A novel anticoagulant peptide discovered from Crassostrea gigas by combining bioinformatics with the enzymolysis strategy: inhibitory kinetics and mechanisms.

A novel anticoagulant peptide (IEELEEELEAER) derived from oyster (Crassostrea gigas) was discovered by combining the emerging bioinformatics with the classical enzymolysis approach. The anticoagulant peptide drastically reduced the extrinsic clotting activity (49% residual PT activity) and impaired the intrinsic clotting activity (77% residual PT activity). Consistent with the clotting data, the thrombin peak height reduced to 88.7 from 123.4 nM, and the thrombin generation time delayed to 5.32 from 4.42 min when an extrinsic trigger was applied. The inhibitory kinetics of FXIa, FIXa, FXa, FIIa, and APC in a purified component system rationally explained the reduction of the extrinsic clotting activity and impairment of thrombin generation. Besides the inhibition of FXa and FIIa activity, the activation processes of FX and FII by an intrinsic/extrinsic tenase complex and prothrombinase were also damaged. The anticoagulant activity in the plasma system was the result of comprehensive inhibition of various factors. The research provided a frame for anticoagulant evaluation and inhibitory mechanism of bioactive peptides from food products.

Polygalactan from bivalve Crassostrea madrasensis attenuates nuclear factor-κB activation and cytokine production in lipopolysaccharide-activated macrophage.

A polygalactosamino-glucopyranosyl fucopyranose →4)-ß-GlcAp{(3→1)-α-Fucp}-ß-GalNAcp-(4,6-SO3-)-(1→ isolated from the bivalve Crassostrea madrasensis exhibited prospective anti-inflammatory activity against cyclooxygenase-2 and 5-lipoxygenase (IC50 < 50 µg mL-1) on lipopolysaccharide-induced macrophages. The polygalactan attenuated inducible nitric oxide synthase (IC50 65.7 µg mL-1) in lipopolysaccharide-prompted inflammation leading to the reduction of pro-inflammatory cytokine nitric oxide (236.2 µg mL-1 lysate), nuclear factor-κB, tumor necrosis factor-α, and interleukins (0.19-0.22 units mg-1 protein at 100 µg mL-1) by inhibiting cyclooxygenase-2. The polygalacatan suppressed the mRNA of nuclear factor-κB and cyclooxygenase-2 in lipopolysaccharide-induced macrophages. Western blot experiment revealed that the polygalactan attenuated the migration of nuclear factor-κB-p65 to the nucleus from cytoplasm, and suppressed the phosphorylation of α-subunit of κB inhibitor. Greater selectivity index of sulfated polygalactan (3.93) towards inducible cyclooxygenase-2 as compared with the anti-inflammatory agent ibuprofen (1.11), and the potential to inhibit nuclear factor-κB cascade to generate chemokine production manifested its utilization in the development of functional food attenuating inflammation-related disorders.

Optimization of Oyster (Crassostrea talienwhanensis) Protein Hydrolysates Using Response Surface Methodology.

Oyster (Crassostrea talienwhanensis) protein was hydrolyzed by trypsin to produce peptides with different response values, and response surface methodology (RSM) was applied to optimize the hydrolysis conditions. The highest degree of hydrolysis (DH) of the oyster peptide (OP) was obtained at an enzyme concentration of 1593.2 U/g, a pH of 8.2, a hydrolysis temperature of 40.1 °C, a hydrolysis time of 6.0 h, and a water/material ratio of 8.2. The greatest hydroxyl-radical-scavenging activity of OP was obtained at an enzyme concentration of 1546.3 U/g, a pH of 9.0, a hydrolysis temperature of 50.2 °C, a hydrolysis time of 5.1 h, and a water/material ratio of 5.6. The largest branched-chain amino acid (BCAA) content of OP was obtained at an enzyme concentration of 1323.8 U/g, a pH of 8.3, a hydrolysis temperature of 41.7 °C, a hydrolysis time of 6.7 h, and a water/material ratio of 4.8. The three experimental values were significantly in agreement with the predicted values within the 95% confidence interval. Furthermore, ultrafiltration and chromatographic methods were used to purify the OP, and 13 peptides that were rich in Lys, Arg, His, and Thr were identified by LC-MS/MS. The results of this study offer different optimum hydrolysis conditions to produce target peptides from oyster protein by using RSM, and this study provide a theoretical basis for the high-value utilization of oyster protein.

