Analysis of camelid IgG for antivenom development: Immunoreactivity and preclinical neutralisation of venom-induced pathology by IgG subclasses, and the effect of heat treatment

Antivenom is the most effective treatment of snake envenoming and is manufactured from the IgG of venom-immunised horses and sheep. Camelids have a unique IgG structure which may account for the report that camel IgG is less immunogenic and less likely to activate complement than equine or ovine IgG. Camelid IgG therefore offers potential safety advantages over conventional IgGs used for antivenom manufacture. The reported thermostability of camelid IgG also holds promise in the inclusion of a relatively inexpensive anti-microbial heat step in antivenom manufacture. However, these potential benefits of camelid IgG would be much reduced if any one of the three camel IgG subclasses dominated, or under-performed, the serological response of camels to venom immunisation because of the prohibitive manufacturing costs of having to purify, or exclude, one or more IgG subclasses. This study compared the titre, antigen-specificity, relative avidity and ability to neutralise the haemorrhagic and coagulopathic effects of Echis ocellatus venom of each IgG subclass from the venom-immunised camels. The results demonstrated that no one IgG subclass consistently out-performed or under-performed the others in their immunoreactivity to venom proteins and ability to neutralise venom-induced pathologies. We concluded therefore that IgG taken from a pool of immunised camels could be processed into antivenom without requiring the implementation of expensive chromatographic separations to select, or indeed to exclude, a specific IgG subclass. The immunoreactivity of the heavy and light chain, IgG1 subclass, was markedly more vulnerable to extreme heat treatment than the heavy chain-only IgG2 and IgG3 subclasses.     

Purification and partial characterization of the phospholipase A2 and co-lytic factor from sea anemone (Aiptasia pallida) nematocyst venom

Functional nematocysts of one specific morphological class, the penetrant microbasic mastigophores, were isolated from the sea anemone, Aiptasia pallida. These nematocysts contain a multicomponent venom composed of several proteins, including those with neurotoxic, hemolytic, and lethal activities. Hemolytic activity is produced by at least three synergistic venom proteins. One of these proteins is identified as a phospholipase A₂ (EC 3.1.1.4) which exists in two isozymic forms, α and β, with molecular weights of 45,000 and 43,000, respectively. The β isozyme has been purified to homogeneity. It is a single-chained glycoprotein with an isoelectric point (pI) of 8.8 and represents 70% of the phospholipase activity of the venom. The activity of the β isozyme is relatively labile and is inactivated by 3.5 M urea or by heating at 45°C. It is most stable at pH 4.0 and loses 50% of its activity at pH values below 3.5 and above 8.0. A second venom protein has also been purified. It is essential for the hemolytic activity of the venom and is termed co-lytic factor (CLF). It is a monomeric glycoprotein having a pI of 4.5. CLF has a molecular weight of approximately 98,000, a sedimentation coefficient of 4.8 S, and is prolate in shape, having a frictional ratio of about 1.6. CLF constitutes about 1.25% of the total venom protein and is assayed by reversing fatty acid inhibition of the venom hemolysis activity.     

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.     

中华眼镜蛇毒C组分对荷人胶质细胞瘤株U-251裸鼠血清抗氧化酶活性的影响

目的探讨中华眼镜蛇毒C组分对荷人胶质细胞瘤株U-251裸鼠血清抗氧化酶活性的影响。方法取荷人胶质细胞瘤株U-251的Balb/c裸鼠,经腹腔注射低、中、高浓度的FCNNAV1mg/L、5mg/L、10mg/L,采用比色法检测裸鼠血中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-PX)活性。结果荷人胶质细胞瘤株U-251裸鼠血清SOD、GSH-PX、CAT活性显著高于正常裸鼠组(P<0.05),MDA含量显著低于正常裸鼠组(P<0.05)。结论中华眼镜蛇毒C组分的抑瘤作用可能与其提高荷瘤鼠血血清抗氧化酶活性的影响有关。     

Proteins, lethality and in vivo effects of Iurus dufoureius asiaticus scorpion venom.

Iurus dufoureius asiaticus, Birula, 1903 scorpions were collected in Mugla province located in the Aegean region, Turkey. There are few number of publications about I.d. asiaticus, and there are no data regarding minimal lethal dose and effects of the scorpion venom till now. This is the first study about toxicity and effects of I.d. asiaticus scorpion venom in mice. Previously, most of the proteins in venom of I.d. asiaticus from Aydin region in Turkey were reported to be between 14 and 205 kDa in size. In this study, we determined the electrophoretic protein pattern of the venom taken from Mugla province to be between 29 and 116 kDa by SDS-PAGE. Intracerebroventricular (i.c.v.) was determined instead of s.c. injection since there were no deaths in any s.c. test groups. The LD(50) of I.d. asiaticus scorpion venom was found to be 47.7 microg/20 g mouse by i.c.v. injection route. After s.c. injection venom, mice were shown any intoxication symptoms. On the other hand, after i.c.v. administration of venom, mice showed symptoms such as excitability, hyper salivation, weakness, paralysis, coma and resulting in death. The possible cause of death could be due to multi-system organ failure depending on the toxic effect of the venom. These both results showed that the venom was not lethal on s.c. injection, but it was lethal on i.c.v. injection. This may imply that the scorpion is of little danger to humans.     

