytotoxic at the tested concentration. Also, for the analysis of glucose uptake activity in L6 cells, the 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxy- d -glucose assay was performed in the presence of wortmannin and genistein inhibitors. WL15 demonstrated antidiabetic activities through a dose-dependent increase in glucose uptake (64%) and glycogen storage (7.8 mM). The optimal concentration for the maximum activity was found to be 50 µM. In addition, studies of gene expression in L6 myotubes demonstrated upregulation of antioxidant genes and genes involved in the pathway of insulin signaling. In cell-based assays, WL15 peptide decreased intracellular reactive oxygen species levels and demonstrated insulin mimic activity by enhancing the primary genes involved in the insulin signaling pathway by increased glucose uptake indicating that glucose transporter type 4 (GLUT4) is regulated from the intracellular pool to the plasma membrane.

Biochemical and histopathological effects of the stonefish (Synanceia verrucosa) venom in rats.

The Reef Stonefish (Synanceia verrucosa) is one of the most dangerous venomous fish known, and has caused occasional human fatalities. The present study was designed to examine some of the pathological effects of the venom from this fish in Sprague Dawley rats. Crude venom was extracted from venom glands of the dorsal spines of stonefish specimens collected from coral reefs in the Gulf of Aqaba (in the northeastern branch of the Red Sea). The rats were given intramuscular injections of the venom and acute toxicity and effect on selected serum marker enzymes as well as normal architecture of vital organs were evaluated. The rat 24 h LD50 was 38 µg/kg body weight. The serum biochemical markers; alanine transaminase (ALT), lactate dehydrogenase (LDH) and creatine kinase (CK) increased after 6 h of administration of a sub lethal dose of the venom and remained significantly raised at 24 h. Amylase levels also significantly increased after venom injection. The venom caused histological damage manifested as an interstitial hemorrhage, inflammatory cell infiltration, and necrosis. The demonstrated rises in the levels of different critical biochemical parameters in the serum may have led to the observed abnormal morphological changes in these organs. These results may account for some of the clinical manifestations observed in victims of stonefish envenomation. Thus, the presented data provide further in vivo evidence of the stonefish toxic effects that may threaten human life and call for the need for special measures to be considered.

Identification of C-type isolectins in the venom of the scorpionfish Scorpaena plumieri.

Chemical analyses of the hemagglutinating fraction from Scorpaena plumieri venom revealed that it contains five components (Sp-CL 1-5) with similar chromatographic elution profiles (35-38% of acetonitrile), molecular masses (16,800-17,000 Da) and N-terminal sequences, suggesting that they are isoforms of the same protein. The amino acid sequence of Sp-CL4 was determined and shown to have homology with fish C-type lectins. These data demonstrate for the first time the presence of C-type isolectins in a scorpionfish venom.

Clinical manifestations and experimental studies on the spine extract of the toadfish Porichthys porosissimus.

Toadfish are fish from the family Batrachoididae that are found in marine and brackish environment around the world. Among the toadfish, Porichthys genus is very common, where Porichthys porosissimus, also called Atlantic Midshipman is found in Southwest Atlantic, from Rio de Janeiro, Brazil to eastern Argentina. There was no consensus about the classification of the genus Porichthys as venomous fish because so far there are no published studies regarding human envenomations and/or toxic activities induced in animal models. Herein, we report two conclusive envenoming in human beings caused by P porosissimus spines, with clear signs and symptoms that were very important for the development of our experimental studies. We demonstrated that the P. porosissimus spine extract, now venom, can induce nociceptive and edematogenic responses in mice as well an induction of an inflammatory response elicited by intravital microscopy and leukocyte migration. Finally, we identified in the P. porosissimus spine extract, through analysis by mass spectrometry, the presence of proteins previously detected in the venoms of other fish species and other venomous animals. We believe that based on our studies we will dismiss the non-venomous nature of this fish and clarify this issue.

Structural and biological characterization of Nattectin, a new C-type lectin from the venomous fish Thalassophryne nattereri.

