Search

Abstract: ESI-MS can only be accepted as a quantification method when using standards with a high resemblance to the analyte(s). Unfortunately, this is usually not applicable to metallothioneins (MTs), a superfamily of singular metal-binding cysteine-rich proteins, present in all living organisms, since the absence of suitable reference material due to the high diversity among metal–MT species precludes their quantification by molecular mass spectrometry. Even thus, it is widely assumed that the intensities of the ESI-MS peaks of similar species are directly correlated with their relative concentration in the sample, and this has been extended to the determination of different MT proteins coexisting in a sample. Practically all organisms contain several MT isoforms, some of them exhibiting highly similar sequences, with conserved coordinating Cys residues. For the current analysis, we used as a model system the MT isoforms of two terrestrial snails (Helix pomatia and Cornu aspersum). Hence, distinct samples were prepared by mixing, at different molar ratios, the recombinant HpCuMT and HpCdMT isoforms from H. pomatia, or the recombinant CaCuMT, CaCdMT and CaCdCuMT isoforms from C. aspersum, and they were analyzed by ESI-MS both at neutral pH (for Zn-loaded MT forms) and at acidic pH (for the corresponding apo-forms). The results here presented reveal that the ESI-MS peak intensity of a single MT species strongly depends on its sensitivity to be ionized, and thus, on the presence or absence of metal ions bound. Furthermore, our data demonstrate that very similar MT isoforms of the same organism with similar pI (ranging from 7.9 to 8.3) can show a clear different sensitivity to ES ionization, something that cannot be readily predicted only by consideration of their amino acid content. In conclusion, even in this optimum case, deductions about quantity features of MT samples drawn from ESI-MS measurements should be carefully considered. [ABSTRACT FROM AUTHOR]

Farming potential of the most popular edible terrestrial snails, it was investigated within the context of efficient agricultural land use of for maxinnun animal protein production. The stress induced by climate changes spon the snail's development is a considering factor, therefore snails are tested for fitness. Sel-PlexTM is used as concentrated forage additive in both 2009and 2010 experiments involving Cornu aspersum (Helix aspersa Muller) and Helix pomatia edible terrestrial snails. Young snails of 4-7 days were tested for crawling speed as fitness component, an expression of vitality and resistance during prolonged heat waves and drought season. Using proper white led based cold lighting sources and high definition highly sensitive video recording equipment snail's activity has been recorded. Snails crawling on graded paper allowed for easuring their speed. Seeing thru the snail's shell due to proper light positioning, heartbeat was counted as well during the crawling. While the heartbeat rate of the snails is relatively high on all snail batches, respectively 97-102 bpm (bits per minute) due to the high temperature, the snails fed with Sel-PlexTM 0.10% have the highest speed, namely 1.5694±0.0504 mm/s followed by the snails fed with Sel-PlexTM 0.04%, averaging 1.4403±0.0323 mm/s respectively. The paper introduces for the first time the concept of Gain to the Proposed Norm" (GPN), presented as a development indicative, a progress indices towards meeting a predetermined goal target. The yield upon slaughter is a factor that piays a major roil as to recommend Cornu aspersum species for farming as opposed to its counterpart Helix pomatia. [ABSTRACT FROM AUTHOR]

Abstract: Terrestrial snails are able to transform themselves into inactivity ceasing their behavioral activity under unfavorable environmental conditions. In the present study, we report on the activity-dependent changes of the peptide and/or polypeptide profile in the brain and hemolymph of the snail, Helix pomatia, using MALDI TOF and quadrupole mass spectrometry. The present data indicate that the snails respond to low temperature by increasing or decreasing the output of selected peptides. Average mass spectra of the brain and hemolymph revealed numerous peaks predominantly present during the active state (19 and 10 peptides/polypeptides, respectively), while others were observed only during hibernation (11 and 13). However, there were peptides and/or polypeptides or their fragments present irrespective of the activity states (49 and 18). The intensity of fourteen peaks that correspond to previously identified neuropeptides varied in the brain of active snails compared to those of hibernating animals. Among those the intensity of eight peptides increased significantly in active animals while in hibernated animals the intensity of another six peptides increased significantly. A new peptide or peptide fragment at m/z 1110.7 was identified in a brain of the snail with the following suggested amino acid sequence: GSGASGSMPATTS. This peptide was found to be more abundant in active animals because the intensity of the peptide was significantly higher compared to hibernating animals. In summary, our results revealed substantial differences in the peptide/polypeptide profile of the brain and hemolymph of active and hibernating snails suggesting a possible contribution of peptides in the process of hibernation. [Copyright &y& Elsevier]

A novel sialic acid-specific lectin (MCsialec) was detected from an expressed sequenced tag (EST) sequence from Manila clam haemocytes infected with Perkinsus olseni. The cDNA of the lectin was cloned using gene-specific primers based on a previously determined EST and characterized. The full-length cDNA of MCsialec is 603 bp in length and encodes a polypeptide of 200 amino acids with a calculated molecular mass of 21.928 kDa. Sequence alignment and protein motif analyses showed that MCsialec shares identity with sialic acid-specific invertebrate lectins from Cepaea hortensis, Helix pomatia and Haliotis discus discus. The lectin was expressed in Escherichia coli M15 cells and purified using a Ni-NTA His-binding resin matrix for antibody production. The presence of the lectin in various tissues of Perkinsus-infected and uninfected Manila clams was analysed by both PCR and immunohistochemical localization assays. MCsialec was detected in each tissue of the clams; however, upon infection, the level of expression of the lectin increased in each tissue. Vibrio tapetis infection also induced high-level expression of MCsialec in the haemocytes. These data suggest that MCsialec plays a crucial role in the immune system of the Manila clam during pathogenic infection. [ABSTRACT FROM AUTHOR]

