Mojave toxin affects fusion of myoblasts and viability of myotubes in cell cultures.

Mojave toxin is a neurotoxic, heterodimeric phospholipase isolated from the venom of Crotalus scutulatus scutulatus. Responses of primary rat muscle cell cultures and clonal muscle cell lines to treatment with Mojave toxin and its constituent subunits were examined. Continuous exposure of cells to 0.5 microM or 1.0 microM Mojave toxin or the basic subunit, added 24 hr after plating, prevented fusion of primary myoblasts and C2 myoblasts to multinucleate myotubes. Under the same experimental conditions, some myotube formation was observed when RMo cells were used, but the number and size of the myotubes were reduced substantially compared to untreated controls. The addition of Mojave toxin to established myotubes that arose from differentiation of primary myoblasts or C2 myoblasts essentially led to total disappearance of the myotubes from the cell layer within 48 hr. Myotubes from RMo cells treated in the same manner, however, did not disappear, but they were smaller and less numerous than comparable controls. Similar results were generated by exposure of myotubes to the basic subunit of Mojave toxin under the same conditions. The underlying layer of mononucleate cells was retained in both instances. Toxin-free cultures continued to develop in the usual manner. Treatment with 1.0 microM concentrations of the acidic subunit, pancreatic phospholipase A2 or a non-neurotoxic phospholipase from Naja naja atra gave results indistinguishable from untreated control cultures.     

Mojave toxin: rapid purification, heterogeneity and resistance to denaturation by urea

This report establishes that purified Mojave toxin prepared from the snake venom of Crotalus scutulatus scutulatus contains multiple heterogeneous dimers (isoforms) differing slightly in isoelectric points. This conclusion is based upon chromatographic, immunological, sodium dodecyl sulfate--polyacrylamide gel electrophoretic and polyacrylamide isoelectric focusing experiments. The Mojave toxin-related proteins were rapidly purified from venom via a single chromatography step. Generation of Mojave toxin-related proteins from isolated subunits and immunoblots of these proteins subsequent to electrophoretic separation demonstrate that each of the proteins consists of acidic and phospholipase basic subunits. The analysis of venom in narrow range polyacrylamide isoelectric focusing gels at varying concentrations of urea, in conjunction with immunoblots utilizing antibodies specific to the basic subunit, demonstrates that the isoforms of Mojave toxin are native and not artifacts from isolation procedures. Analyses of venoms from Crotalus scutulatus scutulatus individuals indicate that each snake produces multiple isoforms of the neurotoxin. Additionally, the same predominant isoform of Mojave toxin is present in both individual and commercial venoms. The heterogeneity of the Mojave toxin-related proteins is largely due to differences in the acidic subunits and some of the forms may reflect post-translational processing of the protein. The Mojave toxin-related proteins demonstrate a resistance to urea denaturation by characteristically entering and focusing in polyacrylamide isoelectric focusing gels containing 0-6 M urea, but dissociating to constituent subunits in 8 M urea. Experimental evidence suggest that salt bridges may be important in stabilization of the Mojave toxin complex.     

Crotoxin: A Biochemical Analysis of Its Mode of Action

Abstract

Crotoxin, the major toxic component of the South American Rattlesnake, Crotalus durissus terrificus, is a potent neurotoxin which possesses a phospholipase A₂ activity and blocks neuromuscular transmission primarily at the presynaptic level, although at higher doses it also reduces the postsynaptic response to acetylcholine by stabilizing the cholinergic receptor in an inactive conformational state.

Crotoxin, which is in fact a mixture of very similar isoforms, consists of two non identical subunits. The basic component-B carries the phospholipase A₂ activity of the toxin and possesses a low toxicity and the acidic component-A has no enzymatic activity although it resembles a phospholipase A₂ in its primary structure. Component-A, is not toxic by itself but considerably enhances the lethal potency of the phospholipase ccmponent-B. Upon interaction with biological or artificial membranes, the two subunits dissociate: component-A is released free in solution and component-B is bound. The isolated phospholipase component-B binds in a non saturable manner to either erythrocyte or postsynaptic membranes. Component-A which does not bind to membranes, considerably reduces the non specific adsorption of the phospholipase subunit, without preventing its saturable (specific) binding to a limited nunber of binding sites on the synaptic membrane.

The isolated component-B possesses a low affinity for unilamellar vesicles constituted of zwitterionic (neutral) phospholipids, but binds with a high affinity to negatively charged phospholipids. The non enzymatic component-A enhances the selectivity of component-B for negatively charged phospholipids since it completely inhibits the low affinity binding of component-B to vesicles of zwitterionic phospholipids. These observations strongly suggest that negatively charged phospholipids are the physiological target of crotoxin or at least an important part of this target. This hypothesis implies that, at variance with other plasma membranes, the presynaptic plasma membrane (or some specialized area of the plasma membrane) exposes negatively charged phospholipids on its external surface.     

Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms

Immunochemical responses of a number of pit viper venoms to antibodies derived separately from the acidic and basic subunits were investigated by enzyme linked immunosorbent assay (ELISA) and Ouchterlony immunodiffusion. The polyclonal antisera to the basic subunit were generated in rabbits, whereas mouse hybridoma cell cultures were used to produce antibodies to the acidic subunit. The immunochemical response of a venom correlated well with published values for LD50 dose for the test venom. Many venoms that elicited a positive response with antiserum to the basic subunit also reacted strongly with the hybridoma derived antibodies to the acidic subunit. The data support the conclusion that crotalid venoms which are more lethal have in common a potent venom component that is immunochemically related to Mojave toxin.     

Cross-reactivity and neutralization by rabbit antisera raised against crotoxin, its subunits and two related toxins

Antisera were raised against intact crotoxin (Crotalus durissus terrificus), Mojave toxin (Crotalus scutulatus scutulatus) and concolor toxin (Crotalus viridis concolor), as well as the subunits of crotoxin. Double immunodiffusion and enzyme-linked immunosorbent assays (ELISA) demonstrated antigenic similarity between these three purified toxins and their subunits. Additionally, when crotoxin antisera were pre-incubated with each of the three toxins before injection, the lethal activity of all were neutralized equally well. Antiserum was considerably more effective in neutralizing crotoxin in vivo when the toxin was injected i.m. than when injected i.v. Antisera against both intact crotoxin and its basic subunit were an order of magnitude more effective than crotoxin acidic subunit antiserum in crotoxin neutralization. Purified phospholipase A2 from Crotalus adamanteus and Crotalus atrox showed weak cross-reactivity with antisera raised against intact crotoxin and its subunits in the ELISA. Our results suggest that crotalid neurotoxins can be detected and neutralized by polyclonal antibodies raised against any intact toxin or basic subunit in this group of homologous toxins.     

Venom characteristics as an indicator of hybridization between Crotalus viridis viridis and Crotalus scutulatus scutulatus in New Mexico

One hundred and thirteen venoms from 46 populations of Crotalus viridis viridis were screened by immunodiffusion for protein toxins antigenically similar to the phospholipase A2 (PLA) toxin 'Mojave toxin', using a polyclonal antibody to it's basic PLA subunit. Venom i.p. LD50 values in mice were recorded from 22 of the 46 populations. The venoms of three of 14 specimens from southwest (S.W.) New Mexico and one specimen from northern Arizona were immunologically positive by the immunodiffusion tests and produced low LD50 values (0.38-0.65 mg/kg) compared to all immunologically negative venoms (0.9-5.5 mg/kg). These four specimens were morphologically typical for C. v. viridis and their venoms were the only samples of 15 southern New Mexico specimens examined by reverse phase HPLC to exhibit peaks corresponding to the acidic and basic subunits of Mojave toxin. Alkaline polyacrylamide gel electrophoresis (PAGE) analysis of the recombined subunit peaks from the C.v. viridis venom from the S.W. New Mexico specimens showed more similarity to Mojave toxin from C.s. scutulatus venom than to similar toxins in C.v. concolor venom. The combined results of the immunodiffusion, lethal toxicity tests, HPLC profiles and PAGE analysis strongly suggest that the venoms of the three New Mexico specimens contain Mojave toxin(s), as a result of some previous hybridization with C.s. scutulatus. The northern Arizona specimen likely contains 'concolor toxin' through integration with C.v. concolor in its' genetic background.     

A neurotoxic complex from the venom of the Bulgarian viper (Vipera ammodytes ammodytes) and partial amino acid sequence of the toxic phospholipase A2

The basic toxic component of the neurotoxic complex from the venom of the Bulgarian viper (Vipera ammodytes ammodytes) possessed phospholipase A₂ activity (EC 3.1.1.4) and the acidic component partially inhibited the enzymatic activity of the basic component. The partial amino acid sequence at the N- and C-terminus of the toxic phospholipase A₂ was compared with the sequence of several phospholipases and neurotoxins with weak phospholipase activity.     

Mojave toxin in venom of Crotalus helleri (Southern Pacific Rattlesnake): molecular and geographic characterization.