Lectins are glycan-binding receptors that recognize glycan epitopes on foreign pathogens and in the host systems. They can be involved in functions that include innate immunity, development, immune regulation and homeostasis. Several lectins have been purified and characterized from fish species. In this work, using cation-exchange chromatography, a galactose-specific lectin belonging to the family of C-type lectins was isolated from the venom of the Brazilian venomous fish Thalassophryne nattereri. Nattectin is a basic, non-glycosilated, 15 kDa monomeric protein. It exhibits hemagglutination activity that is independent of Ca(2+). We also demonstrated a lectin activity for Nattectin in the innate immune system, especially in neutrophil mobilization in mice, indicating that marine organisms are source of immunomodulator agents.

[Anti-angiogenic activities of Lj-RGD3 toxin protein from Lampetra japonica and its mutation protein Lj-112 without RGD motifs].

Arg-Gly-Asp (RGD)-toxin protein Lj-RGD3 of Lampetra japonica shares homologous with a Histidine-rich glycoprotein (HRG), and both RGD-toxin protein and HRG have antiangiogenic activities with different targets. To study the relationship between the function and the structure of Lj-RGD3, we studied the anti-angiogenic characteristics of both Lj-RGD3 and the mutation named Lj-112 of which three RGD motifs of Lj-RGD3 were deleted. We synthesized the gene of Lj-112, constructed it to the plasmid pET23b, and expressed the recombinant proteins in Escherichia coli BL21. Both recombinant proteins with the C-terminal his-tag were 15 kDa soluble proteins. Then we purified rLj-RGD3 and rLj-112 using the His-Bind affinity chromatography. To examine the effect of both proteins on bFGF-induced proliferation of ECV304 cell, we carried out the 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assays. For cell migration and invasion assays, we used Transwell containing insert filter and Matrigel to imitate the in vivo environment. To examine whether both proteins were capable of interrupting the angiogenesis in vivo, we used the chick chicken embryonic chorioallantoic membrane (CAM) as an angiogenesis model. We used Integrin-linked kinase1 (ILK1) ELISA method to study functionary mechanisms of rLj-RGD3 and rLj-112. Both rLj-RGD3 and rLj-112 inhibited bFGF-induced proliferation of ECV304 cells in a dose-dependent manner with IC50 at 0.889 micromol/L and 0.160 micromol/L, respectively. The results of migration and invasion assays revealed that both rLj-RGD3 and rLj-112 showed significant inhibition on bFGF induced migration and invasion of ECV304; and rLj-112 was more active than rLj-RGD3. The result of CAM angiogenesis assay demonstrated that both proteins inhibited the angiogenesis in chick CAM, and rLj-112 was more active than rLj-RGD3. ELISA assay of ILK1 showed that both rLj-RGD3 and rLj-112 down-regulated ILK1 expression of ECV304 cell. The fact of rLj-112 was more active than rLj-RGD3 on anti-angiogenesis indicate that rLj-112 was likely with histidine-rich glycoprotein (HRG), and the factor of sequence homologous between rLj-RGD3 and HRG cannot enhance antiangiogenic activities of rLj-RGD3, the signal pathway of anti-angiogenesis of rLj-RGD3 and rLj-112 are differently.

Insights into the local pathogenesis induced by fish toxins: Role ofnatterins and nattectin in the disruption of cell–cell and cell–extracellularmatrix interactions and modulation of cell migration