The kinetics of the hydrolysis of small aryl substrate p-nitrophenyl sulfate (p-NPS) catalyzed by arylsulfatase from Helix pomatia was studied at a wide range of temperatures as well as at ambient and elevated pressures. The employed kinetic assay techniques allow for the determination of activation entropy ( [image omitted]), activation enthalpy ( [image omitted]), as well as activation volume ( [image omitted]) which suggested associative interchange character of the sulfate ester hydrolysis. The pH dependence obtained for catalytic parameters suggests that two protonation/deprotonation reactions can occur and the optimum pH for the catalytic activity was also detected. [ABSTRACT FROM AUTHOR]

Abstract: The specific isoflavone composition of nutritional supplements is commonly not-labeled, although the stated amounts are strongly dependent on the present isoflavone conjugates. Hence, 11 soy-based dietary supplements were characterized via a newly established ultra performance liquid chromatography (UPLC™) method, on both their native conjugated isoflavone spectra, as well as on quantitative amounts derived as total aglycones after enzymatic hydrolysis utilizing Helix pomatia juice. Capitalizing on sub-2 μm particles, the established RP-UPLC™ technique facilitated efficient chromatographic separation of all 12 soy intrinsic isoflavone forms within 10min. Derived native isoflavone profiles implied a certain variability, comprising conjugated forms, especially glycosides, as the predominant isoflavonic constituents throughout the majority of supplements, whereas only two samples indicated the more bioavailable free aglycones as prevailing compounds. Moreover, the robust quantification as total aglycones subsequent to enzymatic hydrolysis, unexceptionally yielded negative deviations referring to the labeled specifications, thus implying that stated amounts were typically calculated on basis of the high molecular isoflavone conjugates. Thus, especially in regard to better comparability, regulations concerning an uniform labeling basis are needed. [Copyright &y& Elsevier]

In terrestrial pulmonate snails, cadmium (Cd) uptake leads to the induction of a Cd-specific metallothionein isoform (Cd-MT) that protects against adverse interactions of this toxic metal ion. Increasing concentrations of Cd cause increased individual mortality possibly linked to pathological alterations in the snail midgut gland. Histological, immuno-histochemical, and electron-microscopic methods in combination with tissue metal analyses and quantification of MT induction parameters were applied to the midgut gland of Cd-exposed Roman snails ( Helix pomatia). Conspicuous concentration-dependent alterations occurred in this organ, including the metal-induced increase of Cd-MT concentration and manifestation of Cd-MT mRNA precipitations in all midgut gland cell types. The most evident alteration was an increase of cellular turnover reflected by enhanced cell proliferation. Intensified vesiculation of endoplasmic reticulum was noted in basophilic cells and an increasing formation of lipofuscin granules in excretory cells. At the highest Cd concentrations, mitochondrial membranes were disrupted in basophilic cells, and lipofuscin granules were released from excretory cells into the midgut gland tubular system. Some of these alterations (e.g., increased cell proliferation rate, vesiculation of endoplasmic reticulum) detected at low Cd concentrations were interpreted as adaptive response processes enhancing the tolerance of exposed individuals to metal stress. Cellular alterations at higher Cd concentrations (e.g., mitochondrial structural damage) clearly represented ongoing irreversible cellular disruption. Combined evaluation of cellular biomarkers and MT saturation levels indicated that the transition from stress resistance to depletion of resistance capacity occurred above a threshold of 0.8 µmol Cd/g dry weight in the midgut gland of H. pomatia. At these Cd concentrations, Cd-MT was saturated with Cd2+ ions, whereas at the cellular level, structural alterations turned into pathological deterioration. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, the ability of a range of endogenous neuropeptides to modulate neuromuscular transmission was examined in the salivary duct neuromuscular preparation of the terrestrial snail, Helix pomatia. Immunohistochemical and physiological techniques were used to localize the neuropeptides (GSPYFVamide, CARP, FMRFamide and APGWamide) and to investigate whether contractions elicited by the stimulation of the salivary nerve or by exogenously applied 5-HT are subject to peptidergic modulation. All of the neuropeptides studied decreased the tonus by a direct action on the muscle fibers in a concentration dependent manner in a range of 10−9 to 10−6 M. Neuropeptides distinctly affected the 5-HT evoked contraction or relaxation and GSPYFVa and APGWa decreased also the amplitude of contractions elicited by the stimulation of the salivary nerve. All four neuropeptides facilitated the relaxation phase providing further evidence for the postsynaptic action of neuropeptides. Low Ca2+/high Mg2+ saline abolished the nerve-elicited contractions, however the denervated muscle retained the ability to contract due to the mobilization of the Ca2+ from intracellular stores. It was concluded, that peptides belonging to different peptide families exerted their effects through pre- and postsynaptic mechanisms. The modulatory effect of neuropeptides can be assigned to the partial co-localization of 5-HT and neuropeptides in the nerves innervating muscles of the salivary duct, as it was demonstrated by double-labeling immunohistochemistry. A double origin of the 5-HTergic innervation was demonstrated, including efferents originating from both the cerebral and visceral ganglia. [Copyright &y& Elsevier]

