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PubMed1996

Effects of injecting calcium-buffer solution on [Ca2+]i in voltage-clamped snail neurons.

We have investigated why fura-2 and Ca(2+)-sensitive microelectrodes report different values for the intracellular free calcium ion concentration ([Ca(2+)]i or its negative log, pCa(i)) of snail neurons voltage-clamped to -50 or -60 mV. Both techniques were initially calibrated in vitro, using calcium calibration solutions that had ionic concentrations similar to those of snail neuron cytoplasm. Pressure injections of the same solutions at resting and elevated [Ca(2+)]i were used to calibrate both methods in vivo. In fura-2-loaded cells these pressure injections generated changes in [Ca(2+)]i that agreed well with those expected from the in vitro calibration. Thus, using fura-2 calibrated in vitro, the average resting [Ca(2+)]i was found to be 38 nM (pCa(i) 7.42 +/- 0.05). With Ca(2+)-sensitive microelectrodes, the first injection of calibration solutions always caused a negative shift in the recorded microelectrode potential, as if the injection lowered [Ca2+]i. No such effects were seen on the fura-2 ratio. When calibrated in vivo the Ca(2+)-sensitive microelectrode gave an average resting [Ca2+]i of approximately 25 nM (pCa(i) 7.6 +/- 0.1), much lower than when calibrated in vitro. We conclude that [Ca(2+)]i in snail neurons is approximately 40 nM and that Ca(2+)-sensitive microelectrodes usually cause a leak at the point of insertion. The effects of the leak were minimized by injection of a mobile calcium buffer.

Animals↗

PubMed1998

Evidence for the involvement of nitric oxide and nitric oxide synthase in the modulation of opioid-induced antinociception and the inhibitory effects of exposure to 60-Hz magnetic fields in the land snail.

The attenuation of opioid peptide-mediated antinociception is a well-established effect of extremely low frequency (ELF) electromagnetic fields with alterations in calcium channel function and/or calcium ion flux and protein kinase C activity being implicated in the mediation of these effects. The present study was designed to examine the effects of nitric oxide (NO) and calcium ion/calmodulin-dependent nitric oxide synthase (NOS) on opioid-induced antinociception and their involvement in mediating the inhibitory effects of exposure to ELF magnetic fields. We observed that enkephalinase (SCH 34826)-induced, and likely enkephalin-mediated, antinociception in the land snail, Cepaea nemoralis, as measured by the enhanced latency of a foot withdrawal response to a thermal (40 degreesC) stimulus, was reduced by the NO releasing agent, S-nitro-N-acetylpenicillamide (SNP), and enhanced by the NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME). Exposure of snails to an ELF magnetic field (15 min, 60 Hz, 141 microT peak) also reduced the enkephalinase-induced antinociception. The inhibitory effects of the 60-Hz magnetic field were significantly reduced by the NO synthase inhibitor, l-NAME, and significantly enhanced by the NO releasing agent, SNP, at dosages which by themselves had no evident effects on nociceptive sensitivity. These results suggest that: (1) NO and NO synthase have antagonistic effects on opioid-induced analgesia in the snail, Cepaea and (2) the inhibitory effects of ELF magnetic fields on opioid analgesia involve alteration in NO and NO synthase activity.

Animals↗

PubMed2002

The effect of antibiotic treatment on the supercooling ability of the land snail Helix aspersa (Gastropoda: Pulmonata).

The land snail Helix aspersa is a partially freezing tolerant species whose supercooling ability is limited to ca. -3 to -5 degrees C. One hundred adult snails were subjected to the following two experimental conditions: (i) a starved group, provided with water; (ii) an antibiotic-treated group that was provided with a solution containing a mixture of two antibiotics. The antibiotic group exhibited a T(c) significantly lower than the starved group (-3.94 +/- 1.32 degrees C, n = 40 and -3.07 +/- 0.99, n = 30, t test, p < 0.005). This study showed that bacteria of the gut are likely to elevate animal supercooling points. It is also the first report in which a possible ice-nucleating activity of the gut microflora in a land snail has been suggested by the action of antibiotics on the T(c).

Acclimatization↗

PubMed2000

Growing snails used as sentinels to evaluate terrestrial environment contamination by trace elements.

