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Temperature selection in tiger snakes (Notechis scutatus) is strongly influenced by hydration state and this response varies between two distinct neighbouring populations on semi-arid Carnac Island and mainland wetland (Herdsman Lake). Fed and hydrated (control) Carnac Island snakes selected a preferred body temperature of 26.2+/-1.2 degrees C and an average maximum temperature of 32.5+/-0.5 degrees C in a photo-thermal gradient. Dehydrated Carnac Island snakes selected a significantly lower preferred body temperature (19.7+/-1.6 degrees C) and average maximum temperature (27.7+/-1.0 degrees C). Control Herdsman Lake snakes selected a preferred body temperature of 27.5+/-0.6 degrees C and an average maximum temperature of 33.3+/-0.4 degrees C. Dehydrated Herdsman Lake snakes selected a significantly lower preferred body temperature (23.3+/-1.1 degrees C) and a lower average maximum temperature (31.8+/-0.6 degrees C). Thermal depression (decreased preferred body and average maximum temperatures) in response to dehydration was greater for Carnac Island than Herdsman Lake snakes. As decreases in temperature and activity can reduce water loss, our laboratory data suggest that the survival of the relict population of tiger snakes on Carnac Island is associated with thermoregulatory modifications, which may have the effect of enhancing water conservation in this waterless habitat.

Selective cloning of the cDNA coding for a weak neurotoxin (WTX) from cobra N. kaouthia including the 5'- and 3'-non-translated regions (NTR) is described. The known amino acid sequence of WTX was used together with the nucleotide sequence of a weak neurotoxin NNAM2 from cobra Naja atra, to design WTX-specific primers for direct amplification of an internal WTX cDNA fragment by RT- PCR. The sequence of the complete WTX cDNA was determined in sequencing runs on internal PCR products, cloned 3'- and 5'-RACE-fragments and several full-length cDNA clones. The cDNA coding sequence is in excellent agreement with the previously determined WTX amino acid sequence, has a high homology with other known weak toxin cDNAs, whereas even higher homology (up to 96%) with several classes of 3-finger toxins was detected in the 59 bp 3'-NTR consensus sequence. A possible function of the highly conserved nucleotide sequence elements is discussed.

The possible involvement of specific regions/loops of cardiotoxin from Naja sputatrix venom in mediating its cytolytic activity is evaluated using a new cytolytic assay. In this assay, the amount of chloramphenicol acetyltransferase (CAT) that is released upon lysis of the cellular membranes by the cytotoxin has been measured as an index of cytolysis. This newly developed CAT system is more sensitive than the traditional haemolysis method utilizing red blood cells or the lactate dehydrogenase assay for cytolysis. Series of chimaeric toxin molecules have been constructed by swapping the loops between highly hydrophilic neurotoxin and highly hydrophobic cardiotoxin molecules from Naja sputatrix, which are known to exhibit structural similarity (three-finger conformation) but to have different functional properties. Comparison of the cytolytic activities of the recombinant chimaeric toxins demonstrated the possible involvement of all three loops of cardiotoxin in its cytolytic potency. However, the first two loops of the protein appear to make the major contribution to its lytic activity. cDNAs encoding cardiotoxin and the chimaeric toxins, when expressed in transfected cultured Chinese hamster ovary cells, resulted in cell lysis, indicating that these cDNAs can be developed as useful cytolytic agents.

Alpha-neurotoxin (alpha-NTX) from the venom of cobra, Naja sputatrix, is a highly lethal post-synaptic toxin that is responsible for the lethality caused by the venom. However, this toxin is found at low levels (3%) in the crude venom. The expression of its gene is determined by a promoter which is 90% similar to the promoter of another three-fingered toxin, cardiotoxin (CTX), which is produced in large amounts (60%) in the same venom. Functional analysis of the NTX-2 gene promoter demonstrated the presence of a silencer element of 24 nucleotides (nt -678 to -655) at its 5(') flanking region. This element has been found to play a major role in the down-regulation of NTX-2 gene expression. A point mutation on this silencer appears to attenuate its repressive property in CTX-2 gene.

The crystal structure of an acidic phospholipase A(2) from Ophiophagus hannah (king cobra) has been determined by molecular replacement at 2.6-A resolution to a crystallographic R factor of 20.5% (R(free)=23.3%) with reasonable stereochemistry. The venom enzyme contains an unusual "pancreatic loop." The conformation of the loop is well defined and different from those in pancreas PLA(2), showing its structural variability. This analysis provides the first structure of a PLA(2)-type cardiotoxin. The sites related to the cardiotoxic and myotoxic activities are explored and the oligomer observed in the crystalline state is described.

