Purification and characterization of a protease from the visceral mass of Mytella charruana and its evaluation to obtain antimicrobial peptides.

This work describes purification of a protease from the visceral mass of the mussel Mytella charruana as well as evaluation of its ability to hydrolyze milk casein to generate antimicrobial peptides. The enzyme showed pI of 4.1 and a single polypeptide band of 83.1 kDa after SDS-PAGE. Sequence similarities with tropomyosin and myosin from mollusks were detected. The protease showed a trypsin-like activity with optimal temperature of 40 °C and stability in a wide pH range (3.0-9.0). Km was 4.28 ±â€¯0.34 mM of the synthetic substrate N-benzoyl-dl-arginyl-ρ-nitroanilide, whereas Vmax was 0.056 ±â€¯0.001 nmol min-1. The enzyme hydrolyzed casein, and the hydrolysate inhibited the growth of Escherichia coli, Micrococcus luteus, Bacillus subtilis, and Klebsiella pneumoniae at a minimal inhibitory concentration of 5.0 µg mL-1. In conclusion, the visceral mass of M. charruana contains a trypsin-like protease that can generate peptides from casein that have a bacteriostatic effect.

Significant association between SNPs in the superoxide dismutase 3, extracellular (SOD3) gene and resistance to Aeromonas hydrophila in the freshwater mussel Hyriopsis cumingii.

Extracellular superoxide dismutase (SOD3) is a major antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). In this study, the SOD3 gene was identified and characterized from the freshwater mussel Hyriopsis cumingii (Hc-SOD3). The cDNA sequence consists of 763 bp, encoding a protein of 208 amino acids. The amino acid sequence possesses two CuZnSOD signature sequences, and amino acids required for binding of Cu (His-93, -95, -110 and -169) and Zn (His-110, -118, -129 and Asp-132) were conserved in Hc-SOD3. The Hc-SOD3 genomic sequence was 9165 bp in length, containing four exons and three introns. Eighteen single nucleotide polymorphisms were detected in the Hc-SOD3 gene from resistant stock (RS) and susceptible stock (SS) of H. cumingii to Aeromonas hydrophila. The genotype and allele distribution were examined in resistant and susceptible stocks. Among them, a C/G substitution at the g.7994C>G locus and G/C substitution at the g.8087G>C locus were significantly associated with resistance/susceptibility of H. cumingii to A. hydrophila, both in genotype (P = 0.017, P = 0.004 respectively) and allele frequency (P = 0.021, P = 0.006 respectively). Linkage disequilibrium analysis revealed that g.7994C>G, g.8001A>G, g.8035G>A, g.8087G>C and g.8191T>A were in linkage disequilibrium. The results suggest that the two polymorphic loci, g.7994C>G and g.8087G>C, could be potential genetic markers for future molecular selection of strains that are resistant to diseases.

The role of catalase in the immune response to oxidative stress and pathogen challenge in the clam Meretrix meretrix.

Catalase (CAT) can effectively eliminate H(2)O(2) and maintain the redox balance of immune system, which is essential for innate immunity. A catalase gene was cloned and its potential role in immune system was investigated in the clam, Meretrix meretrix. The catalase (MmeCAT) gene had an open reading frame of 1533 bp encoding 511 amino acids which showed high identity with that of molluscs. The distribution of MmeCAT in clam tissues was examined and the mRNA, protein expression and CAT activity paralleled with each other, with the highest expression in hepatopancreas. In response to H(2)O(2) challenge, MmeCAT mRNA showed significantly higher expression at 12 h and 24 h post-challenge in experimental clams than in control clams (P < 0.05). Meanwhile, the protein expression in experimental clams was increased to about 3 times as much as that in control clams at 6 h post-challenge. After injection with a Vibrio parahaemolyticus-related bacterium (MM21), the expression of MmeCAT mRNA was significantly up-regulated at 12 h and 24 h post-injection (P < 0.05). It suggested that MmeCAT might be involved in the immune response to Vibrio infection. To better understand the role of MmeCAT in immune system, its mRNA expression was compared between a Vibrio-resistant population and a control population after immersion challenge with MM21. The continuously increased transcription in resistant population suggested MmeCAT could benefit the immune system of clams to defend against pathogen infection. Our study indicated that the redox balance was essential for M. meretrix to resist pathogen infection.

Intracellular Cu/Zn superoxide dismutase (Cu/Zn-SOD) from hard clam Meretrix meretrix: its cDNA cloning, mRNA expression and enzyme activity.

