In Silico Analysis of Relationship between Proteins from Plastid Genome of Red Alga Palmaria sp. (Japan) and Angiotensin I Converting Enzyme Inhibitory Peptides.

Plastid proteins are one of the main components in red algae. In order to clarify the angiotensin I converting enzyme (ACE) inhibitory peptides from red alga Palmaria sp. (Japan), we determined the plastid genome sequence. The genome possesses 205 protein coding genes, which were classified as genetic systems, ribosomal proteins, photosystems, adenosine triphosphate (ATP) synthesis, metabolism, transport, or unknown. After comparing ACE inhibitory peptides between protein sequences and a database, photosystems (177 ACE inhibitory peptides) were found to be the major source of ACE inhibitory peptides (total of 751). Photosystems consist of phycobilisomes, photosystem I, photosystem II, cytochrome complex, and a redox system. Among them, photosystem I (53) and II (51) were the major source of ACE inhibitory peptides. We found that the amino acid sequence of apcE (14) in phycobilisomes, psaA (18) and psaB (13) in photosystem I, and psbB (11) and psbC (10) in photosystem II covered a majority of bioactive peptide sequences. These results are useful for evaluating the bioactive peptides from red algae.

Long-term stability of RNA isolated from muscle of red seabream (Pagrus major) during ice storage.

Recovering high-quality intact RNA from postmortem tissue is of major concern for gene expression studies. However, it is difficult to perform RNA extraction from aquacultured fish immediately after death, as rapid and accurate skills are needed for the procedure. The objective of this study was to quantitatively assess the integrity of total RNA extracted from muscle, liver, and digestive tract tissues of red seabream stored in ice as whole bodies, at a range of time points up to 10 days postmortem, using RNA integrity number (RIN) and quantitative PCR (qPCR). The RIN of total RNA in muscle remained over 8.0 for 5 days postmortem. The RINs in the liver and digestive tract were under 5.0 at 2 days postmortem. The mRNA levels of tissue inhibitor metalloproteinase 2 (TIMP-2) and ß-actin, measured using qPCR in muscle, decreased to 87.8% at 1 day postmortem and to 45.5% at 2 days postmortem, from that at 0 days postmortem. In the liver and digestive tract, the mRNA levels were not significantly changed until 1 day postmortem. These results indicate that RNA, especially from fish muscle, can be maintained at high quality for several days postmortem solely by storing the fish body in ice.

Mangrove crab intestine and habitat sediment microbiomes cooperatively work on carbon and nitrogen cycling.

Mangrove ecosystems, where litter and organic components are degraded and converted into detrital materials, support rich coastal fisheries resources. Sesarmid (Grapsidae) crabs, which feed on mangrove litter, play a crucial role in material flow in carbon-rich and nitrogen-limited mangrove ecosystems; however, the process of assimilation and conversion into detritus has not been well studied. In this study, we performed microbiome analyses of intestinal bacteria from three species of mangrove crab and five sediment positions in the mud lobster mounds, including the crab burrow wall, to study the interactive roles of crabs and sediment in metabolism. Metagenome analysis revealed species-dependent intestinal profiles, especially in Neosarmatium smithi, while the sediment microbiome was similar in all positions, albeit with some regional dependency. The microbiome profiles of crab intestines and sediments were significantly different in the MDS analysis based on OTU similarity; however, 579 OTUs (about 70% of reads in the crab intestinal microbiome) were identical between the intestinal and sediment bacteria. In the phenotype prediction, cellulose degradation was observed in the crab intestine. Cellulase activity was detected in both crab intestine and sediment. This could be mainly ascribed to Demequinaceae, which was predominantly found in the crab intestines and burrow walls. Nitrogen fixation was also enriched in both the crab intestines and sediments, and was supported by the nitrogenase assay. Similar to earlier reports, sulfur-related families were highly enriched in the sediment, presumably degrading organic compounds as terminal electron acceptors under anaerobic conditions. These results suggest that mangrove crabs and habitat sediment both contribute to carbon and nitrogen cycling in the mangrove ecosystem via these two key reactions.

A close relationship between androgen levels and eumelanogenesis in the teleost red seabream (Pagrus major): Quantitative analysis of its seasonal variation and effects of oral treatment with methyl-testosterone.

