[Effects of copper and cadmium ions on protective enzyme activity in Oncomelania hupensis].

OBJECTIVE: To evaluate the effects of Cu2+ and Cd2+ at different concentrations on superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity in Oncomelania hupensis. METHODS: Cu2+- and Cd2+-containing solutions were prepared at 7 concentrations, and O. hupensis snails were exposed to the solutions for 24 h, of 15 snails in each concentration. Then, the snail body was collected following removal of the snail shell and homogenated, and the SOD, CAT and POS activities were detected in the supernatants. RESULTS: With the increase of the Cu2+ concentration, the SOD activity appeared a rise followed by a reduction in O. hupensis snails, and the CTA activity appeared a decline-rise-decline tendency, while the POD activity showed a tendency towards rise followed by decline. With the increase of the Cd2+ concentration, the SOD activity appeared a rise followed by a reduction in O. hupensis snails, and the CTA activity appeared a decline- rise- decline tendency, while the POD activity showed a decline-rise-decline tendency. CONCLUSIONS: Exposure to Cu2+ and Cd2+ at high concentrations results in a decline in the activity of SOD, CAT and POD in O. hupensis at the same time.

In vivo evaluation of oxidative stress and biochemical alteration as biomarkers in glass clover snail, Monacha cartusiana exposed to zinc oxide nanoparticles.

Oxidative stress is considered a main commonly reported mechanism of nanoparticles toxicity, so this study aimed to evaluate oxidative stress and biochemical alterations in the haemolymph and digestive gland of snail, Monacha cartusiana exposed to sublethal concentrations of zinc oxide nanoparticles (ZnONPs) for 14 days (d). The results indicated that, ZnONPs induced significant increases in lipid peroxidation (LPO) and lactate dehydrogenase (LDH) in treated animals and did not return to normal levels after recover period. A significant decline of glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, and glutathione (GSH) content in the haemolymph and digestive gland of snails was observed when compared with control. A significant increase was observed in catalase (CAT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities of treated animals. In general, nano-materials are able to induce oxidative stress in exposed animals. The present findings indicate that, alterations of antioxidant enzyme activities, increase of LPO, LDH, and reducing of GSH content and GST, GPx activities are recognized to oxidative stress and cell damage. This species could be considered a good bioindicator to assess nano-materials exposure.

Molecular Diversity and Gene Evolution of the Venom Arsenal of Terebridae Predatory Marine Snails.

Venom peptides from predatory organisms are a resource for investigating evolutionary processes such as adaptive radiation or diversification, and exemplify promising targets for biomedical drug development. Terebridae are an understudied lineage of conoidean snails, which also includes cone snails and turrids. Characterization of cone snail venom peptides, conotoxins, has revealed a cocktail of bioactive compounds used to investigate physiological cellular function, predator-prey interactions, and to develop novel therapeutics. However, venom diversity of other conoidean snails remains poorly understood. The present research applies a venomics approach to characterize novel terebrid venom peptides, teretoxins, from the venom gland transcriptomes of Triplostephanus anilis and Terebra subulata. Next-generation sequencing and de novo assembly identified 139 putative teretoxins that were analyzed for the presence of canonical peptide features as identified in conotoxins. To meet the challenges of de novo assembly, multiple approaches for cross validation of findings were performed to achieve reliable assemblies of venom duct transcriptomes and to obtain a robust portrait of Terebridae venom. Phylogenetic methodology was used to identify 14 teretoxin gene superfamilies for the first time, 13 of which are unique to the Terebridae. Additionally, basic local algorithm search tool homology-based searches to venom-related genes and posttranslational modification enzymes identified a convergence of certain venom proteins, such as actinoporin, commonly found in venoms. This research provides novel insights into venom evolution and recruitment in Conoidean predatory marine snails and identifies a plethora of terebrid venom peptides that can be used to investigate fundamental questions pertaining to gene evolution.

Identification and characterization of a goose-type lysozyme from sewage snail Physa acuta.

