The Helix aspersa (brown garden snail) allergen repertoire

BACKGROUND: Ingestion of snails can induce strong asthmatic or anaphylactic responses, mainly in house-dust-mite-sensitized patients. The aim of this study was to identify the Helix aspersa (Hel a), Theba pisana (The p) and Otala lactea (Ota l) allergens and the extent of their cross-reactivity with the Dermatophagoides pteronyssinus (Der p) mite. PATIENTS AND METHODS: In 60 atopic patients, skin prick tests (SPT) to snail and D. pteronyssinus, total and specific IgE, specific IgE immunoblots, RAST and immunoblot inhibition assays were performed. RESULTS: Mean total IgE was >1,000 kU/l. Mean specific IgE (class 6 for Der p and class 2 for Hel a) SPT were positive in 44 patients for snail and in 56 for mite. Isoelectric focusing (IEF) and SDS-PAGE followed by immunoblotting of H. aspersa extract enabled the identification of 27 and 20 allergens, respectively. Myosin heavy chains from snails (molecular weight >208 kDa) disclosed two major allergens. Hel a and Der p RAST were strongly inhibited by their homologous extracts, with Hel a RAST being inhibited by the Der p extract to a much greater extent (72.6%) than the inverse (5.6%). A complete inhibition of the immunoblots by their homologous extract was obtained. However, Hel a extract did not inhibit Der p IEF separated recognition. On the other hand, mite extract extensively inhibited snail immunoblots from both IEF and SDS-PAGE separations. Immune detection on chicken, pig, rabbit, cow and horse myosins did not reveal any IgE cross recognition with snail. CONCLUSIONS: In most cases of snail allergy, mite appeared to be the sensitizing agent. Nevertheless, snails may also be able to induce sensitization by themselves. This hypothesis is supported by the finding of specific IgE to Hel a in 2 patients who did not show specific IgE to Der p, and one of them was suffering from asthma after snail ingestion.     

PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHev b 8)

BACKGROUND: Profilin (Hev b 8) in natural rubber latex (NRL) has been assumed to be an important allergen. Since latex profilin has a molecular mass similar to two other latex allergens (Hev b 1 and Hev b 6.03) in the 14-kDa range, it is difficult to obtain sufficient amounts of purified native profilin for investigations and diagnostics. The present study aimed to produce recombinant latex profilin (rHev b 8) and study its IgE-binding reactivity. METHODS: A profilin-specific cDNA encoding the latex profilin from Hevea brasiliensis leaves was synthesized and subcloned, and the rHev b 8 was overexpressed in fusion with the maltose-binding protein (MBP) in E. coli. The IgE-binding reactivity of rHev b 8 was studied by immunoblotting, immunoblot inhibition experiments, and the Pharmacia CAP method, with 25 sera from health-care workers with latex allergy and 17 sera from latex-sensitive spina bifida patients. RESULTS: rHev b 8 was found to have 131 amino acids and a sequence identity of 75% with birch profilin (Bet v 2). Analysis by the CAP system revealed the presence of rHev b 8-specific IgE antibodies in two out of 17 sera from spina bifida patients and in five out of 25 sera (20%) from health-care workers. Two subjects of the latter group with rHev b 8-specific IgE showed negative results in the skin prick tests with tree-pollen extracts and had no IgE to rBet v 2, indicating the presence of IgE-binding epitopes on the Hev b 8-molecule which do not cross-react with birch profilin. Immunoblot inhibition assays using MBP-rHev b 8 as inhibitor confirmed the presence of latex profilin in the NRL extract. IgE binding to the native latex profilin could be completely inhibited by the MBP-rHev b 8. CONCLUSIONS: Latex profilin represents a minor allergen in NRL and may have IgE-binding epitopes different from Bet v 2.     

