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Background: Specific desensitisation to food allergens, which produce anaphylaxis after ingestion, has not been considered as a treatment for food allergy until recently. The purpose of this study was to assess if a parenteral immunotherapy program, using a partially characterised crude peanut extract, could induce a state of immunological tolerance in a patient who exhibited anaphylaxis, asthma and urticaria on exposure to peanut and other legumes. A further aim was to measure the serum antibody responses to the immunotherapy., Methods and Results: We report the successful desensitisation towards all of the legumes tested of a male patient on parenteral immunotherapy using a partially characterised peanut extract. The immunologic parameters measured during treatment included specific IgE and IgG4 for peanut, soybean, pea and lentil extracts. Immunoblots of specific IgE and IgG4 were made before and after therapy. The antibody response followed the same pattern seen in successful desensitisation of patients with bee venom anaphylaxis. The IgG4 levels increased strongly from a low pre-treatment level in proportion to the antigen dose received. The antigen-specific IgE levels gradually fell from a high pretreatment level, but remained significantly elevated. Immunoblotting for specific IgE and IgG4 demonstrated that acquisition of clinical tolerance after therapy was associated with declines in the number and intensity of bands in IgE blots and the development of more bands of increasing density in the IgG4 blots., Conclusions: Parenteral immunotherapy may offer an alternative treatment to lifelong dietary restriction and epinephrine injections in patients who exhibit life-threatening IgE-mediated anaphylaxis to peanut. Cross desensitisation to other legumes appears to have occurred in this study. The quality and potency of the extract used is an important factor in achieving the desired acquisition of clinical tolerance. In our patient this tolerance correlated with his ability to maintain high levels of specific IgG4, which acted as a marker of protection against anaphylaxis. The use of IgG4 immunoblotting may provide an improved level of discrimination in the assessment of correlation of clinical efficacy with the immunologic response.

Background: There appears to be a lack of agreement in the literature on the allergenicity of hen egg proteins. This may be partly due to the use of impure proteins in some cases. Egg yolk proteins have also been largely ignored in such studies. We therefore set out to determine, using especially purified proteins, their relative allergenicity, and to observe whether there were any relationships between their potency and the sensitivity of patients to them., Methods and Results: The sera of 40 patients with clinically observed hen egg hypersensitivity were tested for specific IgE binding to purified egg white and egg yolk proteins using the radioallergosorbent test (RAST). Statistical treatment by correspondence analysis of the percent radioactive uptakes in the RAST to the 8 proteins demonstrated that there were four distinct groups of patients reacting in a similar way to four discrete sets of proteins., Conclusions: The first three sets of allergens consisted of egg white proteins as follows: firstly, lysozyme and ovalbumin; secondly, ovomucoid; and thirdly, ovomucin. The fourth set contained the egg white protein ovotransferrin and the egg yolk proteins apovitellenins I and VI and phosvitin. The existence of patient groups may explain why various workers have reported different allergens to be important in egg hypersensitivity. A sufficiently large number of patients must be examined so as to give a representative distribution across each group, otherwise the results may be biased towards one allergen.

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

The omega-atracotoxins are a family of 36 to 37-residue peptide neurotoxins that block insect but not mammalian voltage-gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure-function relationships in the omega-atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel-web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of omega-atracotoxin-Hv1a (omega-ACTX-Hv1a), we have isolated and sequenced five additional omega-atracotoxins. Five of the six omega-atracotoxins isolated from the venom of H. versuta (omega-ACTX-Hv1a to -Hv1e) differ from one another by only 1-3 residues and have similar insecticidal potencies. In contrast, omega-ACTX-Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N-terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N-terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N-terminal charge, rather than the chemical properties of the N-terminal residues themselves, may be critical for the activity of omega-atracotoxin on insect calcium channels.