Young garden snails (Helix aspersa) reared in standard conditions (aged two months, mean weight 4.6 +/- 0.5 g) set as sentinels in cages laid on the soil for four weeks, give data for biomonitoring the environmental impact of chemicals on soil ecosystems in the field. The survival and the growth of the snails are influenced by the nature of the biotope and the level of the pollutants. Assay of cadmium, copper, lead and zinc bioaccumulated in the tissues of the sentinel snails provides information on the bioavailability of metals in the environment. The encagement model, little used for terrestrial species, can be useful in monitoring (specific and global endpoints) metal pollution of the environment in reference to the trophic level of the primary consumers. Active biomonitoring is positively compared with the passive biomonitoring.

Animals↗

PubMed1995

Combinatorial peptide libraries in drug design: lessons from venomous cone snails.

Many present-day drugs are derived from compounds that are natural products, a traditional source of which is fermentation broths of microorganisms. The venoms of cone snails are a new natural resource of peptides that may have a pharmaceutical potential equivalent to those from traditional sources, particularly for developing drugs that target cell-surface receptors or ion channels. In effect, cone snails have used a combinatorial library strategy to evolve their small, highly bioactive venom peptides. The methods by which the snails have generated thousands of peptides with remarkable specificity and high affinity for their targets may provide important lessons in designing combinatorial libraries for drug development.

Amino Acid Sequence↗

PubMed1997

Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of cytoplasmic second messengers.

The role of the intracellular second messengers on the bursting firing of action potentials in central snail neurons elicited by d-amphetamine was studied in the identified RP4 neuron of the African snail Achatina fulica Ferussac. Oscillation of membrane potential and bursting firing of action potentials were elicited by d-amphetamine in a concentration dependent manner. The bursting firing of action potentials was decreased following extracellular application of (1) H8 (N-(2-methyl-amino) ethyl-3-isoquinoline sulphonamide dihydrochloride), a specific protein kinase A inhibitor and (2) anisomycin, a protein synthesis inhibitor. However, the bursting firing of action potentials were not affected after (1) extracellular application of H7 (1,(5-isoquinoline-sulphonyl)-2-methylpiperasine dihydrochloride), a specific protein kinase C (PKC) inhibitor, or (2) intracellular application of GDPbetaS, a G protein inhibitor. The oscillation of membrane potential of the bursting activity was blocked after intracellular injection of 3'-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by d-amphetamine in snail neurons may be associated with the cyclic adenosine monophosphate (cAMP) second messenger system: on the other hand it may not be associated with the G protein and protein kinase C activity.

Action Potentials↗

PubMed2001

The increasing complexity of the Snail gene superfamily in metazoan evolution.

The Snail family of zinc-finger transcription factors is involved not only in the development of vertebrate and invertebrate embryos, but also in tumour progression. Following the identification of eight new members, we have analysed the evolutionary history of these genes and found that they constitute a superfamily that groups two independent families, Snail and Scratch. We propose that the duplication of an ancestral gene at the time of the metazoan radiation (1000-500 Myr ago) gave rise to Snail and Scratch, and that independent duplications in protostomes and deuterostomes led to the present situation. We discuss the implications of the distinct duplication events on the acquisition of new functions.

Animals↗

PubMed1997

The NMDA receptor antagonist, NPC 12626, reduces the pronociceptive effects of orphanin FQ and kappa opiate antinociception in the land snail, Cepaea nemoralis.

The heptadecapeptide, orphanin FQ or nociceptin (Phe-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln), originally isolated from rat brain has been identified as an endogenous ligand for the orphan opioid-like receptor. Although orphanin FQ shares some sequence and structural homology with kappa-opioid peptides, it has been speculated to exert its effects through novel nonopioid mechanisms. Kappa opioids have also been suggested to have nonopioid actions in rodents involving the N-methyl-D-aspartate (NMDA) receptor. The present study examined the effects of the competitive NMDA antagonist, NPC 12626, on the antinociceptive effects of the specific kappa-opiate receptor agonist, U69,593, and the pronociceptive effects of orphanin FQ in an invertebrate system, the land snail, Cepaea nemoralis. NPC 12626 had no effect on the basal nociceptive sensitivity of snails, as measured by the latency of response to a thermal (40 degrees C) surface. As reported for rodents, NPC 12626 dose-dependently reduced U69,593-induced antinociception in a manner comparable to that produced by the specific kappa-opiate antagonist, nor-binaltorphimine, while slightly enhancing the antinociceptive effects of the predominately mu-opiate agonist, morphine. Similarly, NPC 12626 dose-dependently reduced the pronociceptive effects of orphanin FQ. These findings with the snail, Cepaea, indicate that NMDA systems/receptors are associated with the mediation of the nociceptive effects of both kappa opioids and orphanin FQ. They suggest an early evolutionary development and phylogenetic continuity of NMDA opioid and related neuropeptide interactions in the mediation of nociception.