Hard clam (Meretrix meretrix) is an economically important bivalve in China. In the present study, a gene coding for an intracellular Cu/Zn-SOD was cloned and characterized from hard clam. The full-length cDNA of this Cu/Zn-SOD (designated as Mm-icCuZn-SOD) consisted of 1,383 bp, with a 462-bp of open reading frame (ORF) encoding 153 amino acids. Several highly conserved motifs, including the Cu/Zn binding sites [H(46), H(48), H(63), and H(119) for Cu binding; H(63), H(71), H(80), and D(83) for Zn binding], an intracellular disulfide bond and two Cu/Zn-SOD signatures were identified in Mm-icCu/Zn-SOD. The deduced amino acid sequence of Mm-icCu/Zn-SOD has a high degree of homology with the Cu/Zn-dependent SODs from other species, indicating that Mm-icCu/Zn-SOD should be a member of the intracellular Cu/Zn-dependent SOD family. Real-time PCR analysis showed that the highest level of Mm-icCu/Zn-SOD expression was in the hepatopancreas, while the lowest level occurred in the hemocytes. Hard clam challenged with Vibrio anguillarum showed a time-dependent increase in Mm-icCu/Zn-SOD expression that reached a maximum level after 6 h. Mm-icCu/Zn-SOD purified as a recombinant protein expressed in E. coli retained a high level of biological activity, 83 % after 10 min incubation at 10-50 °C, and more than 87 % after incubation in buffers with pH values between 2.2 and 10.2. These results indicated that Mm-icCu/Zn-SOD may play an important role in the innate immune system of hard clam.

Characterisation of nucleosides and nucleobases in Mactra veneriformis by high performance liquid chromatography coupled with diode array detector-mass spectrometry (HPLC-DAD-MS).

Mactra veneriformis has been used as sea food and traditional Chinese medicine (TCM) for thousands of years in China. In the present study, a high performance liquid chromatograph coupled with photodiode array detector and electrospray ionisation-mass spectrometer (HPLC-DAD-ESI-MS) method was established for detection of the nucleosides and nucleobases in M. veneriformis from four aquaticultural area of Jiangsu during different harvest time of one year. The validated method was successfully applied to identifying 10 nucleosides and nucleobases in 48 M. veneriformis samples. Quantitative analysis showed that nucleosides and nucleobases are rich in all M. veneriformis samples. However, their contents vary in different areas and harvest times. Principal component analysis (PCA) was used to classify the 48 samples based on the contents of the nucleosides and nucleobases. As a result, the samples could be mainly clustered into four groups, which was similar as aquaticultural areas classification. Based on the results, present method might be applicable for the quality control of M. veneriformis, or even other marine shellfish aquiculture and their products, and the quality of M. veneriformis might be more related with aquaticultural areas.

A manganese superoxide dismutase (MnSOD) from Ruditapes philippinarum: comparative structural- and expressional-analysis with copper/zinc superoxide dismutase (Cu/ZnSOD) and biochemical analysis of its antioxidant activities.

Superoxide dismutases (SODs), antioxidant metalloenzymes, represent the first line of defense in biological systems against oxidative stress caused by excessive reactive oxygen species (ROS), in particular O(2)(•-). Two distinct members of SOD family were identified from Manila clam Ruditapes philippinarum (abbreviated as RpMnSOD and RpCu/ZnSOD). The structural analysis revealed all common characteristics of SOD family in both RpSODs from primary to tertiary levels, including three MnSOD signatures and two Cu/ZnSOD signatures as well as invariant Mn(2+)- and Cu/Zn(2+)-binding sites in RpMnSOD and RpCu/ZnSOD, respectively. Putative RpMnSOD and RpCu/ZnSOD proteins were predicted to be localized in mitochondrial matrix and cytosol, respectively. They shared 65.2% and 63.9% of identity with human MnSOD and Cu/ZnSOD, respectively. Phylogentic evidences indicated the emergence of RpSODs within molluscan monophyletic clade. The analogous spatial expression profiles of RpSODs demonstrated their higher mRNA levels in hemocytes and gills. The experimental challenges with poly I:C, lipopolysaccharide and Vibrio tapetis illustrated the time-dependent dynamic expression of RpSODs in hemocytes and gills. The recombinant RpMnSOD was expressed in a prokaryotic system and its antioxidant property was studied. The rRpMnSOD exhibited its optimum activity at 20 °C, under alkaline condition (pH 9) with a specific activity of 3299 U mg(-1). These outcomes suggested that RpSODs were constitutively expressing inducible proteins that might play crucial role(s) in innate immunity of Manila clam.