Male and female teleost seabream (Pagrus major) were examined for seasonal variation of eumelanin, pheomelanin, 11-ketotestosterone (11KT, fish androgen), lightness (L* value) and Gonad Somatic Index (GSI: gonad mass/body massx100). In males, levels of pyrrole-2,3,5-tricarboxylic acid (a marker of eumelanin), 11KT and the GSI increased sharply from September and plateaued in March and April when the fish are sexually mature. These results are consistent with the lightness of their body color. Using the data from males, a high correlation was observed for all combinations of those four variables (PTCA, 11KT, lightness and GSI). In females, little change was observed in those variables except for the GSI. 4-Amino-3-hydroxyphenylalanine (a marker of pheomelanin) was also analyzed, but it was below the detection limit at all times. Oral treatment of juvenile red seabream with synthetic androgen methyl-testosterone for 2 months induced eumelanin accumulation about 3 times higher than the control. These data show that there is a close relationship between androgen levels and eumelanin accumulation in teleosts. This is the first report that androgen affects melanin accumulation in a dose-dependent manner.

Correlation of proline, hydroxyproline and serine content, denaturation temperature and circular dichroism analysis of type I collagen with the physiological temperature of marine teleosts.

Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.

Complete sequence of mitochondrial DNA of red alga dulse Palmaria palmata (Linnaeus) Weber & Mohr in Japan.

Red algae contain high amount of proteins compared to the other algae. Red algae dulse is one of the protein rich species and a good candidate for protein sources. In this study, the complete mitochondrial genome of Palmaria palmata in Japan was determined. It had a circular mapping molecular with the length of 31,399 bp and contained 53 genes including 27 protein-coding, 2 rRNA, and 24 tRNA. Phylogenetic analysis showed that Palmaria palmata in Japan was separated with Atlantic dulse. This is the first report of complete mitochondrial genome from Pacific dulse.

Biochemical study of type I collagen purified from skin of warm sea teleost Mahi mahi (Coryphaena hippurus), with a focus on thermal and physical stability.

Acid- and pepsin-soluble collagen were purified from the skin of mahi mahi (mmASC and mmPSC). The Pro+Hyp content of the latter (185/1,000 residues) was highest among all marine teleost fishes. Fourier transform infrared spectroscopy and Circular Dichroism (CD) analysis showed the typical structure of type I collagen. The ratio of positive over negative peak intensity calculated from the CD spectrum was approximately 1.19 in mmPSC, which is remarkably high, and indicates the stability of the triple helix. The denaturation temperatures (Td ) of mmASC and mmPSC were the highest (29.5 and 28.8°C, respectively) among the marine teleost fishes previously studied. atomic force microscope and scanning electron microscope images showed that even after pretreatment, the fibrils presented their structure and fiber orientation. These results indicate the robustness of both collagens, which can be attributed to the high value of Pro+Hyp stabilizing the helix structure of the collagen molecule. Practical applications While Mahi mahi is highly valuable for its meat, other parts such as skin is not fully utilized in seafood industry. On the contrary, it has been empirically shown that the skin of Mahi mahi has high thermal stability, thus, the skin has been used for leather products in some areas located in the tropical and subtropical zones. In this study, we focused on collagen a major component in skin and investigated the structure and the biochemical characteristics of it. Some results showed that collagen from skin has high physical stability. The collagen from skin of Mahi mahi will be a new fishery resource which could be used as a material for collagen products.

Close-up observations on the spawning behavior of a captive Japanese flying squid (Todarodes pacificus).

The spawning behavior of a Japanese flying squid (Todarodes pacificus) is described based on up-close observation of a captive female. The squid was first transferred from a 10-ton tank to a polystyrene plastic box containing 45 liters of seawater. About one hour later, the mantle-contraction rate increased rapidly, followed by a brief convulsion of the mantle and arms and a whitening of the body. The mantle contractions become shallow and rapid, and several seconds later, semitransparent jelly presumably from the nidamental glands emerged from the funnel and passed between the ventral pair of arms. Approximately 90 seconds after the egg mass first emerged, the female began ejecting oocytes through the funnel into the egg mass using rapid, powerful mantle contractions. Soon after the oocytes were ejected, translucent strands (presumably sperm) emanated from the buccal membrane. The female continued to eject oocytes for approximately two minutes, after which the mantle convulsed, and the mantle-contraction rate decreased slowly for about one minute until the contractions stopped. The squid died soon afterwards.