Mojave toxin (MT) was detected in five of 25 Crotalus helleri (Southern Pacific rattlesnake) sampled using anti-MT antibodies and nucleotide sequence analysis. All of the venoms that were positive for MT were collected from Mt San Jacinto in Riverside Co., California. Since this population is geographically isolated from C. scutulatus scutulatus (Mojave rattlesnake), it is unlikely that this finding is due to recent hybridization. MT concentration differences between C. helleri and C. s. scutulatus reflected the presence of 'isoforms' of the toxin in the venom. Whereas C. s. scutulatus generally has several isoforms of the toxin (detected by Western blotting), only one 'isoform' that focused at pI 5.1 was detected in C. helleri. Both acidic and basic subunits of MT sequences were obtained from C. helleri DNA with primers specific for MT, but only from snakes that had MT in their venom. The sequence identity of the C. helleri acidic subunit to the C. s. scutulatus subunit was 84.9%, whereas the sequence identity of the C. helleri basic subunit was 97% to the C. s. scutulatus basic subunit. Using casein, fibrin, and hide powder azure as substrates, assays for proteolytic activity suggested that C. helleri possesses several different types of metalloproteinases in their venom. However, proteolytic activity was not detected, or present in reduced amounts, in specimens having MT. Clinical neurotoxicity following envenomation by certain populations of C. helleri may be due to MT.     

Synergism of the two subunits of crotoxin

Crotoxin, a potent neurotoxin from the venom of $\text{Crotalus}$$\text{durissus}$$\text{terrificus}$, is composed of an acidic subunit which is non-toxic and enzymatically inactive and a basic subunit which possesses a phospholipase activity and low toxicity.It is shown that crotoxin very efficiently blocks the cholinergic post-synaptic response of the isolated electroplaque from $\text{Electrophorus}$$\text{electricus}$ and of cellular microsacs from $\text{Torpedo}$$\text{marmorata}$. This post-synaptic effect was investigated by studying the binding of crotoxin and its isolated subunits to acetylcholine receptor-rich membranes from $\text{Torpedo}$ and by analysing the relationship between its catalytic activity and its pharmacological effects on $\text{Electrophorus}$ electroplaque. The mechanism of action of crotoxin could be divided into two distinct steps: a quasi irreversible binding step, which has no blocking action by itself, and a catalytic step, which irreversibly inhibits the postsynaptic response.These results suggest that the non-enzymatic subunit of crotoxin enhances the pharmacological efficiency of the phospholipase by preventing its adsorption to non-saturable binding sites, restricting its binding to specific critical target sites. These sites are distinct from the cholinergic receptor sites, but probably closely related to them, as suggested by their approximately equal number and by the fact that after crotoxin action the receptor appears to be blocked in a molecular form very similar to the desensitized state. The mechanism proposed explains in simple terms the synergistic action of the two subunits of crotoxin at the level of the cholinergic receptor-ionophore assembly.     

Isolation and characterization of a presynaptic neurotoxin,P-elapitoxin-Bf1a from Malaysian Bungarus fasciatus venom

Presynaptic neurotoxins are one of the major components in Bungarus venom. Unlike other Bungarus species that have been studied, β-bungarotoxin has never been isolated from Bungarus fasciatus venom. It was hypothesized that the absence of β-bungarotoxin in this species was due to divergence during evolution prior to evolution of β-bungarotoxin. In this study, we have isolated a β-bungarotoxin isoform we named P-elapitoxin-Bf1a by using gel filtration, cation-exchange and reverse-phase chromatography from Malaysian B. fasciatus venom. The toxin consists of two heterogeneous subunits, subunit A and subunit B. LCMS/MS data showed that subunit A was homologous to acidic phospholipase A₂ subunit A3 from Bungarus candidus and B. multicinctus venoms, whereas subunit B was homologous with subunit B1 from B. fasciatus venom that was previously detected by cDNA cloning. The toxin showed concentration- and time-dependent reduction of indirect-twitches without affecting contractile responses to ACh, CCh or KCl at the end of experiment in the chick biventer preparation. Toxin modification with 4-BPB inhibited the neurotoxic effect suggesting the importance of His-48. Tissue pre-incubation with monovalent B. fasciatus (BFAV) or neuro-polyvalent antivenom (NPV), at the recommended titer, was unable to inhibit the twitch reduction induced by the toxin. This study indicates that Malaysian B. fasciatus venom has a unique β-bungarotoxin isoform which was not neutralized by antivenoms. This suggests that there might be other presynaptic neurotoxins present in the venom and there is a variation in the enzymatic neurotoxin composition in venoms from different localities.     

Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera

Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxin's two subunits - basic and toxic phospholipase A₂ (PLA₂) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 10⁹ pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones - D₁₂, E₃, F₆, D₁₀ and G₅ exhihest binding affinity for the toxic component. Clones A₁, D₁₂ and C₁₂ recognized preferentially vipoxin's acidic component. Clones E₃, G₅ and H₄ inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E₃, G₅, H₄, C₁₂, D₁₀ and A₁₁) inhibited direct hemolytic activity of vipoxin and its pure PLA₂ subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A₂ activity for the vipoxin toxicity and its effective neutralization.     

Action on phosphatidylcholine of the toxic phospholipase A2 from the venom of Bulgarian viper (Vipera ammodytes ammodytes)

The action of the neurotoxic complex and its components from the venom of the Bulgarian viper (Vipera ammodytes ammodytes) on phosphatidylcholine was studied. The nontoxic acidic component partially inhibited the phospholipase A₂ activity of the strongly toxic basic component. The basic component, separated from the acidic, was unstable and in the course of 12–14 days lost its enzymatic activity. The Michaelis constant, K_m = 1 × 10⁻³M was the same for the free phospholipase A₂ and the neurotoxic complex. Temperature optimum was 23–26°C and pH optimum was 9.9–10. A concentration of 1–3 × 10⁻³ M CaCl₂ was required for maximum enzyme activity. The influence of divalent cations on the initial velocity of the enzyme hydrolysis was studied. Although composed of a basic toxic phospholipase A₂ and a nontoxic acidic component the neurotoxic complex exhibited an insignificant enzyme activity.     

Preparation of a crotoxin neutralizing monoclonal antibody

Crotoxin is a heterodimeric protein composed of an acidic and basic subunit from the venom of Crotalus durissus terrificus and is representative of a number of presynaptically acting neurotoxins found in the venom of rattlesnakes. Four different monoclonal antibodies, typed as IgG1 subclass, were raised against the basic subunit of this toxin. One was a potent neutralizing antibody of intact crotoxin, which could neutralize approximately 1.6 moles of purified crotoxin per mole of antibody. The monoclonal antibody enhanced the neutralizing ability of commercial polyvalent crotalid antivenom against the lethality of crude C. d. terrificus venom four-fold. Paradoxically, this monoclonal antibody by itself was ineffective against the lethality of crude C. d. terrificus venom. Using an enzyme-linked immunosorbent assay, we tested various proteins for competitive inhibition of binding of biotinylated-crotoxin to plates coated with the four individual monoclonal antibodies. Concolor toxin, vegrandis toxin, intact crotoxin, Mojave toxin, and the basic subunit of crotoxin showed increasing effectiveness as displacers of crotoxin from the neutralizing monoclonal antibody. None of the monoclonal antibodies reacted with purified phospholipase A2 enzymes from Crotalus atrox or Crotalus adamanteus, nor any of the components present in the crude venoms from four different elapids known to contain presynaptically acting neurotoxins, which show some sequence identity to crotoxin.     

Specificity and binding affinity of an anti-crotoxin combinatorial antibody selected from a phage-displayed library

A crotoxin-specific, monoclonal, high-affinity, single-chain antibody variable region (scFv) was generated by combinatorial methods using Pharmacia's Recombinant Phage Antibody System. A high-affinity clone, designated A10G, was selected, and its DNA sequence was determined. Protein A10G showed high reaction specificity, with only the closely related rattlesnake neurotoxins, concolor toxin and Mojave toxin, showing cross-reactivity out of eleven group II phospholipase A₂s (PLA₂s) screened. No group I PLA₂s cross-reacted in enzyme-linked immunosorbent assays. The gene coding for A10G was subcloned into an expression vector, and the resulting expressed nonfusion protein, designated A10GPE, was renatured and purified to apparent homogeneity. Dissociation constants of A10G with intact crotoxin and crotoxin basic subunit were determined to be 7 × 10⁻¹⁰ and 6.8 × 10⁻⁹ M, respectively. When A10GPE was preincubated with either the basic subunit or intact crotoxin at molar ratios of up to 5:1, no inhibition of phospholipase activity was observed. Expressed protein, however, could partially neutralize the lethality of Mojave toxin, a crotoxin homolog, in mice.     

The two-component toxin of Vipera palaestinae: Contribution of phospholipase A to its activity

The heat stable toxic fraction of Vipera palaestinae venom contains a basic (pI 10) non-enzymic component and an acidic (pI 4·5) phospholipase A. The purified phospholipase appears as a single protein band in SDS-gel electrophoresis corresponding to a mol. wt of 15,000. An antitoxic factor previously isolated from the blood serum of Vipera palaestinae inhibits both lethality of the toxic fraction and phospholipase activity of the acidic component. Heterologous phospholipases from the venoms of Pseudocerastes fieldi and Walterinnesia aegyptia, but not from the porcine pancreas could be substituted for the original enzyme in the Vipera two-component toxic system. It is thus evident that phospholipase is an integral part of the V. palaestinae toxin, and it is either species- or organ-specific.     

