Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells

Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors.     

Akt mediates Rac/Cdc42-regulated cell motility in growth factor-stimulated cells and in invasive PTEN knockout cells

Growth factors promote cell survival and cell motility, presumably through the activation of Akt and the Rac and Cdc42 GTPases, respectively. Because Akt is dispensable for Rac/Cdc42 regulation of actin reorganization, it has been assumed that Rac and Cdc42 stimulate cell motility independent of Akt in mammalian cells. However, in this study we demonstrate that Akt is essential for Rac/Cdc42-regulated cell motility in mammalian fibroblasts. A dominant-negative Akt inhibits cell motility stimulated by Rac/Cdc42 or by PDGF treatment, without affecting ruffling membrane-type actin reorganization. We have confirmed a previous report that Akt is activated by expression of Rac and Cdc42 and also observed colocalization of endogenous phosphorylated Akt with Rac and Cdc42 at the leading edge of fibroblasts. Importantly, expression of active Akt but not the closely related kinase SGK is sufficient for increasing cell motility. This effect of Akt is cell autonomous and not mediated by inhibition of GSK3. Finally, we found that dominant-negative Akt but not SGK reverses the increased cell motility phenotype of fibroblasts lacking the PTEN tumor suppressor gene. Taken together, these results suggest that Akt promotes cell motility downstream of Rac/Cdc42 in growth factor-stimulated cells and in invasive PTEN-deficient cells.     

Rule-based spatial modeling with diffusing,geometrically constrained molecules

Background  

We suggest a new type of modeling approach for the coarse grained, particle-based spatial simulation of combinatorially complex chemical reaction systems. In our approach molecules possess a location in the reactor as well as an orientation and geometry, while the reactions are carried out according to a list of implicitly specified reaction rules. Because the reaction rules can contain patterns for molecules, a combinatorially complex or even infinitely sized reaction network can be defined.     

Genetic and thermal effects on the latitudinal variation in the timing of fin development of a fish Oryzias latipes

Heterochrony, an evolutionary change in developmental processes, is one of the major proximate causes of morphological diversity of organisms. It has been reported in the medaka Oryzias latipes that higher-latitude larvae have a genetic tendency to complete fin ray formation at larger body sizes, which results in relatively shorter anal and dorsal fins in adults. However, this latitudinal, heterochronic variation in fin length in the wild may be partially explained by latitudinal differences in thermal environments, if temperatures affect the timing of fin ray formation. Common-environment experiments revealed that the body size at which fin pterygiophore (a basal skeleton of fin rays) formation was completed was larger in higher-latitude larvae than in lower-latitude larvae at all temperatures examined, supporting the proposal that fin ray formation of the former is genetically delayed. However, phenotypic plasticity in response to temperature was also evident; lower temperatures caused delayed fin ray formation until a larger body size had been achieved in both high- and low-latitude larvae. These observations suggest that habitat temperatures also contribute to the latitudinal difference in the timing of fin development, magnifying phenotypic variation in fin length across latitudes. We discuss reasons for this positive covariance between genetic and environmental effects on the latitudinal, heterochronic variation, from the viewpoint of local adaptation and evolution of phenotypic plasticity.     

Effects of Dietary Supplementation with Fish Scales-Derived Collagen Peptides on Skin Parameters and Condition: A Randomized,Placebo-Controlled,Double-Blind Study

Fish scales-derived collagen peptides (CPs) are characterized by their specific amino acid composition with a high concentration of glycine, proline and hydroxyproline. These amino acids have been known to exert beneficial effects on human skin. The aim of the present study was to examine the effects of collagen peptides obtained from fish scales on changes in periorbital wrinkles, facial skin hydration, and skin elasticity in healthy women aged 30–60 years. In the present randomized, placebo-controlled, double-blind trial, 71 subjects consumed a 20 mL beverage containing 3000 mg of CPs or placebo once per day over 12 weeks. Significant decreases in periorbital wrinkles (p?<?0.05) were observed in the treatment group after 12 weeks of CPs ingestion compared to the control group. This study also demonstrated a consistent trend of enhanced facial skin moisture (p?<?0.001) and skin elasticity (p?<?0.001) by dietary intake of CPs without any side effects or adverse events. These findings indicate that fish-derived CPs hold great promise as a natural supplement with cutaneous anti-aging properties.     

Highly sensitive analytical method for aluminum movement in soybean root through lumogallion staining

We present highly sensitive aluminum detection method in root using fluorescent lumogallion. Roots treated with 100 μM AlCl₃ including 0.2 mM CaCl₂ (pH 4.5) were stained for 60 min with 10 μM lumogallion fluorescence solution and fluorescence from aluminum complex in root was observed under confocal laser microscope. There was a good correlation between the intensity of fluorescence and aluminum content. When the amount of aluminum lost during each step in staining process was measured, it was found that about 10% of aluminum was lost only at staining stage. Through lumogallion staining method, aluminum accumulation especially at an early stage of aluminum treatment in root was shown. At the beginning (2 hr), aluminum began to be accumulated in root cap. After 4 hr treatment, the aluminum distribution was spread to about 3 mm from root apex in the root cap and outer cortex. When aluminum was found in the outer cortex in 3–5 mm from the root apex, the viability was tended to be decreased in the same area (6 hr). At the same time, aluminum amount in meristem was increased. However the comparison of lumogallion staining method with that of morin, which has been widely used to detect aluminum in root, the sensitivity of lumogallion method was found to be much higher.     

The influence of floral symmetry and pollination systems on flower size variation

We compared the amount of variation in flower size between autogamous and insect-pollinated species to examine the hypothesis that pollinator-mediated selection stabilizes flower size in plant populations. One would expect the flower size variation to be larger in selfing species that are less affected by pollinator-mediated stabilizing selection than in insect-pollinated species. The results of phylogenetic comparisons between autogamous and insect-pollinated flowers supported the pollinator-mediated stabilizing selection hypothesis, although the non-phylogenetic comparison did not. According to our results, we discuss the factors influencing the flower size variation.     

Rapid extraction of high-quality genomic DNA from Porphyra yezoensis (Bangiales, Rhodophyta)

We developed a simple, rapid and stable method for extraction of high molecular weight DNA from the marine red alga Porphyra yezoensis Ueda using both guanidium treatment and QIAGEN? kit (Funakoshi, Tokyo, Japan). The method does not require expensive equipment and complex steps. The DNA yield averaged 1.5 μg 100 mg^?1 of Porphyra tissue and the A260/A280 and A230/A260 ratios of the DNA were approximately 1.8 and 0.4, respectively. It was of sufficient quality to be used for not only polymerase chain reactions but also other DNA manipulation techniques such as restriction digestion and construction of genomic libraries.     

A Mono-2-Ethylhexyl Phthalate Hydrolase from a Gordonia sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate

Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K_m and V_max values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.