Amino Acids↗

PubMed1998

Analgesic effects of a specific pulsed magnetic field in the land snail, Cepaea nemoralis: consequences of repeated exposures, relations to tolerance and cross-tolerance with DPDPE.

It has been demonstrated previously that a short acute exposure to a specific extremely low frequency pulsed magnetic field (Cnp) can induce significant partly opioid-mediated analgesia in the land snail, Cepaea nemoralis. Here, this Cnp-induced analgesia is examined for the development of tolerance to daily repeated acute exposures of 15 or 30 min duration. Acute cross-tolerance to the delta opioid receptor directed agonist DPDPE, [D-Pen2, D-Pen5]enkephalin, was also found. Before (pre-exposure) and after (0, 15, 30 and 60 min) exposure to either a sham or Cnp magnetic field, snails were tested for an aversive reaction to a warmed surface (40 degrees C), and the latency time to the aversive reaction was recorded. Snails that were exposed to the Cnp showed a significant increase in the latency time (F1.55 = 2856.4; p < 0.001; Eta2 = 0.95), which may be interpreted as an induction of analgesia. During the daily (9 day) repeated acute exposures, the induction of analgesic response was significantly reduced, but not ablated. Altering the environmental conditions of the Cnp exposure restored a significant proportion of the partly developed tolerance, consistent with previous reports of environmental specificity in the development of opioid tolerance. These findings suggest that the partial development of tolerance to the opioid-mediated portion of Cnp-induced analgesia may be countered by altering the specific environmental Cnp exposure conditions.

Analgesia↗

PubMed1997

Antinociceptive effects of a pulsed magnetic field in the land snail, Cepaea nemoralis.

Pulsed magnetic fields (patent pending) consisting of approximately 100 microT (peak), frequency modulated, extremely low frequency magnetic fields (ELFMF) were shown to induce a significant degree of antinociception ('analgesia') in the land snail Cepaea nemoralis. Fifteen minute exposures to a specific magnetic field both increased enkephalinase inhibitor induced opioid analgesia and induced analgesia in untreated snails. Injection of the prototypic opioid antagonist naloxone, attenuated, but did not completely block, the pulsed magnetic field induced analgesia. Two other pulsed waveform designs failed to induce analgesia in untreated snails. These findings suggest that specific magnetic field exposure designs may be tailored to produce significant behavioral effects including, but certainly not limited to, the induction of analgesia.

Analgesia↗

PubMed2000

Detection and quantitation of Ehrlichia risticii genomic DNA in infected horses and snails by real-time PCR.

A real-time quantitative PCR using the TaqMan fluorogenic detection system (TaqMan PCR) was established for identification of Ehrlichia risticii, the agent of Potomac horse fever (PHF). The TaqMan PCR identified an 85 base pair section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for eight tested E. risticii strains. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of E. risticii. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The TaqMan PCR was evaluated on horses with infectious colitis and on freshwater stream snails collected from regions with a history of PHF. E. risticii could be detected in 22 of 153 (14.4%) horses with infectious colitis and in 25 of 234 (10.7%) snails in the TaqMan PCR. The same results were obtained in the conventional nested PCR. The Ehrlichia-load was in the range of 10,000-9,000,000 and 35,000-680, 000,000 Ehrlichia equivalents per microg leukocyte DNA and snail DNA, respectively.

Animals↗

PubMed1998

Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails.

