Purification and characterization of parvalbumins, the major allergens in red stingray (Dasyatis akajei)

Fish has received increasing attention because it induces IgE-mediated food allergy. Parvalbumin (PV) represents the major allergen of fish, and IgE cross-reactivity to PV in various teleost fish species has been shown, while little information is available about allergens in elasmobranch fish. In this study, two PV isoforms (named as PV-I and PV-II) from red stingray (Dasyatis akajei) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose and Sephacryl S-200. Purified PVs revealed a single band on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of PV-I and PV-II were 12.29 and 11.95 kDa, respectively, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot using antifrog PV monoclonal antibody (PARV-19) showed positive reactions to the two proteins, confirming that they were PVs, although their immunological reactivities were weaker than those of PV from silver carp. The N-terminal amino acid sequence of PV-I was determined, and comparison with PVs from other fish species showed low homology between teleost and elasmobranch fish. The isoelectric points of PV-I and PV-II were 5.4 and 5.0, respectively, as determined by two-dimensional electrophoresis (2-DE), suggesting that both isoforms belong to the α-group. IgE immunoblotting analysis showed that sera from fish-allergic patients reacted to both PV-I and PV-II from red stingray. Thermal stability revealed that PV-I easily formed oligomers than PV-II, which might contribute to the maintenance of its allerginicity during heat processing.     

Baking reduces prostaglandin, resolvin, and hydroxy-fatty acid content of farm-raised Atlantic salmon (Salmo salar)

The consumption of seafood enriched in n-3 polyunsaturated fatty acids (PUFA) is associated with a decreased risk of cardiovascular disease. Several n-3 oxidation products from eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (22:6n-3) have known protective effects in the vasculature. It is not known whether the consumption of cooked seafood enriched in n-3 PUFA causes appreciable consumption of lipid oxidation products. We tested the hypothesis that baking Atlantic salmon (Salmo salar) increases the level of n-3 and n-6 PUFA oxidation products over raw salmon. We measured the contents of several monohydroxy-fatty acids (MHFA), prostanoids, and resolvins. Our data demonstrate that baking did not change the overall total levels of MHFA. However, baking resulted in selective regioisomeric loss of hydroxy fatty acids from arachidonic acid (20:4n-6) and EPA, while significantly increasing hydroxyl-linoleic acid levels. The contents of prostanoids and resolvins were reduced several-fold with baking. The inclusion of a coating on the salmon prior to baking reduced the loss of some MHFA but had no effect on prostanoid losses incurred by baking. Baking did not decrease n-3 PUFA contents, indicating that baking of salmon is an acceptable means of preparation that does not alter the potential health benefits of high n-3 seafood consumption. The extent to which the levels of MHFA, prostanoids, and resolvins in the raw or baked fish have physiologic consequence for humans needs to be determined.     

Detection, accumulation, distribution, and depletion of furaltadone and nifursol residues in poultry muscle, liver, and gizzard

Nitrofurans were broadly used as an extremely effective veterinary antibiotic especially in pig and poultry production farms. Because of fears of the carcinogenic effects on humans, the nitrofurans were banned from use in livestock production in many countries, including the European Union. The present study examines the accumulation, distribution, and depletion of furaltadone and nifursol and of their tissue-bound metabolites [3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and 3,5-dinitro-salicylic acid hydrazine (DNSAH), respectively, in poultry edible tissues (muscle, liver, and gizzards) following administration to chickens of therapeutic and subtherapeutic concentrations of both compounds. Nitrofurans determination was performed by high-performance liquid chromatography-diode array detection and liquid chromatography-tandem mass spectrometry, respectively, for feeds and for poultry tissues. Furaltadone and nifursol, in very low concentrations, were found in samples of muscle, liver, and chicken's gizzard collected from slaughtered animals after 5 weeks of treatment and no withdrawal time period. When a withdrawal time period of 3 weeks was respected, no detectable nitrofuran parent compounds was observed in all of the studied matrices. For AMOZ, concentrations of 270 μg/kg in meat, 80 μg/kg in liver, and 331 μg/kg in gizzard were determined after administration of a medicated feed with furaltadone (132 mg/kg), 3 weeks after withdrawal of treatment. For DNSAH, the concentration values obtained are much lower than those observed for AMOZ. For meat, liver, and gizzard, DNSAH concentrations of 2.5, 6.4, and 10.3 μg/kg, respectively, were determined, after administration of a medicated feed with nifursol (98 mg/kg), 3 weeks after withdrawal of treatment. The gizzard could be considered a selected matrix for nitrofuran residues evaluation in poultry, due to its capacity of retaining either nitrofuran parent compounds or metabolites in higher concentrations, regardless of the administered dose or of the respected withdrawal time period.     

Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution

A leucine aminopeptidase was purified for the first time from marine fish red sea bream ( Pagrus major) skeletal muscle to homogeneity with 4850-fold and a yield of 7.4%. The purification procedure consisted of ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, and phenyl-Sepharose. The enzyme was approximately 96 kDa as estimated by SDS-PAGE and gel filtration and preferentially hydrolyzed substrate Leu-MCA. The enzymatic activity was optimal at 45 degrees C and pH 7.5. The K m and k cat values of the enzyme for Leu-MCA were 1.55 microM and 26.4 S (-1) at 37 degrees C, respectively. Activation energy ( E a) of the enzyme was 59.6 kJ M (-1). The enzyme was specifically inhibited by metal-chelating agents, and Zn (2+) and (or) Mn (2+) seemed to be its metal cofactor(s). In addition, bestatin strongly inhibited its activity, and K i was 1.44 microM. Using a highly specific polyclonal antibody, the location of enzyme was demonstrated intracellularly and distributed in different tissues.     

Minor Elements and Trace Metals in the Shell of Marine Gastropods from a Shore in Tropical East Africa

The concentrations of minor elements (magnesium and strontium)and trace metals (iron, manganese, copper and zinc) in the shellof fifty-nine species of marine gastropod collected from a shorein tropical East Africa are reported. Mean trace metalconcentrations and range of concentrations in the fifty-threespecies having an aragonitic shell were: iron, 17.5 μg g^-1, range 7.2 to 30.6 μg g^-1; manganese 2.4 μgg^-1, range 1.8 to 3.4 μg g^-1; copper, 1.2 μgg^-1, range 0.6 to 2.4 μg g^-1 and zinc, 1.5 μgg^-1, range 0.8 to 2.6 μg g^-1. The six species ofthe Nerita included in the study all had a shell of mixedmineralogy, a high magnesium concentration (2550 to 3407 μgg^-1) and relatively elevated concentrations of the tracemetals, manganese, copper and zinc (mean concentrations, 7.5,3.4 and 5.5 μg g^-1, respectively). Highestconcentrations of all three metals occurred in the shell of N. albicilla. Manganese, copper and zinc concentrations inshells of a population of N. albicilla (n = 20) taken fromthe Kenyan coast were higher than those in a similar populationof shells taken from Aldabra Atoll (Seychelles) by factors of29, 10 and 2.6, respectively. Of the fifty-nine species ofmarine gastropod included in this study the shell of N.albicilla is identified as having the greatest potential as abiomonitoring tissue for trace metals.     

Fatty acid pattern, oxidation product development, and antioxidant loss in muscle tissue of rainbow trout and Dicentrarchus labrax during growth

The levels of hydrophilic, lipophilic, and enzymatic antioxidants, the oxidative damage to lipids and proteins, and the fatty acid patterns of triglyceride and phospholipid fractions were assayed in fresh muscle tissue of rainbow trouts (Oncorhynchus mykiss) and sea basses (Dicentrarchus labrax) during aging, to investigate the correlation between oxidative stress and aging processes in fish. The present studies suggests that lipid peroxidation and accumulation of oxidized proteins during in vivo aging are most likely to be linked with an age-dependent decline of lipophilic antioxidants (CoQH(2), CoQ, and vitamin E) and vitamin C contents in muscle tissue, whereas fish aging is not linked to a decline in antioxidant enzymes and reduced glutathione levels. Lipophilic antioxidant and vitamin C levels represent a reliable marker of oxidative stress during aging, and their determination might be useful for the assessment of fish age.     

Fatty acid, triacylglycerol, phytosterol, and tocopherol variations in kernel oil of Malatya apricots from Turkey