Dendroaspis natriuretic peptide (DNP) is a recently isolated 38 amino acid peptide that shares structural and functional properties with the other members of the natriuretic peptide family. The present study demonstrates the presence of DNP-like immunoreactivity in sections of rat aorta, carotid artery and renal vasculature and tubules. DNP-like immunoreactivity was detected in culture aortic vascular smooth muscle cells and medium and is regulated by endothelin-1, angiotensin II and sodium nitroprusside but not by transforming growth factor-beta. Our observations indicate that DNP elicits a marked inhibitory effect on DNA synthesis in culture rat aortic vascular smooth muscle cells.

L-Amino acid oxidase (LAO) widely exists in snake venoms. Purification of LAO from the Naja naja kaouthia (monocellate cobra) venom has been reported (Tan and Swaminathan, 1992), but its structural characterization and physiological function remained to be determined. The function of snake venom LAOs in hemostasis, especially their effect on platelet aggregation, has been controversial. We determined the N-terminal amino acid sequence of the N. n. kaouthia LAO named K-LAO to be DDRRSPLEECFQQNDYEEFLEIAKNGLKKTxNPKHVXxV (38 residues). The protein data base search revealed that the enzyme had high similarities with other snake venom LAOs. Further, platelet aggregation studies revealed that K-LAO functionally did not induce platelet aggregation in a platelet-rich plasma system, but that it inhibited platelet aggregation induced by agonists such as ADP, collagen and ristocetin in a dose-dependent manner. K-LAO diminished platelet aggregation more intensely under low than high shear stress. This inhibitory activity of K-LAO on either ristocetin-induced or shear-induced platelet aggregation was quenched by addition of catalase. These results indicate that K-LAO functions as an inhibitor to platelet aggregation through the formation of hydrogen peroxide. The enzyme may contribute to the development of a severe hematological disorder due to cobra envenomation.

Coral snakes are the only Elapids in America. They are represented by three genera: Leptomicrurus, Micruroides and Micrurus, of which the latter are the most abundant and diversified group. Little is known about the biochemistry of Micrurus venoms due to low availability. Here, we present a study on the cross reactivity of different specific Micrurus antivenom with homologous and heterologous snake venoms in order to contribute to the generation of more efficient antiserum for therapeutic purposes. The three specific antisera tested, anti-Micrurus corallinus, anti-Micrurus frontalis, and anti-Micrurus spixii, as well as the bivalent anti-elapid venom sera, raised against a mixture (50% each) of Micrurus frontalis and Micrurus corallinus venoms, were assayed by Western Blot against Micrurus and non-Micrurus elapid venoms. An antisera raised against a recombinant alpha-neurotoxin-like protein from Micrurus corallinus venom, only reacted in Western blot with its homologous venom, indicating that this protein is specific for Micrurus corallinus coral snake.

A PCR technique was used in this study to identify and distinguish monocellate cobra snake bites using snake venoms and swab specimens from snake bite-sites in mice from bites by other common Thai snakes. The sequences of nucleotide primers were selected for the cobrotoxin-encoding gene from the Chinese cobra (Naja atra) since the sequences of monocellate cobra (Naja kaouthia) venom are still unknown. However, the 113-bp fragment of cDNA of the cobrotoxin-encoding gene was detected in the monocellate cobra venom using RT-PCR. This gene was not found in the venoms of Ophiophagus hannah (king cobra), Bungarus fasciatus (banded krait), Daboia russelii siamensis (Siamese Russell's Viper, and Calloselasma rhodostoma (Malayan pit viper). Moreover, direct PCR could detect a 665-bp fragment of the cobrotoxin-encoding gene in the monocellate cobra venom but not the other snake venoms. Likewise, this gene was only observed in swab specimens from cobra snake bite-sites in mice. This is the first report demonstrating the ability of PCR to detect the cobrotoxin-encoding gene from snake venoms and swab specimens. Further studies are required for identification of this and other snakes from the bite-sites on human skin.

This paper discusses the properties of the three most specific ligands found for the extracellular faces of M1, M2 and M4 muscarinic receptors (m1-toxin1, m2-toxin and m4-toxin, respectively). The primary goal of this paper is to show the known and potential usefulness of these toxins and their biotinylated, radioactive, fluorescent and mutated derivatives.