Transcriptional regulation of selenium-dependent glutathione peroxidase from Venerupis philippinarum in response to pathogen and contaminants challenge.

Glutathione peroxidases (GPx) are key enzymes in the antioxidant systems of living organisms by catalyzing the reduction of peroxides to non-reactive products. In the present study, the full-length cDNA encoding a selenium-dependent GPx was identified from Venerupis philippinarum (designated as VpSe-GPx), and the spatial and temporal expression patterns post-Vibrio anguillarum, heavy metals and benzo[a]pyrene (B[a]P) challenge were also investigated. VpSe-GPx possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase. The VpSe-GPx mRNA was found to be most abundantly expressed in hepatopancreas. Vibrio challenge could significantly up-regulate the mRNA expression of VpSe-GPx, and the highest expression level was detected at 24 h post-infection with 6.5-fold increase compared with that in the control group. For heavy metals exposure, the expression of VpSe-GPx was significantly induced by 20, 40 µg L(-1) Cd and 10, 20 µg L(-1) Cu but depressed by 10 µg L(-1) Cd and 40 µg L(-1) Cu. With regards to B[a]P exposure, the expression of VpSe-GPx mRNA was significantly induced at 48 and 96 h post challenge. All these results suggested that VpSe-GPx was potentially involved in mediating the immune response and antioxidant defense in V. Philippinarum.

[New sources of organic form zinc].

The Comparative analysis of the efficiency of the enzymatic hydrolysis of the mussels meat has been carried out, using enzymatic preparations with proteolytic action called "Protozim", "Flavoenzyme" and "Pancreatine". Complexes of fermentolyzates of the mussels meat with zink have been obtained by the reaction of chelating. Prepared fermentolyzates and their complexes with zink have been determined by physicochemical methods. Composition of amino acids has been determined in fermentolyzates and in their complexes with zink has been characterized the content of zink. The valuation of molecular-mass distribution of peptide fractions of fermentolyzates and their complexes with zink has been given.

Assessment of contaminant impacts in a semi-enclosed estuary (Amvrakikos Gulf, NW Greece): bioenergetics and biochemical biomarkers in mussels.

A combination of bioenergetics and biochemical biomarkers in mussels was applied to assess possible pollution impacts in a protected semi-enclosed estuary (Amvrakikos Gulf, NW Greece) that receives pesticide discharges through riverine transport. Scope for growth, a physiological condition index representing the energy budget of the organism, was applied to detect general stress effects on the health status of mussels. The low energy budgets of mussels revealed stress conditions and provided early warning signals of possible consequences at higher levels of biological organization. Biochemical markers of exposure confirmed a risk of pesticide contamination. Decreased acetylcholinesterase activities indicated exposure to organophosphate and carbamate pesticides. Responses of the antioxidant enzyme glutathione peroxidase suggested the presence of contaminants capable of reactive oxygen species production that could be related to organochlorine pesticide contamination in the area. On the other hand, metallothionein levels implied low metal contamination.

Antioxidant responses of the Mediterranean mussel, Mytilus galloprovincialis, to environmental variability of dissolved oxygen.

Physiological responses of Mytilus galloprovincialis against environmental dissolved oxygen partial pressure (pO(2)) variation were studied in terms of the modulated induction of the main antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT) and selenium-dependent glutathione peroxidase (GPX). Field in vivo studies were performed at two sites of the Lagoon of Venice, characterized by different aquatic environmental conditions implying different pO(2). SOD and GPX are more active in gills, and their complementary role is discussed. CAT is more active in the digestive gland, where the enzyme dismutates H(2)O(2) derived from divalent reduction of O(2) performed by various oxidases in peroxisomes. Antioxidant enzyme activities are correlated with water dissolved oxygen (DO), especially in the gills. This tissue, because of its anatomical localization and its physiological role, responds to DO variations modulating the induction of the antioxidant enzymes as a protection mechanism against potential toxicity due to increases in ROS formation.

The occurrence of NADPH-ferrihemoprotein reductase in Corbula caribea, from a natural oil seep at La Brea, Trinidad.