Freshwater snail Physa acuta has been considered as an important invasive species and medical mollusc. Field investigation has shown that this snail could survive better than other snails in polluted water bodies. To understand the immune mechanisms of P. acuta, suppression subtractive hybridization hepatopancreas cDNA library has been constructed with bacterial challenge. In this study, a full-length cDNA of a novel goose-type lysozyme (PALysG) has been identified from P. acuta by EST and RACE technique. The conservative structure domains share high homology with other molluscan g-type lysozymes including the SLT domain, the substrate binding sites, the catalytic residues, three alpha-helices structures and six molluscan specific cysteines. Meanwhile, PALysG is the first record of goose-type lysozyme in Gastropoda. Real-time PCR indicated that PALysG mRNA had been expressed significantly at high levels in hepatopancreas for 8-48 h. PALysG recombinant protein displayed the lytic activity of g-type lysozyme with other organisms against Micrococcus lysodikicus.

Immune response of apoptosis-related cysteine peptidases from the red abalone Haliotis rufescens (HrCas8 and HrCas3): molecular characterization and transcription expression.

Caspases play an important role in the different stages of programmed cell death, or apoptosis, which has been related to the immune response in multicellular organisms. The present study characterized an initiator caspase (HrCas8) and an effector caspase (HrCas3) from the red abalone Haliotis rufescens using the RACE method and qPCR analysis. HrCas8 showed a complete sequence of 2529 base pairs (bp) with an open-reading frame (ORF) of 1911 bp, a 5'UTR of 201 bp, and a 3'UTR of 417 bp. The estimated molecular mass for the 636 amino acids from HrCas8 was 71.5 kDa with an isoelectric point of 6.2. The HrCas8 sequence had two death-effector domains (DEDs) and the subunits p20 and p10, in addition to an active site characteristic of cysteine proteins. Meanwhile, the effector caspase HrCas3 showed a complete sequence of 1404 bp, a 5'UTR of 82 bp, and a 3'UTR of 574 bp. The ORF of this caspase had 747 bp that coded for 248 residues. Moreover, the predicted molecular mass of HrCas3 was 29.4 kDa; the theoretical isoelectric point was 5.70, and the sequence evidenced a conserved caspase recruitment domain (CARD). The distribution of the caspases in distinct tissues revealed that HrCas8 was principally expressed in the hemolymph, while HrCas3 had a higher expression in the gills. A basal level of expression was found for both caspases in muscle tissue. The immune response of caspases in H. rufescens was evaluated through an injection of Vibrio anguillarum. The results showed an increase in the transcription of HrCas8 post-challenge, as well as an activation of HrCas3, which together suggest the initiation of apoptosis as a response to bacterial infection in H. rufescens.

Toxicity of imidazolium-based ionic liquids on Physa acuta and the snail antioxidant stress response.

In the present study, the acute and developmental toxicities of imidazolium ionic liquids (ILs) with different alkyl chain lengths, as well as the antioxidant response and lipid peroxidation levels were evaluated in the snail, Physa acuta. Longer alkyl chains corresponded to increased IL toxicity in snails. Long-term IL exposure at lower concentrations inhibited snail growth and reproduction. We also found that IL inhibited the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), promoted the activity of catalase (CAT), and increased the glutathione content. However, SOD, GST, and CAT activities returned to control levels after 96 h of recovery. In addition, malondialdehyde levels were increased in treatment groups compared with the control and did not return to control levels even after a recovery period, indicating that ILs induced lipid peroxidation in snail viscera. These results suggest that oxidative stress and lipid peroxidation may be involved in the mechanism of toxicity for ILs.

Purification, spectroscopic characterization and o-diphenoloxidase activity of hemocyanin from a freshwater gastropod: Pila globosa.

Hemocyanins are multi-subunit oxygen carrier proteins, found in select species of arthropoda and mollusca. Here, we have purified native hemocyanin from Pila globosa, a freshwater gastropod, verified using mass spectrometry and determined its molecular weight, secondary structure and the spectral properties, using Ultraviolet/visible, Fourier transform infra-red and Circular dichroism spectroscopy. Our results reveal the oligomeric and glycosylated nature of the protein, comprising of 400 kDa subunits, organized predominantly into a thermo-stable, alpha-helical conformation. Further, biochemical assays confirm catecholoxidase-like activity in hemocyanin, which has been used to develop a first-generation optical sensor, for the detection of phenols.