Abstract: Glycosyltransferases from the albumen gland of Helix pomatia could be used in tandem mode for the chemoenzymatic synthesis of β,1-3/β,1-6-linked oligogalactans. By employing recombinant trans-sialidase of Trypanosoma cruzi (TcTS) the formation of a range of modified Galβ,1-3GalNAc derivatives could be terminally α,2-3 sialylated. Biacore studies indicated the binding of these modified trisaccharides to myelin-associated glycoprotein (MAG). Using an eight-step synthetic route N-acyl-modified sialyl donor structures could be obtained. TcTS was used to transfer these structures to an isolactoside, and Michaelis constants of the donors indicated the kind and size of modifications allowed at the 5-nitrogen site. A number of sialic acid C-glycosides could be obtained via the C-allyl sialoside and subsequent metathesis. Biacore measurements showed derivatives substituted with aromatic residues to give KD values in the mM range. Benzaldehyde-functionalized glycosides of mono and disaccharides were synthesized by metathesis and could be used for the formation of novel glyco-self assembled monolayers (glyco-SAMs) employing various tether structures and attached to gold surfaces. Initial experiments were performed with concanavalin A and manno-SAMs. By atomic force microscopic measurements of tethered glycosides attached to gold-coated tips and surfaces weak forces in the nN range could be detected. Structure activity correlation of forces suggested rationales for complex interactions of various glycosides including minor stereochemical variations. [Copyright &y& Elsevier]

The article offers research information on transcription patterns of Cd-specific metallothionein isoform (Cd-MT) expression in Helix pomatia and Cantareus aspersus through experimentation, gene assessment and transient evaluation. It presents the implication and role of the distal metal responsive elements (MRE) in the inhibitory action by the Helix pomatia. It focuses on several ways using cadmium exposure in two helicid snails in metal transcription factor-2 evaluation,and the implication of the promoter oganization and the Cd-metallothionein genes transcription.

Abstract: Estivation enables land snails to survive a prolonged dryness but the return to active state imposes conditions of oxidative stress on internal organs due to a transient large increase in oxygen consumption, which augments mitochondrial production of reactive oxygen species. Therefore, activities of antioxidant enzymes, concentrations of reduced glutathione (GSH) and TBARS as an index of lipid peroxidation, were evaluated in Helix pomatia snails (i) during summer activity, (ii) during estivation, which was induced experimentally, (iii) at the start of arousal from estivation, and (iv) being aroused for 24 h. Estivation induced significant decreases in activity of catalase in the kidney and hepatopancreas and glutathione peroxidase in the kidney. Activity of glutathione reductase was unaffected by estivation/arousal cycle. Summer-active and estivating snails maintained high activity of glutathione transferase. Concentration of GSH was organ-dependent and was positively affected by estivation. Lack of increase in TBARS concentration during estivation/arousal cycle suggests that antioxidant defence system of H. pomatia snails is highly efficacious. In conclusion, permanent maintenance of relatively high activities of the antioxidant enzymes and the high concentration of GSH in H. pomatia snails indicate that they have well-developed strategy of defence against oxidative injury. [Copyright &y& Elsevier]

Being much appreciated all over the world for their high nutritional values, escargots or terrestrial snails are farmed in many countries. Within the last few years, snail fanning started to become a very popular activity in Romania too. It represents an ecological type of agricultural activity that can also be certified as biological, organic farming if the soil's conditions and the technology are as such. Extensive amount of research offers details on the physiology of the edible snail species as well as regarding their biochemical content and their nutritional value. No much research reflects snail's ability to bioaccumulate selenium. In the same time, a lot of research demonstrated the snail's ability to accumulate contaminants such as heavy metals. Using fluorimetric analysis, we investigated selenium accumulation in meat and shell of edible terrestrial snails Helix pomatia and Eobania vermiculata Muller gathered from different regions of Moldova Republic, Ukraine and Russia. The meat selenium concentration in terrestrial snails reflects the ability of those invertebrates to accumulate high selenium contents. Based on the intake recommendations, snails can be a very good source of selenium for human consumption. Depending upon the soil mineral content and level of contamination, the snails will accumulate large quantities of heavy metals such as lead and cadmium, therefore making the snails a very high-risk food product. Therefore, it is very important a careful selection of the agricultural land designated for snail farming. Additives containing selenium may be a way to supplement snail's diet. [ABSTRACT FROM AUTHOR]

Abstract: In this study we examined changes in a persistent sodium current (I NaP) after behavioral aversive classical conditioning in the snail Helix pomatia. We trained animals by pairing food with a mild electric shock that triggered the whole-body withdrawal reflex. This aversive training resulted in transcription dependent long-term associative memory. Isolated central nervous system preparations were set up from trained, random control and naive animals and using two-electrode voltage clamp methods, I NaP was activated and measured in identified body withdrawal interneurons RPa3 and LPa3. We show here that in preparations from conditioned animals I NaP is increased, suggesting that modifications in intrinsic cellular properties contribute to the formation of the memory trace. Blocking RNA synthesis by systemic injection of actinomycin D (0.12μM) suppressed both memory consolidation in intact animals and the learning-induced increase of I NaP in withdrawal interneurons, suggesting that aversive classical conditioning affects sodium channel expression at the transcriptional level. [Copyright &y& Elsevier]