Ameliorative Effects of Peptides from the Oyster (Crassostrea hongkongensis) Protein Hydrolysates against UVB-Induced Skin Photodamage in Mice.

Chronic exposure to ultraviolet B (UVB) irradiation is a major cause for skin photoaging. UVB induces damage to skin mainly by oxidative stress, inflammation, and collagen degradation. This paper investigated the photo-protective effects of peptides from oyster (Crassostrea hongkongensis) protein hydrolysates (OPs) by topical application on the skin of UVB-irradiated mice. Results from mass spectrometry showed that OPs consisted of peptides with a molecular weight range of 302.17-2936.43 Da. In vivo study demonstrated that topical application of OPs on the skin significantly alleviated moisture loss, epidermal hyperplasia, as well as degradation of collagen and elastin fibers caused by chronic UVB irradiation. In this study, OPs treatment promoted antioxidant enzymes (SOD and GPH-Px) activities, while decreased malondialdehyde (MDA) level in the skin. In addition, OPs treatment significantly decreased inflammatory cytokines (IL-1ß, IL-6, TNF-α) content, and inhibited inflammation related (iNOS, COX-2) protein expression in the skin. Via inhibiting metalloproteinase 1(MMP1) expression, OPs treatment markedly decreased the degradation of collagen and elastin fibers as well as recovered the altered arrangement of extracellular matrix network in the dermis of skin. Our study demonstrated for the first time that OPs protected against UVB induced skin photodamage by virtue of its antioxidative and anti-inflammatory properties, as well as regulating the abnormal expression of MMP-1. The possible molecular mechanism underlying OPs anti-photoaging is possibly related to downregulating of the MAPK/NF-κB signaling pathway, while promoting TGF-ß production in the skin.

Low molecular polypeptide from oyster hydrolysate recovers photoaging in SKH-1 hairless mice.

When the human skin is chronically exposed to external stimuli such as ultraviolet (UV) radiation, the skin tissue suffers damage and the structure of the extracellular matrix (ECM) in the skin is disrupted. This eventually causes symptoms such as wrinkles loss of elasticity, skin sagging, and skin cancer. We previously found that hydrolysate extracted from pacific oyster (Crassostrea gigas) is effective in improving wrinkle formation. In this study, we selected a pentapeptide that was expected to have the most wrinkle reduction effect among the various peptides in oyster hydrolysate through preliminary in vitro screening and examined whether the pentapeptide derived from oyster hydrolysate (OHP) is effective in reducing wrinkles in vivo. We investigated the wrinkle-reducing effect of the OHP through 18-week SKH-1 hairless mice model. Our results showed that the OHP reduces wrinkles lengths, depths, and epidermal thickness which were increased by UVB radiation, and restores the amount of collagen. The OHP recovered the activity of antioxidant enzymes and regulated the expression of proinflammatory cytokines. We also found that OHP increases the expression of type I collagen through stimulating the TGFß/Smad signaling pathway and inhibits the MMPs expression by regulating the MAPK/AP-1 signaling pathway. This study has shown that the OHP plays crucial roles in collagen production and wrinkle reduction in hairless mice and we proved the possibility of the OHP as a component for inhibiting wrinkle formation which was induced by photoaging.

Anti-Inflammatory Activity of ß-thymosin Peptide Derived from Pacific Oyster (Crassostrea gigas) on NO and PGE2 Production by Down-Regulating NF-κB in LPS-Induced RAW264.7 Macrophage Cells.