Combined proteomic and transcriptomic approaches to study the composition of the venom of Thalassophryne nattereri venomous fish revealed the primary structures of the major toxins as a family of proteases natterins, never described on venoms and a C-type lectin nattectin. To gain new insights into the mechanisms of venom pathogenesis and to further elucidate the role of its major toxins, the natterins and nattectin, we undertookin vitro investigations using these isolated toxins. Here we demonstrated the specific ability of the nattectin to bind types I and V collagen and natterins to bind and cleave type I collagen as well as type IV collagen, disrupting cell attachment and HeLa cells survival. Natterins have cytotoxic effect on both adherent cells or at in suspension, showing direct induction of necrosis that is followed by cell detachment. Nattectin improves integrinmediated HeLa cell adhesion and resistance to apoptosis by its binding to RGD dependent integrins, especially the b1 subunit. Based on our studies we now report that extracellular matrix (ECM) components as well as the integrin b1 subunit are targets for the natterins and nattectin. The ECM degradation or remodeling activities exerted by these toxins affect cell–cell and cell–ECM adhesion and survival and impair inflammatory cell migration into inflamed tissues.

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 (EC(50) 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 microg/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.

[Expression and bioactivity effects to Hela of recombinant toxin protein rLj-RGD3 from Lampetra japonica].

Lj-RGD3 was a toxin from the saliva gland of Lampetra japonica. To study the anti-tumor function of rLj-RGD3 and confirm its biological status and significance, we extracted total RNA from the saliva gland and amplified the cDNA of Lj-RGD3 by RT-PCR. The cDNA of Lj-RGD3 was 357 bp long and encoded a polypeptide composed of 118 amino acids including 2 cysteines, 17 histidines and 3 RGD (Arg-Gly-Asp) motifs. We cloned the cDNA into the plasmid pET23b, and expressed the recombinant protein rLj-RGD3 in Escherichia coli BL21. Fusion rLj-RGD3 with the C-terminal his-tag was a 15 kD soluble protein. Using the His-Bind affinity chromatography, we purified rLj-RGD3. Furthermore, we determined the biological activities of rLj-RGD3. To examine the ability of rLj-RGD3 inhibiting Hela cells proliferation, we used MTT assay. The results showed that, rLj-RGD3 inhibited bFGF induced proliferation of Hela cells in a dose-dependent manner, the IC50 value was 2.6 micromol/L. Hoechst staining assay revealed that, the nuclei of the cells treated with rLj-RGD3 were stained much brighter than that of untreated cells due to chromatin condensation. Furthermore, the DNA ladder patterns from the cells treated with rLj-RGD3 were also observed. These results demonstrated that rLj-RGD3 could induce apoptosis of Hela cells. Cell adhesion, migration and invasion are critical processes in tumor metastasis. rLj-RGD3 significantly inhibited adhesion of Hela cells to vironectin in a dose-dependent manner. In order to determine the effect of rLj-RGD3 on Hela cells migration toward bFGF, we used Transwell containing insert filter. rLj-RGD3 showed a significant inhibition on Hela cells migration, the inhibition rate was 60%. In the invasion assay, the Matrigel and Transwell were used to imitate environment in vivo. The results of invasion assay revealed that, rLj-RGD3 significantly inhibited bFGF induced invasion of Hela cells. Taken together, these results revealed that rLj-RGD3 had typical functions of RGD toxin protein and will be valuable in developing anti-tumor recombinant medicine.

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom.

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.

Purification, properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom.

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of alpha-subunit (702 amino acid residues) and beta-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX alpha- and beta-subunits share as high as 87 and 95% sequence identity with the SNTX alpha- and beta-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX beta-subunit with the reported VTX beta-subunit is 90%, being lower than that with the SNTX beta-subunit.

Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom.

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

The effects of L-arginine, D-arginine, L-name and methylene blue on channa striatus-induced peripheral antinociception in mice.