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes

Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guinea-pig (Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, (Kd1 = 4 nM and Kd2 = 87 nM, Bmax1 = 2 and Bmax2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau (Kd = 2 nM and Bmax = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a Kd-value of 3 nM. The Kd value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, beta-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.     

Crotoxin: Novel activities for a classic β-neurotoxin

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric β-neurotoxin that consists of a weakly toxic basic phospholipase A₂ and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure-function studies and investigations that have led to the identification of novel pharmacological activities for the toxin.     

Inhibition of calcium channel dihydropyridine receptor binding by purified Mojave toxin

Mojave toxin, the principal toxic component of the venom of the Mojave rattlesnake Crotalus scutulatus scutulatus, is a protein complex of about 22,000 mol. wt. The mechanism of action of this potent (LD50 = 0.039 micrograms/g, mouse, IV) neurotoxin is a matter of conjecture, but physiologic data suggest a presynaptic site of action with disruption of stimulus-secretion coupling and neurotransmitter release. The selectivity of Mojave toxin's effect on several ion channels involved in neurotransmission was assessed in the present study using competitive radioisotopic binding procedures. Synaptic membranes from rat brain were used to assess the toxin's interaction with Ca++ and Cl- channels while membrane fragments from the Torpedo fish electric organ were used to determine toxin interaction with the nicotinic acetylcholine receptor-coupled Na+ channel. Mojave toxin was found to irreversibly inhibit 3H-nitrendipine binding to dihydropyridine receptors associated with Ca++ channels in rat brain, but had no effect on radioligand binding in the Na+ and Cl- channel assays. Saturation analysis of the binding further showed that the effects of MoTX on dihydropyridine binding were noncompetitive, with MoTX producing a decrease in both the affinity and density of 3H-nitrendipine sites. These results are consistent with the hypothesis that MoTX acts selectively on Ca++ channel function and that this interaction occurs via an allosteric mechanism in which MoTX binds to a membrane site that is topologically distinct from the dihydropyridine receptor.     

Inhibitory effect of phospholipase A2 isolated from Crotalus durissus terrificus venom on macrophage function

Recent work demonstrated that crotoxin, the main toxin of Crotalus durissus terrificus venom, inhibits macrophage spreading and phagocytic activities. The crotoxin molecule is composed of two subunits, an acidic non-toxic and non-enzymatic polypeptide named crotapotin and a weakly toxic basic phospholipase A₂ (PLA₂). In the present work, the active subunit responsible for the inhibitory effect of crotoxin on macrophage function was investigated. Peritoneal macrophages harvested from naive rats were used. Crotapotin (2.12, 3.75, or 8.37 nM/ml), added for 2 h to the medium of peritoneal cell incubation, did not modify the spreading and phagocytic activities of these cells. On the other hand, the PLA₂ (1.43, 2.86, or 6.43 nM/ml) subunit caused a significant reduction (30, 33, and 35%, respectively) of the spreading activity. The PLA₂ also inhibited the phagocytosis of opsonised zymosan, opsonised sheep erythrocytes, and Candida albicans, indicating that this inhibitory effect is not dependent on the type of receptor involved in the phagocytosis process. The inhibitory effect of PLA₂ was not due to loss of cell membrane integrity, since macrophage viability was higher than 95%. These findings indicate that the inhibitory effect of crotoxin on macrophage spreading and phagocytic activities is caused by the phospholipase A₂ subunit.     

Cultured cells of the nervous system,including human neurones,in the study of the neuro-degenerative disorder,Alzheimer's disease: an overview

1. Human nervous-system cells in culture are a suitable model for the study of the degenerative changes associated with Alzheimer's disease.

2. Alzheimer-diseased brain contains a factor which induces the formation of paired helical filaments (PHF) in cultured cells, similar to that seen in Alzheimer's disease.

3. The excitotoxic amino acids, glutamate and aspartate, induce similar PHE formation in cultured cells.

4. The neurotoxic element aluminium is present in high concentrations in the brain in several human neurological disorders, including Alzheimer's disease.

5. In cultured-cell systems, aluminium interacts with acidic nuclear proteins, decreases steroid binding, produces a form of neurofibrillary degeneration and alters nucleoside metabolism.