Identification of a direct hemolytic effect dependent on the catalytic activity induced by phospholipase‐D (dermonecrotic toxin) from brown spider venom

Brown spiders have world‐wide distribution and are the cause of health problems known as loxoscelism. Necrotic cutaneous lesions surrounding the bites and less intense systemic signs like renal failure, DIC, and hemolysis were observed. We studied molecular mechanism by which recombinant toxin, biochemically characterized as phospholipase‐D , causes direct hemolysis (complement independent). Human erythrocytes treated with toxin showed direct hemolysis in a dose‐dependent and time‐dependent manner, as well as morphological changes in cell size and shape. Erythrocytes from human, rabbit, and sheep were more susceptible than those from horse. Hemolysis was not dependent on ABO group or Rhesus system. Confocal and FACS analyses using antibodies or GFP‐phospholipase‐D protein showed direct toxin binding to erythrocytes membrane. Moreover, toxin‐treated erythrocytes reacted with annexin‐V and showed alterations in their lipid raft profile. Divalent ion chelators significantly inhibited hemolysis evoked by phospholipase‐D , which has magnesium at the catalytic domain. Chelators were more effective than PMSF (serine‐protease inhibitor) that had no effect on hemolysis. By site‐directed mutation at catalytic domain (histidine 12 by alanine), hemolysis and morphologic changes of erythrocytes (but not the toxin's ability of membrane binding) were inhibited, supporting that catalytic activity is involved in hemolysis and cellular alterations but not toxin cell binding. The results provide evidence that L. intermedia venom phospholipase‐D triggers direct human blood cell hemolysis in a catalytic‐dependent manner. J. Cell. Biochem. 107: 655–666, 2009. © 2009 Wiley‐Liss, Inc.     

Structural and functional characterization of N-terminally blocked peptides isolated from the venom of the social wasp Polybia paulista

Two novel peptides were isolated from the crude venom of the social wasp Polybia paulista, by using RP-HPLC under a gradient of MeCN from 5 to 60% (v/v) and named Polybine-I and -II. Further purification of these peptides under normal phase chromatography, rendered pure enough preparations to be sequenced by Edman degradation chemistry. However, both peptides did not interact with phenylisothiocyanate reagent, suggesting the existence of a chemically blocked N-terminus. Therefore, the sequences of both peptides were assigned by ESI-MS/MS under CID conditions, as follows: Polybine-I Ac-SADLVKKIWDNPAL-NH₂ (Mr 1610 Da) and Polybine-II Ac-SVDMVMKGLKIWPL-NH₂ (Mr 1657 Da). During the tandem mass spectrometry experiments, a loss of 43 a.m.u. was observed from the N-terminal residue of each peptide, suggesting the acetylation of the N-terminus. Subsequently, the peptides with and without acetylation were synthesized on solid phase and submitted to functional characterizations; the biological activities investigated were: hemolysis, chemotaxis of polymorphonucleated leukocytes (PMNL), mast cell degranulation and antibiosis. The results revealed that the acetylated peptides exhibited more pronounced chemotaxis of PMNL cells and mast cell degranulation than the respective non-acetylated congeners; no hemolytic and antibiotic activities were observed, irrespective to the blockage or not of the -amino groups of the N-terminal residues of each peptide. Therefore, the N-terminal acetylation may be related to the increase of the inflammatory activity of both peptides.     

Novel biopesticide based on a spider venom peptide shows no adverse effects on honeybees

Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a ‘carrier’ protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a ‘carrier’, is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD₅₀ > 100 µg bee⁻¹) or injection of fusion protein. Bees fed acute (100 µg bee⁻¹) or chronic (0.35 mg ml⁻¹) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.     

Effects of opioid peptides and naloxone on tissue from the central and peripheral nervous system in culture

A study was made of the effects of opioid peptides (leu-enkephalin and dalargin AE-1, its synthetic analog) and of naloxone, an opiate receptor blocker, on organotypic cultures of spinal cord and spinal ganglia cells. The cellular composition and size of explant outgrowth was estimated according to in vitam morphological observations. It was found that all the opioid peptides tested at concentrations of 10^–9-10^–10M exercise a clear-cut growth-promoting effect on cultures from the spinal cord as well as those from the peripheral nervous system [4, 5]. Naloxone at a concentration of 10^–5-10^–6 M does not block peptide action, but itself stimulates growth. It was also proved that opioid peptides act as trophic factors for spinal ganglia nerve cells, increasing their survival in culture. Endorphins can thus serve as growth factors for tissues of the peripheral as well as the central nervous system. The likely processes lying at the root of the growth-promoting and trophic effects of endorphins on nerve tissue are discussed.Institute of Experimental Cardiology of the All-Union Cardiological Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 227–233, March–April, 1986.     