Involvement of Na+/K+ pump in fine modulation of bursting activity of the snail Br neuron by 10 mT static magnetic field.

The spontaneously active Br neuron from the brain-subesophageal ganglion complex of the garden snail Helix pomatia rhythmically generates regular bursts of action potentials with quiescent intervals accompanied by slow oscillations of membrane potential. We examined the involvement of the Na(+)/K(+) pump in modulating its bursting activity by applying a static magnetic field. Whole snail brains and Br neuron were exposed to the 10-mT static magnetic field for 15 min. Biochemical data showed that Na(+)/K(+)-ATPase activity increased almost twofold after exposure of snail brains to the static magnetic field. Similarly, (31)P NMR data revealed a trend of increasing ATP consumption and increase in intracellular pH mediated by the Na(+)/H(+) exchanger in snail brains exposed to the static magnetic field. Importantly, current clamp recordings from the Br neuron confirmed the increase in activity of the Na(+)/K(+) pump after exposure to the static magnetic field, as the magnitude of ouabain's effect measured on the membrane resting potential, action potential, and interspike interval duration was higher in neurons exposed to the magnetic field. Metabolic pathways through which the magnetic field influenced the Na(+)/K(+) pump could involve phosphorylation and dephosphorylation, as blocking these processes abolished the effect of the static magnetic field.

Genetic markers for studies on the systematics and population genetics of snails, Bithynia spp., the first intermediate hosts of Opisthorchis viverrini in Thailand.

Snails are the critical amplifying hosts of the liver fluke Opisthorchis viverrini, the causative agent of hepatobiliary disease and cholangiocarcinoma in the Mekong area of Southeast Asia. Bithynia funiculata, B. siamensis goniomphalos and B. s. siamensis are the first intermediate hosts of O. viverrini in Thailand. Morphological similarity between Bithynia species and subspecies creates problems for their taxonomic identification and an understanding of Bithynia systematics. In this study, multilocus enzyme electrophoresis (MEE) was applied to define genetic markers that could prove useful for investigating the systematics and population genetics of this genus in Thailand. Of the 34 enzymes examined, 20 encoding a presumptive 24 loci showed sufficient staining intensity and resolution for genetic interpretation. Of these, three loci were monomorphic and eight loci were diagnostic among the three Bithynia taxa. The remaining 13 loci were diagnostic between combinations of the three taxa. Fixed genetic differences were detected at 67-73% of loci among these taxa which in turn differed from a closely related species, Hydrobioides nassa, at 88% of loci. Seventy three percent fixed genetic differences were detected between B. funiculata and the two sub-species B. s. siamensis and B. s goniomphalos. Our data reveals similarly large genetic divergence, 67% fixed genetic differences, between B. s. siamensis and B. s. goniomphalos, which may well represent different species rather than subspecies as currently defined. The genetic markers detected will form the basis for subsequent comprehensive studies on the systematics and population genetics of Bithynia snails as well as for their role in the transmission of O. viverrini and opisthorchiasis.

Biomarkers of oxidative stress in the land snail, Theba pisana for assessing ecotoxicological effects of urban metal pollution.

This study investigates the biomarkers of oxidative stress and heavy metal accumulation for assessing ecotoxicological effects of urban metal pollution in Alexandria city, Egypt. This investigation was performed in the digestive gland of roadside Theba pisana snails obtained from six different sites in the city. Relationships between heavy metal (Zn, Cu, Pb and Cd) concentrations and oxidative stress indicators were also examined. The results showed that mean concentrations of the measured elements (mugg(-1) dry weight) were higher in polluted sites when compared to the background levels of the reference site. The pattern of metals accumulation at all sites was Zn>Cu>Pb>Cd. In the metal polluted sites, snails displayed higher mean of catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities, lower reduced glutathione (GSH) content and higher levels of lipid peroxidation (LPO) compared to snails from the reference site. All oxidative stress parameters were positively correlated to heavy metal concentrations except GSH content which exhibits negative correlation with the concentrations of these metals. Our results suggest that the tested antioxidant defenses are sensitive parameters that could be useful as oxidative stress biomarkers in snails exposed to the actual metals in the environment. These biomarkers when complemented with metal accumulation in the digestive gland of snails may provide a powerful biomonitor for the assessment of environmental metal pollution.