Cloning and characterisation of two IgE-binding proteins,homologous to tropomyosin and alpha-tubulin,from the mite Lepidoglyphus destructor

BACKGROUND: The dust mite Lepidoglyphus destructor is a major source of mite allergy in European rural environments, but it also causes allergy in urban populations around the world. We have previously cloned, sequenced and expressed several allergens from L. destructor (Lep d 2, Lep d 5, Lep d 7 and Lep d 13). The aim of this study was to identify and clone additional allergens from L. destructor, and to evaluate their IgE-binding reactivities. METHODS: PCR and screening with sera from L. destructor-sensitised individuals were used to isolate new clones from a phage display L. destructor cDNA library. The complete coding sequences of the clones were determined and expressed as His(6)-tagged recombinant proteins in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE, immunoblotting and mass spectrometry. RESULTS: Two new clones, showing homology to tropomyosin and alpha-tubulin in several species, were isolated from the phage display L. destructor cDNA library. Due to its homology to group 10 dust mite allergens, the tropomyosin clone was named Lep d 10. The IgE-binding frequencies of the recombinant Lep d 10 and alpha-tubulin were 13% (18/136) and 12% (11/95), respectively, among subjects with IgE reactivity to mites and/or crustaceans. CONCLUSIONS: Two new allergens from L. destructor have been identified and can now be added to the repertoire of recombinant L. destructor allergens. In addition, both these allergens belong to highly conserved protein families and may be important for evaluation of allergenic cross-reactivity.     

Ontogenesis of the snail, Helix aspersa: embryogenesis timetable and ontogenesis of GABA-like immunoreactive neurons in the central nervous system

Late stages of embryogenesis in the terrestrial snail Helix aspersa L. were studied and a developmental timetable was produced. The distribution of gamma-aminobutyric acid-like immunoreactive (GABA-ir) elements in the CNS of the snail was studied from embryos to adulthood in wholemounts. In adults, approximately 226 GABA-ir neurons were located in the buccal, cerebral and pedal ganglia. The population of GABA-ir cells included four pairs of buccal neurons, three neuronal clusters in the pedal ganglia, two clusters and six single neurons in the cerebral ganglia. GABA-ir fibers were observed in all ganglia and in some nerves. The first detected pair of GABA-ir cells in the embryos appeared in the buccal ganglia at about 63–64% of embryonic development. Five pairs of GABA-ir cell bodies were observed in the cerebral ganglia at about 64–65% of development. During the following 30% of development three more pairs of GABA-ir neurons were detected in the buccal ganglia and over fifteen cells were detected in each cerebral ganglion. At the stage of 70% of development, the first pair of GABA-ir neurons was found in the pedal ganglia. In the suboesophageal ganglion complex, GABA-ir fibers were first detected at about 90% of embryonic development. In the posthatching period, the quantity of GABA-ir neurons reached the adult status in four days in the cerebral ganglia, and in three weeks in the pedal ganglia. In juveniles, transient expression of GABA was found in the pedal ganglia (fourth cluster).     

Up- and down-regulation of Helix command-specific 2 (HCS2) gene expression in the nervous system of terrestrial snail Helix lucorum

A novel gene named Helix command-specific 2 (HCS2) was shown to be expressed predominantly in four giant parietal interneurons involved in withdrawal behavior of the terrestrial snail Helix lucorum L. and several single neurons in other ganglia. Decrease in spontaneous electrophysiological activity of neurons in the isolated CNS by 24h incubation in saline with elevated Mg(2+) concentration significantly decreased the number of HCS2-expressing neurons. Five short-term serotonin applications (each of 10microM), during a 24h incubation of the nervous system in saline induced expression of the HCS2 gene in many cells in cerebral, parietal, pleural and pedal ganglia. Dopamine applications under similar conditions were not effective. Application of anisomycin or cycloheximide, known to block protein synthesis, did not prevent the induction of HCS2 expression under serotonin influence. Skin injury elicited a significant increase in the number of HCS2-expressing cells 24h later in pleural and cerebral ganglia. Incubation of the isolated nervous system preparations for three days in culture medium elicited close to a maximum increase in number of HCS2-expressing cells. Elevation of the normal Mg(2+) concentration in the culture medium significantly decreased the number of cells demonstrating HCS2 expression. Application of the cAMP activator forskolin (10microM) increased the expression under Mg(2+), indicating that cAMP was involved in the up-regulation of HCS2. Application of thapsigargin (10microM), known to release Ca(2+) from intracellular stores, was also effective in increasing expression, suggesting participation of Ca(2+) in regulation of HCS2 expression. Cellular groups expressing the HCS2 gene under different conditions seem to be functionally related since it was demonstrated earlier that some neurons constituting these clusters are involved in the withdrawal behavior and the response of the organism to stress stimuli. From these results we suggest that the HCS2 pattern of expression can be down-regulated by a decrease in synaptic activity in the nervous system, and up-regulated by external noxious inputs, as well as the application of neurotransmitters and second messengers known to be involved in the withdrawal behavior and maintenance of isolated ganglia in culture medium. When up-regulated, the HCS2 expression appears, at least in part in neurons, to be involved in the withdrawal behavior.     

