Molecular Functions of Oxygen-Evolving Complex Family Proteins in Photosynthetic Electron Flow

Oxygen-evolving complex (OEC) protein is the original name for membrane-peripheral subunits of photosystem (PS) Ⅱ. Recently,multiple isoforms and homologs for OEC proteins have been identified in the chloroplast thylakoid lumen, indicating that functional diversification has occurred in the OEC family. Gene expression profiles suggest that the Arabidopsis OEC proteins are roughly categorized into three groups: the authentic OEC group, the stressresponsive group, and the group including proteins related to the chloroplast NAD(P)H dehydrogenase (NDH) complex involved in cyclic electron transport around PSI. Based on the above gene expression profiles, molecular functions of the OEC family proteins are discussed together with our current knowledge about their functions.     

Anti-metatype peptides, a molecular tool with high sensitivity and specificity to monitor small ligands

To develop a new immunological detection system of gibberellins (GAs), a class of phytohormones, peptides that interact with an antibody against GA₄ in a GA₄-dependent manner, were screened from phage display random peptide libraries. The biopanning procedure yielded peptides designated as anti-metatype peptides (AM-peps), which showed specific binding to the complex of the antibody and its ligand GA₄; that is, the antibody could not be replaced with the other anti-GA₄ antibody, and GA₄ could not be replaced with GA₁, another ligand of the antibody. Together with computational analyses such as analysis of structural propensity of the AM-peps and docking simulation of the AM-peps and the 8/E9-GA₄ complex, it was suggested that AM-peps formed a helix in their central region and interacted with a part of the 8/E9-GA₄ complex located in close proximity to the GA₄ molecule. Based on the property of AM-peps to make a ternary complex with antibody and its ligand, a noncompetitive enzyme-linked immunosorbent assay (ELISA) system corresponding to sandwich ELISA was developed to detect GA₄. GA₄ as low as 30 pg, which could not be achieved by conventional competitive ELISA, could be detected by the new system, demonstrating the feasibility of this system.     

NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells

Nitric oxide (NO) has been implicated in pancreatic β-cell death in the development of diabetes. The mechanisms underlying NO-induced β-cell death have not been clearly defined. Recently, receptor-interacting protein-1 (RIP1)-dependent necrosis, which is inhibited by necrostatin-1, an inhibitor of RIP1, has emerged as a form of regulated necrosis. Here, we show that NO donor-induced β-cell death was inhibited by necrostatin-1. Unexpectedly, however, RIP1 knockdown neither inhibited cell death nor altered the protective effects of necrostatin-1 in NO donor-treated β-cells. These results indicate that NO donor induces necrostatin-1-inhibitable necrotic β-cell death independent of RIP1. Our findings raise the possibility that NO-mediated β-cell necrosis may be a novel form of signal-regulated necrosis, which play a role in the progression of diabetes.     

Carbon cycling and budget in a forested basin of southwestern Hokkaido,northern Japan

Quantification of annual carbon sequestration is very important in order to assess the function of forest ecosystems in combatting global climate change and the ecosystem responses to those changes. Annual cycling and budget of carbon in a forested basin was investigated to quantify the carbon sequestration of a cool-temperate deciduous forest ecosystem in the Horonai stream basin, Tomakomai Experimental Forest, northern Japan. Net ecosystem exchange, soil respiration, biomass increment, litterfall, soil-solution chemistry, and stream export were observed in the basin from 1999–2001 as a part of IGBP-TEMA project. We found that 258 g C m^–2 year^–1 was sequestered annually as net ecosystem exchange (NEE) in the forested basin. Discharge of carbon to the stream was 4 g C m^–2 year^–1 (about 2% of NEE) and consisted mainly of dissolved inorganic carbon (DIC). About 43% of net ecosystem productivity (NEP) was retained in the vegetation, while about 57% of NEP was sequestered in soil, suggesting that the movement of sequestered carbon from aboveground to belowground vegetation was an important process for net carbon accumulation in soil. The derived organic carbon from aboveground vegetation that moved to the soil mainly accumulated in the solid phase of the soil, with the result that the export of dissolved organic carbon to the stream was smaller than that of dissolved inorganic carbon. Our results indicated that the aboveground and belowground interaction of carbon fluxes was an important process for determining the rate and retention time of the carbon sequestration in a cool-temperate deciduous forest ecosystem in the southwestern part of Hokkaido, northern Japan.     

The adapter protein Crkl links Cbl to C3G after integrin ligation and enhances cell migration

Crkl, an SH2-SH3-SH3 adapter protein, is one of the major tyrosine phosphoproteins detected in cells from patients with chronic myelogenous leukemia. Crkl binds to BCR/ABL through its N-terminal SH3 domain and is known to interact with several signaling proteins that have been implicated in integrin signaling, including Cbl, Cas, Hef-1, and paxillin. We have previously shown that overexpression of Crkl enhances adhesion to extracellular matrix proteins through beta(1) integrins. In this study, the effects of Crkl on spontaneous and chemokine-directed migration of the hematopoietic cell line Ba/F3 were examined. Full-length, SH2-, and SH3(N)-domain deletion mutants of Crkl were expressed transiently as fusion proteins with green fluorescent protein. Successfully transfected cells were isolated by fluorescence-activated cell sorting. The ability of these cells to migrate across a fibronectin-coated membrane, either spontaneously or in response to the chemokine stromal-derived factor-1alpha, was determined. Cells expressing green fluorescent protein alone were not distinguishable from untransfected or mock transfected Ba/F3 cells. However, Ba/F3 cells overexpressing full-length Crkl were found to have an increase in spontaneous migration of 2.8 +/- 0.6-fold in seven independent assays. The enhancement of migration required both the SH2 domain and the N-terminal SH3 domain. Migration in response to stromal-derived factor-1alpha was not significantly enhanced by overexpression of Crkl. Overexpression of Crkii also augmented spontaneous migration but to a lesser degree than did Crkl. Because the SH2 domain was required for enhanced migration, we looked for changes in phosphotyrosine containing proteins coprecipitating with Crkl, but not Crkl DeltaSH2, after integrin cross-linking. Full-length Crkl, but not CrklDeltaSH2, coprecipitated with a single major tyrosine phosphoprotein with an M(r) of approximately 120 kDa, identified as Cbl. The major Crkl SH3-binding protein in these cells was found to be the guanine nucleotide exchange factor, C3G. Interestingly, overexpression of C3G also enhanced migration, suggesting that a Cbl-Crkl-C3G complex may be involved in migration signaling in Ba/F3 cells. These data suggest that Crkl is involved in signaling pathways that regulate migration, possibly through a complex with Cbl and C3G.     