Acquisition and use of nematocysts by cnidarian predators

Although toxic, physically destructive, and produced solely by cnidarians, nematocysts are acquired, stored, and used by some predators of cnidarians. Despite knowledge of this phenomenon for well over a century, little empirical evidence details the mechanisms of how (and even why) these organisms use organelles of cnidarians. However, in the past 20 years a number of published experimental investigations address two of the fundamental questions of nematocyst acquisition and use by cnidarian predators: (1) how are cnidarian predators protected from nematocyst discharge during feeding; and (2) how are the nematocysts used by the predator?     

Role of IgG(T) and IgGa isotypes obtained from arachnidic antivenom to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms.

The ability of IgG(T) and IgGa subclasses--isolated by liquid chromatography from equine arachnidic antivenom (AAV)-to neutralize toxic activities of Loxosceles gaucho, Phoneutria nigriventer and Tityus serrulatus venoms as well as to remove venom toxins from circulation was investigated. These subclasses showed similar antibody titers against L. gaucho, P. nigriventer and T. serrulatus venoms, and by immunoblotting few differences were observed in the recognition pattern of venom antigens. IgG(T) and IgGa neutralized 100% lethality induced by L. gaucho and 50% of P. nigriventer venom, but IgGa failed to neutralize T. serrulatus venom, in contrast to IgG(T). Both subclasses neutralized local reactions and dermonecrosis induced by L. gaucho venom in rabbits. In mice, IgG(T) and IgGa partially neutralized the edematogenic activity induced by P. nigriventer and T. serrulatus venoms, but only IgG(T) neutralized (ca. 81%) the nociceptive activity induced by T. serrulatus venom. Both subclasses failed to neutralize nociceptive activity induced by P. nigriventer venom. IgG(T) reduced the serum venom levels of animals injected with L. gaucho, P. nigriventer or T. serrulatus venoms, while IgGa solely reduced L. gaucho and P. nigriventer venoms levels. Our results demostrate that IgG(T) and IgGa subclasses neutralize toxic activities induced by P. nigriventer, T. serrulatus and L. gaucho venoms with different efficacies, as well as depurate these venoms from circulation.     

Re-investigation of venom chemistry of Solenopsis fire ants. I. Identification of novel alkaloids in S. richteri

Dialkylpiperidines are characteristic of fire ants in the genus Solenopsis (Hymenoptera: Formicidae). Workers of the black imported fire ant, S. richteri produce cis and trans stereoisomers of 2,6-dialkylpiperidines with the trans isomer predominating. We used silica gel short column chromatography to separate both stereoisomers (cis and trans) of S. richteri venom alkaloids and coupled gas chromatography mass spectrometry (GC-MS) to identify novel minor components. The identities of various peaks in GC-MS analyses of the venom fractions were based on relative retention times and mass spectral data. GC profiles verified the presence of both cis and trans stereoisomers of C_15:1 and C₁₅ in S. richteri. The GC trace of the cis stereoisomers of S. richteri alkaloids was presented for the first time. In addition to the previously described components of S. richteri venom, seven novel 2,6-dialkyl-Δ^1,2-piperideines and 2,6-dialkyl-Δ^1,6-piperideines were detected. The chemical identities of these minor components were determined by comparing with fragmentations of known compounds. Possible biosynthetic pathways for the production of cis and trans solenopsins by S. richteri are discussed.     

The evolution of venom-conducting fangs: insights from developmental biology.

The present study of the origin of the various types of fang represented among colubroid snakes (i.e., tubular, grooved, and ungrooved) attempts to reconcile the morphology of adult fangs with current phylogenetic hypotheses. Observations of growth series of developing tubular fangs were hypothesised to shed light on the evolutionary origin of fangs in snakes. While molecular phylogenies and evolutionary studies of venom proteins and of other anatomical components of the venom-delivery system reconstruct a consistent evolutionary scenario, the character of a tubular venom-conducting fang does not fit in this scenario. The present review offers a series of possible scenarios to resolve this anomaly. Of these, a new idea argues that a heterochronic mechanism (alteration of the timing of developmental events) may provide the answer that the ungrooved and grooved teeth of colubrid snakes evolved from an ancestral tubular fang by means of attachment of replacement tubular fangs to the maxilla at an earlier developmental stage than usual (precocial ankylosis).