Cloning,isolation, and IgE-binding properties of Helix aspersa (brown garden snail) tropomyosin

BACKGROUND: Gastropod consumption is quite frequent in the Mediterranean countries and cross-reactivities with crustaceans have been described, but the mechanism of this allergenic cross-reactivity has not been studied in detail. This study aimed to produce recombinant Helix aspersa (brown garden snail) tropomyosin and investigate its implication for cross-reactivity among invertebrates. METHODS: A tropomyosin-specific cDNA encoding H. aspersa tropomyosin was synthetized, and recombinant allergen was overexpressed in Escherichia coli as nonfusion protein. IgE-binding reactivity was studied by immunoblotting and immunoblot inhibition experiments with sera from snail-allergic patients. RESULTS: Cloned brown garden snail tropomyosin shares high homology with other edible mollusk tropomyosins (84-69% identity) as well as with those from arthropods (65-62%), and less homology with vertebrate ones (56% identity). Tropomyosin reacted with 18% of the sera from patients with snail allergy. Inhibition experiments, using natural and recombinant tropomyosins, showed different degrees of cross-reactivity between invertebrate tropomyosins. Sera from snail-allergic subjects recognized tropomyosins in both mollusks and crustacean extracts. CONCLUSIONS: Tropomyosin represents a minor allergen in snail extracts, but it is clearly involved in invertebrate cross-reactivity.     

The sensitivity of Helix aspersa neurones to injected calcium ions

1. When calcium chloride was injected into Helix aspersa neurones there was a fall in membrane resistance and the membrane potential became hyperpolarized.2. The reversal potential of the response was dependent on the concentration of potassium in the external solution.3. Injection of a calcium-EGTA buffer containing 9 x 10(-7)M free calcium reduced the membrane resistance by 25%. When calcium chloride was injected it was necessary to increase the total intracellular calcium concentration by about 10(-3)M to produce similar change of resistance.4. In sodium-free (Tris) solution there was a slow fall of membrane resistance as if the intracellular calcium concentration had increased. There was a similar resistance change in the presence of 2,4-dinitrophenol and iodoacetate.5. A series of repetitive depolarizing pulses produced a long lasting reduction in membrane resistance which was enhanced by 2,4-dinitrophenol and iodoacetate.6. It is concluded that (a) injection of calcium causes an increase in potassium permeability, (b) the injected calcium is rapidly pumped from the cytoplasm by a sodium-dependent mechanism and by mitochondria, and (c) 1-2 msec depolarizing pulses stimulate an influx of calcium. This influx is rapid enough to trigger potassium activation during an action potential.     

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).     

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.     

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.     

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.

     

The antagonistic actions of calcium and magnesium on the superfused ventricle of the snail Helix pomatia

1. The isolated, superfused half-ventricle of the snail (Helix pomatia) maintains a degree of tonic activity even when not beating, since the membrane is depolarized beyond the tension threshold. Beating may be initiated by lateral stretch of the ventricle and by 5-hydroxytryptamine.2. Ca and Mn each have a hyperpolarizing action, while K and to a lesser extent Na cause depolarization: the tonic activity of the ventricle is affected accordingly.3. An increase in the extracellular concentration of Mg also causes the tension to fall, but without change in membrane potential. It can also initiate beating.4. Contractures induced by 30 mM-K are steadily maintained, but start with a more-or-less distinct twitch-like contraction. A contracture induced by a concentration of K above about 50 mM is poorly sustained and is followed by a further brief contracture when the K concentration is reduced to normal.5. Relations between contracture tension and the concentrations of Ca and Mg accord with the hypothesis that the two ions compete for attachment to a binding site on the cell surface and that tension is proportional to the amount of bound Ca. On this hypothesis, the apparent dissociation constant of the Mg complex is 11.4 mM and that of the Ca complex 0.15 mM or less.6. This effect of Mg is like that of Na on frog ventricle and some of the differences in the behaviour of snail and frog ventricles are abolished by appropriate adjustment of the extracellular concentrations of these ions.     

MHC class II (I-A) region control of the IgE antibody repertoire to the ABA-1 allergen of the nematode Ascaris

ABA-1 is an approximately 14,000 molecular weight (MW) allergen which is among the most abundant proteins synthesized by the nematode parasite Ascaris. IgG and IgE responses to it are major histocompatibility complex (MHC)-restricted in rodents and have only been found to occur in rats of the RT1u haplotype and mice of the H-2s haplotype. Humans infected with the parasite vary substantially in their immune response to the allergen, but the genetic basis for this unknown. H-2 recombinant mice were used to identify the region within the MHC controlling antibody responses to the allergen. IgG antibody to immunoaffinity purified ABA-1 was assayed by radio-immunoassay and IgE by passive cutaneous anaphylaxis. This showed that the restriction element is the I-A molecule and that there was some evidence for I-E modulation of the level of response.     