ß-thymosin is known for having 43 amino acids, being water-soluble, having a light molecular weight and ubiquitous polypeptide. The biological activities of ß-thymosin are diverse and include the promotion of wound healing, reduction of inflammation, differentiation of T cells and inhibition of apoptosis. Our previous studies showed that oyster ß-thymosin originated from the mantle of the Pacific oyster, Crassostrea gigas and had antimicrobial activity. In this study, we investigated the anti-inflammatory effects of oyster ß-thymosin in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells using human ß-thymosin as a control. Oyster ß-thymosin inhibited the nitric oxide (NO) production as much as human ß-thymosin in LPS-induced RAW264.7 cells. It also showed that oyster ß-thymosin suppressed the expression of prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, oyster ß-thymosin reduced inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Oyster ß-thymosin also suppressed the nuclear translocation of phosphorylated nuclear factor-κB (NF-κB) and degradation of inhibitory κB (IκB) in LPS-induced RAW264.7 cells. These results suggest that oyster ß-thymosin, which is derived from the mantle of the Pacific oyster, has as much anti-inflammatory effects as human ß-thymosin. Additionally, oyster ß-thymosin suppressed NO production, PGE2 production and inflammatory cytokines expression via NF-κB in LPS-induced RAW264.7 cells.

Abundance and characteristics of microplastics in market bivalves from South Korea.

Microplastic contamination in marine organisms is a growing environmental issue with implications for seafood safety. Among marine organisms, shellfish are considered to be an important route of human exposure to microplastics because they filter a large volume of seawater while feeding and, thus, accumulate microplastics from seawater; furthermore, they are consumed whole, without gut removal. In this study, a market survey was carried out to understand microplastic contamination in domestic bivalves sold in fishery markets in three major cities of South Korea. Four popular bivalve species, oyster (Crassostrea gigas), mussel (Mytilus edulis), Manila clam (Tapes philippinarum) and scallop (Patinopecten yessoensis), were selected as monitoring species, which together account for 79-84% of total shellfish consumption in Korea. The mean concentration of microplastics in these four species was 0.15 ±â€¯0.20 n/g and 0.97 ±â€¯0.74 n/individual. Fragments and particles smaller than 300 µm were dominant shape and size, accounting for 76% and 65% of total microplastics, respectively. Polyethylene (PE), polypropylene (PP), polystyrene (PS), and polyester were the major polymer types. Interestingly, differing polymer compositions were observed according to the culture methods and habitat characteristics of each species. PS was found in high proportions in oysters and mussels cultured in the upper layer of the water column, while the proportions decreased and those of polyester increased in Manila clams and scallops that were cultured in intertidal sediments or the middle and bottom layers of the water column. The annual dietary intake of microplastics by the Korean population via shellfish consumption was estimated as 212 n/person·year. Our results suggested that microplastic pollution is widespread in commercial bivalves and we recommend a systematic and integrative market-basket survey to clarify the current status of human exposure to microplastics.

Composition characterization of oyster polysaccharides from Crassostrea hongkongensis and their protective effect against H2O2-induced oxidative damage in IEC-6 cells.

The proliferative activity of oyster polysaccharides in intestine epithelial cells (IEC-6) alleviated 5-fluorouracil-induced intestinal inflammation. In this study, we aimed to measure the ability of oyster polysaccharides to promote IEC-6 cell migration and antioxidant activity and further describe their cytoprotective effect on H2O2-challenged IEC-6 cells. The C30-60% fraction of polysaccharides (CHP2) showed rapid stimulation of IEC-6 cell migration after wounding. Then, CHP2 was fractionated into four fractions, namely, CHP2-1, CHP2-2, CHP2-3 and CHP2-4. The CHP2-3 fraction possessed high scavenging activities against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC), in comparison with other fractions. And CHP2-3 was heteropolysaccharide with sulfuric esters, and it was mainly composed of glucose, galactose and arabinose and had an average molecular weight of 41.81 kDa. Pretreatment with CHP2 and CHP2-3 significantly improved the survival rate of H2O2-treated IEC-6 cells, and reduced intracellular reactive oxygen species (ROS) levels. Moreover, CHP2-3 also significantly decreased H2O2-mediated increases in the secretion of interleukin-1ß (IL-1ß) and interleukin-6 (IL-6), and attenuated nuclear factor-κB (NF-κB) p65 activation. These results indicate that CHP2-3 may play a vital role in reducing oxidative damage in IEC-6 cells via radical scavenging, decreasing proinflammatory factors secretion, inhibiting the NF-κB pathway, and thus, reducing cell apoptosis.