Toxicities of crude venoms from 49 coral snake (Micrurus sp.) populations, representing 15 nominal taxa, were examined in both laboratory mice and in native prey animals and compared with data gathered from two non-micrurine elapids and a crotalid, which served as outgroups. These venoms were further compared on the basis of 23 enzymatic activities. Both toxicities and enzymatic activities were analyzed with respect to natural prey preferences, as determined from stomach content analyses and literature reports. Venoms of nearly all Micrurus for which prey preferences are known, are more toxic to natural prey than to non-prey species. Except for amphisbaenians, prey are more susceptible to venoms of Micrurus that feed upon them, than to venoms of those that eat other organisms. All venoms were more toxic i.v.>i.p.>i.m. Route-specific differences in toxicity are generally greatest for preferred prey species. Cluster analyses of venom enzymatic activities resulted in five clusters, with the fish-eating M. surinamensis more distant from other Micrurus than even the crotalid, Bothrops moojeni. Ophiophagous and amphisbaenian-eating Micrurus formed two close subclusters, one allied to the outgroup species Naja naja and the other to the fossorial, ophiophagous Bungarus multicinctus. Prey preference is shown to be the most important determinant of venom composition in Micrurus.

Three new phospholipase A2 (PLA2) isoenzymes were purified from the Micropechis ikaheka venom by successive chromatographies. The homogeneity of them was accessed by capillary zone electrophoresis and mass spectrometry. Their N-terminal sequences showed high identity (94, 88 and 90, respectively) with MiPLA-1, a group IB PLA2 also from this venom. In addition, strong immuno-cross-reaction with anti-MiPLA-1 serum was observed. These results suggested that three newly purified PLA2 belonged to group IB. Beside enzymatic activity, they induced various pharmacological effects, including myotoxic, anticoagulant effects and insulin secretion stimulating effects. Our results indicated that enzymatic activity is essential for their myotoxic and anticoagulant effects. On the other hand, no direct correlation between their insulin secretion stimulating effect and enzymatic activity was observed, suggesting that they may stimulate insulin secretion through a non-enzymatic mechanism.

Four novel cardiotoxins were isolated from Naja naja atra (Taiwan cobra) venom by successive separation on a SP-Sephadex C-25 column and a reverse phase column. Amino acid sequences of the cardiotoxins were determined by Edman degradation and carboxypeptidase digestion. It shows that these cardiotoxins comprise 60 amino acid residues. Comparative analyses on the amino acid sequences of cardiotoxins from the venoms of N. naja atra and other Naja species indicated that amino acid substitutions of cardiotoxin isoforms frequently occurred at positions 7-11, 27-32 and 45-47. The hypervariable segments encoded by the second and third exon of cardiotoxin genes are located at or near the tips of loop structure of cardiotoxin molecules. These results, together with the suggestions that the residues at the tips of cardiotoxins' loop structure were involved in the manifestation of the biological activities of cardiotoxins, reflect that the preferential mutations may contribute to alterations in the function of cardiotoxin molecules. Analysis on the secondary structure of pre-mRNAs of N. naja atra cardiotoxin 4 gene and N. naja sputatrix cardiotoxin 3 gene has shown that the hypervariable regions of the exon 2 pertain to form intra-exon pairings and are not involved in the formation of intron-exon pairings. Since the pairings of splice sites and gene architecture were supposed to be associated with intron-exon recognition, it is likely that the preferred loci of mutations occurring with the evolution of cardiotoxin genes would not affect the processing of cardiotoxin precursors.

The venom of the spitting cobra, Naja naja sputatrix contains highly potent alpha-neurotoxins (NTXs) in addition to phospholipase A2 (PLA2) and cardiotoxin (CTX). In this study, we report the complete characterization of three genes that are responsible for the synthesis of three isoforms of alpha-NTX in the venom of a single spitting cobra. DNA amplification by long-distance polymerase chain reaction (LD-PCR) and genome walking have provided information on the gene structure including their promoter and 5' and 3' UTRs. Each NTX isoform is approximately 4 kb in size and contains three exons and two introns. The sequence homology among these isoforms was found to be 99%. Two possible transcription sites were identified by primer extension analysis and they corresponded to the adenine (A) nucleotide at positions +1 and -45. The promoter also contains two TATA boxes and a CCAAT box. Putative binding sites for transcriptional factors AP-2 and GATA are also present. The high percentage of similarity observed among the NTX gene isoforms of N. n. sputatrix as well as with the alpha-NTX and kappa-NTX genes from other land snakes suggests that the NTX gene has probably evolved from a common ancestral gene.

To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A2-glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A2. Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A2-phospholipid interactions.