Corbula caribea is the most common non-polychaete macrofaunal organism identified at a large natural oil seep at La Brea in south Trinidad. It is hypothesized that these animals may possess (NADPH-ferrihemoprotein reductase) a component of the Mixed Function Oxygenase system (MFO), which may allow them to ameliorate the potentially deleterious effects resulting from exposure to the high levels of petroleum hydrocarbons within this environment. This study was designed to determine whether organisms from the seep site showed greater enzyme activity when compared to organisms from a non-seep reference site. NADPH-ferrihemoprotein reductase activity was determined by incubating 10 microm cryostat sections with nitro-blue tetrazolium. The reaction product was determined by visual assessment and quantified by measuring the relative mean stain intensity. The intense staining, indicative of enzyme activity was evident in the digestive epithelia of seep animals. Observations indicated that organisms from the seep showed more intense staining, indicating greater enzyme activity, when compared to animals from a non-seep reference site. The relative stain intensity of NADPH-ferrihemoprotein reductase determined for organisms from the seep was 61.30. This was significantly higher than the stain intensity determined for organisms from the non-seep reference site (7.11). This supported visual assessments, which suggested that the seep organisms showed higher enzyme activity than organisms from the non-seep site. The results suggest that NADPH-ferrihemoprotein reductase may be present in Corbula caribea from the seep site and not in those from the non-seep site. It is possible that this enzyme may contribute to these animals ability to tolerate chronic exposure to petroleum hydrocarbons and offer then a selective advantage for survival the seep environment.

The 'Coral Bulker' fuel oil spill on the north coast of Portugal: spatial and temporal biomarker responses in Mytilus galloprovincialis.

In December 2000, the ship 'Coral Bulker' ran aground at the entrance of the port of Viana do Castelo (North-west coast of Portugal). A large amount of fuel oil was spilled and part of it reached the shore. To evaluate the spatial and temporal impact of this oil spill, a field study, and several laboratory toxicity tests were performed using Mytilus galloprovincialis as biological indicator of environmental contamination and the biomarkers glutathione S-transferases (GSTs) and acetylcholinesterase (AChE) as indicative criteria. Fifteen days after the oil spill, mussels collected at stations located near the ship presented higher and lower values of GSTs and AChE activity, respectively. These results, and those obtained in the laboratory toxicity tests, evidence that these biomarkers were sensitive indicators of exposure to this kind of pollution and were able to monitor a spatial impact of the oil spill of at least 10 km, confirming the higher level of contamination near the ship and a contamination gradient along the sampling stations. One year after the accident, such a contamination gradient was no longer evident. This study highlight the potential suitability of a biomarker approach for assessing spatial and temporal impacts of marine pollution accidents, such as fuel oil spills, suggesting the inclusion of these biomarkers in risk assessment studies, as cost-effective and early warning recognized tools. Major advantages and limitations of the biomarker approach used in this study are further discussed.

The use of respiratory enzymes as biomarkers of petroleum hydrocarbon exposure in Mytilus edulis planulatus.

The effect of exposure to petroleum hydrocarbons via the water column and through contaminated sediment upon changes in respiratory enzymes in the common mussel (Mytilus edulis planulatus) was investigated. Mussels were exposed to three concentrations of the water-accommodated fraction (WAF) of Bass Strait crude oil, for 24, 48, and 96 h. In a second study mussels were exposed to three concentrations of crude oil-contaminated sediment for 2 weeks and 1, 2, 4 and 6 months. Activities of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured in the gills. In mussels exposed to WAF, a significant decrease in CS activity was observed over time (P<0.05), whereas treatment did not cause a significant change in CS activity (P>0.05); neither treatment nor time had an effect on LDH activity. Exposure to contaminated sediment did not have a significant effect on CS activity, however, time had a significant effect on CS activity (P<0.05). Both time and treatment had an effect on LDH activity (P<0.05). Results demonstrated that changes in gill CS and LDH are not sensitive biomarkers of petroleum hydrocarbon exposure in M. edulis planulatus.

Induction of glutathione-S-transferases in primary cultured digestive gland acini from the mollusk bivalve Pecten maximus (L.): application of a new cellular model in biomonitoring studies.

In the last three decades, marine invertebrates have been used to monitor environmental health conditions and potential pollution, e.g. in the Mussel Watch Program. The whole animal or specific organs are used to determine contamination levels and disturbances. In the present study, a new in vitro cell culture model was validated for pollution monitoring. A commercial species, the scallop Pecten maximus, was tested for the presence and induction of phase II glutathione-S-transferase (GST) enzymes. These activities were monitored for a year, and the results were found to be consistent with those in the literature. Tributyltin, ethylmethane sulfonate and the water-soluble fraction of crude oil were assayed in, in vitro induction studies. A rapid increase of GST activities was observed within 24 h with all compounds tested, and a time- as well as a dose-response was established. This in vitro cell culture model seems suitable for routine use to predict the effects of pollutants on whole organisms within an ecosystem and in fisheries.

