Purification, characterization and cDNA cloning of two natterin-like toxins from the skin secretion of oriental catfish Plotosus lineatus

The oriental catfish Plotosus lineatus is known to contain proteinaceous toxins in the skin secretion as well as in the venom gland. However, detailed properties and primary structures of the skin toxins have not been clarified. In this study, two proteinaceous toxins (toxins I and II) were purified from the skin secretion of oriental catfish by a combination of gel filtration, anion-exchange HPLC and hydroxyapatite HPLC. Toxins I and II are monomeric simple proteins with almost the same molecular mass (35 kDa for toxin I and 37 kDa for toxin II) and are distinguishable from each other in isoelectric point (6.5 for toxin I and 5.1 for toxin II). Both toxins display lethal, edema-forming and nociceptive activities, although toxin I is significantly more potent than toxin II. The primary structures of toxins I and II were elucidated by cloning experiments based on the determined partial amino acid sequences. Toxins I (317 amino acid residues) and II (315 amino acid residues) share as high as 86% sequence identity with each other and are also highly homologous (56–75% identities) with the known fish natterin-like proteins.     

Further studies on pumiliotoxin 251D and hydroquinone content of the skin secretion of Melanophryniscus species (Anura, Bufonidae) from Uruguay.

In whole animal ethanolic extracts from adult specimens of Melanophryniscus atroluteus (27 specimens) and M. devincenzii (16 specimens) as well as of two egg clutches and four tadpole samples from the latter species, the major alkaloid pumiliotoxin (PTX) 251D and hydroquinone were assayed quantitatively by gas chromatography/mass spectrometry. All toad extracts contained high concentrations of PTX 251D and hydroquinone and exhibited considerable variation in the content of these compounds among individual specimens. The extracts of the eggs and tadpoles were entirely free of alkaloids as well as hydroquinone, pointing to a dietary origin of these compounds.     

Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens.

The increase in the emergence of antibiotic-resistant microorganisms and difficult to treat infections caused by these pathogens stimulate research aiming the identification of novel antimicrobials. Skin secretion of amphibian contains a large number of biologically active compounds, including compounds that performance defense mechanisms against microorganisms. In the present work, two antimicrobial bufadienolides, telocinobufagin (402.1609 Da) and marinobufagin (400.1515 Da), were isolated from skin secretions of the Brazilian toad Bufo rubescens. The specimens were collected in Brasilia (Distrito Federal, Brazil), the skin secretions extracted by electric stimulation, and submitted to purification by RP-HPLC. The molecular structure and mass determination were done by (1)H and (13)C NMR and mass spectrometry data, respectively. The antimicrobial activity was performed by liquid growth inhibition against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentrations of telocinobufagin and marinobufagin were, respectively, 64.0 and 16.0 microg/mL for E. coli and both 128 microg/mL for S. aureus. Besides the antimicrobial activity both bufadienolides promoted an increase of the contraction force in isolated frog ventricle strips.     

Muscle and skin necrotizing and edema-forming activities of Duvernoy's gland secretion of the xenodontine colubrid snake Philodryas patagoniensis from the north-east of Argentina.

Philodryas patagoniensis is a colubrid snake spread by all South America, but very little is known about the composition and biological activities of its Duvernoy's gland secretion. In order to characterize it, we studied edematogenic, dermonecrotic and myonecrotic activities. For edematogenic activity, solutions containing different amounts of secretion were injected s.c. in the right foot pad of mice, both feet were subsequently cut off and weighed individually. For myonecrotic activity, mice were injected i.m. with solutions containing 40 microg of secretion, and at various time intervals mice were bled to determine serum creatine kinase activity and gastrocnemius muscles were removed for microscopic examination (Hematoxylin-Eosin stain). For dermonecrotic activity, solutions containing different amounts of secretion were injected into the shaved dorsal skin of mice; the necrotic lesion was measured on the inner surface of the skin and trimmed for microscopic examination (Hematoxylin-Eosin stain). Phospholipase A(2) activity was evaluated using a kinetic method. Results showed that P. patagoniensis Duvernoy's gland secretion exhibits a high edematogenic activity and moderate myonecrotic and dermonecrotic activities, while lacking phospholipase A(2) effect. Regarding edema, a 30% increase in the weight was produced by injecting 0.26 microg of Duvernoy's gland secretion. Microscopically, myonecrosis reached its highest intensity 12 h after injection, which was also demonstrated by serum creatine kinase levels. Dermonecrosis was proportional to the amount of secretion injected, with a minimum necrotizing dose of 15.7 microg. Myonecrotic, edematogenic and dermonecrotic activities were inhibited when the secretion was pre-incubated with 1 mM Na(2)EDTA. This suggests that the enzymes responsible for those activities are mostly metalloproeinases. All the studies carried out up to now demonstrate the potential toxicity of P. patagoniensis Duvernoy's gland secretion (which inhabits the north-east region of Argentina) and that the local lesions caused by this colubrid snake are very similar to those found in bothropic accidents. This latter suggests a more careful evaluation of the victims when considering the medical treatment to be adopted.     

