A hyperosmotic stress-induced mRNA of carp cell encodes Na+- and Cl−-dependent high affinity taurine transporter1The sequence reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with accession no. AB006986.1

AbstractA cDNA clone encoding a Na+- and Cl−-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78–80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na+- and Cl−-dependent taurine transport activity with an apparent Km of 56 μM. The Na+/Cl−/taurine coupling ratio for cloned transporter was estimated as 2:1:1. Uptake of radiolabeled taurine was inhibited by excessive cold taurine, hypotaurine, β-alanine and γ-aminobutyric acid but not by α-alanine, glycine, leucine or glycinebetaine. Taurine transporter mRNA is ubiquitously distributed in carp tissues, and its level decreases in the order: kidney>spleen>heart>fin>eye≈intestine≈gill>skin>brain>muscle>hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH2O. These data suggest the significant role of taurine as an osmolyte in carp cells

Aquatic invertebrate’s Carbohydrate-binding module assists environmental cellulase to immobilize in wetland sediments

Carbohydrate-binding modules (CBMs) are non-catalytic protein domains that bind to carbohydrates, and have been well studied in microorganisms. Endogenous CBMs in aquatic invertebrates, however, have not yet been identified, and little is known about their ecological significance to wetland environments. Using an approach of characterizing a recombinant CBM (CjCel9A) from a brackish bivalve, Corbicula japonica, this work identified CjCel9A-CBM’s cellulose-binding activity. Scatchard plot analysis in the study of CjCel9A-CBM binding to α-cellulose showed a high corresponding partitioning coefficient (Kr) of 20.33, indicating CjCel9A-CBM’s high affinity for cellulose. In addition, this affinity tolerated a high ion concentration buffer system, consistent with C. japonica’s adaption to brackish wetland environments. Moreover, immuno-scanning electron microscopy (immuno-SEM) suggested that CjCel9A-CBM binds to α-cellulose unevenly, which was further determined to be caused by its higher affinity for crystalline cellulose (Cellulose I, mostly seen in plant leaves). Together, these findings suggest that CjCel9A-CBM is capable of immobilizing its associated catalytic domain on environmental crystalline cellulose (i.e., fallen leaves) in wetland sediments. Most importantly, they could provide a reasonable answer to a question recognized broadly in wetland ecologists, namely, why many wetland sediments have constant cellulase activities, although the sediments are being washed almost every day

Function of meiobenthos and microorganisms in cellulose breakdown in sediments of wetlands with different origins in Hokkaido

To validate the mechanism of cellulose breakdown in cold climate wetlands, we investigated cellulase activity in sediments collected from 17 wetland sites in Hokkaido, the northern area of Japan. We evaluated cellulase activity by quantitative analysis of glucose released from carboxymethyl cellulose and found that sediments from peat fens demonstrated high activity, followed by sediments from lagoons and estuaries. Sediments from peat fens also contained greater amounts of organic matter, followed by lagoons and estuaries, thereby suggesting a strong positive correlation between organic matter content and cellulase activity. Evaluation of cellulase activity by qualitative cellulose zymographic analysis showed that various cellulases with different molecular sizes were implicated in cellulose breakdown in wetlands. Among them, cellulose breakdown in Meguma Pond (peat fen), Notsuke Gulf (peat fen), and Lake Utonai (lagoon) was potentially due to microorganism cellulase, while that in Lake Chobushi (lagoon) was ascribed to meiobenthos (Oligochaeta species) cellulase. The findings presented herein suggest that the origin and activity level of cellulase vary depending on the type of cold climate wetland

Sediment-complex-binding cellulose breakdown in wetlands of rivers

We have been assessing the activity level of cellulase in wetland sediments to clarify the significance of cellulase for the turnover of plant cellulose in wetlands. In this study, we investigated the cellulose degrading function of sediment in wetlands to clarify the biochemical breakdown mechanism of cellulose. Specifically, we measured cellulase activities of sediments collected from wetlands of the Chikugo River (Fukuoka Prefecture), Midori River (Kumamoto Prefecture), Hamado River (Kumamoto Prefecture), Yodo River (Osaka Prefecture), and Tanaka River (Mie Prefecture). The results revealed that the activity levels differed significantly among rivers. Additionally, the cellulase activities of the sediment were not completely suppressed in the presence of chloramphenicol. These findings suggested that a portion of the cellulase activities were derived from cellulases binding to the components of sediments. Actually, sediments also showed the ability to bind fungal cellulase. Comparison of the binding ability of clay and plant residues, the main components of sediments, revealed that plant residues had significantly higher abilities to bind cellulase. This finding was supported by the fact that there was a strong correlation between the organic matter content in the sediment and the cellulase-binding ability (R = 0.66). Results of this study show that sediment complexes harboring cellulases might function as a bioreactor to degrade cellulose in wetlands

魚類の筋肉プロテアーゼに関する研究

京都大学0048新制・課程博士農学博士甲第3123号農博第408号新制||農||385(附属図書館)学位論文||S59||N1476(農学部図書室)UT51-59-D228京都大学大学院農学研究科水産学専攻(主査)教授 池田 靜德, 教授 門田 元, 教授 土井 悦四郎学位規則第5条第1項該当Kyoto UniversityDFA

The iron content and ferritin contribution in fresh, dried, and toasted nori, Pyropia yezoensis.

