C.H. Tan

Structure and organization of the cardiotoxin genes in Naja naja sputatrix.

We report the genomic structure, organization and the presence of multiple isoforms of the gene encoding cardiotoxins (CTX) of Naja naja sputatrix. The cardiotoxin gene consists of six CTX isoforms, each (2.2 kb) having three exons and two introns. Two possible transcription initiation sites as well as consensus TATA boxes and transcription factor binding motifs, AP‐2, NFIL‐6/C/EBP, NF‐κB and PuF have been identified in the 5′‐region of the gene. The CTX gene isoforms show nucleotide variations at specific segments in exon 2 and exon 3, which correspond to the functional domains in the three‐finger loop structure of the cardiotoxin molecule. The diverse functions of cardiotoxins together with our findings suggest that the cardiotoxin gene isoforms may have evolved under adaptive pressure through a positive Darwinian selection process.

A major lethal factor of the venom of Burmese Russell's viper (Daboia russelli siamensis) : isolation, N-terminal sequencing and biological activities of daboiatoxin

A major lethal factor, daboiatoxin (DbTx), showing strong PLA2 activity (specific activity 91.7 nmoles/min/mg), was purified to homogeneity from the venom of Burmese Russells viper (Daboia r. siamensis) by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on CM-Sephadex C-25, followed by purification on high-performance gel filtration Shim-pack Diol-150 column. DbTx is a single-chain PLA2 toxin with approximate mol. wt 15,000 as determined by HPLC gel filtration and SDS-PAGE. It constitutes 12% of total venom protein and is the main lethal component of Burmese Russells viper venom with an LD50 i.p. (0.05 mg/kg) 12-fold greater than that of the whole venom (LD50 i.p. 0.6 mg/kg). DbTx produces neurotoxic symptoms in mice and exhibits potent oedema-inducing activity (minimum oedema dose 0.05 microgram), indirect haemolytic activity and a strong myonecrotic activity, but no haemorrhagic activity. DbTx is cytotoxic to HeLa cells causing cytolysis of the cells 24 hr post-exposure to toxin (50 micrograms/ml). The first 20 N-terminal amino acid sequence (NFFQF AEMIV KMTGK EAVHS) shows a significant resemblance to those of the PLA2s from the venoms of Bulgarian viper (V. a. ammodytes) and Taiwan Russells viper (V. r. formosensis).

Cloning and characterization of cDNAs encoding three isoforms of phospholipase A2 in malayan spitting cobra (Naja naja sputatrix) venom

cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.

Effects of sea anemone (Heteractis magnifica and Actinia equina) cytolysins on synaptosomal uptake of GABA and choline

Magnificalysin I and II (HMg I and II) and equinatoxin II (EqTx II) are cytolytic toxins extracted from sea anemones Heteractis magnifica and Actinia equina, respectively. They induced haemolysis in rat red blood cells and inhibited gamma amino butyric acid (GABA) and choline uptake into rat brain synaptosomes. These effects were concentration dependent. The inhibition of GABA and choline uptake could be overcome by the addition of exogenous sphingomyelin, suggesting that there might be interaction between these cytolysins and the phospholipid. Although the precise mechanisms involved in haemolysis and inhibition of GABA and choline uptake are unknown, they appeared to be different.

Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis

Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.

INHIBITION OF SODIUM-DEPENDENT UPTAKE PROCESSES IN PURIFIED RAT BRAIN SYNAPTOSOMES BY LOPHOZOZYMUS PICTOR TOXIN AND PALYTOXIN

To get an insight into the mechanism of neurotoxicity exhibited by Lophozozymus pictor toxin (LPTX) and the toxin isolated from P.caribaeorum (C-PTX) studies were carried out on the effect of these toxins on the uptake of selected substrates (neurotransmitters, amino acids and glucose) in isolated nerve endings. The toxins were found to inhibit the uptake of gamma-aminobutyric acid (GABA), noradrenaline, choline, L-leucine and 2-deoxy-D-glucose in rat brain synaptosomes. LPTX- or C-PTX-induced inhibition of synaptosomal uptake was reduced in the absence of Na+ in the assay medium. Synaptosomes exposed to LPTX and C-PTX release K+ in a dose-dependent manner. Ouabain, a selective inhibitor of the plasma membrane Na+, K(+)-ATPase could inhibit LPTX- and C-PTX-induced K+ efflux from synaptosomes and alleviate the toxin-induced inhibition of synaptosomal GABA uptake. It appears that the induction of ionic flux is the primary cause of toxicity by these toxins leading to the inhibition of Na(+)-dependent uptake processes in synaptosomes. The antagonistic action of ouabain suggests the involvement of the membrane sodium pump in the development of cytotoxicity.