Structure and properties of the egg mass of the ommastrephid squid Todarodes pacificus.

The Japanese flying squid, Todarodes pacificus, is thought to spawn neutrally buoyant egg masses that retain a specific location in the water column by floating at the interface between water layers of slightly different densities. It is important to understand the physical process that determines the vertical distribution of the egg masses to predict their horizontal drift in relation to embryo survival and subsequent recruitment. Here, mesocosm experiments were conducted in a 300 m3 tank by creating a thermally stratified (17-22°C) water column to obtain egg masses. A cage net methodology was developed to sustain egg masses for detailed observation. We measured the density of the egg masses of T. pacificus, and used this information to infer the vertical distribution patterns of the egg masses at the spawning grounds (Tsushima Strait, Japan). When measured separately, the density of the outer jelly of each egg mass was 2.7 σ units higher than that of the surrounding water. The outer jelly and the specific gravity of embedded individual eggs (~1.10) cause the egg masses to have very slight negative buoyancy relative to the water in which they are formed. Analysis of the vertical profile of the spawning ground showed that water density (σθ) increased sharply at ~30 m depth; thus, egg masses might settle above the pycnocline layer. In conclusion, we suggest that T. pacificus egg masses might retain their location in the water column by floating at the interface between water layers of slightly different densities, which happen to be above the pycnocline layer (actual depth varies seasonally/annually) in the Tsushima Strait between Korea and Japan.

Hemocyte components in crustaceans convert hemocyanin into a phenoloxidase-like enzyme.

The functional conversion of hemocyanin (Hc), an oxygen transporter, into an enzyme was investigated in crustaceans. Hc is converted into a phenoloxidase-like enzyme by hemocyte components, which is triggered by beta-1,3-glucan. This activation is severely hampered with leupeptin and E-64 treatment, indicating that the serine/cysteine proteases in the hemocytes are involved in the activation. In a SDS-PAGE-analysis, no change was observed between normal and activated Hc under reduced conditions. However, under non-reduced condition of normal Hc, several minor bands were observed at oligomeric position of Hc subunit, which disappeared upon activation. These results indicate that a split of the reductive bond, such as the disulfide bond between subunits, is essential for Hc activation. This is the first report to show the enzymatic conversion of Hc and the presence of the covalent bond in the Hc subunit of crustaceans.

Transcriptome analysis of the duodenum, pancreas, liver, and muscle from diabetic Goto-Kakizaki rats fed a trypsin inhibitor derived from squid viscera.

Trypsin inhibitors (TIs) have various nutritional effects. However, a detailed mechanism for their effects, especially on the gene expression patterns in various tissues, remains unknown. Here, we used transcriptome techniques and gene ontology (GO) analysis to examine the effects of squid TI (sqTI), a biochemically stable peptide, on diabetic Goto-Kakizaki rats after feeding for 10 weeks. We demonstrated that downregulation of SREBP1c in the liver via duodenal/pancreatic hormones suppresses the blood cholesterol level. Consistently, in GO analysis, the term "cholesterol biosynthetic process" was enriched among downregulated genes. No hypoglycemic or insulinotropic effects were observed, in contrast to the results from our previous studies (single stimulation with the same dose of TI), which can be partly ascribed to the inactive responses of the duodenum and pancreas in this condition.

Selective analysis of lipids by thin-layer chromatography blot matrix-assisted laser desorption/ionization imaging mass spectrometry.

Thin-layer chromatography (TLC) is an essential method for food composition analyses such as lipid nutrition analysis. TLC can be used to obtain information about the lipid composition of foods; however, it cannot be used for analyses at the molecular level. Recently we developed a new method that combines matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) with TLC-blotting (TLC-Blot-MALDI-IMS). The combination of MALDI-IMS and TLC blotting enabled detailed and sensitive analyses of lipids. In this study, we applied TLC-Blot-MALDI-IMS for analysis of major phospholipids extracted from bluefin tuna. We showed that TLC-Blot-MALDI-IMS analysis could visualize and identify major phospholipids such as phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin.

Transcriptome analysis of the duodenum in Wistar rats fed a trypsin inhibitor derived from squid viscera.