Arousal of land snails from torpor is inseparably connected with an increase in oxygen consumption leading to oxidative stress. Therefore, activity of antioxidant defence system (antioxidant enzymes and reduced glutathione) and degree of oxidative damage (concentration of malondialdehyde as an index of lipid peroxidation) in the snail Helix pomatia L., 1758 were tested to check whether torpid snails are able to activate their antioxidative defence against oxidative damage prior to arousal from winter torpor. Snails, which were collected from their natural habitats, were tested at the beginning, in the middle part, and at the end of winter torpor. Active snails collected in autumn and spring were taken as control groups. Snails were immediately killed and their foot, hepatopancreas, and kidney were used for the biochemical assays. Winter torpor induced significant changes in activities of the crucial antioxidant substances. The lowest activities were observed at the beginning of torpor, whereas activity of some of these enzymes was significantly enhanced prior to spring arousal. Reduced glutathione concentration did not show time-dependent changes during winter torpor. MDA (1,3-propanedial) level was elevated in the kidney and foot but was unchanged in the hepatopancreas. In conclusion, the snail H. pomatia is able to maintain REDOX balance necessary to prevent oxidative injury during arousal. [ABSTRACT FROM AUTHOR]

Metallothioneins (MTs) are a family of multifunctional proteins involved, among others, in stress response. The Cadmium (Cd)-MT gene of the Roman snail ( Helix pomatia), for example, encodes for a protein induced upon cadmium exposure. While our previous studies have demonstrated that the expressed Cd-MT isoform of Roman snails assists detoxification of cadmium, the present work focuses on the potential plasticity of this gene in response to a variety of environmental stressors playing a crucial role in the specific ecological niche of H. pomatia. Our hypothesis is based on a bioinformatic approach involving gene sequencing, structural and in silico analysis of transcription factor binding sites (TFBs), and a comparison of these features with other MT genes. Our results show that the Roman snail's Cd-MT gene not only is the largest known MT gene, but also contains ¿ apart from the regulatory promoter region ¿ several intronic repeat cassettes of putative TFBs suggested to be involved in environmental stress response, immune competence, and regulation of gene expression. Moreover, intronic scaffold/matrix attachment regions (S/MARs) and stress-induced duplex destabilization sites confer a high potential for epigenetic gene regulation. This suggested regulatory plasticity is also supported by physiological data showing that Cd-MT in Roman snails can be induced differentially not only after cadmium exposure, but also in response to nonmetallic environmental stressors. It is concluded that structural analysis combined with bioinformatic screening may constitute valuable tools for predicting the potential for plasticity and niche-specific adaptation of stress-responsive genes in populations living under rapidly changing environmental conditions. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh) is one of the main signals regulating nitric oxide synthase (NOS) expression and nitric oxide (NO) biosynthesis in mammals. However, few comparative studies have been performed on the role of ACh on NOS activity in non-mammalian animals. We have therefore studied the cholinergic control of NOS in the snail Helix pomatia and compared the effects of ACh on NO synthesis in the enteric nervous system of the snail and rat. Analyses by the NADPH-diaphorase reaction, immunocytochemistry, purification with ion-exchange chromatography, Western-blot, and quantitative polymerase chain reaction have revealed the expression of neuronal NOS in the rat intestine and of a 60-kDa subunit of NOS in the enteric nerve plexus of H. pomatia. In H. pomatia, quantification of the NO-derived nitrite ions has established that NO formation is confined to the NOS-containing midintestine. Nitrite production can be elevated by L-arginine but inhibited by Nω-nitro-L-arginine. In rats, ACh moderately elevates nitrite production, whereas ACh, the nicotinic receptor agonists (nicotine, acetyl thiocholine iodide, metacholine) and the cholinesterase inhibitor eserine reduce enteric nitrite formation in snails. The nicotinic receptor antagonist tubocurarine also provokes nitrite liberation, whereas the muscarinic receptor agonists or antagonists have no significant effect in snails. In the presence of EDTA or tetrodotoxin, ACh fails to inhibit nitrite production. In pharmacological studies, we have found that ACh contracts the midintestinal muscles and, in snails, simultaneously reduces the antagonistic muscle relaxant effect of L-arginine. Our experiments provide the first evidence for an inhibitory regulation of neuronal NO synthesis by ACh in an invertebrate species. [ABSTRACT FROM AUTHOR]

