Temperature-induced changes in the coenzyme environment of D-amino acid oxidase revealed by the multiple decays of FAD fluorescence.

A temperature-dependent change in the microenvironment of the coenzyme, FAD, of D-amino acid oxidase was investigated by means of steady-state and picosecond time-resolved fluorescence spectroscopy. Relative emission quantum yields from FAD bound to D-amino acid oxidase revealed the temperature transition when concentration of the enzyme was lowered. The observed fluorescence decay curves were well described with four-exponential decay functions. The amplitude of the shortest lifetime (tau 0), approximately 25 ps, was always negative, which indicates that the fluorescence of D-amino acid oxidase at approximately 520 nm appears after a metastable state of the excited isoalloxazine decays. The other components with positive amplitudes were assigned to dimer or associated forms of the enzyme, monomer, and free FAD dissociated from the enzyme. Ethalpy and entropy changes of intermediate states in the quenching processes were evaluated according to the absolute rate theory. The temperature transition was much more pronounced in the monomer than in the dimer or associated forms of the enzyme.     

Multiple pathways of excitation energy flow in the photosynthetic pigment system of a cryptophyte,Cryptomonas sp. (CR-1)*

Energy transfer pathways in a cryptophyte, Cryptomonas sp. (CR-1 strain) were investigated mainly by the steady state fluorescence spectroscopy and the time-resolved spectrum. Cryptomonas sp. (CR-1) contains chlorophyll (Chi) a, Chi c₂, carotenoids and cryptomonad phycoer-ythrin (Cr-PE₅₆₅), the last of which is known to be located in the lumenal side of the thyiakoid membranes. The spectral heterogeneity cf pigments was resolved by fluorescence spectra; there were at least five emission bands of Chi a at -196°C. Chlorophyll C₂ and carotenoids transferred independently to the common Chi a form (Chi a₆₆₃), and Cr-PE₅₆₅, to the different form (Chi a₆₈₂). Chlorophyll c₂ was not an intermediary component of energy transfer from carotenoid to Ch a; this is a common phenomenon to green algae and brown algae. The Chi a₆₆₃ and Chi a₆₈₂ are postulated to be located in the light-harvesting chlorophyll protein (LHC) II; thus, the energy is accumulated on Chi a₆₈₂‘n LHC II. The energy transfer step in Cr-PE_;565, was short, which was shown by a small, time-dependent red-shift of the emission. In the photosystem (PS) II core, two fluorescence components were resolved at 688 and 696 nm. The former was the trap at cryogenic temperatures. A large red-shift induced by the low temperature was explained by an equilibrium between Chi a₆₈₂ in LHC II and Chi a₆₈₈ in PS II core. The presence of Chi a₆₈₂ emission at physiological temperature is a unique feature of this alga. This was also reported in dinophyceae, which contain peridinin-ChI a-protein in the lumenal side of the thyiakoid membrane. Thus, this modification might be common in systems where the antenna complexes bind to the LHC II on the lumenal side. Based on the spectral data, we proposed a model for the molecular organization of PS II and the energy transfer pathways in cryptophyceae.     

Monitoring of the thromboxane A2/prostacyclin ratio in the urine of patients with retinal vascular occlusion through the low-dose-aspirin therapy using the gas chromatography/selected ion monitoring method

We determined the levels of the stable urinary metabolites of thromboxane A2 and prostacyclin, 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 2,3-dinor-6-keto-prostaglandin F1alpha (2,3-dinor-6-keto-PGF1alpha) in patients with retinal vascular occlusion (RVO) to elucidate the change of the thromboxane A2/prostacyclin (TX/PGI) ratio with this disease and the effect of low-dose-aspirin therapy. 11-Dehydro-TXB2 and 2,3-dinor-6-keto-PGF1alpha were converted to 1-methyl ester-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative and 1-methyl ester-6-methoxime-9,12,15-tris-dimethylisopropylsilyl ether derivative, respectively, and applied to a gas chromatography/selected ion monitoring. The average level of 11-dehydro-TXB2 in 30 patients with RVO was 1038 +/- 958 pg/mg creatinine. It was significantly higher than that of 27 healthy volunteers, which was 616 +/- 294 pg/mg creatinine (p < 0.05 with unpaired t-test). However, 2,3-dinor-6-keto-PGF1alpha levels were not significantly different between these two groups. The average ratio of TX/PGI in the RVO patients was 32 +/- 26 and it was significantly higher than that of healthy volunteers, 17 +/- 10 (p < 0.01). Patients with central retinal artery occlusion or branch retinal artery occlusion showed greatly high 11-dehydro-TXB2 levels and TX/PGI ratios, although the number of patients was limited in the current study. After the administration of low-dose aspirin (40 mg/day) for about 1 month, the TX/PGI ratio decreased to around the normal level. Following the levels for up to 10 months, they also remained at the normal level. These observations suggested that the 11-dehydro-TXB2 levels and the TX/PGI ratio reflect the pathological conditions of RVO and are useful markers of the treatment.     