Robustoxin, the lethal neurotoxin from the Sydney funnel web spider Atrax robustus, is a polypeptide of 42 residues cross-linked by four disulfide bonds. This paper describes the sequence-specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of robustoxin in aqueous solution. Several broad backbone amide resonances were encountered in spectra recorded at 27 degrees C, making the assignments at that temperature incomplete. In spectra recorded at lower temperatures these amide resonances became sharper, but others that were sharp at 27 degrees C became broad, indicative of conformational averaging on the millisecond timescale for certain regions of the structure. Nevertheless, it was possible to establish that robustoxin contains a small, triple-stranded, antiparallel beta-sheet and several reverse turns, but no alpha-helix. These observations indicate that this toxin may adopt the inhibitor cystine knot structure found in polypeptides from a diverse range of species, including a number of spiders. Analysis of the pH dependence of the spectrum yielded pKa values for Tyr22 and Tyr25, one of the three carboxyl groups, and the Lys residues.

Background: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta-ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels., Results: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 310 helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-I, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction., Conclusions: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

Background: The house dust mite has been shown to be an important source of domestic allergens associated with immediate hypersensitivities. The Group I mite allergens Der p I from Dermatophagoides pteronyssinus and Der f I from D. farinae display extensive amino acid sequence homology and have similarities with cysteine protease enzymes., Objective: The availability of the complete amino acid sequences for these allergens allowed us to search for the allergic determinants within these molecules. The aim of the present investigation was to identify any continuous IgE-binding epitopes within these amino acid sequences. We also sought to test the validity of previously reported Der p I peptide epitope sequences., Methods: In order to identify any continuous IgE epitopes, the amino acid sequences of Der p I and Der f I were synthesized as decapeptides overlapping in sequence and coupled to plastic pins. The specific IgE-binding capacity of these peptides was assayed using an enzyme-linked biotin-streptavidin procedure and sera from patients known to be sensitive to these allergens. Previously reported Der p I peptide epitopes were synthesized as free peptides and tested for their ability to inhibit specific IgE binding to allergen extract discs., Results: None of the pin-coupled Der p I or Der f I peptides was found by the continuous epitope mapping procedure to bind significantly to specific IgE in the sera of hypersensitive patients. The previously reported Der p I peptide epitopes did not inhibit specific IgE binding to mite extract discs., Conclusion: The specific IgE binding epitopes of the house dust mite allergens Der p I and Der f I are discontinuous in nature.

The present study investigated the action of textilotoxin, isolated from the venom of the Australian common brown snake Pseudonaja textilis, on neuromuscular transmission in isolated toad nerve-muscle preparations. Initial muscle twitch tension experiments revealed a triphasic pattern of changes in muscle tension and a irreversible binding action of textilotoxin (10 micrograms/ml) similar to other snake beta-neurotoxins. This was characterised by an initial depression of twitch tension, followed by a period of enhanced tension, eventually leading to a reduction in tension to complete neuromuscular blockade. These actions on muscle tension were investigated further by assessing the action of textilotoxin on end-plate potential amplitude (EPP). This revealed a similar triphasic alteration of the nerve-evoked release of acetylcholine from the motor nerve terminal. These actions on acetylcholine release were confirmed to be of a presynaptic origin since the modal amplitude of miniature end-plate potentials (MEPPs) was not reduced and in twitch tension experiments the muscle still contracted in response to direct muscle stimulation when nerve-evoked release was completely blocked. Interestingly dramatic effects were observed on the spontaneous release of acetylcholine, including an marked increase in MEPP frequency, a skewing of the MEPP amplitude frequency histogram to the right, and a resultant increase in the number of 'giant' MEPPs. These results indicate that textilotoxin causes a presynaptic blockade of neuromuscular transmission involving a disruption of the regulatory mechanism that controls acetylcholine release.

Robustoxin is the lethal polypeptide toxin in Atrax robustus venom. A monoclonal antibody was produced using synthetic, unfolded robustoxin conjugated to keyhole limpet haemocyanin as the immunogen. This monoclonal antibody did not protect newborn mice against challenge with the crude venom of the male Sydney funnel-web spider, but did slightly prolong their survival time. Western blotted crude venom of the male Sydney funnel-web spider showed two monoclonal antibody binding bands. One band at low M(r) corresponded to robustoxin (M(r) 4854), while the other higher M(r) band (approximately 37,000) may be due to a pre-robustoxin molecule.