Occurrence of antibiotics, estrogenic hormones, and UV-filters in water, sediment, and oyster tissue from the Chesapeake Bay.

Globally, the occurrence of contaminants of emerging concern (CECs) in the environment has raised critical questions on ecological and human health, but few efforts have focused on the Chesapeake Bay, the largest estuary in the United States. Here, 43 antibiotics, 3 estrogenic hormones, and 5 ultraviolet-filters (UV-filters), which are active ingredients in a variety of personal care products, were measured in water, sediment, and oyster tissue from 14 sites along the Eastern Shore of the Chesapeake Bay in Maryland. Fluoroquinolone, macrolide, and sulfonamide antibiotics were detected in water samples. As both human- and animal-labeled antibiotics were found, wastewater effluent and agricultural runoff were identified as potential sources. The highest aqueous-phase concentrations were recorded for norfloxacin (94.1 ng/L), enrofloxacin (17.8 ng/L), sulfamethoxazole (14.8 ng/L), and clarithromycin (9.7 ng/L). Estrone and four UV-filters, namely 2-ethylhexyl-4-methoxycinnamate, benzophenone-3, homosalate, and octocrylene, were frequently detected in Chesapeake Bay water (93-100%), sediment (100%), and oyster tissue (79-100%). High sediment-phase concentrations of estrone (58.4 ng/g) and 17ß-estradiol (11.5 ng/g) were detected at the mouth of the Manokin River. Homosalate and benzophenone-3 were present at concentrations as high as 187.9 and 113.7 ng/L in water, 74.2 and 10.8 ng/g in sediment, and 158.3 and 118.0 ng/g in oyster tissue, respectively. These results demonstrate the ubiquitous presence of CECs in the Chesapeake Bay, confirm UV-filter bioaccumulation in oysters, and suggest the need for improved CEC removal during municipal wastewater treatment and agricultural waste management within the Chesapeake Bay watershed.

Identification and inhibitory activity against α-thrombin of a novel anticoagulant peptide derived from oyster (Crassostrea gigas) protein.

A newly discovered anticoagulant peptide was isolated, purified and identified from the pepsin hydrolysate of oyster (Crassostrea gigas) which could potently prolong the activated partial thromboplastin time and the thrombin time. The anticoagulant peptide with a 1264.36 Da molecular mass was similar to the amino acid sequence of the C-terminal segment (DFEEIPEEYLQ) of hirudin (a potent thrombin inhibitor). The peptide specifically inhibited a vital blood coagulation factor: thrombin. The molecular docking energy scores of the anticoagulant peptide with the active site, exosite-I and exosite-II of thrombin were 132.355 kcal mol-1, 151.266 kcal mol-1 and 147.317 kcal mol-1, respectively. The anticoagulant peptide interacted with thrombin by competing with fibrinogen for an anion-binding exosite I. In the anticoagulant peptide-thrombin complex, there are seven hydrogen bonds and reciprocity exists between hydrogen atoms and oxygen atoms, and electrostatic and hydrophobic interactions are also involved. Such abundant interactions may be accountable for the high affinity and specificity of the anticoagulant peptide.

Photo-enhanced toxicity of undispersed and dispersed weathered Macondo crude oil to Pacific (Crassostrea gigas) and eastern oyster (Crassostrea virginica) larvae.