Effect of cadmium on antioxidant enzyme activities and lipid peroxidation in the gills of the clam Ruditapes decussatus.

Metals are known to influence the oxidative status of marine organisms, and antioxidant enzymes have been often proposed as biomarkers of effect. The clam Ruditapes decussatus is a well-known metal bioindicator. In this species cadmium (Cd) induces metallothionein (MT) synthesis only after 7 days of exposure. Before MT synthesis is induced, the other mechanisms capable of handling the excess of Cd are unknown. In order to identify some of these mechanisms, variations in antioxidant systems (superoxide dismutase, catalase, selenium-dependent glutathione peroxidase and non-selenium-dependent glutathione peroxidase), malondialdehyde (MDA) and MT were studied in the gills of R. decussatus exposed to different Cd concentrations (4, 40 and 100 gl-1) for 28 days. These parameters, together with total proteins and Cd concentrations, were measured in the gills of the clams over different periods of exposure. Results indicate that Cd accumulation increased linearly in the gills of R. decussatus with the increase in Cd concentration. This increase induces an imbalance in the oxygen metabolism during the first days of Cd exposure. An increase in cytosolic superoxide dismutase (SOD) activity and a decrease in mitochondrial SOD activity was observed at the same time as or after a decrease in cytosolic and mitochondrial catalase activity and of selenium-dependent and non-selenium-dependent glutathione peroxidase activity. After 14 days of exposure, Cd no longer affect these enzymes but there was elevation of other cellular activities, such as MDA and MT production. MT bound excess Cd present in the cell. These variations in these parameters suggest their potential use as biomarkers of effects such as oxidative stress resulting from Cd contamination in molluscs.

Isolation and characterization of the carbon-phosphorus bond-forming enzyme phosphoenolpyruvate mutase from the mollusk Mytilus edulis.

The enzyme phosphoenolpyruvate mutase was purified to homogeneity from the mollusk Mytilus edulis. The subunit size of the native homotetramer was determined to be 34,000 Da. The steady-state kinetic constants for catalysis of the conversion of phosphonopyruvate to phosphoenolpyruvate at pH 7.5 and 25 degrees C were measured at kcat = 34 s-1, phosphonopyruvate Km = 3 microM, and Mg2+ Km = 4 microM. The enzyme displayed a broad specificity for divalent metal ion activation; Co2+, Mn2+, Zn2+, and Ni2+ are activators, whereas Ca2+ is not. Analysis of the pH dependence of the Mg2+-activated mutase-catalyzed reaction of phosphonopyruvate revealed one residue that must be protonated (apparent pKa = 8.3) and a second residue that must be unprotonated (apparent pKa = 7.7) for maximal catalytic activity.

Induction of peroxisomal oxidases in mussels: comparison of effects of lubricant oil and benzo(a)pyrene with two typical peroxisome proliferators on peroxisome structure and function in Mytilus galloprovincialis.

Marine mussels are used as bioindicators of water pollution in marine and estuarine environments in the so-called "Mussel Watch" programs because of their capacity to accumulate numerous organic xenobiotics including aromatic hydrocarbons. In this study, we have analyzed the effects of two xenobiotics [benzo(a)pyrene and the water accommodated fraction of a lubricant oil] and two typical (rodent) peroxisome proliferators (clofibrate and dioctyl phthalate) on structure and function of peroxisomes in digestive glands of mussels Mytilus galloprovincialis, either following water exposure (for 1, 7, and 21 days) or after direct injection through the adductor muscle (for 1 and 7 days). The activities of catalase (CAT), acyl-CoA oxidase (AOX), and D-amino acid oxidase were determined in whole homogenates of digestive glands. In addition, stereological methods were applied on sections stained histochemically for demonstration of catalase activity in order to quantify the morphological changes of peroxisomes. The peroxisomal acyl-CoA oxidase and D-amino acid oxidase were increased in mussels injected for 7 days with benzo(a)pyrene, phthalate, and clofibrate and a similar trend was noted for benzo(a)pyrene and lubricant oil in water exposure experiments (21 days). The catalase activity was reduced or unchanged depending on the mode of exposure of animals. By stereology, significant increases of numerical and volume densities of peroxisomes were found in animals injected for 7 days with lubricant oil or clofibrate. These observations indicate that peroxisomal oxidases in mussels are induced at moderate rates in response to different xenobiotics and that their determination could provide a (sensitive) marker for detection of effects of some toxic pollutants, particularly the lubricant oils which in addition induce significant structural alterations of mussel peroxisomes.