Green laser light (532nm) activates a chloride current in the C1 neuron of Helix aspersa

Five hundred and thirty-two nanometers laser light evokes neuron-specific electrical responses in identified neurons of Helix ganglia. Such responses are intensity-dependent over the range 25-1500 mW, readily reversible and repeatable. Detailed experiments on the C1 neuron, which is inhibited by 532 nm light, showed that inhibition results from a selective increase in transmembrane Cl(-) ion conductance. Experiments with calcium-sensitive microelectrodes suggest that the response does not result from an increase in [Ca(2+)](i). The change in Cl(-) ion conductance probably occurs in the extensive plasmalemma infoldings of the proximal axon.     

Vertebrate tropomyosin: distribution,properties and function

Tropomyosin (TM) is widely distributed in all cell types associated with actin as a fibrous molecule composed of two -helical chains arranged as a coiled-coil. It is localised, polymerised end to end, along each of the two grooves of the F-actin filament providing structural stability and modulating the filament function. To accommodate the wide range of functions associated with actin filaments that occur in eucaryote cells TM exists in a large number isoforms, over 20 of which have been identified. These isoforms which are expressed by alternative promoters and alternative RNA processing of four genes, TPM1, 2, 3 and 4, all conform to a general pattern of structure. Their amino acid sequences consist of an integral number, six or seven in vertebrates, of quasiequivalent regions of about 40 residues that are considered to represent the actin-binding regions of the molecule. In addition to the variable regions a large part of the polypeptide chains of the TM isoforms, mainly centrally located and expressed by five exons, is invariant. Many of the isoforms are tissue and filament specific in their distribution implying that the exons expressed in them and the regions of the molecule they represent are of significance for the function of the filament system with which they are associated. In the case of muscle there is clear evidence that the TM moves its position on the F-actin filament during contraction and it is therefore considered to play an important part in the regulation of the process. It is uncertain how the role of TM in muscle compares to that in non-muscle systems and if its function in the former tissue is unique to muscle.     

Dopamine and serotonin receptors mediating contractions of the snail,Helix pomatia,salivary duct

The combination of high performance liquid chromatography, bioassay and immunocytochemistry was applied to study the regulation of the salivary duct muscle of the snail, Helix pomatia. The major function of the duct appears to be to propel the saliva toward the buccal cavity during feeding. It has been established that serotonin and dopamine applied exogenously mimic the effect on the duct exerted by the stimulation of the salivary nerve. Immunohistochemistry revealed the presence of serotonin, but not dopaminergic nerve elements in the nerve and along the duct surface. However, both serotonin (14.9-15.5 pmol/mg) and dopamine (0.38-0.58 pmol/mg), as well as the synthesizing enzymes (tyrozine hydroxylase 0.28 pmol/mg tissue/h and DOPA 0.32 nmol synthesized DA/mg tissue/h) could regularly be assayed in the salivary duct by high performance liquid chromatography. When released following the stimulation of the salivary nerve, both monoamines were shown to interact with distinct membrane receptors. Dopamine elicited a sustained increase of the muscle tone in concentration-dependent manner (K(d)=1.5 microM). Mammalian D(1) receptor antagonist flupenthixol and fluphenazine attenuated, whereas the D(1) receptor agonist SKF-38393 mimicked the effect elicited by exogenous dopamine. Serotonin had a double effect on the salivary duct: a relaxing and a contracting one with different K(d) values 76 nM and 2.4 microM, respectively. 5-HT(2) receptor antagonist ritanserin and ketanserin attenuated the serotonin-induced relaxation. In contrast 5-HT(3) antagonist metoclopramide and MDL2222 decreased and 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide mimicked the serotonin-induced contraction, suggesting that serotonin exerted its action on two different receptor subtypes. The release of radiolabeled serotonin and dopamine upon nerve stimulation was found to be Ca-dependent. Furthermore, the increase in serotonin concentration induced a decrease of the potency of dopamine to elicit sustained contraction.These results provide evidence for the transmitter role of serotonin and dopamine in salivary duct. It is concluded that receptors reveal a pharmacological profile related to vertebrate D(1), 5-HT(2) and 5-HT(3) receptor subtypes. Moreover, it was found that the process of conveying the saliva is modulated by an interaction of dopamine and serotonin.     

