Functional and Structural Properties of Type V Collagen from the Skin of the Shortbill Spearfish (Tetrapturus angustirostris).

Type V collagen is considered to be a crucial minor collagen in fish skin with unique physiological functions. In this research, the cDNAs of three procollagens (Tacol5a1, Tacol5a2, and Tacol5a3) in type V collagen were cloned from the skin of shortbill spearfish (Tetrapturus angustirostris). The open reading frames (ORFs) of Tacol5a1, Tacol5a2, and Tacol5a3 contained 5991, 4485, and 5607 bps, respectively, encoding 1997, 1495, and 1869 amino acid residues. Each of the deduced amino acid sequences of procollagens contained a signal peptide and a fibrillar collagen C-terminal domain (COLFI). A conserved thrombospondin-like N-terminal domain (TSPN) was found at the N-terminus of Tacol5a1 and 5a3 procollagens, whereas a von Willebrand factor (VWC) was found at the N-terminus of Tacol5a2 procollagen. Tacol5a1, Tacol5a2, and Tacol5a3 had their theoretical isoelectric points of 5.06, 6.75, and 5.76, respectively, and predicted molecular weights of 198,435.60, 145,058.48, and 189,171.18, respectively. The phylogenetic tree analysis revealed that Tacol5a1 of shortbill spearfish clustered with that of yellow perch (Perca flavescens) instead of broadbill swordfish (Xiphias gladius). In addition, type V collagen was extracted from the shortbill spearfish skin. The in silico method demonstrated that shortbill spearfish type V collagen has a high potential for angiotensin-converting enzyme (ACE) inhibition activity (79.50%), dipeptidyl peptidase IV inhibition (74.91%) activity, and antithrombotic activity (46.83%). The structural clarification and possible functional investigation in this study provide the foundation for the applications of exogenous type V collagen derived from fish sources.

Preparation, Purification and Characterization of Antibacterial and ACE Inhibitory Peptides from Head Protein Hydrolysate of Kuruma Shrimp, Marsupenaeus japonicus.

Kuruma shrimp (Marsupenaeus japonicus) heads, as the main by-product of the seafood processing industry, are rich in underutilized high-quality protein. After papain hydrolysis at 50 °C for 4 h, the protein hydrolysate of shrimp heads was found to show notable antibacterial and angiotensin I-converting enzyme (ACE) inhibitory activities. After purification using two stages of revered-phase high-performance liquid chromatography (RP-HPLC), the antibacterial peptide VTVP and the ACE inhibitory peptide ARL/I were successfully identified from most active fractions by LC-MS/MS. Peptide VTVP was a desirable hydrophobic peptide, with a MIC value in the range from 1.62 to 8.03 mM against all tested pathogens. Peptide ARL/I exhibited potent ACE inhibitory activity, with an IC50 value of 125.58 µM, and was found to be a competitive inhibitor based on the Lineweaver-Burk plot. Moreover, the result of the molecular docking simulation indicated that the interaction binding between ARL/I and ACE was mainly stabilized by hydrogen bonds, as well as forming a coordinate bond with the Zn2+ site. The purified peptides did not show hemolytic activity toward rabbit erythrocytes. To sum up, the bioactive peptides isolated from shrimp heads could be applicable for food or pharmaceutical areas as promising ingredients.

Isolation and Characterization of Collagen and Collagen Peptides with Hyaluronidase Inhibition Activity Derived from the Skin of Marlin (Istiophoridae).

Type I and V collagens are the major components of fibrillogenic proteins in fish skin, and their hydrolysis products possess hyaluronidase inhibitory activity. In this study, for the first time, type I and V collagens were isolated from the skin of shortbill spearfish and striped marlin. Type I (2α1[I]α2[I]) and type V (α1[V]α3[V]α2[V]) collagens composed of distinct α-peptide chains with comparable structures were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and UV spectrophotometric chromatography. After enzymatic digestion, the collagen peptides were purified by using ultrafiltration (30 KDa) and high-performance liquid chromatography (RP-HPLC) to yield CPI-F3 and CPV-F4 fractions with strong hyaluronidase inhibition rates (42.17% and 30.09%, respectively). Based on the results of simulated gastrointestinal fluid, temperature, and pH stability assays, CPI-F3 and CPV-F4 exhibited stability in gastric fluid and showed no significant changes under the temperature range from 50 to 70 °C (p > 0.05). The results of this first research on the bioactivity of type V collagen peptides provide valuable information for the biomedical industry and show the potential for future bioactivity investigations of type V collagen and its peptides.