The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia

The aim of this study was to show the presence, distribution and function of the pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors in the CNS and peripheral nervous system of the mollusk, Helix pomatia. PACAP-like and pituitary adenylate cyclase activating polypeptide receptor (PAC1-R)-like immunoreactivity was abundant both in the CNS and the peripheral nervous system of the snail. In addition several non-neuronal cells also revealed PACAP-like immunoreactivity. In inactive animals labeled cell bodies were mainly found and in the neuropile of active animals dense immunostained fiber system was additionally detected suggesting that expression of PACAP-like peptide was affected by the behavioral state of the animal. RIA measurements revealed the existence of both forms of PACAP in the CNS where the 27 amino acid form was found to be dominant. The concentration of PACAP27 was significantly higher in samples from active animals supporting the data obtained by immunohistochemistry. In Western blot experiments PACAP27 and PACAP38 antibodies specifically labeled protein band at 4.5 kDa both in rat and snail brain homogenates, and additionally an approximately 14 kDa band in snail. The 4.5 kDa protein corresponds to PACAP38 and the 14 kDa protein corresponds to the preproPACAP or to a PACAP-like peptide having larger molecular weight than mammalian PACAP38. In matrix-assisted laser desorption ionization time of flight (MALDI TOF) measurements fragments of PACAP38 were identified in brain samples suggesting the presence of a large molecular weight peptide in the snail. Applying antibodies developed against the PACAP receptor PAC1-R, immunopositive stained neurons and a dense network of fibers were identified in each of the ganglia. In electrophysiological experiments, extracellular application of PACAP27 and PACAP38 transiently depolarized or increased postsynaptic activity of neurons expressing PAC1-R. In several neurons PACAP elicited a long lasting hyperpolarization which was eliminated after 1.5 h continuous washing. Taken together, these results indicate that PACAP may have significant role in a wide range of basic physiological functions in snail.     

Identification of Dioscorea batatas (sanyak) allergen as an inhalant and oral allergen

Dioscorea batatas is widely used in Asia as a herbal medicine or food product with potential health benefits. There have been several reports of occupational asthma caused by inhalation of D. batatas dust. However, there has been no report of systemic allergic reactions after oral administration of D. batatas. Two patients with D. batatas allergy were enrolled. One had experienced severe urticaria and angioedema after indigestion, and the other had been exposed to D. batatas dust and was diagnosed as having occupational asthma. Both patients had high serum-specific IgE and IgG4 antibodies to D. batatas. And IgE immunoblot demonstrated that both sera bound to a 27-kDa protein with an IgE-binding motif, which was revealed by 2-D-electrophoresis to have the sequence Asn-Val-Glu-Asp-Glu-Phe-Ser-X-Ile- Glu-Gly-Asn-Pro-X-X-Pro-Glu-Asn-X-Gly (pI 6.40, 6.04). In conclusion, discorin from D. batatas (DB3S) was identified as the major allergen of D. batatas in patients sensitized via an oral or inhalant route.     

Inhaled latex allergen (Hev b 1)

BACKGROUND: IgE-mediated responses to natural rubber latex allergens have become a major health problem among recognized risk groups. OBJECTIVE: The purpose of this investigation was to measure the amounts of Hevea brasiliensis latex allergen (Hev b 1) inhaled and deposited on surfaces when latex or vinyl gloves were worn and compare the results with the conventional measures (breathing zone samplers) of occupational exposure. METHODS: Hev b 1 exposure was measured by nasal sampling and breathing zone sampling. Latex allergen exposure was generated by having each subject don a pair of powdered latex examination gloves and continuing his or her normal daily activity for 30 minutes. By means of adhesive tape, surface dust samples were collected from the surfaces of gloves, the subject's hands, and work areas. Sampling was performed with subjects wearing no gloves, subjects wearing powdered vinyl gloves, subjects wearing powdered latex gloves, and nearby colleagues wearing latex gloves. All samples were assayed through use of the HALOgen assay (Inhalix, Sydney, Australia) with a Hev b 1-specific mAb. Particles transporting latex allergen were identified by a surrounding immunostain halo, and these were quantified and reported as total numbers of particles inhaled, airborne, or found on surface areas evaluated. RESULTS: Study subjects inhaled 26 times more allergen when powdered latex gloves were worn than under the "no glove" and powdered vinyl glove conditions. During the same period, Hev b 1 particle levels measured in the ambient air through use of the breathing zone sampler increased by 24-fold. The median numbers of particles carrying Hev b 1 allergen per square centimeter on the surface of the hands after the wearing of latex and vinyl gloves were 1964 and 5, respectively. Latex allergen was physically associated both with cornstarch granules and with larger dust particles having a darker, more irregular appearance. CONCLUSION: In a laboratory where gloves are worn for protection, the use of latex gloves resulted in a 26-fold increase in inhaled latex allergen over background levels measured while vinyl gloves were worn as controls. Low levels of latex exposure also occurred when vinyl gloves or no gloves were worn; the reasons for this are under investigation.