The effects of a neurotoxin (versutoxin VTX), purified from the venom of the Australian Blue Mountains funnel-web spider Hadronyche versuta, on the ionic currents in rat dorsal root ganglion cells were investigated under voltage-clamp conditions using the whole-cell patch-clamp technique. VTX had no effect on tetrodotoxin-resistant (TTX-R) sodium currents or potassium currents. In contrast VTX produced a dose-dependent slowing or removal of tetrodotoxin-sensitive (TTX-S) sodium current inactivation, a reduction in peak TTX-S sodium current but did not markedly slow tail current kinetics of TTX-S sodium currents. This steady-state sodium current was maintained during prolonged depolarizations at all test potentials and the reduction in sodium current amplitude produced by VTX had an apparent Ki of 37 nM. In the presence of 32 nM VTX the voltage dependence of steady-state sodium channel inactivation (h infinity) also showed a significant 7 mV shift in the voltage midpoint in the hyperpolarizing direction, with no change in the slope factor. In addition there was a steady-state or non-inactivating component present (14 +/- 2% of maximal sodium current) at prepulse potentials more depolarized than -40 mV, potentials which normally inactivate all TTX-S sodium channels. Finally, there was an observed increase in the rate of recovery from inactivation in the presence of VTX. These selective actions of VTX on sodium channel gating and kinetics are similar to those of alpha-scorpion and sea anemone toxins.

In this study the immunochemical structure of the heavy chain polypeptide from tetanus toxin was studied. Numerous antigenic determinants were identified by probing a set of overlapping peptides derived from the amino acid sequence of tetanus toxin with polyclonal anti-toxoid antibody preparations. Synthetic antigens representing continuous epitopes were prepared and used to immunize mice. The capacity of the resulting anti-peptide antibodies to react with tetanus toxin in vitro and in vivo was determined. The majority of antibodies bound to tetanus toxin and three epitopes capable of eliciting neutralizing antibodies were identified.

Hexapeptides of sequential overlapping sequences of beta-lactoglobulin (BLG) were used to probe serum from children with immediate-type cow milk allergy for IgE binding to continuous epitopes of BLG in an enhanced enzyme-linked immunosorbent assay (ELISA). Six regions of IgE binding were identified on the BLG molecule and these were synthesized as dodecapeptides. Inhibition of IgE binding to whole BLG was used to confirm the BLG-specific binding of IgE to each of the synthesized peptides. One of the peptides, peptide 4, showed inhibition in an IgE anti-BLG radioimmunoassay to all 16 sera tested. The patterns of inhibition with the native BLG molecule and peptide 4 were significantly correlated (P = 0.005), suggesting that this peptide contains a major continuous IgE binding epitope of BLG.

Neurotoxins isolated from the venoms of Australian funnel-web spiders increase spontaneous action potential activity in a variety of excitable cells. In the present study intracellular recordings were made with microelectrodes (30-60 M omega, 2 M KCl) from locus coeruleus, mesencephalic nucleus of the trigeminal nerve and laterodorsal tegmental neurons in brain slices. Versutoxin, a polypeptide toxin isolated from the venom of Hadronyche versutus produced a profound increase in spontaneous synaptic activity impinging on neurons, which did not fully recover for up to 3 h after washout. The threshold concentration was 1.5 nM in locus coeruleus neurons, with increasing concentrations (up to 50 nM) producing larger effects. A modest increase in synaptic activity was observed in mesencephalic nucleus of the trigeminal nerve neurons during superfusion with 50 nM versutoxin. The increase in spontaneous synaptic activity was reversed by agents which block synaptic potentials impinging on locus coeruleus neurons, i.e., tetrodotoxin (100 nM), Co2+ (3 mM) or the combination of 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and bicuculline (30 microM). Threshold, peak amplitude, maximum rate of rise, duration, amplitude of afterhyperpolarisations and interspike intervals of action potentials in each type of neuron were unaffected by versutoxin. Voltage-current relationships were also unaffected. Calcium-dependent action potentials evoked in locus coeruleus neurons in the presence of tetrodotoxin were unaffected by versutoxin, as were depolarisations produced by exogenously applied glutamate. These results suggest that versutoxin increases spontaneous synaptic activity, but has no effect on the membrane properties of the soma of several types of rat brain neurons.