In vivo evaluation of oxidative stress biomarkers in the land snail, Theba pisana exposed to copper-based pesticides.

The in vivo evaluation of oxidative stress biomarkers in the digestive gland of Theba pisana exposed to sublethal doses (40% and 80% of LD(50) after 48 h) of copper-based pesticides; copper oxychloride, copper hydroxide and copper sulphate was examined. Oxidative individual perturbations were assessed by measuring non-enzymatic (glutathione; GSH) and enzymatic (catalase; CAT, glutathione peroxidase; GPx and glutathione S-transferase; GST) antioxidants in digestive gland of the snails. Lipid peroxidation (LPO) was also evaluated as marker of cell damage. The results indicated that copper sulphate was the most potent compound against this snail followed by copper hydroxide and copper oxychloride where their corresponding LD(50) values were 26.54, 334.54 and 582.18 microg snail(-1), respectively. Copper-based compounds resulted in a significant increase in the level of LPO whereas a significant decline of GSH content in the digestive gland of snails was observed when compared with untreated controls. The CAT, GPx and GST activities of treated snails were significantly higher than those of untreated controls. In general, the activation power of these compounds was in the following order: copper sulphate > copper hydroxide > copper oxychloride. This study suggests that up-regulation of the antioxidant enzyme activities, elevation of LPO and the reduction of GSH content is related to oxidative stress in this species that they could be used as potential biomarkers of copper-based pesticides exposure.

[Effect of Alternanthera philoxeroides on enzymic histochemistry of Oncomelania hupensis].

OBJECTIVE: To investigate the mechanisms of Alternanthera philoxeroides (Mardus) Grisebach in inhibiting Oncomelania snails' locomotivity and killing effect. METHODS: Uninfected snails were divided into four groups and exposed to an aqueous extract of A. philoxeroides and dechlorinated water (as control) for 12 h or 20 h, respectively. The activities of the Mg2+-adenosine triphosphatase (Mg2+-ATPase), cholinesterase (ChE), lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) in the head-foot muscles, centric ganglions, gills and liver of Oncomelania hupensis were analyzed using enzyme histochemistry technology and changes were observed under a light microscope. Statistical quantitative analysis of data of grey values was conducted on the computer-assisted image analyzing system (HPIAS-1000). RESULTS: The color of stained ChE in the head-foot muscles, centric ganglions and gills of the snail lightened evidently, showing a decrease of ChE activity after snails were immersed in the extract of A. philoxeroides for 12 h or 20 h. Results of grey values at different stained parts of snail, measured by the computer-assisted image analyzing system, indicated that there was a significant difference (P<0.01) between the activities of ChE in the head-foot muscles (130.95 +/- 8.08, 129.91 +/- 7.05), centric ganglions (127.43 +/- 7.27, 126.78 +/- 7.38) and gills (121.38 +/- 7.31, 126.41 +/- 8.28) from snails exposed to aqueous extract of A. philoxeroides for 12 h and 20 h and the activities of the enzyme in counter-parts (64.65 +/- 8.54, 65.18 +/- 7.96, 57.86 +/- 6.57, 50.71 +/- 6.15, 88.96 +/- 6.78 and 89.86 +/- 7.01, respectively) from control group. The activities of Mg2+-ATPase also showed a significant difference (P<0.05) in the head-foot muscles (89.91 +/- 5.08), centric ganglions (71.15 +/- 5.43) and liver (112.40 +/- 7.81) of the snail after 20 h of exposure against those in counter-parts (78.81 +/- 8.10, 60.09 +/- 6.05 and 95.50 +/- 8.35, respectively) from the control, but no significant difference (P>0.05) was shown on the activities of Mg2+-ATPase between the snails exposed for 12 h in extract of A. philoxeroides and those of control. No statistical difference (P>0.05) was found between the dechlorinated water group and the extract of A. philoxeroides group in the activities of LDH and SDH after 12 h or 20 h of exposure. CONCLUSION: The extract of A. philoxeroides rapidly inhibits ChE, and then the activity of Mg2+-ATPase, which may suppress the release and utilization of ATP in the Oncomelania snails and finally causes death of snails.

