Daniel A. Coury

Effect of a seaweed mixture on serum lipid level and platelet aggregation in rats

To assess the effect of a seaweed mixture on lipid levels in serum as well as platelet aggregation in rats, Eisenia bicyclis (‘Arame’), Hizikia fusiformis (‘Hijiki’) and Undaria pinnatifida sporophylls (‘Mekabu’), all brown seaweeds, and Porphyra yezoensis (‘Susabinori’), a red seaweed, were powdered and mixed in a ratio of 45:30:20:5 (w/w). When rats were fed a cholesterol-rich diet containing this mixture of seaweeds (9–10% w/w) for 28 days, serum total cholesterol, LDL-cholesterol, free cholesterol, and triglyceride levels declined significantly to 49.7%, 48.1%, 49.0% and 74.8%, respectively, of those of the control. Serum HDL-cholesterol, however, was unchanged. Though activated partial thromboplatin time, prothrombin time, antithrombin III activity, and fibrinogen levels in plasma were unchanged, the maximal ADP- and collagen-induced platelet aggregation decreased significantly to 89.0% and 85.5% control levels, respectively. These results indicate that this mixture of E. bicyclis, H. fusiformis, U. pinnatifida sporophylls, and P. yezoensis, is useful for the prevention of hyperlipidemia and thrombosis in rats.

Isolation and characterization of the rbcS genes from a sterile mutant of Ulva pertusa (Ulvales, Chlorophyta) and transient gene expression using the rbcS gene promoter

We isolated two different genomic DNAs (UprbcS1 and UprbcS2) encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and portions of the 5′- and 3′-flanking regions from sterile Ulva pertusa Kjellman. The UprbcS1 and UprbcS2 genes had three introns in the coding region. Each predicted UprbcS polypeptide was a 180-amino-acid (AA) residue including a 38-AA transit peptide, although the 104th AA residue was replaced. The nucleotide sequences of UprbcS cDNAs isolated from a cDNA library corresponded to that of the UprbcS1 gene, suggesting that the UprbcS1 gene was predominantly expressed in sterile U. pertusa compared to UprbcS2. Southern blot analysis showed that each UprbcS gene was a single-copy gene in the sterile U. pertusa genome. Northern hybridization indicated that the expression of UprbcS was induced and repressed by dark and light treatments, respectively. When sterile U. pertusa cells were transformed with an expression vector containing the UprbcS1 promoter and terminator sequences fused with the green fluorescent protein (GFP) gene, GFP fluorescence was observed in the cells transformed. These results suggest that the UprbcS1 gene promoter is light regulated and highly active in the sterile U. pertusa cells and is available for genetic transformation system in the alga.

cDNA cloning and expression analysis of two heat shock protein genes, Hsp90 and Hsp60, from a sterile Ulva pertusa (Ulvales, Chlorophyta)

Molecular chaperones are synthesized and accumulated under a variety of unfavorable conditions in all organisms. Heat shock protein 90 (Hsp90) and Hsp60, which are classified into the major classes of molecular chaperones, play important roles in cellular stress responses. In this study, we characterized sterile Ulva pertusa Hsp90 (UpHsp90) and UpHsp60 genes which may be involved in tolerance to thermal and heavy metal stresses in this alga. The UpHsp90 cDNA consisting of 2,118 nucleotides encoded a polypeptide of 705 amino acids (AA). On the other hand, the UpHsp60 cDNA consisting of 1,722 nucleotides encoded a protein whose predicted length was 573 AA. The AA sequence alignment and phylogenetic analyses showed that the UpHsp90 and UpHsp60 proteins were more similar to cytoplasmic Hsp90s and mitochondrial Hsp60s, respectively, than to other types of the respective Hsps. Southern blot analysis indicated that the sterile U. pertusa genome had at least two cytoplasmic Hsp90-encoding genes and two mitochondrial Hsp60-encoding genes. The UpHsp90 and UpHsp60 mRNA levels were significantly affected by diurnal and temperature changes, and slightly affected by exposure to heavy metals. These results suggest that UpHsp90 and UpHsp60 genes play particularly important roles in adaptation to diurnal and temperature changes.

