The presence of free d-aspartate in marine macroalgae is restricted to the Sargassaceae family.

The presence of d-aspartate (d-Asp), a biologically rare amino acid, was evaluated in 38 species of marine macroalgae (seaweeds). Despite the ubiquitous presence of free l-Asp, free d-Asp was detected in only 5 species belonging to the Sargassaceae family of class Phaeophyceae (brown algae) but not in any species of the phyla Chlorophyta (green algae) and Rhodophyta (red algae). All other members of Phaeophyceae, including 3 species classified into the section Teretia of Sargassaceae did not contain d-Asp. These results indicate that the presence of free d-Asp in marine macroalgae is restricted only to the Sargassaceae family, excluding the species in the section Teretia.

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method.

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the "two-step labelling method," is effective for the simultaneous determination of d- and l-amino acids.

Characterization of differentially expressed genes in liver in response to the rearing temperature of rainbow trout Oncorhynchus mykiss and their heritable differences.

To characterize thermal-responsive genes in fish, firstly, juvenile rainbow trout were reared in four different temperature conditions (average temperatures were 10, 14, 18, and 22 °C, respectively) and differentially expressed genes were identified. Gene expression in the liver was analyzed by the differential display method, followed by validation using real-time PCR. Subsequently, to examine whether the identified genes show heritable differences, the gene expression levels were compared among juveniles of three genetically distinct lines of rainbow trout (a strain and two closed colonies) by rearing at two different temperature conditions (average 14 and 22 °C). By rearing at 22 °C, growth retardation was observed compared with fish reared at 14 and 18 °C, and six genes were identified as differentially expressed genes in response to the rearing temperature in the gene expression analyses. With the increase in rearing temperature, gene expressions of a complement C1q and two ribosomal proteins were significantly up-regulated. On the other hand, three metabolic genes (betaine homocysteine methyltransferase, triosephosphate isomerase, and glucose-6-phosphatase) were down-regulated, indicating a metabolic depression due to high temperature. In the subsequent analyses, in response to the rearing temperature (14 and 22 °C), there was a trend that the complement C1q and glucose-6-phosphatase genes showed different expression patterns among the three rainbow trout lines, suggesting heritable differences in these genes. Our study provides information on thermal-responsive genes in fish, and we anticipate it will facilitate further investigation in the thermal biology of fish.

Immunohistochemical localization of endogenous D-Aspartate in the marine brown Alga Sargassum fusiforme.

Immunohistochemical localization (cellular localization) of endogenous D-aspartate in the marine brown alga Sargassum fusiforme was investigated by the use of a specific polyclonal antibody raised against D-aspartate. D-Aspartate immunoreactivity was evident in the medullary layer in the blade of the alga, and weak staining was found in the cortical layer, whereas epidermal cells were found to lack D-aspartate. Within the cells of the layers, immunoreactivity was confirmed only in the cytosol and not in the cell wall, chloroplast, or vacuole. These results suggest that D-aspartate is present in S. fusiforme cells, and excludes the possibility that it is derived from attached or symbiotic organisms such as marine bacteria. This is the first report describing the localization of free D-aspartate in plant cells.

The effects of 2-bromopalmitate on the fatty acid composition in differentiating adipocytes of red sea bream (Pagrus major).

To determine whether external factors affect the adipogenic function of fish adipocytes, the effects of 2-bromopalmitate (a PPAR agonist) on the fatty acid composition in differentiating adipocytes of red sea bream were investigated in vitro. In the presence of 2-bromopalmitate, the red sea bream adipocytes were differentiated and the effects on the fatty acid composition and the adipogenic gene expression were analyzed. With the level of 2-bromopalmitate, the content of 16:1n-7, a delta-9 desaturation product, increased in association with the increase in a stearoyl CoA desaturase (SCD) gene expression level while the triglyceride accumulation was not affected. Subsequently, the effects on the bioconversion of the n-3 and n-6 fatty acids, which are main series of dietary essential fatty acids, were examined. In the presence of 300 microM of 18:3n-3 or 18:2n-6, red sea bream stromal-vascular cells accumulated the lipid in the cytoplasm within 3 days by the fatty acid uptake with the increase of corresponding fatty acid contents. Furthermore, in both the 18:3n-3 and 18:2n-6 stored cells, the products of delta-6 desaturation (18:4n-3 and 18:3n-6, respectively) and C(18-20) elongation (20:3n-3 and 20:2n-6, respectively) were detected. However, neither the delta-6 desatutration nor C(18-20) elongation of 18:3n-3 and 18:2n-6 were enhanced by 2-bromopalmitate treatment. In conclusion, the results indicate that the adipocyte function in fish, e.g. adipogenic gene expression and fatty acid composition, can be modified by external factors and a main effect of 2-bromopalmitate is the increase in the content of delta-9 desaturation product by stimulating the SCD gene expression.

Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major.

Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.