Abstract: By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between α-macroglobulin (αM) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: αM (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: αD-HpH, αN-HpH and β-HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation. (i) With β-HpH, which is richer in carbohydrates than αD-HpH and αN-HpH, mainly due to a higher 3-O-methyl-d-galactose content, the cross-reaction with HpαM was highest. (ii) From the 8 functional units, designated a–h, isolated from β-HpH, two that lack carbohydrates (c and f) were not recognized by antibodies against HpαM, while the six glycosylated ones were strongly cross-reacting. The nearly complete loss of the cross-reactivity upon deglycosylation of functional units d and g and the inhibition in competitive ELISA experiments by glycopeptides isolated from both β-HpH and HpαM are further evidence that glycans are involved in the immunological relationship between HpH and HpαM. Carbohydrate analyses indicated that the glycan structures present on HpαM are very similar (or identical) to those found on HpH, suggesting that glycans are common epitopes on both proteins. Especially d-xylose and 3-O-methyl-d-galactose seem to be responsible for the cross-reactivity since the α-macroglobulin and hemocyanin of the cephalopod Sepia officinalis, which lack these two monosaccharides in their glycan structures, do not immunologically cross-react. [Copyright &y& Elsevier]

The substrate specificity of snail (Helix pomatia and Helix aspersa), limpet (Patella vulgata), and bovine glucuronidases was examined by using p-nitrophenyl glucuronide (GlcA-O-pNP) and p-nitrophenyl 6-O-sulfo-β-D-glycopyranosides as the glycosyl donor and acceptors, respectively. When the donor was treated with these enzymes in the absence of the acceptors, β (1 → 3) glucuronyl disaccharides were obtained as the major products together with β (1 → 2) isomers as the result of an enzymatic “self-transglycosylation” reaction. When p-nitrophenyl 6-O-sulfo-β-D-glucopyranosides (6-O-sulfo-Glc-O-pNP and 6-O-sulfo-Glc-S-pNP) were applied as acceptor substrates, every glucuronidase transferred the GlcA residue to either the O-3 or O-2 position in 6-O-sulfo-Glc to yield a mixture of GlcAβ (1 → 3)- and GlcAβ (1 → 2)-linked disaccharides in a ratio of 12:1 ∼ 1:1. On the other hand, when p-nitrophenyl 6-O-sulfo-β-D-galactopyranosides (6-O-sulfo-Gal-O-pNP and 6-O-sulfo-Gal-S-pNP) were applied, limpet and bovine glucuronidases gave a GlcAβ (1 → 3)-linked disaccharide regioselectively, while the snail enzymes showed no reactivity. [ABSTRACT FROM AUTHOR]

Abstract  Despite frequent reports on the presence of Blastocystis hominis in human intestinal tract, its pathogenicity remains a matter of intense debate. These discrepancies may be due to the varying pathogenic potential or virulence of the isolates studied. The present study represents the first to investigate both phenotypic and genotypic characteristics of B. hominis obtained from symptomatic and asymptomatic individuals. Symptomatic isolates had a significantly greater size range and lower growth rate in Jones’ medium than asymptomatic isolates. The parasite cells of symptomatic isolates exhibited rougher surface topography and greater binding affinity to Canavalia ensiformis (ConA) and Helix pomatia (HPA). The present study also identifies further phenotypic characteristics, which aided in differentiating the pathogenic forms from the non-pathogenic forms of B. hominis. Blastocystis subtype 3 was found to be correlated well with the disease. [ABSTRACT FROM AUTHOR]

Abstract: The effects of 2.7 mT and 10 mT static magnetic fields were investigated on two identified neurons with different bioelectric properties of the snail Helix pomatia. Membrane resting potential, amplitude, spiking frequency, and duration of action potential were measured. The two neurons of H. pomatia, parabolic burster Br and silent N1, showed different responses to a static magnetic field. The magnetic field of 2.7 mT intensity caused changes in the amplitude and duration of action potential of the Br neuron, whereas the 10 mT magnetic field changed the resting potential, amplitude spike, firing frequency, and duration of action potential of the Br neuron. Bioelectric parameters measured on the N1 neuron did not change significantly in these magnetic fields. [Copyright &y& Elsevier]

The aim of present work is to explore the influence of extremely low-frequency electromagnetic fields (8.34 and 217 Hz) utilized in cell phones on habituation of the mollusk single neuron to intracellular stimuli. The isolated nervous system of the mollusk Helix Pomatia was used in the experiments. Helmholtz coils were used to expose brain ganglia to the low-frequency electromagnetic fields. Peak values of the extremely low-frequency fields were between 1 and 6 mT. Neuron electrophysiology was investigated using a standard microelectrode technique. Exposure of the neuron to the low-frequency electromagnetic fields caused dehabituation to intracellular stimulus. The effect was proportional to the magnetic induction peak value. The observed dehabituation occurs by degradation of the signal to noise ratio and by alteration of the neuron's normal function. [ABSTRACT FROM AUTHOR]

The mechanisms of cAMP action on the contractility of the isolated heart were studied in the snail Helix pomatia. Serotonin is a powerful activator of heart contractility in this animal. Preincubation of the isolated heart ventricle with the activator of protein kinase A (PKA) Sp-8-bromoadenosine-3′,5′-cyclic monophosphothioate (200 μM) or the activator of Epac proteins 8-(4-chlorophenylthio)-2′- O-methyladenosine-3′,5′-cyclic monophosphate (100 μM) proved to enhance the amplitude of contractions induced by serotonin. Two types of changes in the contractile response were observed: each agent caused either a uniform increase in the amplitude of heart contractions at all concentrations of serotonin or an abrupt increase in the response to the first minimum dose of serotonin. These results indicate that Epac proteins along with PKA are involved in the transmission of cAMP effect on heart contractility. [ABSTRACT FROM AUTHOR]