Histochemical localisation of FMRFamide-gated Na+ channels in Helisoma trivolvis and Helix aspersa neurones

FMRFamide-gated Na⁺ channels of molluscan neurones belong to the ENa/Deg family of channels which have diverse functions. FMRFamide (Phe-Met-Arg-Phe-NH₂) Na⁺ channels were detected electrophysiologically in specified neurones of Helix (Helix aspersa) and Helisoma (Helisoma trivolvis), and clones (FaNaCs) subsequently identified. We have now made a study to determine the distribution of mRNA for the clones HaFaNaC (Helix) and HtFaNaC (Helisoma) in the nervous systems of these species using standard in situ hybridization techniques. Immunohistochemical experiments were also made using an HtFaNaC antibody to detect the channel protein in Helisoma neurones. Many neurones in the central ganglia, including those which exhibit the FMRFamide Na⁺ current, stained for FaNaC-mRNA, suggesting a much wider distribution of the channel than was indicated by the earlier work. An immunoreactive response to the channel antibody was also observed in some Helisoma neurones, such as the giant dopamine neurone of the left pedal ganglion, also shown to possess HtFaNaC-mRNA and to exhibit the FMRFamide Na⁺ current. Taken together, these experiments suggest that the clones HaFaNaC and HtFaNaC are major, if not the only, subunits of the FMRFamide-gated Na⁺ channel detected electrophysiologically in the identified neurones of these species. However, fewer neurones in Helisoma reacted with the HtFaNaC-antibody than those which exhibited message for the channel. This discrepancy may be due to a difference in sensitivity of the two techniques, or because not all of the channel mRNA is normally expressed as a membrane protein.     

In vitro phagocytosis by amoebocytes from the haemolymph of Helix aspersa Muller: I. Evidence for opsonic factor(s) in serum

In vitro phagocytosis of foreign particles by amoebocytes from the haemolymph of Helix aspersa (Müller) was studied.

Results showed the presence of an opsonin in Helix serum. This opsonic effect of serum is due to the adsorption of serum component(s) on to the foreign particles. No phagocytosis occurs in the absence of these factors.

There was some degree of specificity of the opsonin for two different foreign particles: formalized yeast cells (Saccharomyces cerevisiae) and formalized sheep red blood cells.

     

Some functional properties of nonpolymerizable and polymerizable tropomyosin

The binding of 125I-labelled nonpolymerizable (brain or carboxypeptidase A-treated skeletal muscle) and polymerizable (intact skeletal muscle) tropomyosin to muscle F-actin was studied by ultracentrifugation under various conditions. The amount of nonpolymerizable tropomyosin bound to F-actin both in 0.1 M KCl and in 7 mM MgCl2 was much lower than that of the polymerizable one. In the presence of MgCl2 the amount of nonpolymerizable tropomyosin bound to F-actin approached saturation level. Under these conditions, however, the amount of skeletal muscle tropomyosin bound exceeded saturation, suggesting formation of both head-to-tail polymers and side-to-side aggregates. The latter seems to be responsible for the inhibition of acto-heavy meromyosin ATPase activity which is caused by skeletal muscle tropomyosin but not by nonpolymerizable tropomyosin. Nonpolymerizable tropomyosin can substitute for the rabbit skeletal muscle tropomyosin in the regulatory system operating in skeletal muscle. Inhibition of ATPase activity of acto-heavy meromyosin by nonpolymerizable tropomyosin in the presence of troponin and the absence of calcium ions is less than that obtained with polymerizable tropomyosin. The inhibition of ATPase activity is directly correlated with the extent of binding of nonpolymerizable tropomyosin to F-actin under the conditions of the ATPase assay.