To investigate the effects of oral administration of a trypsin inhibitor (TI), normal Wistar rats were fed a TI derived from squid (Todarodes pacificus) for 10 weeks and gene expression profiles in the duodenum, pancreas, liver, and muscle were then analyzed using DNA microarrays. Although no significant changes could be observed in growth, food intake, tissue weight, or blood tests among the tissues tested, the duodenum showed the most remarkable changes in the global gene expression profile. Significant up-regulation of mRNAs encoding gastrin, gastrokine, cholecystokinin and somatostatin in the duodenum was validated by qPCR analysis. In gene ontology (GO) analysis of the up-regulated differentially expressed genes (DEGs), GO terms related to keratinization and innate mucosal defense were enriched (p < 0.001) in the category of biological processes in addition to assumable terms such as regulation of secretion and response to nutrients, vesicle-mediated transport, and so forth. In the same analysis, calcium ion binding was listed at the deepest hierarchy in the category of molecular function. These results indicate that the duodenum responds to TI treatment by a wider range of physiological processes than previously assumed such as keratinocyte differentiation and innate mucosal defense, in which calcium plays a crucial role.

Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization.

We investigated the enzymatic properties and immunohistochemical localization of cuticular hemocyanin, a known oxygen transporter in the prawn Penaeus japonicus. The molecular weight of hemocyanin purified from the cuticle was estimated to be 67-77 k using SDS-PAGE, and the purified protein was effectively converted into a phenoloxidase-like enzyme by an SDS-treatment. The activated enzyme catalyzed the o-hydroxylation of monophenols and the oxidation of o-diphenols and was inhibited by typical inhibitors of phenoloxidase. These characteristics were nearly identical to the enzymatic properties of hemolymph hemocyanin. Immunological detection showed a diffuse distribution of hemocyanin over the exocuticle and endocuticle, and a higher signal level was observed in the latter. Based on these results, roles of hemocyanin in various physiological processes such as immune response and sclerotization of the cuticle were discussed.

An oxygen transporter hemocyanin can act on the late pathway of melanin synthesis.

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) are precursors of eumelanin. The effects of crustacean hemolymph proteins on these eumelanin-related metabolites were investigated. Zymogram analysis indicated that polymers of hemocyanin (Hc) subunits converted DHI into black pigment while no effects were observed using DHICA as a substrate. Spectrum changes for mixtures of purified Hc and DHI showed a profile similar to oxidized DHI by mushroom tyrosinase while Hc had only slight effects on DHICA. Typical inhibitors of tyrosinase and phenoloxidase severely hampered the production of oxidized DHI. Taken together with previous results, these data indicate that Hc plays a crucial role in the conversion of DHI in the hemolymph of crustaceans, which promotes late reactions in the melanin synthetic pathway as well as early reactions (oxidation of tyrosine and DOPA to dopaquinone).

The histological analysis, colorimetric evaluation, and chemical quantification of melanin content in 'suntanned' fish.

Human melanocytes respond to UV irradiation by increasing the synthesis of melanin. While much is now understood of the pathways governing this process and the nature of the melanin synthesized, little is known of melanins produced by lower vertebrates and their capacity to respond to UV. Here we report that a fish, red seabream, can undergo 'suntanning'. Histological, colorimetric and chemical assays were performed for suntanned red seabream fish bred in net cages to analyse the melanins and compared with shaded or wild red seabream fish. For color evaluation, the L* values of suntanned fish were dramatically lower than those in the other two groups. Pyrrole-2,3,5-tricarboxylic acid (PTCA), an indicator of eumelanin, was detected in suntanned fish at five times higher levels than in shaded or wild fish while 4-amino-3-hydroxyphenyl-alanine (4-AHP), a marker for pheomelanin, could not be detected in any of the samples. Histological analysis showed that melanocytes in the suntanned skin enlarged and increased in number to form a monolayer at the surface of the skin. Analysis of L* values and PTCA levels showed quite a high correlation coefficient (r = -0.843). When comparing shaded and wild red seabream fish, the scores were closer but some significant differences were still found in some body areas. These results indicate that eumelanin accumulates in suntanned fish during the increase in skin color, which is induced by sunlight, presumably by ultraviolet radiation.