We consider a Hodgkin–Huxley-type model of oscillatory activity in neurons of the snail Helix pomatia. This model has a distinctive feature: It demonstrates multistability in oscillatory and silent modes that is typical for the thalamocortical neurons. A single neuron cell can demonstrate a variety of oscillatory activity: Regular and chaotic spiking and bursting behavior. We study collective phenomena in small and large arrays of nonidentical cells coupled by models of electrical and chemical synapses. Two single elements coupled by electrical coupling show different types of synchronous behavior, in particular in-phase and antiphase synchronous regimes. In an ensemble of three inhibitory synaptically coupled elements, the phenomenon of sequential synchronous dynamics is observed. We study the synchronization phenomena in the chain of nonidentical neurons at different oscillatory behavior coupled with electrical and chemical synapses. Various regimes of phase synchronization are observed: (i) Synchronous regular and chaotic spiking; (ii) synchronous regular and chaotic bursting; and (iii) synchronous regular and chaotic bursting with different numbers of spikes inside the bursts. We detect and study the effect of collective synchronous burst generation due to the cluster formation and the oscillatory death. [ABSTRACT FROM AUTHOR]

Abstract: 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 ∼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. [Copyright &y& Elsevier]

Abstract: Helix pomatia (Hp) juice is a common enzymatic preparation for deconjugation of urinary steroids. It has been used in many published studies on urinary testosterone (T) in chimpanzees and bonobos, although the ability of Hp juice to convert other urinary steroids into T has been reported for human urine. We developed a protocol for determination of reliable T levels in primate urine using liquid chromatography–mass spectrometry. T levels were determined in a set of human, bonobo and chimpanzee urine samples (A) by measurement of intact testosterone glucuronide (TG) and testosterone sulfate (TS), (B) after hydrolysis/solvolysis with β-glucuronidase from Hp and (C) from Escherichia coli. When samples were hydrolyzed with Hp juice, results were not correlated with the direct assay of TG and TS, and determined T concentrations were considerably higher. By contrast, hydrolysis with E. coli β-glucuronidase yielded a good agreement of T concentrations. We demonstrated the ability of Hp juice to convert androst-5-ene-3β, 17β-diol (A5diol) into T using commercial standards and within the urine of all three species. As A5diol usually is present at higher levels in urine than T, this artifact leads to erroneous results for T concentrations in primate urine. The proportion of T excreted as sulfate (TS) is often neglected as TS can only be cleaved by additional solvolysis. In all three species, we found substantial amounts of TS in the urine of some subjects and a high variance of TS proportion between and within subjects. Therefore the inclusion of solvolysis into the sample preparation seems necessary. [Copyright &y& Elsevier]

The thermal stability of the eight functional units of β-hemocyanin of the gastropodan mollusc Helix pomatia was investigated by FTIR spectroscopy. Molluscan hemocyanin functional units have a molecular mass of approximately 50 kDa and generally contain three disulfide bridges: two in the mainly α-helical N-terminal domain and one in the C-terminal β-sheet domain. They show more than 50% sequence homology and it is assumed that they adopt a similar conformation. However, the functional units of H. pomatiaβ-hemocyanin, designated HpH -a to HpH -h, differ considerably in their carbohydrate content (0–18 wt%). Most functional units are exceptionally stable with a melting temperature in the range 77–83 °C. Two functional units, HpH -b and HpH -c, however, have a reduced stability with melting temperature values of 73 °C and 64 °C, respectively. Although the most glycosylated functional unit ( HpH -g) has the highest temperature stability, there is no linear correlation between the degree of glycosylation of the functional units and the unfolding temperature. This is ascribed to variations in secondary structure as well as in glycan attachment sites. Moreover, the disulfide bonds might play an important role in the conformational stability of the functional units. Sequence comparison of molluscan hemocyanins suggests that the less stable functional units, HpH -b and HpH -c, similar to most of their paralogous counterparts, lack the disulfide bond in the C-terminal domain. [ABSTRACT FROM AUTHOR]

Abstract: Urinary steroid profile analysis requires enzymatic hydrolysis of glucuronide and sulfate conjugates and this is achieved simultaneously using Helix pomatia juice (HPJ), but steroids with 3β-hydroxy-5-ene structure undergo transformation and yield of 5α-reduced corticosteroid glucuronides is poor. We describe the use of sodium ascorbate to solve these problems and provide a basis for its mode of action. Steroid conjugates were extracted from urine, hydrolyzed in acetate buffer with HPJ and sodium ascorbate and analyzed as methyloxime–trimethylsilylether derivatives by gas chromatography–mass spectrometry. Ranges of temperature, pH and ascorbate, substrate and HPJ concentrations were compared for urine and pure standards. Activity of other antioxidants and that of bacterial cholesterol oxidase were examined. Helix pomatia enzyme preparations from different commercial sources were compared. Loss of 3β-hydroxy-5-ene steroids was enzyme-dependant, since it required HPJ, was saturable, subject to substrate competition and heat-inactivated. Products were 3-oxo-4-ene steroids and 4,6-diene and 6-oxy derivatives of these but the latter were not formed from 3-oxo-4-ene precursors. Ascorbate, other antioxidants or oxygen exclusion diminished activity. These characteristics were shared by cholesterol oxidase. Yield of 5α-reduced steroids was diminished by pre-incubation of HPJ before ascorbate addition and this was reversed if ascorbate was added to the pre-incubation mixture. We conclude that transformation of 3β-hydroxy-5-ene steroids by HPJ is due to cholesterol oxidase and is diminished by antioxidants or oxygen denial. Yield of 5α-reduced steroids is low due to oxidative damage of β-glucuronidase during hydrolysis, prevented by ascorbate. These features are shared by most commercial Helix pomatia enzyme preparations tested. [Copyright &y& Elsevier]