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells.

BACKGROUND INFORMATION: PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). RESULTS: SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. CONCLUSION: Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

Kinetic characterization of the inhibition of acyl coenzyme A: steroid acyltransferases by tributyltin in the eastern mud snail (Ilyanassa obsoleta).

Exposure to tributyltin (TBT) has been causally associated with the global occurrence of a pseudohermaphroditic condition called imposex in neogastropod species. TBT elevates free testosterone levels in these organisms, and this upsurge in testosterone may be involved in the development of imposex. We investigated the ability of TBT to inhibit acyl coenzyme A:testosterone acyltransferase (ATAT) activity as well as microsomal acyl-coenzyme A:17beta-estradiol acyltransferase (AEAT) in a neogastropod, the eastern mud snail Ilyanassa obsoleta as a mechanism by which TBT elevates free testosterone. TBT significantly inhibited both ATAT and AEAT activities in vitro at toxicologically relevant in vivo concentrations. Kinetic analyses revealed that TBT is a competitive inhibitor of ATAT (K(i)= approximately 9microM) and is a weaker, noncompetitive inhibitor of AEAT (K(i)= approximately 31microM). ATAT and AEAT activities associated with different microsome preparations were significantly correlated, and 17beta-estradiol competitively inhibited the fatty acid esterification of testosterone suggesting that one enzyme is responsible for biotransforming both testosterone and 17beta-estradiol to their corresponding fatty acid esters. Overall, the results of this study supply the much-needed mechanistic support for the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting their major regulatory process for maintaining free testosterone homeostasis-the fatty acid esterification of testosterone.

Suppression of Na+/K+-ATPase activity during estivation in the land snail Otala lactea.

Entry into the hypometabolic state of estivation requires a coordinated suppression of the rate of cellular ATP turnover, including both ATP-generating and ATP-consuming reactions. As one of the largest consumers of cellular ATP, the plasma membrane Na+/K+-ATPase is a potentially key target for regulation during estivation. Na+/K+-ATPase was investigated in foot muscle and hepatopancreas of the land snail Otala lactea, comparing active and estivating states. In both tissues enzyme properties changed significantly during estivation: maximal activity was reduced by about one-third, affinity for Mg.ATP was reduced (Km was 40% higher), and activation energy (derived from Arrhenius plots) was increased by approximately 45%. Foot muscle Na+/K+-ATPase from estivated snails also showed an 80% increase in Km Na+ and a 60% increase in Ka Mg2+ as compared with active snails, whereas hepatopancreas Na+/K+-ATPase showed a 70% increase in I50 K+ during estivation. Western blotting with antibodies recognizing the alpha subunit of Na+/K+-ATPase showed no change in the amount of enzyme protein during estivation. Instead, the estivation-responsive change in Na+/K+-ATPase activity was linked to posttranslational modification. In vitro incubations manipulating endogenous kinase and phosphatase activities indicated that Na+/K+-ATPase from estivating snails was a high phosphate, low activity form, whereas dephosphorylation returned the enzyme to a high activity state characteristic of active snails. Treatment with protein kinases A, C or G could all mediate changes in enzyme properties in vitro that mimicked the effect of estivation, whereas treatments with protein phosphatase 1 or 2A had the opposite effect. Reversible phosphorylation control of Na+/K+-ATPase can provide the means of coordinating ATP use by this ion pump with the rates of ATP generation by catabolic pathways in estivating snails.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily.