PURPOSE: To determine the involvement of nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway in aqueous supernatant of haruan (Channa striatus) fillet (ASH) antinociception using the acetic acid-induced abdominal constriction test. METHODS: The ASH was prepared by soaking fresh haruan fillet in chloroform:methanol (CM) (2/1 (v/v)) for 72 h followed by evaporation of the upper layer supernatant to remove any solvent residues. The supernatant was then subjected to a freeze-drying process (48 h) followed by doses preparation. RESULTS: Subcutaneous (SC) administration of ASH alone (0.170, 0.426 and 1.704 mg/kg) exhibited a dose-dependent antinociception. On the other hand, 20 mg/kg (SC) of L-arginine and MB exhibited a significant nociception and antinociception, while D-arginine and L-NAME did not produce any effect at all. Pre-treatment with L-arginine was found to significantly reverse the three respective doses of ASH antinociception; pre-treatment with D-arginine did not produce any significant change in the ASH activity; pre-treatment with L-NAME only significantly increased the 0.170 and 0.426 mg/kg ASH antinociception; and pre-treatment with MB significantly enhanced the respective doses of ASH antinociception, respectively. Furthermore, co-treatment with L-NAME significantly enhanced the L-arginine reversal effect on 0.426 mg/kg ASH antinociception. In addition, MB significantly reversed the L-arginine nociception on 0.426 mg/kg ASH. CONCLUSIONS: These finding suggest ASH antinociception involves the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. The presence of NO was found to reverse ASH antinociceptive activity while blocking of cGMP system enhanced it.

Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

Biological properties of the venom from the scorpionfish (Scorpaena plumieri) and purification of a gelatinolytic protease.

In this work we describe some biological properties and a partial biochemical characterization of the Scorpanea plumieri crude venom. The fresh venom induced a decrease in blood pressure, cardiac and respiratory frequency, and exhibited hemorrhagic, hemolytic and proteolytic activities. The LD(50) (i.v. mouse) was 0.28 mg/kg. The pharmacological activities were found to be very unstable and this fact could be associated with proteolytic activity. Enzymes which hydrolyze casein and gelatin were found in this venom. A gelatinolytic protease (Sp-GP) was purified to homogeneity from S. plumieri venom through a combination of three chromatographic steps: gel filtration on Sephacryl S-200; ion exchange on DEAE-cellulose and reverse-phase/HPLC on a Vydac C4 column. The purified protease was approximately 2% of the whole protein in the soluble crude venom. The molecular mass of the Sp-GP scorpionfish gelatinase estimated by SDS-PAGE was around 80,000 Da under reducing conditions and 72,000 Da under non-reducing conditions. Attempts to determine the N-terminal sequence by automatic Edman degradation were unsuccessful, probably due to blockage of the N-terminal group. Gelatinolytic activity was optimal at pH 7-8. This is the first report of the isolation and characterization of a scorpionfish venom protease.

Isolation and partial structural evaluation of a cardiotoxic factor from Indian common murrel (Channa striatus L.) skin extract.

It was earlier reported from this laboratory that, Channa striatus, L a common edible fish, whose skin extract (CSSE) was pharmacologicaclly potent and contains several bioactive compounds. In the present communication a cardiotoxic factor was isolated and purified by thin layer chromatography followed by silica gel and neutral alumina column chromatography. Spectroscopic studies (UV, IR, 1H and 13C NMR, FAB-MS) indicated that the lethal cardiotoxic factor (CSS-CTF II) was an aromatic alkaloid compound with -NH, > C = C < and -OH functional groups. The molecular weight was found to be 413 dalton. LD50 of CSS-CTF II was found to be 42.5 mg/kg (i.v) in Swiss albino male mice. Pharmacological studies showed that CSS-CTF II possesses hypotensive and cardiotoxic activities and produced death through apnoea in experimental animals but had no effect on nerve muscle preparations. The haematological and biochemical data also indicated the toxic nature of CSS-CTF II, through significant fall in haemoglobin, total RBC, WBC, platelet count and increased cardiac marker enzyme CPK and CPK-MB value in experimental animals. The present investigation thus established the toxic nature of CSS-CTF II isolated from edible fish C. striatus skin extract. Further work is needed to identify CSS-CTF II's mechanism of action and its antagonism for therapeutic purpose.

Isolation of a haemorrhagic protein toxin (SA-HT) from the Indian venomous butterfish (Scatophagus argus, Linn) sting extract.

