Expressed sequence tags from eyestalk of kuruma prawn, Marsupenaeus japonicus

We analyzed the expressed sequence tags (ESTs) obtained from a cDNA library of the eyestalk of the kuruma prawn, Marsupenaeus japonicus, to examine gene expression profile with special focus on female reproduction. The assembly of 1988 ESTs created 136 contigs from 738 ESTs; however 1250 ESTs remained singletons. Significant similarities (blast score > or = 50 bits) to the DNA sequences in the databank were found for only 16.7% of the 1386 sequences (136 contigs plus 1250 singletons), suggesting that the eyestalk library contains many unknown genes. Ribosomal RNA and mitochondrial respiration enzymes with significant similarities were found abundantly in the ESTs, whereas genes related to maturation or endocrine systems were scarce. Three ESTs were assumed to encode novel eyestalk hormones with marked similarities to pigment-dispersing hormone, molt-inhibiting hormone and crustacean hyperglycemic hormone. Sequences encoding a product highly homologous to farnesoic acid O-methyltransferase, an enzyme that produces methyl farnesoate, were also found.     

The NuRD component Mbd3 is required for pluripotency of embryonic stem cells

Cells of early mammalian embryos have the potential to develop into any adult cell type, and are thus said to be pluripotent. Pluripotency is lost during embryogenesis as cells commit to specific developmental pathways. Although restriction of developmental potential is often associated with repression of inappropriate genetic programmes, the role of epigenetic silencing during early lineage commitment remains undefined. Here, we used mouse embryonic stem cells to study the function of epigenetic silencing in pluripotent cells. Embryonic stem cells lacking Mbd3 - a component of the nucleosome remodelling and histone deacetylation (NuRD) complex - were viable but failed to completely silence genes that are expressed before implantation of the embryo. Mbd3-deficient embryonic stem cells could be maintained in the absence of leukaemia inhibitory factor (LIF) and could initiate differentiation in embryoid bodies or chimeric embryos, but failed to commit to developmental lineages. Our findings define a role for epigenetic silencing in the cell-fate commitment of pluripotent cells.     

The effect of surface charge property on Escherichia coli initial adhesion and subsequent biofilm formation

Polyethylene (PE) sheets were modified by radiation-induced graft polymerization (RIGP) of an epoxy-group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8 h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom-shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000 s(-1)), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications.     

Membrane Protein Rim21 Plays a Central Role in Sensing Ambient pH in Saccharomyces cerevisiae

External alkalization activates the Rim101 pathway in Saccharomyces cerevisiae. In this pathway, three integral membrane proteins, Rim21, Dfg16, and Rim9, are considered to be the components of the pH sensor machinery. However, how these proteins are involved in pH sensing is totally unknown. In this work, we investigated the localization, physical interaction, and interrelationship of Rim21, Dfg16, and Rim9. These proteins were found to form a complex and to localize to the plasma membrane in a patchy and mutually dependent manner. Their cellular level was also mutually dependent. In particular, the Rim21 level was significantly decreased in dfg16Δ and rim9Δ cells. Upon external alkalization, the proteins were internalized and degraded. We also demonstrate that the transient degradation of Rim21 completely suppressed the Rim101 pathway but that the degradation of Dfg16 or Rim9 did not. This finding strongly suggests that Rim21 is the pH sensor protein and that Dfg16 and Rim9 play auxiliary functions through maintaining the level of Rim21 and assisting in its plasma membrane localization. Even without external alkalization, the Rim101 pathway was activated in a Rim21-dependent manner by either protonophore treatment or depletion of phosphatidylserine in the inner leaflet of the plasma membrane, both of which caused plasma membrane depolarization like the external alkalization. Therefore, plasma membrane depolarization seems to be one of the key signals for the pH sensor molecule Rim21.     