Iron is one of the essential trace elements for humans. In this study, the iron contents in fresh, dried, and toasted nori (Pyropia yezoensis) were analyzed. The mean iron content of fresh, dried, and toasted nori were 19.0, 22.6, and 26.2 mg/100 g (dry weight), respectively. These values were superior to other food of plant origin. Furthermore, most of the iron in nori was maintained during processing, such as washing, drying, and toasting. Then, the form of iron in fresh, dried, and toasted nori was analyzed. As a result, an iron storage protein ferritin contributed to iron storage in raw and dried nori, although the precise rate of its contribution is yet to be determined, while ferritin protein cage was degraded in the toasted nori. It is the first report that verified the ferritin contribution to iron storage in such edible macroalgae with commercial importance

A new cleaning method for accurate examination of freshwater gastropod shell specimens covered with iron-rich deposits

Sodium hypochlorite has been used for cleaning specimens of freshwater and brackish water snails that are covered with deposits. Our experiments using specimens of two freshwater snail species, Semisulcospira niponica (Smith, 1876) and S. reticulata Kajiyama and Habe, 1961, showed that this traditional method could remove thin deposit layers, including algae, but was not useful for obstinate deposits. We found that a new method using ammonium thioglycolate could be applied to remove obstinate iron-rich deposits. Though ammonium thioglycolate treatment caused loss of gloss inside the aperture, this loss could be prevented by plugging a kneaded eraser into an aperture. Moreover, the new method could clean specimens with little damage of the periostracum. So as to remove deposits with the least damage to shells, 3% w/v sodium hypochlorite was useful for deposits including algae, and 20% w/v ammonium thioglycolate was suitable for cleaning specimens with iron-rich deposits. Degeneration of the microstructure of inner whorls can be avoided by plugged shell apertures with a kneaded eraser in both methods. Shell deposits that are composed of both algae and iron should be treated first with 20% w/v ammonium thioglycolate, and then with 3% w/v sodium hypochlorite to remove the deposits. Appropriate cleaning methods enable accurate examination and long-term preservation of shell specimens

Cellulase activity in meiobenthos in wetlands

To validate the involvement of meiobenthos in cellulose breakdown in wetlands, meiobenthos were collected from sediments of Lake Furen and the Biwase River in Hokkaido Prefecture, the Kako River in Hyogo Prefecture, and the Chinai River in Shiga Prefecture. Cellulase activities of the meiobenthos were measured by cellulose zymographic analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels containing 0.5% carboxymethyl cellulose. The results showed that most of the Turbellaria, Nematoda, Harpacticoida, and Oligochaeta species exhibited cellulase activity. The molecular sizes of the cellulase-active bands of the sediments in Lake Furen, the Biwase River, and the Chinai River coincided with those of meiobenthos. The findings suggest that meiobenthos might play a major function in cellulose breakdown in these wetlands. This paper is the first to report cellulase activity in meiobenthos and that they are possibly involved in the breakdown of cellulose in wetlands

Further records of introduced Semisulcospira snails in Japan (Mollusca, Gastropoda): implications for these snails’ correct morphological identification

Seven species of the freshwater snail genus Semisulcospira, which are indigenous taxa of the largest lake in Japan, Lake Biwa, have been introduced into 17 localities, including five newly recorded localities. Among these species, S. dilatata Watanabe and Nishino, 1995, S. habei Davis, 1969, and S. rugosa Watanabe and Nishino, 1995 were firstly recorded as introduced species. The alien Semisulcospira snails could be identified based on the combination of three morphological characters: longitudinal ribs number of penultimate whorl of adult shell, spire angle of adult shell, and shell height of the largest embryonic shell. Accordingly, morphological identification of introduced Semisulcospira populations recorded in previously published studies was revisited. The introductions of Semisulcospira snails have been suggested to have occurred due to contamination of seedlings of other shellfish and fishes at most sites. Taking into account the potential impacts of accidental species introductions, seedlings should be carefully managed so as not to unintentionally introduce Semisulcospira snails