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Acid phosphatase complex from the freshwater snail Viviparus viviparus L. under standard conditions and intoxication by cadmium ions.

Acid phosphatases differing in both subcellular localization and substrate specificity were isolated for the first time from the liver of the freshwater snail Viviparus viviparus L. by preparative isoelectrofocusing. One of five characterized phosphatases is highly specific to ADP and the others can hydrolyze (at variable rate) a series of natural substrates. A scheme is proposed for the involvement of the studied phosphatases in carbohydrate metabolism. We have also studied some peculiarities of the effect of Cd2+ in vitro and in vivo on the activities of individual components of the acid phosphatase complex and corresponding changes in metabolism of the freshwater snail as a new test-object allowing the estimation of toxicity in water.

Vacuolar-type ATPase in the accessory boring organ of Nucella lamellosa (Gmelin) (Mollusca : Gastropoda): role in shell penetration.

The structure and function of the accessory boring organ (ABO) of muricid gastropods has been described in numerous studies, and the ABO of Nucella lamellosa was found to be similar to those of other muricid species. The active cap region of the ABO is composed of tall, mitochondria-rich cells with distinct brush borders at their apicies, surrounding a hemolymph-containing central sinus. Using antibodies specific for vacuolar-type ATPase (V-ATPase), enzyme immunoreactivity was found to be limited to the brush border of the epithelial cells. Electron immunohistochemistry revealed that V-ATPase immunoreactivity resides in the plasma membranes of the microvilli. Immunodot blotting using yeast V-ATPase as a positive control confirmed the specificity of the reactions. SDS-PAGE of membrane suspensions from the ABO revealed protein bands of the requisite molecular weight for V-ATPase subunits. Western blots suggest that antibodies raised against mammalian V-ATPase subunits recognize subunits of the molluscan V-ATPase. The molecular weights of these identified subunits are similar to those in mammals. The V-ATPase-specific inhibitor bafilomycin A1 inhibited ATPase activity in samples of ABO homogenate by about 10% relative to control, providing further evidence for the presence of V-ATPase. Specific V-ATPase activity was about 67 picomoles of inorganic phosphate per microgram of protein per minute in the homogenate. Collectively this evidence strongly suggests that a vacuolar-type proton transporting ATPase is present in the brush border of the accessory boring organ of Nucella lamellosa, and is responsible for acidifying secretions from this gland. Similarities between the ABO, osteoclasts, and the mantle of freshwater bivalves also suggest that the mechanism for decalcification of calcareous substrates is conserved.

A novel endopeptidase with a strict specificity for threonine residues at the P1' position.

An endopeptidase was purified from Archachatina ventricosa by chromatography on columns of gel filtration, DEAE-Sepharose and phenyl-Sepharose. The preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and capillary electrophoresis. The purified enzyme displayed two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular weights of 90,000 and 121,000. The protease exhibited maximum proteolytic activity at 55 degrees C and at pH 8.0, but it retained more than 85% of its activity in the pH range 7.5 to 8.5. It was completely inactivated by the chelating agents EDTA and 1,10-phenanthroline which are metalloprotease inhibitors. Studies on substrate specificity showed that only the amide bonds of peptide substrates having a threonine residue at the P1' position were hydrolyzed by the purified protease. This endopeptidase constitutes a novel tool for the study of proteins in view of its narrow and unique substrate specificity.

Neutral endopeptidase-24.11 (NEP)-like activity in molluscan hemocytes.

Using a spectrofluorimetric procedure, we found that the plasma membrane from hemocytes of two freshwater snails, Planorbarius corneus and Viviparus ater, shows neutral endopeptidase-24.11 (NEP)-like activity. Moreover, the addition of ACTH(1-24) to the hemolymph provokes an increase in NEP-like activity. This increased NEP-like activity is blocked by phosphoramidon, a potent inhibitor of mammalian NEP. These findings suggest that this peptidase has been well conserved in the course of evolution and plays an important role in immune-neuroendocrine mechanisms.