1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri). 2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots. 3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

Cysteine-containing peptides corresponding to the putative hinge region connecting the heavy and light polypeptide chains of tetanus toxin were synthesized utilising a solid phase with an acid hyper-labile linkage agent. Both the single-chain cysteine peptides, as well as a disulphide-bonded double-chain peptide, obtained by selective iodine-oxidation of S-trityl and S-acetamidomethyl protected peptides, were conjugated to carrier proteins for the purpose of immunisation and immunoassay. Comparison of the immunochemical specificity of mouse antibodies raised against these constructs, as well as antibodies against tetanus toxoid, permitted the assignment of the location of the inter-chain disulphide bond of tetanus toxin.

The complete amino-acid sequences of subunits A, B, C and D of textilotoxin, the presynaptic neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis, were determined. These confirmed that it is structurally the most complex of any of the known snake venom neurotoxins. Textilotoxin consists of 623 amino-acid residues in five subunits (subunit A, 118 residues; subunit B, 121 residues; subunit C, 118 residues; subunit D, two chains of 133 residues each). All subunits A, B, C and D contain the putative phospholipase A2 active site. Only subunit A showed any lethality on its own (4 mg/kg i.v. in mice). Subunit D contained two identical covalently-linked subunits and was weakly glycosylated. All subunits were necessary for maximum lethality at 1 micrograms/kg mice intraperitoneally. Details of the sequences of the subunits A, B and C are reported and interesting homology with other snake venom phospholipase A2 neurotoxins indicated.

One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid liability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8- pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have liability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.

A peptide containing four threonine residues was synthesised by the solid-phase method using fluorenyl-methoxycarbonylamino acid reactive esters or coupling by preactivation with 1-hydroxybenzotriazole and Castro's reagent. In two separate experiments the synthesis was carried out with or without protection of the side-chain hydroxyl group of threonine as the tert.-butyl ether. Comparison of the crude peptides after deprotection and detachment from the synthesis resin suggests that side-chain protection of threonine is unnecessary under the synthetic conditions employed.

The allergenicity of cow's milk whey proteins, purified by high performance liquid chromatography (HPLC), was examined by the radio-allergosorbent test (RAST) against the sera of children immediately hypersensitive to milk. beta-lactoglobulin and alpha-lactalbumin bound specific IgE in the sera of 63% and 75% of these patients respectively. These allergens were tested for cross reactivity with each other by RAST inhibition. Both inhibited the binding of IgE, in the sera of allergic patients, to the other protein. Two possible determinant peptides, one from beta-lactoglobulin and one from alpha-lactalbumin, were selected by computer prediction of antigenic sites and synthesized by the fluorenylmethoxycarbonyl (FMOC)-polyamide method. The peptides were adsorbed to nitrocellulose discs and used in further RAST studies with sera from the allergic children. Both peptides bound specific IgE in the RAST assay.

Textilotoxin is a presynaptic neurotoxin in the venom of the Australian common brown snake, Pseudonaja textilis. It has the highest lethality and is structurally the most complex of any known snake venom neurotoxin. Reverse-phase HPLC was used to resolve textilotoxin into subunits A, B, C and D. Subunit D consists of two identical covalently linked polypeptide chains. Its sequence is now reported. It is an acidic, slightly glycosylated polypeptide of 133 amino acid residues in each chain. Although it is not itself neurotoxic, it was found to be essential for the neurotoxicity of textilotoxin.

The binding of textilotoxin and its subunits A, B, C and D to polyclonal rabbit antisera directed against textilotoxin, Australian common brown snake (Pseudonaja textilis) and tiger snake (Notechis scutatus scutatus) venoms was studied by ELISA. Subunit D showed greatest reactivity with antisera to textilotoxin and brown snake venom. The phospholipase A2 active subunit A reacted more strongly with antisera to tiger snake venom in keeping with the high degree of homology between the amino acid sequences of subunit A and notexin from tiger snake venom. Subunit A was the only subunit lethal to mice, but at doses 1000-fold greater than for textilotoxin.

