Involvement of acetaldehyde in seed deterioration of some recalcitrant woody species through the acceleration of aerobic respiration

The rate of acetaldehyde (Ald) evolution in the deterioration of recalcitrant woody seeds was investigated. Four plant species, Ligustrum japonicum, Quercus serrata, Quercus myrsinaefolia and Camellia japonica, were used for the experiments. Similar to orthodox seeds, all of the recalcitrant seeds used contained Ald in addition to methanol and ethanol, although the amount of Ald in Camellia, a typical oil seed, was very small. These volatiles were accumulated in a container in which Ligustrum and Q. serrata seeds were stored for a short period. Moreover, all of the seeds that had been previously exposed to Ald for only 6 d at 3 or 13 degrees C lost their vigor rapidly in proportion to the concentration of Ald. The occasional removal by decompression of Ald accumulated in the container prolonged the life span of Q. serrata seeds from 4 to 6 months. These findings suggest that a short life span of the hydrated recalcitrant seeds may involve Ald synthesis as in the orthodox seeds. However, the action mechanism of Ald in Ligustrum and Quercus seeds in which storage substances were polysaccharides seems to differ slightly from that in orthodox seeds, because their aerobic respiration was significantly stimulated by exposure to exogenously applied Ald. It was, therefore, thought that the rapid deterioration of some recalcitrant seeds in woody species may result from a decline in vigor, not only due to the denaturation of functional proteins by Ald as in the orthodox seeds but also due to the rapid consumption of direct substrates for the Ald-stimulated aerobic respiration and related co-enzymes within seeds. In contrast, in the oil-bearing Camellia seeds, Ald was slightly produced and their aerobic respiration was not enhanced by Ald, although they were very sensitive to Ald. Desiccation storage of Camellia seeds caused the deterioration of their outer part, which was accelerated by exogenously applied Ald, which suggests that in Camellia Ald acts only to denature the functional proteins as in orthodox seeds. Thus, the short longevity of these woody recalcitrant seeds is discussed in relation to the actions of Ald produced endogenously.     

Synthesis of oligodeoxynucleotides containing a single diastereoisomer of alpha-(N2-2'-deoxyguanosinyl)-N-desmethyltamoxifen

A phosphoramidite chemical synthesis of oligodeoxynucleotides containing a diastereoisomer of (E)-alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen, a major tamoxifen (TAM)-derived DNA adduct in animal and women treated with TAM, was described. The site-specifically modified oligodeoxynucleotide can be used for mutagenesis, DNA repair, and 3D structural studies and also as standard for quantitative analysis of TAM-DNA adducts in animal and human.     

Wavelength-selective and anisotropic light-diffusing scale on the wing of the Morpho butterfly

We have found that cover scales on the wing of the butterfly Morpho didius possess specially designed microscopic structures for wavelength-selective reflection and contribute considerably to the brilliant blue colour of the wing. In addition, the cover scale functions as an anisotropic optical diffuser which diffuses light only in one plane, while it makes the range of reflection narrower in the orthogonal plane. The quantitative analyses for the wavelength-selection mechanism and the peculiar optical diffuser are given and the role of such a special optical effect is discussed from physical and biological viewpoints.     

Crystal structure of an ADP-dependent glucokinase from Pyrococcus furiosus: implications for a sugar-induced conformational change in ADP-dependent kinase

ADP-dependent kinases are used in the modified Embden-Meyerhoff pathway of certain archaea. Our previous study has revealed a mechanism for ADP-dependent phosphoryl transfer by Thermococcus litoralis glucokinase (tlGK), and its evolutionary relationship with ATP-dependent ribokinases and adenosine kinases (PFKB carbohydrate kinase family members). Here, we report the crystal structure of glucokinase from Pyrococcus furiosus (pfGK) in a closed conformation complexed with glucose and AMP at 1.9A resolution. In comparison with the tlGK structure, the pfGK structure shows significant conformational changes in the small domain and a region around the hinge, suggesting glucose-induced domain closing. A part of the large domain next to the hinge is also shifted accompanied with domain closing. In the pfGK structure, glucose binds in a groove between the large and small domains, and the electron density of O1 atoms for both the alpha and beta-anomer configurations was observed. The structural details of the sugar-binding site of ADP-dependent glucokinase were firstly clarified and then site-directed mutagenesis analysis clarified the catalytic residues for ADP-dependent kinase, such as Arg205 and Asp451 of tlGK. Homology search and multiple alignment of amino acid sequences using the information obtained from the structures reveals that eucaryotic hypothetical proteins homologous to ADP-dependent kinases retain the residues for the recognition of a glucose substrate.     

Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.     

Aspolin, a novel extremely aspartic acid-rich protein in fish muscle, promotes iron-mediated demethylation of trimethylamine-N-oxide

Trimethylamine-N-oxide (TMAO) is abundant in marine fish. Formaldehyde synthesis by TMAO demethylation during storage markedly deteriorates fish meat. In the present work, we cloned the extremely aspartic acid-rich proteins from skeletal muscle of a commercially important species, walleye pollack, in the course of molecular identification of trimethylamine-N-oxide demethylase (TMAOase). One of the cDNAs, designated as aspolin1, encodes an extremely aspartic acid-rich protein of 228 amino acids which is converted to the TMAOase after processing between Ala42 and Asp43. Mature aspolin1/TMAOase protein contains 179 Asp in 186 total amino acids. The other cDNA, designated as aspolin2, has a common nucleotide sequence with aspolin1 in the 5' part and encodes a protein which has an additional Asp polymer and a C-terminal cysteine-rich region. The amino acid sequence of the C-terminal cysteine-rich region of aspolin2 is highly homologous to the mammalian histidine-rich Ca2+-binding protein. Aspolin1/TMAOase and aspolin2 mRNA was most abundant in the skeletal muscle. A lower level of the mRNA was also detected in kidney, heart, spleen, and brain. Synthetic Asp polymer showed marked TMAOase activity in the presence of Fe2+, whereas a monomer and oligomers did not. Purified TMAOase protein bound to Fe2+ with low affinity, which may be responsible for the catalytic activity. Poly aspartic acid-Fe2+ complex generated after death would be involved in formaldehyde synthesis by the demethylation of TMAO during the storage of fish meat.     

Biological activities of homologous loop regions in the laminin alpha chain G domains

Laminin alpha chains (alpha1-alpha5 chains) have diverse chain-specific biological functions. The LG4 modules of laminin alpha chains consist of a 14-stranded beta-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse alpha chains (EF-1: DYATLQLQEGRLHFMFDLG, alpha1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, alpha2 chain 2808-2826; EF-3: RDSFVALYLSEGHVIFALG, alpha3 chain 2266-2284; EF-4: DFMTLFLAHGRLVFMFNVG, alpha4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, alpha5 chain 3304-3323). These homologous peptides showed chain-specific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted alpha2beta1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin alpha1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins.     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.