Major biological effects induced by the skin secretion of the tree frog Phyllomedusa hypochondrialis

Amphibian skin secretions contain several bioactive compounds such as biogenic amines, alkaloids, steroids, proteins and peptides; being peptides a continuously growing field of interest. This work aims to describe the main physiopathological properties of the tree frog Phyllomedusa hypochondrialis skin secretion, obtained by manual stimulation of the dorsal skin surface. Intravenous skin secretion administration provoked lethal effect in mice after 5 min. Low doses induced significant systemic and local effects like edema and nociception in mice and topic administration induced myonecrosis in the endothelium of cremaster mice. The presence of phospholipase A₂ activity, proteolytic activity and creatine kinase activity (in the plasma of treated mice) are reported and are very likely to be related to the physiopathological (edematic and myotoxic) activities observed. These data provide in vivo evidence of the complex toxic effects of the P. hypochondrialis skin secretion as well as possible mechanisms of action for these effects.     

Studies on the poisonous skin secretion of individual red bellied toads, Melanophryniscus montevidensis (Anura, Bufonidae), from Uruguay.

Toads belonging to the genus Melanophryniscus contain toxic alkaloids in their skin. From six locations in south-eastern Uruguay 81 specimens of Melanophryniscusmontevidensis were collected. In whole animal methanolic extracts of individual specimens, alkaloids of the pumiliotoxin (PTX) group and indolizidines were identified by gas chromatography/mass spectrometry; the predominant component PTX 251D was assayed quantitatively. The PTX-content of the various toad populations was found to be highly variable among individual specimens as well as among the populations. Very high levels of PTX 251D were detected in toads of the western part of the collection area, whereas very low levels of this alkaloid were assayed in toads near the Brazilian border. Remarkably high concentrations of the non-alkaloid hydroquinone were found to be present in all toads. The analysis of extracts from 125 arthropod samples (Arachnida and Insecta, including termites, ants and beetles), which may represent a potential food source, revealed no alkaloids of the PTX group.     

Purification and characterization of an irreversible serine protease inhibitor from skin secretions of Bufo andrewsi.

Amphibian skin secretions contain many bioactive compounds. In the present work, an irreversible serine protease inhibitor, termed baserpin, was purified for the first time from the skin secretions of toad Bufo andrewsi by successive ion-exchange and gel-filtration chromatography. Baserpin is a single chain glycoprotein, with an apparent molecular weight of about 60 kDa in SDS-PAGE. Baserpin is an irreversible inhibitor and effectively inhibits the catalytic activity of trypsin, chymotrypsin and elastase. SDS-stable baserpin-trypsin complex could be seen in SDS-PAGE indicates that it possibly belongs to the serpin superfamily. According to the association rates determined, baserpin is a potent inhibitor of bovine trypsin (4.6 x 10(6) M(-1) s(-1)), bovine chymotrypsin (8.9 x 10(6) M(-1) s(-1)) and porcine elastase (6.8 x 10(6) M(-1) s(-1)), whereas it shows no inhibitory effect on thrombin. The N-terminal sequence of baserpin is HTQYPDILIAKPXDK, which shows no similarity with other known serine protease inhibitors.     

Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin.

A novel trypsin inhibitor termed BATI was purified to homogeneity from the skin extracts of toad Bufo andrewsi by successive ion-exchange, gel-filtration and reverse-phase chromatography. BATI is basic single chain glycoprotein, with apparent molecular weight of 22 kDa in SDS-PAGE. BATI is a thermal stable competitive inhibitor and effectively inhibits trypsin's catalytic activity on peptide substrate with the inhibitor constant (K(i)) value of 14 nM and shows no inhibitory effect on chymotrypsin, thrombin and elastase. The N-terminal sequence of BATI is EKDSITD, which shows no similarity with other known trypsin inhibitors.     

Unusual profile of leukocyte recruitment in mice induced by a skin secretion of the tree frog Phyllomedusa hypochondrialis

Phyllomedusa hypochondrialis skin secretion can cause both systemic and local effects. In this study, we describe the pattern of local acute inflammatory response after P. hypochondrialis skin secretion injection. The inflammatory reaction in the mice footpad was analysed, including the leukocyte recruitment into local tissue from the peripheral blood, in a mouse model of tissue injury. We also investigated the release of the cytokines IL-1, IL-6 and TNF-α, chemokines KC and MCP-1 and the eicosanoids LTB 4 and PGE₂ in mice. The present findings support the ability of P. hypochondrialis skin secretion to induce local inflammation. In addition, these skin secretion components play a role in the initial rolling and slowing of recruited leukocytes and the transition from rolling to adhesion. Levels of the proinflammatory cytokine IL-6, chemokines KC and MCP-1 as well as the eicosanoid PGE₂ were significantly increased after injection of a skin secretion of P. hypochondrialis (0.6 μg/30 μl intraplantar), whereas no changes in other parameters were observed. Finally, the mechanisms involved in the local inflammatory process induced by P. hypochondrialis skin secretion is one of the questions of relevance related to the complex pathophysiology induced by this particular secretion and other toxins.     

Peptidomic dissection of the skin secretion of Phasmahyla jandaia (Bokermann and Sazima, 1978) (Anura, Hylidae, Phyllomedusinae)

The systematic investigation of the peptidic composition of the skin secretion of Phasmahyla jandaia, a phyllomedusine anuran endemic to the southern region of the Espinhaço range in Brazil, is herein reported. By means of de novo interpretation of tandem mass spectrometric data, Edman N-terminal sequencing and similarity searches, 57 peptides - including phylloseptins, dermaseptins stricto sensu, dermatoxins, hyposins, tryptophyllins, caerulein-related, bradykinin-related, bradykinin potentiating, tyrosine-rich, and opioid peptides - were sequenced. Moreover, five peptide families without significant similarity to other known molecules were verified. Differently from most Phyllomedusinae genera, the molecular diversity in the skin of representatives of Phasmahyla remained unprospected until now. Therefore, besides disclosing novel natural variants of number of bioactive peptides, the present study contributes to the understanding of the evolution of biochemical characters of the phyllomedusines.     

Isolation and characterization of a trypsin inhibitor from the skin secretions of Kaloula pulchra hainana

Amphibian skin secretions contain many bioactive compounds. A trypsin inhibitor termed KPHTI was purified from the skin secretions of frog Kaloula pulchra hainana by successive ion-exchange and gel-filtration chromatography. KPHTI is a single chain glycoprotein, with an apparent molecular weight of 23 kDa in SDS-PAGE. It is a competitive inhibitor and effectively inhibits trypsin catalytic activity on peptide substrate with the inhibitor constant (K_i) value of 27 nM. KPHTI shows no inhibitory effect on chymotrypsin, thrombin, elastase, and subtilisin. The N-terminal sequence of KPHTI is DHEVTS, which shows no similarity with other known trypsin inhibitors. DTT apparently affected the inhibitory activity of KPHTI. But it was not sensitive to temperature and pH range, which suggested that it possessed stable trypsin inhibitory activity in natural environment, and maybe play an important role in against predators.     