The effect of stimulation frequency on the timecourse of neuromuscular blockade, following the administration of textilotoxin (20 micrograms/kg) or beta-bungarotoxin (50 micrograms/kg), was examined in the interdigital muscles of the hindlimb in anaesthetized mice. While the time of death was variable, neuromuscular blockade of the interdigital muscles occurred at the same time as respiratory failure with both textilotoxin and beta-bungarotoxin only at stimulation rates of 0.5 Hz and above. Textilotoxin (50 micrograms/kg) produced an increase in the heart rate prior to death but no change in the shape of the electrocardiogram.

1. An endogenous antitoxin fraction was isolated from non-immune rabbit sera by affinity chromatography with robustoxin bound to the solid support. 2. Robustoxin is the sole lethal toxin in the venom of the male funnel web spider, Atrax robustus. 3. The fraction was found to contain IgG and IgM immunoglobulins. 4. This fraction prevented or reversed the lethal actions of the crude venom in newborn mice, in mouse phrenic nerve-hemidiaphragm preparations, and in anaesthetized monkeys. 5. The antitoxin fraction is of potential value in the therapy of human envenomation by A. robustus.

A stable toxoid was prepared from robustoxin (the lethal polypeptide neurotoxin in the venom of the male funnel-web spider, Atrax robustus) by polymerization with glutaraldehyde. This material was non-toxic in new-born mice. Administration of the toxoid to three Macaca fascicularis monkeys (50-80 micrograms/kg s.c. at 14-day intervals for 8-12 weeks) produced no toxic effects; anti-robustoxin antibodies were detected in serum samples by immunodiffusion tests within 13-27 days. In vivo evidence of successful protection with the toxoid was obtained by challenging the monkeys with male A. robustus venom (50 micrograms/kg i.v.) under anaesthesia with pentobarbitone (one monkey), or with ketamine, halothane and nitrous oxide, 1-26 weeks after the last injection of the toxoid. Only minor respiratory, cardiovascular and skeletal motor disturbances were produced, and all monkeys recovered fully and uneventfully. Challenge with the same dose of venom in non-immunized or robustoxin N-terminal decapeptide ovalbumin conjugate-treated monkeys resulted in typical lethal neurotoxic effects, culminating in severe hypotension or death from circulatory and respiratory failure within 280 min.

1. A lethal neurotoxin (acanthophin d) was isolated from the venom of the Australian death adder snake Acanthophis antarcticus. 2. Acanthophin d consisted of a single polypeptide chain of 74 amino acid residues cross-linked by five disulphide bridges. 3. The results of neurophysiological experiments on murine phrenic nerve hemi-diaphragm preparations were consistent with irreversible post-synaptic blockage of neuromuscular transmission by acanthophin d.

Synthetic peptides were prepared by a solid-phase method and biotinylated selectively and in high yield at the amino terminus prior to peptide deprotection and detachment from synthesis resin. It was shown that peptides biotinylated in this manner could be bound by avidin immobilized on a plastic surface and used to detect anti-peptide antibodies in an enzyme-linked immunosorbent assay. The advantages of this method compared to conventional immunoassay techniques for anti-peptide antibodies are discussed.

Two cytochrome c allergens were isolated from extracts of the pollens of the dicotyledons English plantain (Plantago lanceolata) and Paterson's Curse (Echium plantagineum) by ion exchange chromatography, gel filtration and preparative isoelectric focusing. They were characterized by their absorption spectra, mol. wt, pI and amino acid composition. The cytochromes c bound specific IgE in the sera of hypersensitive patients by RAST. Preliminary evidence for allergenic cross-reactivity between them was obtained by RAST inhibition.

A method is reported for the purification of type D botulinum toxin using a combination of low and high pressure ion exchange chromatography. The procedure produced homogeneous toxin in its free form in 3 days, with a specific toxicity in mice of 5.4 x 10(7) LD50/mg protein. Polyclonal antibodies against the pure toxin were raised in rabbits and detected the toxin in both ELISA and western blotting. The antibodies also detected type C1 botulinum toxin using these techniques, confirming the presence of cross-reacting antigenic determinants in these two proteins.

