Morphological and physiological differences among cultivation lines of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> in a common garden experiment using a tank culture system

Undaria pinnatifida is grown for food and industrial materials worldwide; therefore, advanced breeding is needed to meet quality and productivity requirements. In this study, we examined regional lines of U. pinnatifida from five cultivation sites in Japan with different environmental conditions: Oga (OGA, the northern Sea of Japan coast), Hirota Bay (HRT, the northeastern Pacific coast), Matsushima Bay (MAT, the northeastern Pacific coast), Naruto (the Seto Inland Sea coast) and Shimonoseki (SIM, the southern Sea of Japan coast). The sporophytes of these lines were cultured in a tank culture system under controlled environmental conditions, and their morphological characteristics, nutrient uptake kinetics (V _max, K _s and V _max/K _s ), and carbon, nitrogen and phosphorus contents were determined. Sporophytes from MAT grew faster, whereas those from SIM were smaller than those from the other sites. Although the blade thickness of sporophytes cultivated in the sea significantly differs among cultivation sites in the previous study, there was no significant difference in blade thickness among the regional lines cultivated in the tank. Sporophytes from OGA had the greatest V _max/K _s values and significantly greater nitrogen contents than the other lines. Therefore, the morphological characteristics of MAT and SIM sporophytes, and the nutrient uptake kinetics of OGA sporophytes may have a genetic origin. This indicates that these lines may represent useful resources for selective breeding, with MAT sporophytes providing faster growth and OGA sporophytes being well-adapted to low-nutrient conditions.     

15-Cis-carotenoids found in the reaction center of a green sulfur bacterium Chlorobium tepidum and in the Photosystem I reaction center of a cyanobacterium Synechococcus vulcanus

Carotenoids were extracted, at 4 °C in complete darkness and under nitrogen atmosphere, from the reaction center (RC) of a green-sulfur bacterium and the Photosystem (PS) I RC of a cyanobacterium; each extract was subjected to high-performance liquid chromatography (HPLC) using an apparatus equipped with a two-dimensional diode-array detector in order to spectroscopically identify cis–trans carotenoids while performing HPLC analysis. In the extract from the RC of Chlorobium tepidum, 15-cis and all-trans--carotenes as well as 13-cis-, 15-cis- and all- trans-chlorobactenes (in the order of elution) were identified, whereas in the extract from the PS I RC of Synechococcus vulcanus, 15-cis-, all-trans- and 9-cis--carotenes were found. Thus, the universal presence of 15- cis carotenoids in the 'iron sulfur-type' RCs has been shown in addition to the previous cases of the 'quinone-type' RCs.     

Molecular Evolution of the Domain Structures of Protein Disulfide Isomerases

Protein disulfide isomerase (PDI) is an enzyme that promotes protein folding by catalyzing disulfide bridge isomerization. PDI and its relatives form a diverse protein family whose members are characterized by thioredoxin-like (TX) domains in the primary structures. The family was classified into four classes by the number and the relative positions of the TX domains. To investigate the evolution of the domain structures, we aligned the amino acid sequences of the TX domains, and the molecular phylogeny was examined by the NJ and ML methods. We found that all of the current members of the PDI family have evolved from an ancestral enzyme, which has two TX domains in the primary structure. The diverse domain structures of the members have been generated through domain duplications and deletions.     

A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes

Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).     

Effects of Sika Deer (Cervus nippon) and Dwarf Bamboo (Sasa senanensis) on Trillium Populations in Akan National Park,Eastern Hokkaido,Japan

The competition between Trillium spp. and other species could modify the utility of Trillium spp. as indicators of the impact of deer on forest vegetation. To evaluate whether Trillium spp. (T. camschatcense and T. tschonoskii) are appropriate indicators of the impact of sika deer (Cervus nippon) in the presence of dwarf bamboo (Sasa senanensis), which competes with Trillium populations, we examined the size class of large‐growth‐stage Trillium plants, the frequency of the presence of deer pellets and the coverage of S. senanensis in deer‐excluded sites (where deer have been eliminated since 1995) and unfenced control sites in Akan National Park, eastern Hokkaido, Japan. The result of a cumulative link mixed model (CLMM) suggests that size class at deer‐excluded sites is influenced by competitive understory species, even without deer grazing. The CLMM result for unfenced control sites suggests that C. nippon negatively influences the size class of Trillium populations; however, the negative effect weakened with increasing S. senanensis coverage. Thus, S. senanensis can function as an unpalatable neighbor that makes it difficult for C. nippon to detect Trillium plants. We conclude that the size class structure of Trillium spp. can be a useful index of C. nippon usage under sparse or medium S. senanensis understories; however, it is difficult to use these indices under dense S. senanensis understories because of the weakened negative effect of C. nippon grazing.     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.     

Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans

Protein glycosylation is a central issue for post-genomic (proteomic) sciences. We have taken a systematic approach for analyzing soluble glycoproteins produced in the nematode Caenorhabditis elegans. The approach aims at assigning (i) genes that encode glycoproteins, (ii) sites where glycosylation occurs, and (iii) types of attached glycan structures. A soluble extract of C. elegans, as a starting material, was applied first to a concanavalin A (ConA) column (specific for high-mannose type N-glycans), and then the flow-through fraction was applied to a galectin LEC-6 (GaL6) column (specific for complex-type N-glycans). The adsorbed glycoproteins were digested with lysylendopeptidase, and the resultant glycopeptides were selectively recaptured with the same lectin columns. The glycopeptides were separated by reversed-phase chromatography and then subjected to sequence determination. As a result, 44 and 23 glycopeptides captured by the ConA and GaL6 columns, respectively, were successfully analyzed and assigned to 32 and 16 corresponding genes, respectively. For these glycopeptides, 49 N-glycosylation sites were experimentally confirmed, whereas 21 sites remained as potential sites. Of the identified genes, about 80% had apparent homologues in other species, as represented by typical secreted proteins. However, the two sets of genes assigned for the ConA and GaL6-recognized glycopeptides showed only 1 overlap with each other. Proof of the practical applicability of the glyco-catch method to a model organism, C. elegans, directs us to explore more complex multicellular organisms.