Osmotic stimulation activates both estivated and inactivated specimens of Helix pomatia and increases their central arousal. High-pressure liquid chromatography has shown that, during activation, the level of both serotonin and dopamine decreases in the central nervous system (CNS) but increases in the foot and heart, organs that are involved in the eversion of the body. In isolated CNS from activated animals, the firing frequency of the heart-modulator serotonergic (RPas) neurons is significantly higher than that in the CNS of estivated or inactivated animals. These neurons innervate both the heart and the anterior aorta. In semi-intact preparations, distilled water (an osmotic stimulus) applied to the mantle collar increases their firing frequency, whereas tactile stimulation evokes their inhibition. Extracellularly applied monoamines mimic the effect of peripheral stimuli: serotonin (0.1–10 μM) increases the activity of the RPas neurons, whereas dopamine (0.1–10 μM) inhibits their activity. Tyrosine-hydroxylase immunocytochemistry and retrograde neurobiotin tracing have revealed similar bipolar receptor cells in the mantle collar and tail, organs that are exposed to environmental stimuli in estivated animals. Serotonin immunocytochemistry carried out on the same tissues does not visualize receptor cells but labels a dense network of fibers that appear to innervate neurobiotin-labeled receptor cells. The combination of neurobiotin-labeling of RPas neurons and immunolabeling suggests that RPas neurons receive direct dopaminergic inputs from receptor cells and serotonergic inputs from central serotonergic neurons, indicating that central serotonergic neurons are interconnected. Thus, the RPas neurons may belong to neuronal elements of the arousal system. [ABSTRACT FROM AUTHOR]

The effects of 4 Hz extremely low frequency Electromagnetic Fields (ELF EMF), Infrasound (IS) and hydrogen peroxide (H2O2) on Na+–K+ pump-induced transient inhibition of heart muscle contractility was studied. It was documented that EMF-treated and H2O2-containing physiological solution had synergic depressing effect on Na+–K+ pump-induced inhibition of muscle contractility, while the IS had elevation effect on it. On the basis of the obtained data the H2O2 could be suggested as the messenger through which the stimulation effect of EMF on heart muscle is realised, while the relaxing effect of IS on heart contractility is due to the decrease of CO2 solubility of muscle bathing aqueous medium. [ABSTRACT FROM AUTHOR]

Abstract: Electrophysiological experiments were performed on in vitro neuronal preparations from terrestrial snail and rat brain slices, to determine the effect of the insecticide bensultap. Although bensultap has low toxicity in mammals, our results showed that bensultap altered the synaptic transmission in the vertebrate as well as in the invertebrate central nervous system. Bensultap caused a significant decrease of the ACh-induced current. The effect depended on the preapplication time and the concentration of the chemical. Bensultap also had an effect on the kinetic parameters of the ACh-induced current; the desensitization time was altered in a concentration-dependent manner. In the rat brain slice preparations, we observed an increase in the amplitude of the evoked responses after a 30min treatment. There was no effect on paired-pulse depression, but LTP-induction was significantly inhibited by bensultap. The efficacy of synaptic transmission was modified by bensultap through effects on both input integration and output organization. [Copyright &y& Elsevier]

The detection of Listeria monocytogenes in Helix pomatia, both raw and cooked is presented here. Polymerase chain reaction (PCR) is a rapid procedure with both sensitivity and specificity for quick detection and identification of Listeria monocytogenes from raw and cooked Helix pomatia. A total of 30 bags (10 g each) of H. pomatia samples were investigated for the Listeria monocytogenes inlB gene with the PCR method. PCR amplification products demonstrated that 18 of the 30 samples showed positive reactions to Listeria monocytogenesin the PCR. All PCR positive samples showed specific amplification of the 343 bp fragment for Listeria monocytogenes. [ABSTRACT FROM AUTHOR]

Abstract: Distinguishing features of biological constructions are high stability and adaptation to their environment. Beside biocompatibility, nontoxicity and degradability these characteristics are demanded for new biomaterials in the field of tissue engineering. This study investigated the chemical composition, the organization and the in vitro osteoconductive potential of the terrestrial gastropod shell (Helix pomatia) on CAL72 and human osteoblast-like cells. Chemical composition of the biomaterial was examined by X-ray diffraction (XRD) and scanning electron microscopy (SEM) was performed to analyze the architecture of the snail shell and the morphology of the seeded cells. A double staining procedure (FDA/PI) and a proliferation test (EZ4U) assessed the viability of the cells. Microscopical images showed the multilayered architecture of the aragonite shell with hexagonal crystals on the inner side. The cells spread well on the biomaterial and the highest proliferation rate could be measured with CAL72 cells on the inner shell surface. The osteoconductive effects of this natural biomaterial could encourage further experiments in the field of tissue engineering. [Copyright &y& Elsevier]

