Chemical synthesis,iron redox interactions and charge transfer complex formation of alkylitaconic acids from Ceriporiopsis subvermispora

In 1999, we first reported that a white rot fungus, Ceriporiopsis subvermispora produced a series of novel alkylitaconic acids (ceriporic acids). In the present paper we synthesized the metabolite, 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B) by Grignard reaction to analyze chemical properties of the alkylitaconates. Mass spectrometer (MS) and nuclear magnetic resonance (NMR) spectra of the synthetic compound was identical to those of the fungal metabolite isolated. The dicarboxylic acid inhibited autoxidation of Fe²⁺ to Fe³⁺ as well as reduction of Fe³⁺ to Fe²⁺ by the strong natural reductants, cysteine, glutathione, and ascorbic acid. The formation of charge transfer complexes (CTCs) between 1-heptadecene-2,3-dicarboxylic acid and oxidized intermediates from phenolic substrates were also observed. Thus, we herein report that the new class of lipid-related metabolites produced by C. subvermispora are potential metabolites participating in the control of iron redox reactions and CTCs formation from oxidized lignin fragments.     

Redox-coupled conformational alternations in cytochrome c(3) from D. vulgaris Miyazaki F on the basis of its reduced solution structure

Heteronuclear NMR spectroscopy was performed to determine the solution structure of (15)N-labeled ferrocytochrome c(3) from Desulfovibrio vulgaris Miyazaki F (DvMF). Although the folding of the reduced cytochrome c(3) in solution was similar to that of the oxidized one in the crystal structure, the region involving hemes 1 and 2 was different. The redox-coupled conformational change is consistent with the reported solution structure of D. vulgaris Hildenborough ferrocytochrome c(3), but is different from those of other cytochromes c(3). The former is homologous with DvMF cytochrome c(3) in amino acid sequence. Small displacements of hemes 1 and 2 relative to hemes 3 and 4 were observed. This observation is consistent with the unusual behavior of the 2(1)CH(3) signal of heme 3 reported previously. As shown by the (15)N relaxation parameters of the backbone, a region between hemes 1 and 2 has more flexibility than the other regions. The results of this work strongly suggest that the cooperative reduction of hemes 1 and 2 is based on the conformational changes of the C-13 propionate of heme 1 and the aromatic ring of Tyr43, and the interaction between His34 and His 35 through covalent and coordination bonds.     

Synthesis of the (17R)- and (17S)-isomers of volicitin,an elicitor of plant volatiles contained in the oral secretion of the beet armyworm

Both the (17R)- and (17S)-isomers of volicitin, which is contained in the oral secretion of the beet armyworm and induces corn seedlings to emit a blend of volatile compounds to attract the natural enemy of the herbivore, were synthesized via the semi-hydrogenation of an intermediary diyne and (Z)-selective olefination as the key steps. They were both obtained as crystalline compounds.     

Inhibition by ajoene of skin-tumor promotion in mice

Ajoene, a major compound containing sulfur in oil-macerated garlic products, inhibited in a two-stage carcinogenesis test on mouse skin. Treatment with ajoene suppressed skin tumor formation, depending on the amount. In particular, the group treated with 250 microg of ajoene had only 4.9% the number of tumors per mouse compared with the control group at 18 weeks.     

3-(4-Methyl-3-pentenyl)-2(5H)-furanone, alpha,alpha-acariolide and 4-(4-methyl-3-pentenyl)-2(5H)-furanone, alpha,beta-acariolide: new monoterpene lactones from the astigmatid mites, Schwiebea araujoae and Rhizoglyphus sp. (Astigmata: Acaridae)

A new monoterpene lactone from the acarid mite, Schwiebea araujoae, was elucidated without its isolation by GC/FT-IR and GC/MS analyses to be 3-(4-methyl-3-pentenyl)-2(5H)-furanone (1) and tentatively named as alpha,alpha-acariolide. The structure of 1 was identified by its synthesis from alpha-bromo-gamma-butyrolactone via 4 reaction steps. The synthesized compound gave the same GC/MS and GC/FT-IR spectra as those of the natural product. The other monoterpene lactone was likewise elucidated from the unidentified Rhizoglyphus mite to be 4-(4-methyl-3-pentenyl)-2(5H)-furanone (2) and named as alpha,beta-acariolide; it was also identified by its synthesis in 5 reaction steps from the same butyrolactone as the starting material. GC/MS and GC/FT-IR spectra of the preparation were identical to those of the natural product.     

Drosophila myosin phosphatase and its role in dorsal closure

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.     

Role of the conserved DRY motif on G protein activation of rat angiotensin II receptor type 1A

To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp-Arg-Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT(1A)) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP(3) production, and the acidification of the medium in response to Ang II were determined. The effects of GTPgammaS on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP(3) production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP(3) and acidify medium comparable with wild type AT(1A). These results indicate as follows; Asp(125) is essential for intracellular signal transduction involving G protein coupling, Arg(126) is essential for coupling of G(q) protein but not other G proteins, and Tyr(127) is not important for G protein coupling.     

Phenylpropanoid amides of serotonin accumulate in witches' broom diseased bamboo

Aciculosporium take (Ascomycota; Clavicipitaceae), causes the witches' broom disease in bamboo, particularly Phyllostachys bambusoides. Since it was observed that endogenous indole-3-acetic acid is reduced in the twigs of the diseased bamboo, the symptoms (bushy appearance) may be induced by reduction in auxin levels. Furthermore, two indolic compounds accumulated in diseased twigs, these being identified as N-p-coumaroylserotonin and N-feruloylserotonin by LC-MS, 1H NMR and 13C NMR spectroscopic analyses. N-p-Coumaroylserotonin possesses antifungal activity against A. take.     

Simple detection of point mutations in DNA oligonucleotides using SYBR Green I

A novel and simple method for detection of mutations in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) is reported. The SYBR Green I is bound specifically with a duplex DNA oligonucleotide (intercalation). This intercalation induces fluorescent emission at 525 nm with excitation at 494 nm. The fluorescence intensity of mismatched oligonucleotides (40-mer) decreases (by more than 13%) in comparison with the perfectly matched oligonucleotides. Moreover, fluorescence measurement of the SYBR Green I can distinguish various types of single-base mismatches, except for the T-G terminal mismatch. The addition of 20% (v/v) formamide, however, to an oligonucleotide solution improved the sensitivity of detection and also enabled the detection of the T-G terminal-mismatch. This detection method requires only a normal fluorescence spectrophotometer, an inexpensive dye and just 50 pmol of sample DNA.     

Genetic manipulation of gibberellin metabolism in transgenic rice

The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA 2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.