Seawater activates l-amino acid oxidase from the serum of the red-spotted grouper Epinephelusakaara.

l-amino acid oxidases (LAOs) catalyze the oxidative deamination of l-amino acid and generate α-keto acid, ammonia, and hydrogen peroxide as byproducts. LAOs showed the variety of bioactivity by the resulting hydrogen peroxide. The serum of the red-spotted grouper Epinephelus akaara contains an LAO (Ea-LAO) with the potential to kill bacterial pathogens Aeromonas salmonicida and Vibrio anguillarum via hydrogen peroxide. However, it is unknown how the grouper tolerates the harmful effects of the serum Ea-LAO byproducts. In this study, we analyzed the kinetics of fish LAOs to understand how they escape the toxicity of byproducts. The LAO activity of grouper serum was suppressed in low-salt solutions such as NaCl, CaCl2, MgCl2, and diluted seawater. The activity was non-linearly increased and fitted to the four-parameter log-logistic model. The EC50 of the seawater was calculated to have a 0.72-fold concentration. This result suggested that the Ea-LAO could be activated by mixing with seawater. The results of circular dichroism spectroscopy showed that the α helix content was estimated to be 12.1% and 5.3% in a salt-free buffer (inactive condition) and the original concentration of seawater (active condition), respectively, indicating that the secondary structure of the Ea-LAO in the active condition was randomized. In addition, the Ea-LAO showed reversible LAO activity regulation according to the salt concentration in the environment. Taken together, this indicates that the Ea-LAO is normally on standby as an inactive form, and it could activate as a host-defense molecule to avoid pathogen invasion via a wound when mixed with seawater.

[Tetramine Contents in the Salivary Glands from 16 Species of Marine Carnivorous Gastropods Collected along Japanese Coasts].

Tetramine (tetramethyl ammonium ion), a neurotoxin, is present at high levels in the salivary glands of buccinid gastropods and is responsible for human intoxication due to consumption of the gastropods. We used LC-MS/MS to examine the tetramine contents of salivary glands from 16 species of carnivorous gastropods collected along Japanese coasts. Tetramine was detected in all specimens except for Babylonia japonica. High levels of tetramine were detected in whelks, Neptunea lamellosa (1,380-9,410 µg/g of salivary gland) and N. purpurea (1,190-7,400 µg/g of salivary gland). Although consumption of N. lamellosa is well-known cause of foodborne tetramine poisoning, it was newly discovered that N. purpurea has tetramine. In addition, we found 7 other species of gastropods containing tetramine: Siphonalia cassidariaeformis (117-135 µg/g), S. fusoides (204 µg/g), Buccinum inclytum (2.94-3.40 µg/g), and B. aniwanum (0.700 µg/g) of the family Buccinidae, and Fusinus perplexus (397 µg/g), F. ferrugineus (105 µg/g), and F. forceps salisburyi (67.5 µg/g) of the family Fasciolariidae. The present study, together with previous studies, shows that gastropods with salivary glands containing more than 1,000 µg tetramine/g of salivary gland, including the genus Neptunea as well as Fusitriton oregonesis and Hemifusus tuba, carry a high risk of tetramine poisoning, and their salivary glands should be removed before consumption to prevent food poisoning.

Cholesterol-binding ability of saponin from Japanese starfish.

Steroidal gylcosides are the predominant metabolites of starfish and are responsible for various biological activities. Some of these activities are recognized as a part of self-defense mechanism of starfish. Cholesterol-binding ability was evaluated with seven starfish crude extracts, where significantly (p < 0.05) highest ability (34%) was observed in Asterias amurensis and the lowest (16%) was attributed in Distolasterias nippon. To characterize the active compound exists in crude saponin from A. amurensis, the extract was subjected to thin layer chromatography following silica gel column chromatography. As the results, seven fractions (fr. A-G) were separated and frs. D and F demonstrated the highest cholesterol-binding ability (32% and 33%, respectively), equivalent to that of the A. amurensis extract. The isolated component (fr. F) was further separated (fr. F1-F3) for structural analysis. Based on cholesterol-binding ability result (29%), fr. F2 was analysed by matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS) and then nuclear magnetic resonance spectroscopy (NMR). The compound was identified as thornasteroside A, one of the major bioactive compounds already found in A. amurensis. The discovery of a saponin with cholesterol-binding ability has important implications not only for the utilization of starfish but also for food and pharmaceutical research.

Functional and structural properties of red color-related pigment-binding protein from the shell of Litopenaeus vannamei.