A haemorrhagic protein toxin (SA-HT) was isolated and purified from the spine extract of the Indian venomous butterfish, S. argus Linn, by two step ion exchange chromatography. The toxin was homogeneous in native and SDS-PAGE gel. SDS-molecular weight of the toxin was found to be 18.1 +/- 0.09 kDa. SA-HT produced severe haemorrhage on stomach wall but devoid of cutaneous haemorrhage. UV, EDTA, trypsin, protease, cyproheptadine, indomethacin, acetylsalicylic acid and BW755C treatment significantly antagonized the haemorrhagic activity of SA-HT. The toxin produced dose and time dependent oedema on mice hind paw, which was significantly encountered by cyproheptadine, indomethacin and BW755C. SA-HT increased capillary permeability on guinea pig dorsal flank. On isolated guineapig ileum, rat fundus and uterus, SA-HT produced slow contraction which was completely antagonised by prostaglandin blocker SC19220. On isolated rat duodenum, SA-HT produced slow relaxation. SA-HT significantly increased plasma plasmin, serum MDA level and decreased serum SOD level indicating the possible involvement of cyclooxygenase and lipooxygenase pathway.

Fishing for bioactive substances from scorpionfish and some sea urchins.

Venom proteins from the dorsal spine of two scorpionfish, Hypodytes rubripinnis and Synanceia verrucosa were assayed for mitogenicity and cytotoxicity. The two venoms had both mitogenic and cytotoxic activity on murine splenocytes and murine P388 leukemic cells. In H. rubripinnis, the second gel chromatographic fraction showed cytotoxic activity on P388 leukemic cells. On native PAGE, the glycoprotein isolated by concavalin A sepharose chromatography appeared to have a molecular mass of 110 kDa. In addition, two D-galactose-binding lectins (SUL-I and SUL-II) and a heparin-binding lectin (TGL-I) were purified from the globiferous pedicellariae of the toxopneustid sea urchins, Toxopneustes pileolus and Tripneustes gratilla, respectively. SUL-I (Nakagawa et al., 1999a) had mitogenic activity and cytotoxic activity but SUL-II and TGL-I did not. SUL-I did not show sequence homology to SUL-II. A hemolytic lectin with a molecular mass of 29 kDa was isolated from the coelomic fluid of T. gratilla. The hemolytic activity of the lectin was dependent on Ca2+ concentration and inhibited by lactose. The present results suggest that some species of scorpionfish and sea urchins may be novel sources for biologically active substances such as anti-tumor compounds or new lectins.

Evidence for a neurotoxic activity in crude venom of the stonefish (Synanceia verrucosa).

The neurotoxic effects of the Synanceia verrucosa venom were investigated in rodents. After intracranial injection in mice (50-125 ng/g), venom induced constant symptoms such as ataxia, circling, partial or complete reversible limbs paralysis, scratching, rolling, sleep-like periods and violent clonic seizure conducing in few seconds to death. EEG alterations occurring in rat brain after intracerebroventricular injection (50-100 microg) were precised. An initial phase was characterized by short repetitive tonic seizure periods together with a significant rise of the relative power in the delta band, no significant modification of the theta II rhythm (4-7 Hz), a decreasing of energy in theta I (7-12 Hz) and 15-40 Hz bands. A second phase was characterized by a marked generalized slowing with transient drastic decreasing of the amplitude and flattening of cortical EEG (comatose state) as the main elements. Propanolol did not reverse the EEG effects of the venom except a slight decrease of the slow wave amplitude. Previous intracerebroventricular administration of a K+(ATP) blocker generally decrease the delay of death. Histopathologic examination of the brain of surviving animals did not reveal any microscopic lesions. These results suggest (1) a complex mechanism of the venom in its neuropathologic expression; (2) at the doses tested, symptoms are not related to adrenergic pathways, K+(ATP) channel opener (verrucotoxin) is not implied in the neurotoxic effect, and the effect of the venom, which not affecting the theta II rhythm, seemed not to be exerted through cholinergic pathway.

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.