Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.     

Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.     

Biotransformation of 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra.

Two biotransformation products formed from 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra were isolated and their structures determined by chemical and spectral data as 3-O-[alhpa-L-arabinopyranosyl-(1----2)-beta-D-Glucuronopy ranosyl]-24- hydroxy-18 beta-glycyrrhetinic acid and 30-O-beta-D-glycopyrano-syl-18 beta-glycyrrhetinic acid. The formation of glycyrrhizin, the main triterpene glucuronide of the licorice root, was not detected among the biotransformation products. This is the first report of the glucuronylation of an exogenous triterpene in plant cell cultures.     

Ghrelin在小鼠体内早期胚胎发育中的表达

为了探讨Ghrelin是否在昆明系小白鼠(Mus musculus)体内早期胚胎发育中发挥作用。运用激素超排卵技术及动物解剖学方法获取小鼠体内不同发育阶段的早期胚胎,应用实时定量PCR技术检测了Ghrelin mRNA的相对表达量。结果表明,Ghrelin mRNA在小鼠各期胚胎中均有表达;同时,GhrelinmRNA的表达量呈现一定规律性变化,2-细胞期表达量最低,8-细胞期表达量达到最高值,总体趋势是先降低后升高之后又降低。研究结果揭示,Ghrelin可能在小鼠早期胚胎发育中发挥一定的作用。     

Two Plastoquinone A Molecules Are Required for Photosystem II Activity: Analysis in Hexane-Extracted Photosystem II Particles

Prenylquinones were extracted with hexane from lyophilized oxygen-evolvingphotosystem II particles prepared from spinach chloroplasts.Determination by high performance liquid chromatography showedthat two molecules of plastoquinone A remained per reactioncenter after the extraction, in contrast to the presence ofthree to four plastoquinone A molecules before the extraction.Electron transfer from water to phenyl-p-quinone was not inhibitedby the extraction. Measurement of EPR signal II and microsecondchlorophyll fluorescence kinetics showed that hexane did notextract quinones which were acting as the secondary electrondonor (Z) and the primary electron acceptor (Q_A) in photosystemII. These results, as well as the effect of quinone extractionon oxygen evolution, indicate that two molecules of plastoquinoneA acting as Z and Q_A are essential for the activity of photosystemII. An artificial donor phenyl-p-quinone probably accepts electronfrom Q_A at the same site as the intrinsic secondary electronacceptor (Q_B). Q_A and Z seem to be surrounded by special microenvironmentswhich differ from that of bulk quinones, and are resistant tohexane treatment. (Received November 27, 1984; Accepted April 30, 1985)     

Pigment formation in callus cultures of Lithospermum erythrorhizon

Undifferentiated callus tissues of Lithospermum erythrorhizon are capable of synthesizing shikonin derivatives, which are normally formed in the cork cells of the roots. Their biosynthesis in cultured cells is controlled by auxin and light. The pigment content increased linearly with time after a lag phase when callus tissues were grown on culture medium containing IAA in the dark, whereas it markedly decreased when 2,4-D was substituted for IAA or when cultures were irradiated with blue light.     

Mechanism of Inhibition by C-terminal α-Helices of the ? Subunit of Escherichia coli FoF1-ATP Synthase

The ϵ subunit of bacterial F_oF₁-ATP synthase (F_oF₁), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal α-helices of ϵ, and the “extended” but not “hairpin-folded” state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of ϵ has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli F_oF₁ (EF_oF₁) mutant in which the ϵ subunit lacked the C-terminal domain (F_oF₁^ϵΔC), and ATP synthesis driven by acid-base transition (ΔpH) and the K⁺-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (F_oF₁^ϵWT). The turnover numbers (k_cat) of F_oF₁^ϵWT were severalfold lower than those of F_oF₁^ϵΔC. F_oF₁^ϵWT showed higher Michaelis constants (K_m). The dependence of the activities of F_oF₁^ϵWT and F_oF₁^ϵΔC on various combinations of ΔpH and ΔΨ was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of ϵ. Solubilized F_oF₁^ϵWT also showed lower k_cat and higher K_m values for ATP hydrolysis than the corresponding values of F_oF₁^ϵΔC. These results suggest that the C-terminal domain of the ϵ subunit of EF_oF₁ slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit.     

Changes in intracellular cAMP level and activities of adenylcyclase and phosphodiesterase during meiosis of lily microsporocytes.

In the yeasts, Saccharomyces cerevisiae and Schyzosaccharomyces pombe, reduction of intracellular cyclic adenosine monophosphate (cAMP) is known to trigger the sporulation processes by activating various meiosis specific genes. In order to ascertain whether a similar mechanism is operative in higher plants, we carried out preliminary studies on lily microsporocytes. Measurement of cAMP levels as well as the activities of adenyl cyclase and phosphodiesterase in somatic cells and different stages of meiosis, and arrest of its in protoplasts cultured under conditions of high cAMP provided direct evidence that similar phenomena occur in plant meiocytes as earlier documented in yeasts.