A phospholipase A2 (PLA2) inhibitor was purified from the blood plasma of a sea krait, Laticauda semifasciata, by sequential chromatography on Sephadex G-200, Mono Q, and Phenyl Sepharose columns. The purified inhibitor was found to be the same type as the PLA2 inhibitors, named PLIgamma, that had been purified from the blood plasma of the Thai cobra Naja naja kaouthia [Ohkura et al. (1994) Biochem. Biophys. Res. Commun. 200, 784-788] and Chinese mamushi Agkistrodon blomhoffii siniticus [Ohkura et al. (1997) Biochem. J. 325, 527-531]. Like other PLIgammas, the L. semifasciata inhibitor (LsPLIgamma) inhibited equally all of the PLA2s investigated including Elapid venom PLA2s (group I), Crotalid and Viperid venom PLA2s (group II), and honeybee PLA2 (group III). The LsPLIgamma was a 100-kDa glycoprotein composed of two distinct subunits, LsPLIgamma-A and LsPLIgamma-B, with an approximate molar ratio of 2:1. The amino acid sequences of the two subunits were determined by alignment of the peptides obtained by lysyl endopeptidase, endoproteinase Asp-N, and staphylococcal V8 protease digestions. LsPLIgamma-A and LsPLIgamma-B were composed of 182 and 181 amino acid residues, respectively; and the former subunit was a glycoprotein containing one asparagine-linked sugar chain at the position 157. The sequences of LsPLIgamma-A and LsPLIgamma-B showed 65 and 74% homology, respectively, to those of the corresponding subunits of N. naja kaouthia PLIgamma, and had two tandem patterns of cysteine residues, characteristic of the urokinase-type plasminogen activator receptor (uPAR) and members of the Ly-6 superfamily.

Specificity of two different types of phospholipase A2 (PLA2) inhibitory proteins from the blood plasma of venomous snakes was investigated. Two Crotalidae inhibitors, having a carbohydrate recognition domain (CRD) in their sequences, inhibited specifically the group-II acidic PLA2s of their own snake venom. On the other hand, Elapidae inhibitor, having two tandem patterns of cysteine residues found in proteins of the Ly-6 superfamily, inhibited not only the group-I PLA2 from its own snake venom but also the group-I, -II, and -III PLA2s from other snake venom. Amino acid sequences of PLA2s that were specifically inhibited by the inhibitors were compared with those of the other PLA2s. A unique aromatic patch structure appeared on the group-II acidic PLA2s was suggested to be involved in the binding to the Crotalidae inhibitors; and residues located in or close to the Ca2+ binding loop of PLA2, in the binding to the Elapidae inhibitor.

Synaptosomes were incubated with bovine serum albumin (BSA) to examine whether the presynaptic action of snake venom phospholipase A2 (PLA2) toxins is due either to the release of fatty acids resistant to extraction by BSA or to the liberation of a specific fatty acid type. In the presence of BSA (0.5% or 1.0%) two PLA2 enzymes from Naja naja atra and Naja naja kaouthia snake venoms that do not have a predominant presynaptic action at the neuromuscular junction (PS-) did not stimulate acetylcholine (ACh) release from synaptosomes. In contrast, two PLA2 enzymes (beta-bungarotoxin, scutoxin) that do have a predominant presynaptic action at the neuromuscular junction (PS+) did stimulate ACh release. BSA did not antagonize PS- enzymes by more efficiently extracting the fatty acids produced by these enzymes relative to PS+ enzymes. While absolute amounts of total and unsaturated fatty acid produced overlapped for the PS- and PS+ enzymes, the two PS+ enzymes produced a significantly greater absolute amount and relative percentage of palmitic acid (16:0) than did either of the PS- enzymes. However, the levels of free palmitic acid remaining in the synaptosomes where they would exert effects on ACh release were similar for the N. n. kaouthia PLA2 (PS-) and beta-bungarotoxin (PS+). Therefore, the total (supernatant plus synaptosomal) amount of palmitic acid produced per se did not account for stimulation of ACh release, since the greater amounts produced by the PS+ enzymes were removed from the synaptosomes by BSA. The production of higher levels of palmitic acid suggests either that PS+ enzymes gain access to sites containing phospholipid substrates unavailable to the PS- enzymes, or that they have a different substrate preference. These findings suggest new possibilities for the mechanism of PS+PLA2 action, including site-directed enzymatic activity and protein acylation.