The fatty acid, sn-2 fatty acid, triacyglycerol (TAG), tocopherol, and phytosterol compositions of kernel oils obtained from nine apricot varieties grown in the Malatya region of Turkey were determined ( P<0.05). The names of the apricot varieties were Alyanak (ALY), Cataloglu (CAT), C?loglu (COL), Hacihaliloglu (HAC), Hacikiz (HKI), Hasanbey (HSB), Kabaasi (KAB), Soganci (SOG), and Tokaloglu (TOK). The total oil contents of apricot kernels ranged from 40.23 to 53.19%. Oleic acid contributed 70.83% to the total fatty acids, followed by linoleic (21.96%), palmitic (4.92%), and stearic (1.21%) acids. The s n-2 position is mainly occupied with oleic acid (63.54%), linoleic acid (35.0%), and palmitic acid (0.96%). Eight TAG species were identified: LLL, OLL, PLL, OOL+POL, OOO+POO, and SOO (where P, palmitoyl; S, stearoyl; O, oleoyl; and L, linoleoyl), among which mainly OOO+POO contributed to 48.64% of the total, followed by OOL+POL at 32.63% and OLL at 14.33%. Four tocopherol and six phytosterol isomers were identified and quantified; among these, gamma-tocopherol (475.11 mg/kg of oil) and beta-sitosterol (273.67 mg/100 g of oil) were predominant. Principal component analysis (PCA) was applied to the data from lipid components of apricot kernel oil in order to explore the distribution of the apricot variety according to their kernel's lipid components. PCA separated some varieties including ALY, COL, KAB, CAT, SOG, and HSB in one group and varieties TOK, HAC, and HKI in another group based on their lipid components of apricot kernel oil. So, in the present study, PCA was found to be a powerful tool for classification of the samples.     

At-sea distribution of breeding Tristan albatrosses Diomedea dabbenena and potential interactions with pelagic longline fishing in the South Atlantic Ocean

The endangered Tristan albatross Diomedea dabbenena is restricted to Gough and Inaccessible Islands. The species is killed as bycatch by longline fisheries in the South Atlantic, but the impact of this mortality is unknown. We satellite tracked 38 breeding Tristan albatrosses and assessed the seasonal and annual at-sea distribution of these birds in relation to reported pelagic longline fishing effort. These birds ranged across the South Atlantic from 50°W to 15°E with most (97%) daytime satellite fixes between latitudes 30°S and 45°S. Considerable fishing effort occurred within the same latitudes. Although there was no correlation between their at-sea distributions, there was a broad overlap between birds and fishing effort. Estimated bycatch rates for Tristan albatross and other Diomedea species in the South Atlantic, and the spatio-temporal overlap between birds and hooks, yield a predicted annual mortality of 471-554 birds, sufficient to cause population decreases of 3.6-4.3% per year. An index of bird × hook interactions (proportional density of birds multiplied by number of hooks by decadal period for each 5° square of longitude and latitude) indicated that 47% of annual interactions occurred in areas around Gough Island, and 11% and 15% of interactions in areas of the west and east Atlantic, respectively. There were also within seasonal differences in the key areas of overlap. The fishing fleets of Taiwan and Japan are likely to be responsible for most interactions based upon the reported magnitude of effort expended in the South Atlantic by these fleets. Ensuring that licensed fishing vessels within the Tristan da Cunha Exclusive Economic Zone (EEZ) operate using best-practise mitigation measures and with fisheries observer programs, could reduce the potential bycatch mortality of breeding Tristan albatrosses in this region by nearly one third. Thorough implementation of international agreements is required in areas of the high seas where most remaining interactions are predicted to occur.     

Annual evolution of fatty acid profile from muscle lipids of the common carp (Cyprinus carpio) in Madagascar inland waters

Annual evolution of muscle lipids fatty acid (FA) from common carp (Cyprinus carpio) has been determined in 2001 through monthly samplings in the reserve pond of Sisaony (SIS series) and Itasy Lake (ITA series) of the Madagascar highlands. Total lipids from muscle were extracted and quantified according to the Bligh and Dyer method. FA identification was performed by GC-MS of FA methyl esters and FA pyrrolidides and led to the identification of 41 FA; routine analyses of FA were made by capillary GC. Principal component analysis (PCA) was performed on the data set to compare FA profiles. Lipid content is low, ranging from 0.91 to 1.73% of wet muscle, with a low stage during the hot season (January-April) and a higher stage during the cold season (July-October). Three FA dominated the FA composition: oleic acid (17.0-21.5%), palmitic acid (13.1-16.1%), and linoleic acid (9.6-13.2%). Polyunsaturated fatty acids (PUFA) were present in appreciable amounts: arachidonic acid (AA; 2.9-5.9%), docosahexaenoic acid (DHA; 2.9-6.7%), eicosapentaenoic acid (EPA; 1.9-3.4%), and docosapentaenoic acid (DPA; 1.9-4.3%). Two opposite evolution schemes appear within two groups of FA; on the one hand PUFA (both n-3 and n-6 series) show a maximum in August-October and a minimum in January-April, and, on the other hand, oleic, palmitic, and linoleic acids show the opposite maxima and minima. PCA results give confirmation of these evolution schemes, the two groups of FA giving opposite high factor loadings on axis 1. The SIS and ITA series are differentiated by axis 2 by mean of minor FA, mostly odd- and branched-chain. Results indicate that common carp, the second most abundant freshwater fish in Madagascar highlands waters, may be an interesting source of dietary PUFA.     