Ethylene-induced putrescine accumulation modulates K+ partitioning between roots and shoots in barley seedlings

We investigated the cause and effect relationships among ethylene, polyamines, and K⁺ in barley ( Hordeum vulgare L. cv. Amagi) seedlings. Application of 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, to the growth medium caused a decrease in K⁺ concentration in roots and an increase in shoots. Addition of ACC induced putrescine accumulation in roots, while spermidine and spermine levels remained unchanged. Exogenous supply of putrescine led to putrescine accumulation and reduced K⁺ concentration. Application of Co²⁺, an inhibitor of ethylene biosynthesis, together with ACC, inhibited putrescine accumulation with a decrease in K⁺ concentration in roots. ACC-treated roots showed K⁺ uptake capacity equivalent to that of control roots, implying that the majority of K⁺ is translocated to shoots. These results suggest that ethylene regulates K⁺ partitioning between roots and shoots through the level of accumulation of putrescine in barley seedlings.     

A Novel Method for In Situ Hybridization in Fungal Cells based on Pricking Micro–injection of Photobiotin Labelled Probes

A novel method, a combination of a micro–injection and in situ hybridization cytochemistry, was developed for the examination of gene expression in Erysiphe graminis f. sp. hordei. In view of its high cellular content, hence high probability of hybridization with corresponding probes, cytoplasmic rRNA was chosen as the target and hybridized with a micro–injected photobiotin–labelled nucleic acid probe. The rDNA sequence was isolated from a genomic library of the fungus by the, use of cDNA derived from 28S RNA of Fusarium oxysporum f. sp. lycopersici , and the complementry RNA strand was synthesized in vitro for a probe. Since neither intact nor fixed conidial cells took up FITC–labelled albumin, the photo, biotin–conjugated RNA probe was introduced into cytoplasm of conidiospores by a pricking method. Positive hybridization was visualized by the colour–generating reaction catalyzed by biotinylated enzyme (alkaline phosphatase) which was first bound to the hybridized photobiotinlabelled probe. Specific hybridization was detected in cytoplasm of more than 80% of pricked conidiospores. A similar result was obtained when a probe was introduced into appressoria and haustoria formed on/in barley coleoptile epidermal cells. Hybridization was also observed in these structures when a double–stranded rDNA probe was introduced by pricking.     

Phased A-tracts bind to the alpha subunit of RNA polymerase with increased affinity at low temperature.

Previously we showed that the expression of a Clostridium perfringens phospholipase C gene (plc) is activated by promoter upstream phased A-tracts in a low temperature-dependent manner. In this paper we characterize the interaction between the alpha subunit of C. perfringens RNA polymerase and the phased A-tracts. Hydroxyl radical footprinting and fluorescence polarization assaying revealed that the alpha subunit binds to the minor grooves of the phased A-tracts through its C-terminal domain with increased affinity at low temperature. The result provides a molecular mechanism underlying the activation of the plc promoter by the phased A-tracts.     

Wnt signal amplification via activity, cooperativity, and regulation of multiple intracellular PPPSP motifs in the Wnt co-receptor LRP6

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/beta-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of beta-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors.     

Suppression of adipocyte differentiation by Cordyceps militaris through activation of the aryl hydrocarbon receptor

Mycelial extracts have a wide range of biological activities that modulate functions of mammalian cells. In this report, we sought to identify antiadipogenic mycelia with the use of 3T3-L1 cells and found that the extract of Cordyceps militaris exclusively suppressed differentiation of 3T3-L1 preadipocytes into mature adipocytes without affecting cell viability. This inhibitory effect was dose dependent, reversible, and associated with 1) a decrease in lipid accumulation, 2) blunted induction of adipocyte markers including adiponectin, peroxisome proliferator-activated receptor-gamma, and CCAAT/enhancer binding protein-alpha, and 3) sustained expression of a preadipocyte marker, monocyte chemoattractant protein-1. C. militaris also significantly decreased accumulation of lipid and hypertrophy in mature adipocytes and preserved their response to insulin (phosphorylation of Akt) during prolonged culture. Subsequent experiments revealed that C. militaris has the potential to activate the aryl hydrocarbon receptor (AhR). In 3T3-L1 cells, treatment with AhR agonists including benzo[a]pyrene and 3-methylcholanthrene reproduced the antiadipogenic effect of C. militaris. Furthermore, dominant-negative inhibition of AhR abrogated the suppressive effect of C. militaris on adipocyte differentiation. These results suggest that C. militaris has the potential to interfere with adipocyte differentiation through activation of AhR.