BACKGROUND: A novel red color-related pigment-binding protein named LvPBP75 isolated from the shell of Litopenaeus vannamei has recently been identified as hemocyanin. However, information on the functional and structural properties of LvPBP75 is insufficient. This study aimed to elucidate the thermal properties and pigment-binding ability of LvPBP75. RESULTS: LvPBP75 showed significant red color change after heat treatment with high concentrations of NaCl (>0.1 mol L-1 ), acidic (<5) or alkaline (>9) pH values and alcohols. LvPBP75 mRNA expression analysis revealed that expression level was highest in hepatopancreas and weakest in muscle. Reconstruction and structural analysis revealed that astaxanthin could bind to hemocyanin derived from the shell of L. vannamei but not to hemocyanins derived from the hepatopancreas or hemolymph of other invertebrates. Three-dimensional models of hemocyanin monomer displayed significant structural differences between native LvPBP75 and hemocyanin derived from shrimp hepatopancreas. CONCLUSION: The results suggest a novel function of hemocyanin as binding with pigment and its involvement in L. vannamei shell color change. The pigment-binding ability of hemocyanins has species and tissue specificity, and their unique structural features play an important role in binding ability. © 2018 Society of Chemical Industry.

Structural and dynamical characteristics of tropomyosin epitopes as the major allergens in shrimp.

Ingestion of marine invertebrates often causes food allergy, where the major allergens have been reported to be derived from tropomyosin (TM). Intact or the digestive fragments of food allergens generally show resistance to digestion, which is usually attributable to the structural stability (or rigidity). The difference in the structural and dynamical characteristics between the epitope and the non-epitope regions in TM has not yet been well understood. In the present study, molecular dynamics simulation was performed at constant pHs for shrimp TM. By analyzing the main-chain dihedral angle fluctuations and local α-helix contents, we found that the epitope regions are more stable than the non-epitope counterparts, providing a possible physical reason for the resistance to digestion in the epitopes regions. The difference of the structural stability between the epitope and the non-epitope regions was largest at low pHs, even though pH dependence of the structural stability in itself was not significant in both regions. The lower content of the Ala cluster in the epitope region is considered to cause the higher stability of the epitope region.

Difference in Uptake of Tetrodotoxin and Saxitoxins into Liver Tissue Slices among Pufferfish, Boxfish and Porcupinefish.

Although pufferfish of the family Tetraodontidae contain high levels of tetrodotoxin (TTX) mainly in the liver, some species of pufferfish, boxfish of the family Ostraciidae, and porcupinefish of the family Diodontidae do not. To clarify the mechanisms, uptake of TTX and saxitoxins (STXs) into liver tissue slices of pufferfish, boxfish and porcupinefish was examined. Liver tissue slices of the pufferfish (toxic species Takifugu rubripes and non-toxic species Lagocephalus spadiceus, L. cheesemanii and Sphoeroides pachygaster) incubated with 50 µM TTX accumulated TTX (0.99-1.55 µg TTX/mg protein) after 8 h, regardless of the toxicity of the species. In contrast, in liver tissue slices of boxfish (Ostracion immaculatus) and porcupinefish (Diodon holocanthus, D. liturosus, D. hystrix and Chilomycterus reticulatus), TTX content did not increase with incubation time, and was about 0.1 µg TTX/mg protein. When liver tissue slices were incubated with 50 µM STXs for 8 h, the STXs content was <0.1 µg STXs/mg protein, irrespective of the fish species. These findings indicate that, like the toxic species of pufferfish T. rubripes, non-toxic species such as L. spadiceus, L. cheesemanii and S. pachygaster, potentially take up TTX into the liver, while non-toxic boxfish and porcupinefish do not take up either TTX or STXs.

Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK1.

This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs).

Molecular Localization of Health-Promoting Peptides Derived from Fish Protein Hydrolyzates on Fish Muscle Proteins.

The four previously reported health-promoting dipeptides, valine-tyrosine, lysine-tryptophan, methionine-phenylalanine, and arginine-isoleucine, found in the fish muscle hydrolyzates, were mainly located in the myosin subfragment-1 heavy chain, whereas the health-promoting tripeptide, alanine-lysine-lysine, was found in the fibrous rod consisting of the myosin subfragment-2 and light meromyosin with a regular coiled-coil structure of α-helix, irrespective of the fish species. Furthermore, the localization of these peptides either in the random coil, ß-sheet, or α-helix was also examined in the three-dimensional image, showing no specific tendency. Surprisingly, the same trend was observed even for the mammalian rabbit fast muscle myosin heavy chain. Since a trade-off between myofibrillar ATPase and structural stability has been reported for fish living at low environmental temperatures, it is speculated that fish muscle proteins, when ingested, are easily digested by various proteases in the human digestive tract and provide various health-promoting peptides also in vivo. While fish actin contained only two dipeptides, methionine-phenylalanine and valine-tyrosine, glyceraldehyde 3-phosphate dehydrogenase, one of the major components of fish muscle water-soluble protein, contained all of the four dipeptides and one tripeptide mentioned above.