Fatty acid profiles, tocopherol contents, and antioxidant activities of heartnut (Juglans ailanthifolia Var. cordiformis) and Persian walnut (Juglans regia L.)

The fatty acid and tocopherol compositions of three heartnut (Juglans ailanthifolia var. cordiformis) varieties (Imshu, Campbell CW1, and Campbell CW3) were examined and compared with those of two Persian walnut (Juglans regia L.) varieties (Combe and Lake). The major fatty acids found in heartnuts and walnuts were identified by gas chromatography as linoleic (18:2n-6), alpha-linolenic (18:3n-3), oleic (18:1n-9), palmitic (16:0), and stearic acid (18:0). Polyunsaturated fatty acids were the main group of fatty acids found in both heartnut and walnut, ranging from 73.07 to 80.98%, and were significantly higher in heartnut than in Persian walnuts (P < 0.001). In addition, heartnuts had significantly higher levels of 18:2n-6 and lower levels of 18:3n-3 compared to the Persian walnuts. gamma-Tocopherol was the main tocopherol homologue present in both types of nuts, followed by delta- and alpha-tocopherol. The highest concentration of gamma-tocopherol was found in Combe Persian walnut at 267.87 mug/g, followed by Lake Persian walnut and Imshu, Campbell CW1, and CW3 heartnut at 205.45, 187.33, 161.84, and 126.46 mug/g, respectively. Tocopherols, particularly the gamma-tocopherol, were found to contribute the most to the strong total antioxidant activities of both walnut and heartnut oils using either the free radical 2,2-diphenyl-1-picrylhydrazyl assay or the photochemiluminescence method.     

Trace Metal Content Trend of Mussel Perna Perna (Linnaeus, 1758) from the Atlantic Coast of Southern Brazil

The concentration assessment of Hg, Pb, Cd, Cu, Zn, Cr, Mnand Fe in marine mussels Perna perna, sampled at 15stations along the 800 km Southern Brazilian Coast, has beencarried out. Samplings were taken twice, in summer andwinter of 2000. The concentrations found were lower thansimilar studies carried out along the Central Coast of Brazil andlower than the maximum limits permitted in bivalve molluscsdesignated for human consumption (Brazilian, Spanish andLuxembourg legislation sets). The data also showed lowervalues compared with other regions of the world. Thecomparison of present data with the data obtained 10 years agoat the same stations did not show significant differences,consequently the concentrations found could be used asbaseline values.     

Uptake by dietary exposure and elimination of aflatoxins in muscle and liver of rainbow trout (Oncorhynchus mykiss)

Uptake and elimination of aflatoxins (AFs) by rainbow trout ( Oncorhynchus mykiss ) during a long-term (21 days) dietary exposure were studied to assess contamination by AFs in aquaculture fish fed AF-containing feed. The uptake factor (UF) of aflatoxin B(1) (AFB(1)) in muscle ranged from 0.40 × 10(-3) to 1.30 × 10(-3). AFB(1) concentrations in liver were 165-342 times higher than in muscle. AFs from feed were more highly accumulated in liver than in muscle. Aflatoxicol (AFL) and aflatoxin M(1) (AFM(1)) were detected in muscle and liver and also in the rearing water. AFL concentrations were higher than AFM(1) by 2 orders of magnitude in muscle, and AFL was a major metabolite of AFB(1). The elimination rate constants (α) of AFB(1) and AFL in muscle (1.83 and 2.02 day(-1), respectively) and liver (1.38 and 2.41 day(-1), respectively) were very large. The elimination half-life (t(1/2)) of AFB(1) was 0.38 days (9.12 h) in muscle and 0.50 days (12.00 h) in liver. The elimination half-life of AFL in muscle and liver was 0.34 day (8.16 h) and 0.29 day (6.96 h), respectively. These data show that AFs are eliminated rapidly and are not biomagnified in fish. Thus, AFB(1) concentration in muscle of fish fed AFB(1)-containing feed (ca. 500 μg/kg) decreased to below the detection limit (20 ng/kg) of the most sensitive analytical method at 1.54 days (36.96 h) after the change to uncontaminated feed.     