Abstract: The phenoloxidase (PO) activity of the hemocyanins (Hcs) from two molluscan species, the gastropod Helix pomatia (Hp) and the cephalopod Sepia officinalis (So), was studied. With catechol as substrate the Hcs showed a weak o-diPO activity, which was moderately enhanced on limited proteolysis with subtilisin. The sites in the Hc molecules mainly responsible for this activity were identified. The highest intrinsic o-diPO activity and also by far the highest level of induction were found in the functional units (FUs) Hp f and So g, isolated from Hp β-Hc and So Hc (subunit 2), respectively. The results thus support the earlier conclusion, made on the basis of sequence homology between molluscan Hcs, that Hp f and So g are functional and structural analogues. The subtilisin treatment of Hp f also induced monoPO activity, considered to be at the origin of browning of the sample. [Copyright &y& Elsevier]

Cytoplasmic loop 4–5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518–1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606–12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4–5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested. [ABSTRACT FROM AUTHOR]

Helix pomatia L., the Roman snail, is a species faced with growing commercial interest in Moldova. Its life history characteristics (slow maturation and recruitment, high mortality among juveniles and low fecundity) along with its strong spatial aggregation, makes it especially vulnerable to exploitation. In this study, differences in density, shell size and age distribution were assessed in 7 unexploited and 10 exploited sites in the northern and central parts of Moldova. A significant impact of exploitation on snail population densities, shell size of adult snails and age distribution was revealed. Exploited sites had much lower densities than unexploited ones and in two places no live snails were found. This may suggest that exploitation is currently carried out at an unsustainable level, but additional information on the demography of populations and intensity of exploitation is required in order to make inferences regarding sustainability and long-term population management. There was a higher proportion of adult snails in exploited sites than in nonexploited, because of the collection strategies: not only adults, but also all other age groups are gathered. Bigger adult shell size in exploited sites may be related to lower population density, but further study is required to confirm this. Establishing of well-organized population monitoring systems and development of snail breeding enterprises are proposed in order to conserve the species in Moldova. [ABSTRACT FROM AUTHOR]

The article identifies the isoflavones in rat plasma using liquid chromatography/mass spectrometry combined with photodiode array detection. The rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia that contains glucuronidase and sulfatase to convert the isoflavone ß-glycosides daidzein, genistein, and glycitein to their active aglycone forms.

Abstract: The β-hemocyanin (β-HpH) is one of the three dioxygen-binding proteins found freely dissolved in the hemolymph of the gastropodan mollusc Helix pomatia. The didecameric molecule (molecular mass 9 MDa) is built up of only one type of subunits. The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Upon excitation of the hemocyanins at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper–dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-β-HpH. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-form. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the β-HpH and its subunits exist in different conformations. The thermal stability of the oxy- and apo-β-HpH is characterized by a transition temperature (T m) of 84 °C and 63 °C, respectively, obtained by differential scanning calorimetry. Increase of the temperature influences the active site at lower temperatures than the environments of tryptophans and tyrosines causing a loss of oxygen bound to the copper atoms. This process is, at least partially, reversible as after cooling of the protein samples, around 60% reinstatement of the copper-peroxide band has been observed. The results confirm the role of the copper–dioxygen complex for the stabilization of the hemocyanin structure in solution. The other important stabilizing factor is oligomerization of the hemocyanin molecule. [Copyright &y& Elsevier]

Abstract: Phytoestrogens occur in a variety of foods and are thought to offer a protective effect against a number of complex diseases. Due to the diversity of phytoestrogen conjugates formed in the human body, most assays include an enzymatic hydrolysis step prior to analysis. β-Glucuronidase from Helix pomatia, which also contains sulfatase activity, is popular for this task but contains appreciable levels of some phytoestrogens and related compounds, which could affect accurate quantification at low concentrations. Use of solid phase extraction on a polymeric resin has been found to remove the majority of these compounds from the enzyme, without affecting the enzyme activity for almost all of the analytes tested. [Copyright &y& Elsevier]

Abstract: This study has been conducted to determine the major contamination sources during frozen snail meat processing. Seventeen different control points and/or sample types (live snail, and snail meats after steaming, after shell removal, after first boiling, after gutting, after second boiling, after packaging and as frozen snail meat; air samples from gutting room, boiling room and packaging room; samples from gutting counter tops, package surfaces, scissors used during processing, forks used during processing, personnel hands, and potable water) have been examined for the enumeration of total aerobic mesophilic bacteria, coliforms, Escherichia coli, Enterobacteriaceae, coagulase positive staphylococci, Salmonella spp., Listeria spp., and yeast and molds. From the control points examined, raw material and environmental air were found as the primary contamination sources. Personnel hands and equipment used were determined as the secondary contamination sources. Second boiling and freezing stages during processing were determined to reduce the overall contamination rate, and therefore had positive effects on the microbiological quality of the final product. Programs approving the acceptance of snails only with low initial microbial counts to the plant, giving emphasis to processing in proper hygiene conditions with sufficient sanitary applications is strongly recommended. [Copyright &y& Elsevier]