Maturation of fermented rice-koji miso can be monitored by an increase in fatty acid ethyl ester

A mixture of steamed soybean and boiled rice with seeded Aspergillus oryzae was naturally fermented without addition of yeasts or Lactobacilli, and kept matured for 12 months at room temperature. Chemical analysis of this rice-koji miso sample for lipid changes during maturation showed that triacylglycerol was gradually decomposed into free fatty acid, with distinct formation of fatty acid ethyl ester which, six months after the start of fermentation, came to account for 35.0% of total lipid. The ester was constituted primarily with linoleic acid (ca. 50%) and oleic acid (ca. 20%), no appreciable change in this proportion being observed during maturation. Also, the proportion was unique in that this did not reflect the fatty acid composition in a mixture of the two materials. It is possible to monitor the maturation of the rice-koji miso by following up the increase with time in fatty acid ethyl ester.     

Development of damage function of acidification for terrestrial ecosystems based on the effect of aluminum toxicity on net primary production

Background  Acidification is one of the important impact categories for life cycle impact assessment. Although its characterization has progressed during this decade through the employment of midpoint approaches, only limited studies of endpoint approaches have been performed. Objective. This study aimed at developing damage function of acidification for terrestrial ecosystems in Japan. Damage function expresses a quantitative relationship between the inventory and endpoint damage. Methods  The geographical boundary was limited in Japan both for emission and impact. In this study, sulfur dioxide (SO₂), nitrogen monoxide (NO), nitrogen dioxide (NO₂) (NO and NO₂ collectively mean NO_x), hydrogen chloride (HC1), and ammonia (NH₃) were considered as major causative substances of acidification. Net primary production (NPP) of existing vegetation was adopted as an impact indicator of terrestrial ecosystems. The aluminum toxicity was adopted as the major factor of effect on terrestrial ecosystems due to acidification. The leachate concentration of monomeric inorganic aluminum ions was selected to express the plant toxicity of aluminum. Results and Discussion  The results of damage function gave utilizable factors both for a midpoint approach and an endpoint approach; Atmospheric Deposition Factor (ADF) and Damage Factor (DF) applicable to the former and the latter, respectively. The ADF indicates an increase of H⁺ deposition per unit area to an additional emission of causative sustance. The additional emission corresponds to some alternatives in industry, not the baseline emission. The DF indicates the total NPP damage in all of Japan due to the additional emission of causative substances. The derived NPP damage is on the order of one millionth of the NPP itself. HC1 and NH3 showed larger ADFs and DFs than that of SO₂ and NO_x. The reason was ascribed to the relatively large source-receptor relationships (SRR) of HC1 and NH₃. However, since the method applied to determine the SRR of HC1 and NH₃ has larger uncertainties than that of SO₂ and NO_x, attention is needed to handle the difference. Conclusion  The damage function easily defines the concrete NPP damage due to an additional emission. The impact indica tor, NPP, also has an advantage in its mass unit that is directly summable through the entire impact categories. Expansion of endpoints, such as in aquatic ecosystems, material degradation, human health, and biodiversity aspects of terrestrial ecosystems, is an important subject for future work. Further, uncertain analyses for major parameters will provide helpful information on the reliability of damage function.     

Formation of N-(hexanoyl)ethanolamine, a novel phosphatidylethanolamine adduct, during the oxidation of erythrocyte membrane and low-density lipoprotein

The primary amino groups of biomolecules such as aminophospholipids, as well as proteins, are the potential targets of covalent modifications by lipid peroxidation products; however, little attention has been paid to the modification of aminophospholipids such as phosphatidylethanolamine (PE). The purpose of this study is to characterize the formation of a novel modified phospholipid, N-(hexanoyl)phosphatidylethanolamine (HEPE), in the reaction of PE with lipid hydroperoxides using mass spectrometric analyses. Upon reaction of egg PE with 13-hydroperoxyoctadecadienoic acid or other oxidized polyunsaturated fatty acids followed by phospholipase D-mediated hydrolysis, the formation of N-(hexanoyl)ethanolamine (HEEA), a head group of HEPE, was confirmed by isotope dilution liquid chromatography/tandem mass spectrometry. Moreover, increasing HEEA was detected in the hydrolysates of oxidized erythrocyte ghosts and low-density lipoprotein with their increasing lipid peroxidation levels. Collectively, these results suggest that the N-hexanoylated product of phospholipid, HEPE, can be generated during lipid peroxidation and may serve as one mechanism for the covalent modification of aminophospholipids in vivo.