Central nervous system effects of peptides, 1980-1985: a cross-listing of peptides and their central actions from the first six years of the journal Peptides

A tabular synopsis is presented for articles concerned with the effects of peptides on the central nervous system that appeared in the journal Peptides from 1980-1985. A table arranged alphabetically by peptide and one arranged by effects, both listing routes of injection, species, direction of change, and qualifying notes, provides easy cross-referencing of peptides and their effects. Over 80 peptides and over 135 effects are listed. The list of peptides includes, but is not limited to: ACTH, angiotensin, bombesin, bradykinin, calcitonin, casomorphin, CCK, ceruletide, CGRP, CRF, dermorphin, DSIP, dynorphin, endorphins, enkephalins, GRF, gastrin, LHRH, litorin, metkephamid, MIF-l, motilin, MSH, NPY, NT, oxytocin, ranatensin, sauvagine, substances P and K, somatostatin, TRH, VIP, vasopressin, and vasotocin. The list of effects includes, but is not limited to: aggression, alcohol, analgesia, attention, avoidance, behavior, cardiovascular regulation, catalepsy, conditioned behavior, convulsions, dopamine binding and metabolism, discrimination, drinking, EEG, exploration, feeding, fever, gastric secretion, GI motility, grooming, learning, locomotor behavior, mating, memory, neuronal activity, open field, operant behavior, rearing, respiration, satiety, scratching, seizure, sleep, stereotypy, temperature, thermoregulation and tolerance.     

Structure-function relationships and site of action of apamin, a neurotoxic polypeptide of bee venom with an action on the central nervous system.

Specific chemical modifications of apamin have been used to study the residues involved in its toxic action. Transformation of Lys4 into homoarginine did not affect toxicity. Modification of the alpha-amino group of Cys1 and of the epsilon-amino group of Lys4 by acetic anhydride or fluorescamine decreased toxicity only by a factor of 2.5-2.8. Modification of the gamma-carboxylate of Glu7 with glycine ethyl ester in the presence of a soluble carbodiimide decreased toxicity by a factor of 2. Diethyl pyrocarbonate treated of the imidazole side chain of His18 decreased toxicity by a factor of 2.6. Thus none of these residues is essential for toxicity. However, combined modification of amino groups and of the imidazole side chain of His-18 completely abolished biological activity. Complete loss of toxicity also resulted from reduction and alkylation of both disulfide bridges, from chemical modification with cyclohexanedione of Arg-13 and Arg-14, and from removal of Arg-14 of acetylated apamin by digestion with trypsin. Incorporation of radioactive acetyl groups on both amino groups of apamin gave an active labeled toxin which has been used to localize the site of action of apamin in the spinal cord, principally in the lumbar part of the neuraxis.     

Investigation on the effect of peptides mixture from tumor cells inducing anti-tumor specific immune response

The peptides mixture was prepared from tumor cells by freezing-thawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide complex, formed by the binding of HSP70 and peptides in vitro, were observed, so was the specific cytotoxicity of the proliferative lymphocytes to tumor cells. The phenotypes of the proliferative lymphocytes were analyzed by a flow cytometer. BALB/c mice inoculated with H22 hepatocarcinoma cells in peritoneal cavity or hind thigh were immunized by injection with HSP70-peptides complex to observe the inhibitory effect of the immunization on tumor and lifetime of tumor-bearing mice. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. The results showed that the peptides mixture from tumor cells contained tumor-specific antigen peptides which could be presented by HSP70 to activate lymphocytes in vitro, the proliferative lymphocyt     

Characterization of two peptides isolated from the venom of social wasp Chartergellus communis (Hymenoptera: Vespidae): Influence of multiple alanine residues and C-terminal amidation on biological effects