Purification, characterization and homology analysis of ocellatin 4, a cytolytic peptide from the skin secretion of the frog Leptodactylus ocellatus

Neobatrachia is the amphibian suborder with the largest number of species and a most important source of bioactive peptides from frog skin secretions. However, 90% of the studies on this subject have been focused on the frog families Hylidae and Ranidae, while very little is known about peptides of other families, like Leptodactylidae. Our work reports for the first time the isolation and characterization of ocellatin 4 (GLLDFVTGVGKDIFAQLIKQI-NH₂), a cytolytic peptide from the skin secretion of the South American frog Leptodactylus ocellatus. While most cytolytic amphibian skin peptides are selective for microorganisms and harmless for mammalian cells, the HC₅₀ of ocellatin 4 against human erythrocytes is 14.3 μM. The interaction between ocellatin 4 and zwitterionic phospholipids in mammalian plasma membranes may be favored by its neutral charge at pH 7.0. Ocellatin 4 also shows some antibacterial activity (MICs_{E. coli and S. aureus}=64 μM) and its sequence shares similarities with the only six leptodactylid peptides previously known and with four peptides from Australian hylid frogs of the genus Litoria.     

Two antimicrobial peptides from skin secretions of Rana grahami.

Two antimicrobial peptides manifested a broad spectrum of antimicrobial activity against various microorganisms have been isolated from skin secretions of Rana grahami. These antimicrobial peptides were named grahamin 1 and grahamin 2. Their primary structures are GLLSGILGAGKNIVCGLSGLC and GLLSGILGAGKHIVCGLSGLC, respectively, determined by Edman degradation and mass spectrometry. They are structurally related to nigrocins identified from skin secretions of the dark-spotted frog, Rana nigromaculata. The cDNA clones encoding the precursor of grahamins were screened and sequenced from the skin cDNA library of R. grahami. The amino sequences deduced from the cDNA sequences match well with the results from Edman degradation. As other antimicrobial peptides from Rana species, grahamins contain a C-terminal loop region delineated by an intra-disulfide bridge named Rana box. Based on structural comparison of grahamin with other known antimicrobial peptides, grahamins could be classified into the family of antimicrobial peptides containing a single intra-disulfide bridge.     

De novo sequencing of peptides from the parotid secretion of the cane toad, Bufo marinus (Rhinella marina)

Amphibian skin secretions are well known as a rich source of bioactive peptides. However, little is known about the presence or role of peptides in the highly toxic, parotid secretion of the cane toad or giant toad, Bufo marinus (Rhinella marina), though small molecule bufadienolides, which act as potent cardiotoxins, have been described. In the current study we used RP-HPLC, MALDI-TOF mass spectrometry and tandem mass spectrometry to analyze and determine the first sequences of peptides from the parotid secretion of B. marinus. We show that peptides in the range of 900-2500 Da are indeed present, however in extremely low abundance. Despite the low abundance, the sequences of 14 peptides were determined, several of which match fragments of larger cellular proteins, yet none share substantial homology with defensive or anti-microbial peptides reported from frog skin secretions. We conclude that peptides are present in the parotid glands of B. marinus only in very low quantities and that they are likely to be breakdown products of proteins involved in cell maintenance. Given these results, we conclude that peptides are unlikely to contribute directly to the high toxicity of the cane toad.     

不同位置注射A型肉毒毒素对除皱后额部皮肤油脂分泌及皮肤质地的影响

目的 研究不同位置注射A型肉毒毒素对除皱后额部皮肤油脂分泌及皮肤质地的影响.方法 60例额部皱纹患者,按照随机数字表法分为对照组和试验组,各30例.对照组给予A型肉毒毒素肌肉下注射治疗,试验组给予A型肉毒毒素真皮下注射治疗.比较两组患者治疗前后额部注射区域(A、B区域)皮肤油脂分泌水平、额部皮肤弹性参数R2、R5、R7...     

Isolation, molecular cloning and functional characterization of a novel beta-toxin from the Venezuelan scorpion, Tityus zulianus.