Gluconic acid,its lactones,and SO(2) binding phenomena in musts from botrytized grapes

Intramolecular gluconic acid esterification reactions led to the formation of two lactones, gamma- and delta-gluconolactone (glucono-1,4-lactone and glucono-1,5-lactone). The presence of the first has not yet been reported in must or wine. These lactones are in equilibrium with gluconic acid, gamma- and delta-gluconolactone representing, respectively, 5.8 and 4.1% of the acid level. Correlations between must SO(2) binding power, gluconic acid, and consequently its lactones are shown. The SO(2) affinity of a mixture containing this acid and gamma- and delta-gluconolactone was determined, and gluconic acid appeared to be indirectly responsible for approximately 8% of the bindable SO(2) in musts from botrytized grapes.     

29 kDa Trypsin from the pyloric ceca of Atlantic Bonito (Sarda sarda): recovery and characterization

Trypsin from the pyloric ceca of Atlantic bonito (Sarda sarda) was purified and characterized with respect to its purity; molecular weight; sensitivity to temperature, pH, and inhibition; and N-terminal sequence. The purified trypsin had a molecular weight of 29 kDa as per sodium dodecyl sulfate polyacrylamide gel electrophoresis, and optimal activity was observed at pH 9 and 65 degrees C with BAPNA as a substrate. The enzyme was stable to heat treatment up to 50 degrees C and within the pH range of 7-12. It was stabilized by calcium ions, but its activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone, and phenyl methyl sulfonyl fluoride. The enzyme exhibited a progressive decrease in activity with increasing NaCl concentration (0-30%). The N-terminal 20 amino acid residues of Atlantic bonito trypsin were determined as IVGGYECQAHSQPWQPVLNS and were homologous with other trypsins.     

Occurrence and distribution of organochlorine pesticides (OCPs) in tomato (Lycopersicon esculentum) crops from organic production

Organochlorine pesticides (OCPs) were quantified by GC-ECD in tomato (Lycopersicon esculentum) during a vegetation period. Plants were harvested at 15, 60, and 151 days after seed germination. Leaves, stem, roots, and fruit (peel and flesh) were analyzed separately. The results showed that tomato plants were able to accumulate OCPs from soils, and a trend to reach the equilibrium among tissues at mature stages was also observed. Endosulfans comprised the main OCP group, probably due to its spray during summer months in the surrounding areas. Banned pesticides such as DDTs, heptachlor, and dieldrin were found. OCPs levels in the fruit were below the maximum residues limits (MRL) considered by the Codex Alimentarius. DDE/DDT and alpha-/gamma-HCH ratios of <1 would indicate recent inputs of DDT and lindane in the environment. The occurrence of OCPs in the study farm, where agrochemicals have never been used, is a result of atmospheric deposition of those pesticides.     

Purification, characterization, and cDNA cloning of a myofibril-bound serine proteinase from the skeletal muscle of crucian carp (Carassius auratus)

A myofibril-bound serine proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by serine proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type serine proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of serine proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of serine proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other serine proteinases, especially in well-conserved regions.     

Purification and characterization of cathepsin L from the muscle of silver carp (Hypophthalmichthys molitrix)

Cathepsin L in silver carp musle was purified to 48.4-fold by acid-heat treatment and ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 30 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64 and insensitive to PMSF and pepstatin A, suggesting that the purified enzyme belongs to a family of cysteine proteinase. Consistent with this conclusion, Zn2+, Cu2+, Co2+, Ni2+, and Fe2+ could strongly inhibit the activity of this enzyme. The optimal pH and temperature were 5.0 and 55 degrees C, respectively. The enzyme catalyzed the hydrolysis of Z-Phe-Arg-MCA with a parameter of K(m) (8.27 microM) and K(cat) (28.7 s(-1)) but hardly hydrolyzed Z-Arg-Arg-MCA, Arg-MCA, and Boc-Val-Leu-Lys-MCA. The microstructure analysis by scanning electron microscopy showed that this proteinase is capable of destroying the network structure of silver carp surimi gels. The enzyme exhibited a higher hydrolytic activity on surimi protein at 65 degrees C than at 40 degrees C.     

Isolation and structure of pulcherrimine, a novel bitter-tasting amino acid, from the sea urchin (Hemicentrotus pulcherrimus) ovaries

A novel sulfur-containing amino acid, pulcherrimine, has been isolated as a bitter principle from ovaries of the sea urchin Hemicentrotus pulcherrimus. The structure was elucidated as 4-(2'-carboxy-2'-hydroxy-ethylthio)-2-piperidinecarboxylic acid by spectroscopic and chemical methods. Absolute stereochemistry was determined by NOE experiments and chiral HPLC analysis. Pulcherrimine exhibited bitterness with a threshold value of 0.306 mM.