Chatergellus communis is a wasp species endemic to the neotropical region and its venom constituents have never been described. In this study, two peptides from C. communis venom, denominated Communis and Communis-AAAA, were chemically and biologically characterized. In respect to the chemical characterization, the following amino acid sequences and molecular masses were identified:Communis: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-COOH (1340.9 Da)Communis-AAAA: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-Ala-Ala-Ala-Ala-Val-Xle-NH₂ (1836.3 Da).Furthermore, their biological effects were compared, accounting for the differences in structural characteristics between the two peptides. To this end, three biological assays were performed in order to evaluate the hyperalgesic, edematogenic and hemolytic effects of these molecules. Communis-AAAA, unlike Communis, showed a potent hemolytic activity with EC₅₀ = 142.6 μM. Moreover, the highest dose of Communis-AAAA (2nmol/animal) induced hyperalgesia in mice. On the other hand, Communis (10nmol/animal) was able to induce edema but did not present hemolytic or hyperalgesic activity. Although both peptides have similarities in linear structures, we demonstrated the distinct biological effects of Communis and Communis-AAAA. This is the first study with Chartegellus communis venom, and both Communis and Communis-AAAA are unpublished peptides.     

Matrix metalloproteinase‐3 in the central nervous system: a look on the bright side

Matrix metalloproteinases (MMPs) are a large family of proteases involved in many cell‐matrix and cell‐cell signalling processes through activation, inactivation or release of extracellular matrix (ECM) and non‐ECM molecules, such as growth factors and receptors. Uncontrolled MMP activities underlie the pathophysiology of many disorders. Also matrix metalloproteinase‐3 (MMP‐3) or stromelysin‐1 contributes to several pathologies, such as cancer, asthma and rheumatoid arthritis, and has also been associated with neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and multiple sclerosis. However, based on defined MMP spatiotemporal expression patterns, the identification of novel candidate molecular targets and in vitro and in vivo studies, a beneficial role for MMPs in CNS physiology and recovery is emerging. The main purpose of this review is to shed light on the recently identified roles of MMP‐3 in normal brain development and in plasticity and regeneration after CNS injury and disease. As such, MMP‐3 is correlated with neuronal migration and neurite outgrowth and guidance in the developing CNS and contributes to synaptic plasticity and learning in the adult CNS. Moreover, a strict spatiotemporal MMP‐3 up‐regulation in the injured or diseased CNS might support remyelination and neuroprotection, as well as genesis and migration of stem cells in the damaged brain.     

Neurochemokines: a menage a trois providing new insights on the functions of chemokines in the central nervous system

Recent observations suggest that besides their role in the immune system, chemokines have important functions in the brain. There is a great line of evidence to suggest that chemokines are a unique class of neurotransmitters/neuromodulators, which regulate many biological aspects as diverse as neurodevelopment, neuroinflammation and synaptic transmission. In physiopathological conditions, many chemokines are synthesized in activated astrocytes and microglial cells, suggesting their involvement in brain defense mechanisms. However, when evoking chemokine functions in the nervous system, it is important to make a distinction between resting conditions and various pathological states including inflammatory diseases, autoimmune or neurodegenerative disorders in which chemokine functions have been extensively studied. We illustrate here the emergent concept of the neuromodulatory/neurotransmitter activities of neurochemokines and their potential role as a regulatory alarm system and as a group of messenger molecules for the crosstalk between neurons and cells from their surrounding microenvironment. In this deliberately challenging review, we provide novel hypotheses on the role of these subtle messenger molecules in brain functions leading to the evidence that previous dogmas concerning chemokines should be reconsidered.     

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A2 formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.     

The effect of a single dose of arecholine and atropine administration on activities of carboxypeptidase H and phenylmethylsulfonyl fluoride-inhibited carboxypeptidase in the nervous system of rats

The effect of a single dose of arecholine and atropine administration on the activities of carboxypeptidase H and phenylmethylsulfonyl fluoride-inhibited carboxypeptidase involved into the final stage of formation of biologically active neuropeptides from precursors has been studied in brain and adrenals. Changes in the enzyme activities were evident during at least 72 h after administration of these drugs to rats. These results suggest that the decrease in the activity of these enzymes may represent one of possible mechanisms involved into reduction of neuropeptide levels during inhibition and activation of cholinergic system.