Sting in children by Tityus zulianus scorpions (western Venezuela) often produces cardiorespiratory arrest and death by pulmonary oedema. To assess its toxicity, lethality in mice of T. zulianus soluble venom was determined. Toxin composition was studied by fractionating the crude venom through reversed-phase HPLC. The most abundant peptide, Tz1, was purified further and its N-terminal sequence, amino acid composition and molecular mass (by electron-spray ionization mass spectrometry) determined. In the presence of Tz1, activation of recombinant rat skeletal muscle sodium channels (Na(V)1.4) was shifted about 35 mV in the hyperpolarizing direction in a prepulse-dependent manner. This typical beta-toxin effect had an apparent EC50 of 3.5 microM A cDNA sequence encoding Tz1 was isolated from T. zulianus venom gland RNA using a combination of 5'- and 3'-RACE PCR. Analysis of the encoded sequence indicated that Tz1 is the processed product of a precursor containing: (i) a 20-residue long leader peptide; (ii) the amino acid sequence of the mature toxin (64 residues); and (iii) an extra Gly-Lys tail at the C-terminus, probably removed post-translationally. A comparison of Tz1 with Tityus serrulatus beta-toxin Ts1 revealed that some of the non-conservative replacements in Tz1 lie in regions potentially involved in receptor recognition.     

Genetic and enzymatic characterization of sphingomyelinase D isoforms from the North American fiddleback spiders Loxosceles boneti and Loxosceles reclusa.

In this study we report the isolation and characterization of several sphingomyelinase D isoforms from the venoms of the North American spiders Loxosceles boneti and Loxosceles reclusa, from Mexico and the United States, respectively. We have measured their enzymatic activity, their capacity to induce necrotic lesions in rabbits, cloned the cDNAs coding for the mature forms of two of the isoforms from L. boneti and two of L. reclusa based on N-terminal sequence information of the purified proteins, and performed a comprehensive comparison of the sequence data generated by us with that reported for other sphingomyelinase genes to date.     

Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina.

Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.     

Duvernoy's gland secretion of Philodryas patagoniensis from the northeast of Argentina: its effects on blood coagulation.

Duvernoy's gland secretion of Philodryas patagoniensis exhibits high hemorrhagic activity, containing enzymes that are able to degrade the vascular wall. In this work we aim to determine if the secretion can also affect the hemostatic system by causing changes in blood coagulation. Procoagulant and coagulant activities were evaluated on plasma and fibrinogen, respectively. The delay in the thrombin clotting time of fibrinogen previously incubated with the secretion was also determined. Specific hydrolysis of fibrinogen and fibrin incubated with the secretion at different time intervals was shown by electrophoresis on polyacrylamide gel. To determine the structural characteristics of the enzymes degrading fibrinogen and fibrin, secretion were incubated in the presence of 45 mM Na(2)EDTA, 40 mM Benzamidine, and/or 2 mM PMSF before the incubation with fibrinogen or fibrin, respectively. The effect in vivo was investigated in adult male rats injected with different dose of secretion, aliquots of blood were withdrawn at different time intervals, and the fibrinogen concentration was determined. Duvernoy's gland secretion of P. patagoniensis did not clot plasma or fibrinogen. It exhibited a potent fibrinogenolytic activity degrading the Aalpha-chain faster than the Bbeta-chain, whereas gamma-chain was resistant. This latter corresponded with a strong delay in the thrombin clotting time of fibrinogen (4 mg/ml) pre-incubated with the secretion, being 9.53 microg the amount of protein from Duvernoy's gland secretion that increased the thrombin clotting time from 20 to 60 s. In vivo, the loss of rat plasma fibrinogen was proportional to the amount of secretion injected. The secretion also hydrolyzed fibrin degrading the alpha-monomer. Inhibition studies with Na(2)EDTA, Benzamidine, and/or PMSF showed that metalloproteinases and serinoproteinases are the main enzymes responsible for the hydrolyzing activity on fibrinogen and fibrin. All these results demonstrate that Duvernoy's gland secretion of P. patagoniensis possesses enzymes able to hydrolyze plasma components playing a relevant role in the blood coagulation. These hydrolyzing activities and those acting on the wall of blood vessels let the secretion exhibit a high hemorrhagic activity, which may result in permanent sequelae or even cause the death of the victims bitten by this colubrid snake.