The specific IgE binding by protein extracts of 11 food legumes, including soybean, was examined by RAST and RAST inhibition. Sera from 15 peanut-sensitive patients were, with very few exceptions, positive in the RAST to all the legumes. RAST-inhibition testing of each extract against RAST discs of the other legumes indicated considerable cross-reactivity of IgE binding between the legumes. Cross-allergenicity was demonstrated to be most marked between the extracts of peanut, garden pea, chick pea, and soybean. The results have important implications for selection of effective hypoallergenic diets and for the diagnosis of patients hypersensitive to foods.

The crude venoms of male and female Sydney funnel web spiders, Atrax robustus, were compared by cation-exchange and high-performance liquid chromatography, lethality to new-born mice, polyacrylamide gel isoelectric focusing, immunoelectrophoresis, phospholipase A analysis, effects on the mouse phrenic nerve hemidiaphragm and passive paw oedema in the rat and, except in the case of rat paw oedema, were found to exhibit quite different properties. Polyacrylamide gel isoelectric focusing and high-performance liquid chromatography proved to be suitable for gender and species determination when applied to the venoms of A. formidabilis, A. infensus, A. robustus and A. versutus. These venoms were also compared by lethality, promotion of muscle fasciculation and phospholipase A activity.

The possible allergenicity of an insect pollinated weed, Echium plantagineum, was investigated in a rural area of Australia. Sixty-one subjects with respiratory allergy were studies. Positive skin test reactions to defatted ammonium bicarbonate extract of pollen were found in over 60% of subjects, and positive RAST tests in a similar number. The question of crossreactivity between weed pollens is discussed. The pollen of E. plantagineum was shown to reach the atmosphere in significant amounts about 1 month before the peak grass pollinating period. Evidence that the pollen of E. plantagineum becomes airborne and elicits an IgE response suggests that further attention should be directed to weed pollens as potential allergens.

Five biologically active daphnane orthoesters have been isolated from Pimelea species (Thymelaeaceae). Four of these possessing antineoplastic activity against in vivo murine P-388 lymphocytic leukemia are gnidimacrin from P. ligustrina, simpleximacrin from P. simplex, linimacrin d and Pimelea factor P3 from P. linifolia. Linimacrin c from P. linifolia and the other four compounds showed piscicidal activity. Gnidimacrin and Pimelea factor P3 have previously been isolated from other members of the Thymelaeaceae. Simpleximacrin and linimacrins c and d are new compounds.

Crude peanut protein fractions from raw and roasted peanuts were examined in the RAST with 10 sera from patients showing clinical peanut sensitivity. The radioactive uptake results, which were generally high, did not reveal any distinguishable pattern. Two commercially available peanut proteins, peanut lectin and phospholipase D, gave poor RAST responses. Three purified peanut proteins, alpha-arachin, conarachin I, and concanavalin A-reactive glycoprotein, all gave significant RAST results that were generally lower than those obtained with the crude extracts. The extent of RAST inhibition obtained with these materials was inversely related to their abundance in the total peanut protein. Crossed immunoelectrophoresis with extracts from raw and roasted peanut indicated the presence of 22 and 10 anodically migrating antigens, respectively. Sixteen IgE binding antigens were revealed for raw peanut and seven for roasted peanut after incubation with a mixed serum from the 10 patients in crossed radioimmunoelectrophoresis (CRIE) using 125I-labeled anti-IgE. CRIE plates treated with individual serum samples showed that all the patients had specific IgE for the major antigen peak, which has been tentatively identified as alpha-arachin. This major storage protein of peanut, which is known to be particularly heat resistant; may be of greater clinical significance than its apparently low RAST activity would seem to indicate.

An 11-amino acid residue peptidyl-linkage agent-polyamide resin complex was synthesized by the fluorenylmethyloxycarbonyl (Fmoc)-polyamide solid-phase system. Mice were immunized with the free peptide, peptidyl-resin and peptide coupled to the carrier proteins ovalbumin (Ova) and keyhole limpet haemocyanin (KLH). The immunogenicity of these materials was assessed by measurement of the capacity of the various antisera to bind the peptide in an enzyme-linked immunosorbent assay (ELISA). The peptidyl-resin exhibited enhanced immunogenicity compared to the free peptide. It is suggested that the time needed for screening for immunogenicity of large numbers of synthetic peptides thus be greatly shortened by using peptidyl-resins for immunization. This method eliminates laborious cleavage of peptide from resin, purification, coupling to carrier and the difficulties of handling peptides of low solubility.