Capillary electrophoresis (CE) with electrospray ionization (ESI) and selected ion-monitoring mass-spectrometric (SIM-MS) detection has been used to provide as much information as possible about the lower molecular-mass fraction (peptides of molecular masses up to 8500 Da) of the venoms of Dendroaspis jamesoni kaimosae (Jameson's Mamba) and Micrurus fulvius (Eastern Coral Snake). Method development was based on the venom of D. jamesoni kaimosae, which contains some previously described peptides, with subsequent application to the completely unknown venom of M. fulvius. CE-ESI-SIM-MS provides a rapid and extremely sensitive method for the detection and molecular-mass determination of peptides present in venoms. It has been utilized to provide molecular masses and thus, by inference, confirmation of the peptide compositions for those toxins which have been previously described in the literature. Our methodology indicates the presence of 83 peptides in the venom of D. jamesoni kaimosae and 49 peptides in the venom of M. fulvius in the molecular-mass range 6000-8500 Da.

For understanding the evolutionary relationships between gonadotropins [GTHs: lutropin (LH) and follitropin (FSH)] and their receptors, we attempted to characterize the extracellular domain of the receptors, which is thought to be a key region of hormone binding, in nonmammalian species, and to compare the information to that of the known mammalian data. For this purpose, we designed two sets of sense and antisense oligonucleotides as polymerase chain reaction (PCR) primers, referring to the known mammalian GTH receptors, such as LH receptors of human, pig, and rat, and FSH receptors of human and rat. All possible combinations of the primers showed the successful amplification of cDNA of LH receptor without contamination of FSH receptor cDNA from rat testicular RNA samples. With these primers, reverse transcription (RT)-PCR was applied to the gonads of nonmammalian species (quail, snake, tortoise, newt, and bullfrog). Only the quail, however, showed the specific amplification when only one set of primers was used. Thus, the PCR product of the quail was used as a probe for Northern blot and in situ hybridization. By Northern blot analysis, a single size of mRNA (3 kb) was identified from quail testicular poly A+ RNA. The distribution of mRNA visualized by in situ hybridization was limited only on Leydig cells of quail testis. These results suggest that a part of the quail LH receptor cDNA was amplified by RT-PCR. The nucleotide and predicted peptide sequences of this amplified cDNA were compared with those of mammalian receptors. The size of characterized cDNA sequence was 519 bp, which is completely identical with those of mammalian LH receptors. The homology of both cDNA and predicted peptide was about 70% of those of mammalian LH receptors (intramammalian, about 80%). In spite of the relatively low homology, the positions of cystein residues and potential N-linked glycosylation sites in the peptide were completely conserved in all species compared (human, pig, rat, and quail). The conserved portions indicate their importance for the molecular conformation and specific ligand binding activity of LH receptors.

1. Dendrotoxins from mamba snake venoms are small proteins that block neuronal K+ channels. In order to investigate structural features associated with their biological activity, partially folded versions of dendrotoxins I and K from black mamba (Dendroaspis polylepis) were prepared by selectively reducing one or more of their three S-S bonds. 2. The modified toxins were tested for ability to compete with 125I-labelled native toxin I to high affinity binding sites on rat brain synaptosomal membranes and for the ability to increase acetylcholine release in a neuromuscular preparation. 3. Binding affinity increased progressively as the toxins folded to the native conformation and the most biologically active of the modified species were those in which only the disulphide bond between residues 14 and 38 was not formed. These intermediates had native-like conformations as determined by circular dichroism but still had about 5-10 times lower affinity than native toxins. 4. Addition of negatively charged groups to block the free sulthydryls at positions 14 and 38 caused a further, marked loss of activity. 5. The results are consistent with the existence of two important regions in the dendrotoxin molecules. The region containing two of the disulphide bonds (around Cys5-Cys55 and Cys30-Cys51) and much of the secondary structure is essential for the binding affinity of the toxins, while the region around Cys14 and Cys38, equivalent to part of the antiprotease site of the homologous protease inhibitor from bovine pancreas (BPTI), plays an important role in the potency of dendrotoxins.

Relationships between the in-vitro permeability of basic compounds through shed snake skin as a suitable model membrane for human stratum corneum and their physiochemical properties were investigated. Compounds with low pKa values were selected to compare the permeabilities of non-ionized forms of the compounds. Steady-state penetration was achieved immediately without a lag time for all compounds. Flux rate and permeability coefficient were calculated from the steady-state penetration data and relationships between these parameters and the physicochemical properties were investigated. The results showed that permeability may be controlled by the lipophilicity and the molecular size of the compounds. Equations were developed to predict the permeability from the molecular weights and the partition coefficients of basic compounds.