The roles of enzymatic antioxidant defenses in the natural tolerance of environmental stresses that impose changes in oxygen availability and oxygen consumption on animals is discussed with a particular focus on the biochemistry of estivation and metabolic depression in pulmonate land snails. Despite reduced oxygen consumption and PO2 during estivation, which should also mean reduced production of oxyradicals, the activities of antioxidant enzymes, such as superoxide dismutase and catalase, increased in 30 day-estivating snails. This appears to be an adaptation that allows the snails to deal with oxidative stress that takes place during arousal when PO2 and oxygen consumption rise rapidly. Indeed, oxidative stress was indicated by increased levels of lipid peroxidation damage products accumulating in hepatopancreas within minutes after arousal was initiated. The various metabolic sites responsible for free radical generation during arousal are still unknown but it seems unlikely that the enzyme xanthine oxidase plays any substantial role in this despite being implicated in oxidative stress in mammalian models of ischemia/reperfusion. We propose that the activation of antioxidant defenses in the organs of Otala lactea during estivation is a preparative mechanism against oxidative stress during arousal. Increased activities of antioxidant enzymes have also observed under other stress situations in which the actual production of oxyradicals should decrease. For example, antioxidant defenses are enhanced during anoxia exposure in garter snakes Thamnophis sirtalis parietalis (10 h at 5 degrees C) and leopard frogs Rana pipiens (30 h at 5 degrees C) and during freezing exposure (an ischemic condition due to plasma freezing) in T. sirtalis parietalis and wood frogs Rana sylvatica. It seems that enhancement of antioxidant enzymes during either anoxia or freezing is used as a preparatory mechanism to deal with a physiological oxidative stress that occurs rapidly within the early minutes of recovery during reoxygenation or thawing. Thus, a wide range of stress tolerant animals display coordinated changes in antioxidant defenses that allow them to deal with oxidative stress that occurs as part of natural cycles of stress/recovery that alter oxygen levels in tissues. The molecular mechanisms that trigger and regulate changes in antioxidant enzyme activities in these species are still unknown but could prove to have key relevance for the development of new intervention strategies in the treatment of cardiovascular ischemia/reperfusion injuries in humans.

Animals↗

PubMed2000

Action potential bursts in central snail neurons elicited by d-amphetamine: roles of ionic currents.

The roles of the ionic currents in the firing of potential bursts elicited by d-amphetamine in central snail neurons were studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage-clamp method. Oscillations of membrane potential bursts were elicited by d-amphetamine. The action potential bursts elicited by d-amphetamine decreased following intracellular injection of either EDTA or magnesium, or extracellular application of lanthanum. Voltage-clamped studies revealed that d-amphetamine decreased the fast Na(+), Ca(2+) and transient outward K(+) currents of the RP4 neuron. It also decreased the steady-state K(+) current and elicited a negative slope resistance in the steady-state I-V curve between -50 and -10 mV. The amplitude of negative slope resistance was decreased if either Na(+)-free saline or Co(2+)-substituted Ca(2+)-free saline was perfused. d-Amphetamine did not increase the amplitude of the slowly inactivating Ca(2+) current or the persistent Na(+) currents of RP4 neuron. Tetraethylammonium, a blocker of the delayed outward K(+) current, elicited action potential bursts and negative slope resistance in the RP4 neuron, while 4-aminopyridine, an inhibitor of transient outward K(+) current (I(A)), did not. These results demonstrate that the delayed outward K(+) current and the negative slope resistance in steady-state I-V curve elicited by d-amphetamine may be responsible for the action potential bursts in central snail neurons elicited by d-amphetamine.

4-Aminopyridine↗

PubMed2000

Identification and analysis of two snail genes in the pufferfish (Fugu rubripes) and mapping of human SNA to 20q.

All members of the snail gene family are zinc-finger transcription factors expressed early in embryonic development and are involved in the formation of tissues such as mesoderm and presumptive neural crest. Here, we report the identification and structural organisation of two snail genes in the compact genome of the pufferfish Fugu rubripes, and examine the phylogenetic relationships between these and other members of the snail gene family. Both genes have a three exon, two intron structure similar to that previously reported for human SLUG. While human SLUG has been mapped to 8q (Cohen, M.E., Yin, M., Paznekas, W.A., Schertzer, M., Wood, S., Jabs, E.W., 1998. Human SLUG organisation, expression and chromosome map location on 8q. Genomics 51, 468-471), the human sna gene SNA, was previously unmapped. We have used sequence similarity to the Fugu genes to identify a human SNA EST and mapped this by radiation hybrid and physical mapping to the distal end of human 20q. This is likely to be the mapping location of the human sna gene (SNA).

Amino Acid Sequence↗

PubMed2000

Burst firing of action potentials in central snail neurons elicited by d-amphetamine: effect of anticonvulsants.

The effect of anticonvulsants on the burst firing of action potentials in snail central neuron elicited by d-amphetamine was studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac. Oscillation of membrane potential and burst firing of action potentials were elicited by d-amphetamine in a concentration-dependent manner. Voltage clamped studies revealed that d-amphetamine elicited a negative slope resistance (NSR) in steady-state I-V curve between - 40 and - 10 mV. The burst firing of action potentials was alleviated following extracellular application of phenytoin, but was not affected after ethosuximide, carbamazepine, and valproic acid. The NSR elicited by d-amphetamine was blocked by phenytoin. However, the NSR was not altered if carbamazepine was added. These results suggest that of the four anticonvulsants tested, only phenytoin could alleviate the burst firing of action potentials elicited by d-amphetamine in snail neuron.