Textilotoxin is a presynaptic neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis. It has the highest lethality and is structurally the most complex of any known snake venom neurotoxin. It was resolved into its five non-covalently linked subunits in a single step by reverse-phase HPLC. Two of the subunits were identical. The N-terminal amino-acid sequence and amino-acid composition of each subunit were determined. Subunit A was the only one found to possess phospholipase A activity. Separation of textilotoxin into its subunits was reversible and reformed textilotoxin had the same Mr and lethality in mice as the native toxin. Experiments with various unnatural combinations of subunits have led to interesting variations in lethality and Mr of the resulting complexes.

A single-column high-performance liquid chromatographic separation of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives, generated during polypeptide sequence analysis by the 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate/phenylisothiocyanate double coupling technique, is described. Recovery of the serine and threonine derivatives was improved by substituting boron trifluoride-diethyl etherate for trifluoroacetic acid in the thiazolinone cleavage reactions. Residues, including the S-carboxymethyl derivative of cysteine, were assigned after a single injection and a cycle time of 30 min. Quantities of 4-N,N-dimethylaminoazobenzene 4'-thiohydantoin amino acid derivatives as low as 100 fmol were detected. Interference of sequencing artefacts with residue assignment was avoided. This technique allows simultaneous manual sequencing of several proteins or peptides at the level of a few picomoles.

Maculotoxin, a potent neurotoxin isolated from the posterior salivary glands of the blue-ringed octopus. Hapalochlaena maculosa, has now been identified as tetrodotoxin. This is the first reported case in which tetrodotoxin has been found to occur in a venom.

There has been unjustified neglect of dicotyledonous (dicot; 'weed') pollens in research directed at isolating pure allergens, since dicot pollens are widespread and frequently important in provoking immediate allergic reactions. Sera from patients who showed positive skin prick test reactions to plantain pollen generally also reacted in the radioallergosorbent test (RAST) with at least one other species of dicot pollen. Fractionation of plantain pollen extracts by ultrafiltration and molecular sieving and examination of the fractions by the RAST revealed a spread of allergenic activity. Using crossed immunoelectrophoresis, at least 16 different antigens were detected in plantain pollen and at least 6 of these antigens may be allergenic. Allergenic glycoproteins that react with concanavalin A were isolated and their complexity examined by electrophoresis and electro-focusing. IgE-binding components were found widely distributed in plantain plants and not confined to the pollen.

The complete amino acid sequence of pseudonajatoxin b, a basic neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis, was determined by automated Edman analysis of the reduced carboxymethylated polypeptide and of peptides derived by digestion of it with Staphylococcus aureus V8 proteinase. Pseudonajatoxin b consists of a single polypeptide chain of 71 amino acids with Mr 7762. The amino acid sequence showed considerable homology with postsynaptic long neurotoxins, but there were striking differences. Pseudonajatoxin b displayed relatively high lethality, LD50 15 micrograms/kg in mice.

The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.

A concanavalin A-reactive glycoprotein allergen has been isolated from peanut (Arachis hypogaea). The allergen was separated by affinity chromatography and purified by gel permeation and ion-exchange chromatography. The monomeric molecular weight is 65,000 and the pI is 4.6. The presence of one cysteine residue per molecule results in some dimer formation. Concanavalin A-reactive glycoprotein is a potent allergen for peanut-sensitive patients in both in vivo and in vitro tests. It is allergenically stable, on in vitro examination, at temperatures of up to 100 degrees C and over the pH range 2.8-10. Removal of the carbohydrate moiety failed to eliminate the allergenicity. Concanavalin A-reactive glycoprotein is identified in the crossed immunoelectrophoretic pattern as a major antigen of peanut protein extract but its structural characteristics indicate that it is probably not a component of the major storage-protein complex, arachin.