Molecular cloning and functional expression of allergenic sarcoplasmic calcium-binding proteins from Penaeus shrimps.

BACKGROUND: Sarcoplasmic calcium-binding proteins (SCPs) have recently been identified as crustacean allergens. However, information on their primary structures is very limited and no recombinant SCP (rSCP) as an alternative of natural SCP (nSCP) is available. This study was aimed to elucidate primary structures of SCPs from two species of Penaeus shrimp (black tiger shrimp and kuruma shrimp) by cDNA cloning and to produce a black tiger shrimp rSCP preparation that is comparable in IgE reactivity to nSCP. RESULTS: The full-length cDNAs encoding black tiger shrimp and kuruma shrimp SCPs were successfully cloned. Both SCPs are composed of 193 amino acid residues and share more than 80% sequence identity with the known crustacean SCPs. The black tiger shrimp SCP was then expressed in Escherichia coli using the pFN6A (HQ) Flexi vector system. Enzyme-linked immunosorbent assay (ELISA) and inhibition ELISA experiments demonstrated that rSCP has the same IgE reactivity as nSCP. CONCLUSION: Our results provide further evidence for the high sequence identity among crustacean SCPs. In addition, rSCP will be a useful tool in studying crustacean allergens and also in the diagnosis of crustacean allergy.

Specific detection by the polymerase chain reaction of potentially allergenic salmonid fish residues in processed foods.

Salmonid fish is one of the allergenic items that are recommended to be labeled in the Japanese allergen-labeling system. This study develops a salmonid-specific polymerase chain reaction (PCR) method. A new primer pair, SKE-F/SKE-R, was designed to specifically detect the salmonid fish gene encoding mitochondrial DNA cytochrome b. Genomic DNAs extracted from 58 kinds of seafood and 11 kinds of processed food were individually subjected to PCR by using the primer pair, and a salmonid-specific fragment of 212 bp was only amplified in the salmonid samples and salmonid-containing processed foods. The detection limit of the PCR method was as low as 0.02 fg/µL of salmonid fish DNA (corresponding to 10 copies). There is no ELISA method for salmonid fish, making our PCR method the only reliable measure for detecting salmonid fish in processed foods.

Purification, characterization and antibacterial activities of red color-related protein found in the shell of kuruma shrimp, Marsupenaeus japonicus.

The well-known red color change plays a significant role in consumer acceptability of crustacean species. In this study, we described the purification of the red color-related protein named MjRCP75 from the shell of Marsupenaeus japonicus. It was a homogeneous monomer with molecular mass of 75 kDa and rich in α-helix conformation. The α-helix content decreased within the increasing of heating temperature and was transformed dominantly to ß types. Identification and structural analysis revealed that MjRCP75 belonged to hemocyanin family. The released pigment from heated MjRCP75 showed a λmax at 483 nm in acetone. MjRCP75 showed clearly antibacterial activity against Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus. These findings identify MjRCP75 as the red color-related protein in M. japonicus shell and reveal its involvement in antibacterial activities.

Pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique.

Marine puffer fish accumulates tetrodotoxin (TTX) in the liver and ovary. In this study, we examined the pharmacokinetics of TTX in Takifugu rubripes by a single administration under general anesthesia at 20 degrees C for 300 min. The blood concentration-time profile showed multiple distinct phases after injection into hepatic portal vein. The area under the blood concentration-time curve (AUC) increased linearly at the dosage of 0.25-0.75 mg TTX/kg body weight, and the total body clearance was 2.06+/-0.17 mL/min/kg body weight. The AUCs following administration into the hepatic portal vein and hepatic vein were closely similar (147+/-33 versus 141+/-1 ng.min/microL), indicating negligible hepatic first-pass effect. Comparison of the AUCs following an administration to the hepatic vein and gastrointestinal tract (0.25 mg TTX/kg body weight) elucidated the bioavailability of TTX to be 62%. There was no significant increase in the AUCs following direct injection into the gastrointestinal tract (0.50 versus 1.0 mg TTX/kg body weight). At the dosage of 0.25 mg TTX/kg body weight into the hepatic vein, hepatic portal vein or gastrointestinal tract, TTX amount in the liver accounted for 84+/-6%, 70+/-9% or 49+/-17% of the total TTX amount applied, respectively. These results demonstrate that TTX is absorbed into the systemic circulation from the gastrointestinal tract by saturable mechanism and finally accumulated in the liver within 300 min.

Evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes.

In this study, we investigated the hepatic uptake clearance (CL(uptake)) of tetrodotoxin (TTX) in the marine puffer fish Takifugu rubripes by integration plot analysis after a single bolus injection of 0.25mg TTX/kg body weight into the hepatic vein at 20 degrees C. The blood concentration of TTX decreased over time after the injection, from 1451+/-45 ng/mL at 10 min to 364+/-59 ng/mL at 60 min. TTX concentrations in the spleen and kidney decreased in parallel with the blood concentrations, whereas those in the muscle and skin remained almost the same throughout the experiment. In contrast, the TTX concentration in the liver gradually increased, reaching 1240+/-90 ng/g liver at 60 min after injection. The amount of TTX that had accumulated in the liver 60 min after injection accounted for 63+/-5% of the administered dose. Integration plot analysis indicated a CL(uptake) of 3.1 mL/min/kg body weight in the liver for TTX, a rate far below that of the hepatic portal vein blood flow rate (at most, 9%). This finding is consistent with negligible extraction of TTX by the liver. The results demonstrated conclusively that the liver-specific distribution of TTX in T. rubripes is achieved by removal from the systemic circulation, but not by the hepatic first-pass effect.

Identification of tropomyosins as major allergens in antarctic krill and mantis shrimp and their amino acid sequence characteristics.

Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the tropomyosins from both species with decapod tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp tropomyosin has high sequence identity (>90% identity) with decapod tropomyosins, especially with fast-type tropomyosins. On the other hand, the Antarctic krill tropomyosin is characterized by diverse alterations in region 13-42, the amino acid sequence of which is highly conserved for decapod tropomyosins, and hence, it shares somewhat lower sequence identity (82.4-89.8% identity) with decapod tropomyosins than the mantis shrimp tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of tropomyosin.

Ani s 1, the major allergen of Anisakis simplex: purification by affinity chromatography and functional expression in Escherichia coli.

Third stage larvae of the nematode Anisakis simplex often infect marine fish and invertebrates. When the larvae are ingested orally via seafood, they can cause IgE-mediated allergic reactions as well as anisakiasis. Of the known A. simplex allergens, Ani s 1 (Kunitz/bovine pancreatic trypsin inhibitor family protein) has been demonstrated to be a major allergen, being expected to be a useful tool for diagnosis of A. simplex allergy. For a diagnostic purpose, sufficient amounts of either natural Ani s 1 (nAni s 1) or recombinant Ani s 1 (rAni s 1) with an IgE-binding capacity should be stably supplied whenever needed. In this study, therefore, we first developed a simple and rapid purification method for Ani s 1 that is based on affinity chromatography using anti-Ani s 1 antibodies as ligands. The method was shown to produce nAni s 1 with a higher yield than the previously reported methods. Then, an attempt was made to express rAni s 1 in Escherichia coli as a His-tagged protein. rAni s 1 obtained as an inclusion body was solubilized in a solvent containing denaturing and reducing reagents and purified by nickel-chelate chromatography. Refolding of rAni s 1 was accomplished by dialysis in the presence of arginine, followed by that in the absence of arginine. Fluorescence ELISA and inhibition ELISA data revealed that rAni s 1 is IgE reactive enough to be used as a diagnostic tool.

Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii.

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.

Isolation and cDNA cloning of a novel red colour-related pigment-binding protein derived from the shell of the shrimp, Litopenaeus vannamei.

Pigment-binding proteins play important roles in crustacean shell colour change. In this study, a red colour-related pigment-binding protein, designated LvPBP75, was purified from the shell of Litopenaeus vannamei. HPLC and PAGE analysis showed that LvPBP75 was a homogeneous monomer with molecular mass of 75kDa. Peptide mass fingerprint analysis revealed that LvPBP75 belonged to hemocyanin, and the released pigment from heated LvPBP75 showed a λmax at 481nm in acetone. The significant red-colour change temperatures were detected at 30 and 80°C, respectively. Based on the determined amino acid fragments, a full-length cDNA of LvPBP75 was cloned and sequenced. The ORF encodes a protein of 662 amino acids having 80% identity with penaeidae hemocyanin. These results strongly suggest a novel function of hemocyanin, namely binding with pigment, and its involvement in L. vannamei shell colour change.