Isolation and identification of gametogenesis-related genes in Porphyra yezoensis (Rhodophyta) using subtracted cDNA libraries

Gametogenesis of Porphyra yezoensis thalli is induced by ageing as well as by changing water temperature and photoperiod. Under laboratory conditions, thalli cultivated at 10 ∘C with a 10:14 h (light: dark) cycle develop vegetatively to adult form without gametogenesis. On the other hand, sexual reproduction, which involves differentiation of vegetative cells and subsequent gametogenesis, is induced by culturing at 15 ∘C with a 16: 8 h (light: dark) cycle. We have constructed subtracted cDNA libraries enriched for differentially expressed transcripts in vegetative and reproductive thalli, and randomly selected 1,152 cDNAs from each subtracted library. Results of the dot blot analyses used for identification of differentially expressed cDNAs indicated that mRNA levels of 176 and 138 cDNAs tended to increase in the vegetative and reproductive thalli, respectively. BLAST analysis of nucleotide and deduced amino acid sequences showed that the cDNAs represented 63 and 59 unique clones for the vegetative and reproductive cDNA enriched subtracted libraries, respectively. Interestingly, some of the cDNAs isolated from the reproductive subtracted library were homologous to genes encoding protein kinases, GTP-binding protein, and heat shock proteins involved in signal transduction and the molecular chaperon system.

Isolation and functional characterization of an ammonium transporter gene, PyAMT1, related to nitrogen assimilation in the marine macroalga Pyropia yezoensis (Rhodophyta).

Ammonium and nitrate are the primary nitrogen sources in natural environments, and are essential for growth and development in photosynthetic eukaryotes. In this study, we report on the isolation and characterization of an ammonium transporter gene (PyAMT1) which performs a key function in nitrogen (N) metabolism of Pyropia yezoensis thalli. The predicted length of PyAMT1 was 483 amino acids (AAs). The AA sequence included 11 putative transmembrane domains and showed approximately 33-44% identity to algal and plant AMT1 AA sequences. Functional complementation in an AMT-defective yeast mutant indicated that PyAMT1 mediated ammonium transport across the plasma membrane. Expression analysis showed that the PyAMT1 mRNA level was strongly induced by N-deficiency, and was more highly suppressed by resupply of inorganic-N than organic-N. These results suggest that PyAMT1 plays important roles in the ammonium transport system, and is highly regulated in response to external/internal N-status.

Isolation and characterization of a new DUR3-like gene, PyDUR3.3, from the marine macroalga Pyropia yezoensis (Rhodophyta)

DUR3 proteins mediate high-affinity transport of exogenous and endogenous urea. Although two DUR3-like genes (PyDUR3.1/3.2) have been identified in Pyropia yezoensis, a BLAST search using these sequences against the P. yezoensis EST database suggested the existence of another DUR3-like gene (PyDUR3.3). In this study, the PyDUR3.3 gene was isolated and characterized and compared to PyDUR3.1/3.2 genes. The predicted length of PyDUR3.3 was 679 amino acids, which included 15 transmembrane domains. An amino acid sequence alignment of algal, plant, and yeast DUR3 proteins showed that PyDUR3.3 was more similar to PyDUR3.2 than to other DUR3 proteins, including PyDUR3.1. Exon–intron structures of PyDUR3.3 and PyDUR3.2 were also closely related to each other, which clearly differed from that of PyDUR3.1. Expression analysis showed that PyDUR3.3 mRNA levels were extremely high in sporophytes, regardless of the nutrient condition, compared to gametophytes. On the other hand, expression of PyDUR3.2 and PyDUR3.1 was high in the gametophytes and sporophytes, respectively, and was induced by nutrient starvation. These results suggest that expression of PyDUR3.3/3.2/3.1 depends on the life history phase as well as the nutrient conditions, and that PyDUR3.3 and PyDUR3.2 are paralogues specifically differentiated in function and life history phase.