During the Deepwater Horizon oil spill rapid natural weathering of Macondo crude oil occurred during the transport of oil to coastal areas. In response to the DWH incident, dispersant was applied to Macondo crude oil to reduce the movement of oil to coastal regions. This study aimed to assess the narcotic and phototoxicity of water-accommodated fractions (WAFs) of weathered Macondo crude oil, and chemically-enhanced WAFs of Corexit 9500 to Pacific (Crassostrea gigas) and eastern (Crassostrea virginica) oyster larvae. Phototoxic effects were observed for larval Pacific oysters exposed to combinations of oil and dispersant, but not for oil alone. Phototoxic effects were observed for larval eastern oysters exposed to oil alone and combinations of oil and dispersant. Corexit 9500 did not exhibit phototoxicity but resulted in significant narcotic toxicity for Pacific oysters. Oyster larvae may have experienced reduced survival and/or abnormal development if reproduction coincided with exposures to oil or dispersant.

Particulate nanocomposite from oyster (Crassostrea rivularis) hydrolysates via zinc chelation improves zinc solubility and peptide activity.

An oyster protein hydrolysates-zinc complex (OPH-Zn) was prepared and investigated to improve zinc bioaccessibility. Zinc ions chelating with oyster protein hydrolysates (OPH) cause intramolecular and intermolecular folding and aggregation, homogeneously forming the OPH-Zn complex as nanoclusters with a Z-average at 89.28 nm (PDI: 0.16 ±â€¯0.02). The primary sites of zinc-binding in OPH were carboxyl groups, carbonyl groups, and amino groups, and they were related to the high number of charged amino acid residues. Furthermore, formation of the OPH-Zn complex could significantly enhance zinc solubility both under specific pH conditions as well as during simulated gastrointestinal digestion, compared to the commonly used ZnSO4. Additionally, after digestion, either preserved or enhanced antioxidant activity of OPH was found when chelated with zinc. These results indicated that the OPH-Zn complex could be a potential functional ingredient with improved antioxidant bioactivity and zinc bioaccessibility.

Biochemical bases of growth variation during development: a study of protein turnover in pedigreed families of bivalve larvae (Crassostrea gigas).

Animal size is a highly variable trait regulated by complex interactions between biological and environmental processes. Despite the importance of understanding the mechanistic bases of growth, predicting size variation in early stages of development remains challenging. Pedigreed lines of the Pacific oyster (Crassostrea gigas) were crossed to produce contrasting growth phenotypes to analyze the metabolic bases of growth variation in larval stages. Under controlled environmental conditions, substantial growth variation of up to 430% in shell length occurred among 12 larval families. Protein was the major biochemical constituent in larvae, with an average protein-to-lipid content ratio of 2.8. On average, 86% of protein synthesized was turned over (i.e. only 14% retained as protein accreted), with a regulatory shift in depositional efficiency resulting in increased protein accretion during later larval growth. Variation in protein depositional efficiency among families did not explain the range in larval growth rates. Instead, changes in protein synthesis rates predicted 72% of growth variation. High rates of protein synthesis to support faster growth, in turn, necessitated greater allocation of the total ATP pool to protein synthesis. An ATP allocation model is presented for larvae of C. gigas that includes the major components (82%) of energy demand: protein synthesis (45%), ion pump activity (20%), shell formation (14%) and protein degradation (3%). The metabolic trade-offs between faster growth and the need for higher ATP allocation to protein synthesis could be a major determinant of fitness for larvae of different genotypes responding to the stress of environmental change.

Protective Effects of Enteral Nutrition Supplemented with Crassostrea hongkongensis Polysaccharides Against 5-Fluorouracil-Induced Intestinal Mucosal Damage in Rats.