Action Potentials↗

PubMed1996

Ultrastructural study of the dorsal lingual epithelium of the Asian snail-eating turtle, Malayemys subtrijuga.

The Asian snail-eating turtle, Malayemys subtrijuga, is classified phylogenetically as a member of the family Emydinae. Members of this family usually live in small rivers or ponds. However, this species is relatively well-adapted to terrestrial life. We describe here the light, scanning electron and transmission electron microscopic appearance of the dorsal lingual epithelium of the snail-eating turtle and we compare the results to those obtained from other freshwater turtles in an attempt to clarify the relationship between the histological and ultrastructural differences in the lingual epithelium and the living circumstances of the turtles. The tongue is triangular with a rounded apex when viewed dorsally but it appears flattened when viewed laterally. Under the scanning electron microscope, no lingual papillae were visible on the dorsal surface of the tongue. Instead, plicae were seen all over the dorsal surface. On the surface of the epithelium of the outermost side, dome-shaped bulges, each of which was coincident with an individual cell, were compactly distributed. At higher magnification, scanning electron microscopy revealed numerous microvilli and microridges on the surface of these cells, and the thickening of cell-margins was clearly seen. Light microscopy revealed that the mucosal epithelium of the tongue was of the non-keratinized, stratified squamous type. Under the transmission electron microscope, the cells of the basal and deep intermediate layers of the epithelium appeared irregularly elliptical in shape. The nucleus was large and also irregularly elliptical, lying in the central region of each epithelial cell. The cytoplasm of these cells contained mitochondria, free ribosomes, rough endoplasmic reticulum and bundles of tonofibrils. Cell membranes formed processes around individual cells. Desmosomes were intercalated between the processes of adjacent cells. In the shallow intermediate layer, the cells were also elliptical, and the elliptical nucleus was located in the central area of each cell. A large part of the cytoplasm was occupied by electron-dense, discoid granules. Filamentous structures filled the spaces between these granules. Small numbers of free ribosomes, mitochondria and rough endoplasmic reticulum were scattered in the cytoplasm. Cell membranes still formed processes around cells. Desmosomes were intercalated between the processes of adjacent cells. The cells of the surface layer were still elliptical, as were their nuclei. Most of the cytoplasm was filled with electron-dense, discoid granules. Fine filamentous structures were dispersed between these granules. Cell membranes formed processes around cells which were coincident with microvilli and microridges. Intercalated desmosomes were also seen. In some cells, many of the electron-dense, discoid granules were secreted into the oral cavity. In conclusion, the histology of the lingual epithelium of the snail-eating turtle is very similar to that of the freshwater turtle, reflecting similarities in the gross morphology of the tongues of these species, in spite of the differences in their life styles.

Animals↗

PubMed2001

Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica.

Acharan sulfate is a glycosaminoglycan (GAG), having the structure -->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose(1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3-5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.

Alcian Blue↗

PubMed2001

Reverse correlation of E-cadherin and snail expression in oral squamous cell carcinoma cells in vitro.

The loss of E-cadherin expression has been shown to correlate to the invasion and metastasis of many types of carcinomas. We established E-cadherin positive (HOC719-PE) and negative (HOC719-NE) clones from an oral squamous cell carcinoma (SCC). HOC719-PE cells showed epithelial morphology with E-cadherin expression in the cell membrane, whereas HOC719-NE cells demonstrated fibroblastic morphology without E-cadherin expression. In invasion assay and three dimensional culture, HOC719-NE showed much higher invasive ability than HOC719-PE cells. These cells expressed similar levels of mRNAs for alpha- and beta-catenin. However, HOC719-NE cells, but not HOC719-PE cells, showed strong expression of snail, a transcription factor implicated in the differentiation of epithelial cells into mesenchymal phenotype. This reverse expression of snail and E-cadherin was further observed in other SCC cells including HOC313, and TSU cells that we previously reported to show no expression of E-cadherin protein. These results indicated that the expression of snail has a key role for the acquisition of more invasive and metastatic phenotypes of SCC and the clones we reported here will be useful tools for understanding the mechanism of the transition from epithelial to mesenchymal SCC cells.

Cadherins↗