Chemotherapeutics, including 5-fluorouracil (5-FU), often damage the intestinal mucosal barrier and cause intestinal mucositis (IM). Supplementation with immunoregulatory polysaccharides from Crassostrea hongkongensis has been shown to positively influence the effectiveness and toxicity of 5-FU. Therefore, we studied the effects of oyster polysaccharides on 5-FU-induced intestinal mucosal damage in rats. The C30-60% ethanol-precipitated fraction of polysaccharides promoted IEC-6 cell proliferation and exhibited a maximal effect at a 0.0195 mg/mL concentration. Moreover, treatment with C30-60% polysaccharide-based nutrition formula (OPNF) partially prevented the 5-FU-induced degenerative changes in the histology and ultrastructure of small intestinal mucosa. In addition, the endotoxin level of rats fed with 5-FU and OPNF decreased to the normal control level. Furthermore, the 5-FU-induced increase of proinflammatory cytokine interleukin (IL)-2 and the decrease of anti-inflammatory cytokine IL-10 level in the peripheral blood were significantly attenuated by OPNF administration. In conclusion, Oyster C30-60% polysaccharides can ameliorate 5-FU-induced IM by partially preventing mucosal damage, reducing inflammation, and promoting immunity.

Cadmium effects on DNA and protein metabolism in oyster (Crassostrea gigas) revealed by proteomic analyses.

Marine molluscs, including oysters, can concentrate high levels of cadmium (Cd) in their soft tissues, but the molecular mechanisms of Cd toxicity remain speculative. In this study, Pacific oysters (Crassostrea gigas) were exposed to Cd for 9 days and their gills were subjected to proteomic analysis, which were further confirmed with transcriptomic analysis. A total of 4,964 proteins was quantified and 515 differentially expressed proteins were identified in response to Cd exposure. Gene Ontology enrichment analysis revealed that excess Cd affected the DNA and protein metabolism. Specifically, Cd toxicity resulted in the inhibition of DNA glycosylase and gap-filling and ligation enzymes expressions in base excision repair pathway, which may have decreased DNA repair capacity. At the protein level, Cd induced the heat shock protein response, initiation of protein refolding as well as degradation by ubiquitin proteasome pathway, among other effects. Excess Cd also induced antioxidant responses, particularly glutathione metabolism, which play important roles in Cd chelation and anti-oxidation. This study provided the first molecular mechanisms of Cd toxicity on DNA and protein metabolism at protein levels, and identified molecular biomarkers for Cd toxicity in oysters.

Integrative biomarker assessment of the effects of chemically and mechanically dispersed crude oil in Pacific oysters, Crassostrea gigas.

The impact of dispersed crude oil and dispersant on adult Pacific oysters, Crassostrea gigas, was evaluated through an integrative biomarker approach including (1) biochemical (plasma catecholase- and laccase-type phenoloxidase and superoxide dismutase), (2) histological (digestive cell lysosomal responses, digestive gland histopathology) and (3) physiological (flesh condition index) endpoints in the haemolymph and digestive gland. Adult oysters were exposed to non-contaminated water (control), chemically-dispersed oil (Brut Arabian Light), mechanically-dispersed oil and dispersant (FINASOL®) alone for 2days, and further depurated in non-contaminated water for 4weeks. After exposure to chemically and mechanically dispersed oil oysters exhibited induction of plasma laccase-type phenoloxidase and superoxide dismutase activities, enlargement of digestive cell lysosomes, lipofuscin accumulation, reduced neutral lipid content and atrophy of digestive gland diverticula; more markedly on exposure to chemically dispersed oil. From the studied biomarkers, only lysosomal biomarkers were significantly affected after exposure to the dispersant alone. This included lysosomal enlargement, neutral lipid depletion and lipofuscin accumulation in the digestive gland epithelium. A recovery of plasma enzyme activities was observed after 4weeks of depuration. The integrative biological response index indicated that chemically dispersed oil caused significantly higher stress to C. gigas than the mechanically-dispersed one or the dispersant alone; nevertheless, the response seems to be reversible after depuration.

Production of immunoregulatory polysaccharides from Crassostrea hongkongensis and their positive effects as a nutrition factor in modulating the effectiveness and toxicity of 5-FU chemotherapy in mice.

Chemotherapy is generally accompanied by undesirable side effects, such as immunosuppression and malnutrition, which reduce tolerance to cancer therapies. Prior studies have shown that immunonutrition improves the clinical outcomes of cancer patients. In this study, immunoregulatory polysaccharides from Crassostrea hongkongensis were included in a nutrition formula that was administered to S180 tumor-bearing mice in combination with 5-fluorouracil (5-FU) treatment. The C30-60% fraction of the polysaccharides was characterized as a branched polysaccharide, with a high amount of d-glucose (96.76% of the total) and the highest uronic acid and sulfate groups' content among all of the polysaccharide fractions. The C30-60% polysaccharide fraction showed a maximal proliferative effect on RAW264.7 cells and T lymphocytes at a concentration of 0.0391 mg mL(-1) and 0.0781 mg mL(-1), respectively. Moreover, the combination treatment of the C30-60% polysaccharide-based nutrition formula (OPNF) with the administration of 5-FU effectively inhibited the growth of tumors and notably increased the leucocyte and lymphocyte counts in S180 tumor-bearing mice. In addition, a slight increase in the erythrocyte and hemoglobin values was observed in the mice treated with the combination of OPNF and 5-FU. These results suggest that supplementation with a C30-60%-based enteral formula would be beneficial for patients undergoing chemotherapy with 5-FU.

Mass Spectrometric Quantification of Amphipathic, Polyphenolic Antioxidant of the Pacific Oyster (Crassostrea gigas).

A novel amphipathic phenolic compound, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), that can be isolated from the Pacific oyster (Crassostrea gigas) has been found to protect human hepatocytes against oxidative stress. This study aims to establish a method for the measurement of DHMBA for industrial application. Liquid chromatography-tandem mass spectrometry using deuterated DHMBA as an internal standard and a polar end-capped ODS (Hypersil GOLD aQ) as the solid phase was validated. The limit of detection was 0.04 pmol (S/N = 5), and the limit of quantitation was 0.1 pmol (S/N = 10). The calibration curve was linear throughout the range of 0.1 - 16 pmol (r(2) = 0.9995). This method successfully quantified DHMBA in oysters from 11 sea areas in Japan. The results showed that the yield of DHMBA was variable from 9.8 to 58.8 µg g(-1) whole oyster meat wet weight but not affected by the seawater temperature. The proposed LC-MS/MS method is useful in quantitative studies for DHMBA and potentially for other amphipathic substances.

Impact of Coastal Pollution on Microbial and Mineral Profile of Edible Oyster (Crassostrea rivularis) in the Coastal Waters of Andaman.

The impact of coastal pollution was studied using edible oysters, Crassostrea rivularis as an indicator at two sites viz., North Wandoor (NW) and Phoenix Jetty (PJ) in Port Blair, Andaman. The hydrographic parameters showed that nitrite, nitrate and phosphate concentration were less and dissolved oxygen were more at NW compared to PJ. The oysters were collected from the study sites and biochemical, microbial, mineral profiles and ATPase activities were estimated. ATPase activity was inhibited in the gill tissue of oysters (p<0.05) of PJ sample. Total microbial load in the water and oyster, and coliform bacteria (MPN) in the water were significantly (p<0.05) higher at PJ compared to the NW. There was no significant difference (p>0.05) in the mineral profile of water collected from both the sites. However, calcium and magnesium were more in the oysters collected from NW (p<0.05), and Cu, Zn and Cd were more in PJ samples (p<0.05).

Pacific oyster polyamine oxidase: a protein missing link in invertebrate evolution.

Polyamine oxidases catalyse the oxidation of polyamines and acetylpolyamines and are responsible for the polyamine interconversion metabolism in animal cells. Polyamine oxidases from yeast can oxidize spermine, N(1)-acetylspermine, and N(1)-acetylspermidine, while in vertebrates two different enzymes, namely spermine oxidase and acetylpolyamine oxidase, specifically catalyse the oxidation of spermine, and N(1)-acetylspermine/N(1)-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylpolyamine oxidases have arisen from an ancestor invertebrate polyamine oxidase with lower specificity for polyamine substrates, as demonstrated by the enzymatic activity of the mollusc polyamine oxidase characterized here. This is the first report of an invertebrate polyamine oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to oxidase both N(1)-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.