Factors That Differentiate the H-bond Strengths of Water Near the Schiff Bases in Bacteriorhodopsin and Anabaena Sensory Rhodopsin

Bacteriorhodopsin (BR) functions as a light-driven proton pump, whereas Anabaena sensory rhodopsin (ASR) is believed to function as a photosensor despite the high similarity in their protein sequences. In Fourier transform infrared (FTIR) spectroscopic studies, the lowest O-D stretch for D₂O was observed at ∼2200 cm⁻¹ in BR but was significantly higher in ASR (>2500 cm⁻¹), which was previously attributed to a water molecule near the Schiff base (W402) that is H-bonded to Asp-85 in BR and Asp-75 in ASR. We investigated the factors that differentiate the lowest O-D stretches of W402 in BR and ASR. Quantum mechanical/molecular mechanical calculations reproduced the H-bond geometries of the crystal structures, and the calculated O-D stretching frequencies were corroborated by the FTIR band assignments. The potential energy profiles indicate that the smaller O-D stretching frequency in BR originates from the significantly higher pK_a(Asp-85) in BR relative to the pK_a(Asp-75) in ASR, which were calculated to be 1.5 and −5.1, respectively. The difference is mostly due to the influences of Ala-53, Arg-82, Glu-194–Glu-204, and Asp-212 on pK_a(Asp-85) in BR and the corresponding residues Ser-47, Arg-72, Ser-188-Asp-198, and Pro-206 on pK_a(Asp-75) in ASR. Because these residues participate in proton transfer pathways in BR but not in ASR, the presence of a strongly H-bonded water molecule near the Schiff base ultimately results from the proton-pumping activity in BR.     

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 °C for 4 h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0 Å. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a Cα atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154–164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the β-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.     

Competitive solubilization of cholesterol and β-sitosterol with changing biliary lipid compositions in model intestinal solution

We used a model intestinal solution to understand the mechanisms of cholesterol lowering by the addition of plant sterols. The experimental results of the competitive solubilization of cholesterol and β-sitosterol in vitro give useful information about these mechanisms. The states of the model intestinal solution as a solubilizer were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) by changing the number of components, and the bile salt and phosphatidylcholine concentrations. There were aggregates of different sizes: liposomes and mixed micelles depending on their components and concentrations. The maximum solubilization of cholesterol increased from 0.2mM to 1.3mM when adding fatty compounds in the pure bile salts system, which is almost the same as the full components model intestinal solution. Therefore, an excessive intake of fatty compounds may also increase cholesterol absorption in vivo. Even if the components of the model intestinal solution were modified from the standard condition, there were not remarkable differences in the selectivity of cholesterol and β-sitosterol in competitive solubilization. With the addition of β-sitosterol, the maximum solubilization of cholesterol decreases to almost half of that in the system with only cholesterol, except for PC-rich systems. In general, the different structures of aggregates considerably influence the maximum solubilization of sterols but not the selectivity of cholesterol and β-sitosterol in the competitive solubilization. The Gibbs energy change (ΔG°) of the solubilization of β-sitosterol showed a more negative value than cholesterol by -4 to -6kJmol(-1), which indicates that β-sitosterol is energetically favored relative to cholesterol in the model intestinal solution, regardless of the different systems.     

The enhancement of phase 2 enzyme activities by sodium butyrate in normal intestinal epithelial cells is associated with Nrf2 and p53

Dietary fiber fermentation by the colonic bacterial flora produces short-chain fatty acids, acetate, propionate and butyrate. Among them, butyrate is considered to be the major energy substrate for colonocytes and, at least in rats, seems to protect against colonic carcinogenesis. In this study, we examined the effect and the mechanisms of short-chain fatty acids on the activity of phase 2 enzymes. Sodium butyrate increased phase 2 enzyme activities in normal rat small intestine epithelial cells, Glutathione S-transferase and NAD(P)H:quinone oxidoreductase (NQO) in a dose-dependent manner_; however, other short-chain fatty acids did not increase them. The mechanism of the induction of phase 2 enzymes with sodium butyrate sodium butyrate, but not other short-chain fatty acids was related to the increase of NF-E2-related factor 2 (Nrf2) nuclear translocation and the decrease in the levels of nuclear fraction p53. Sodium butyrate also caused enhancement of Nrf2 mRNA levels and suppression of p53 mRNA levels. Sodium butyrate enhances the activities of phase 2 enzymes via an increase in the Nrf2 protein levels in the nucleus and a decrease in the mRNA and protein levels of p53.     

Dietary habits of the fanray Platyrhina tangi (Batoidea: Platyrhinidae) in Ariake Bay, Japan

The dietary habits of the fanray Platyrhina tangi were investigated by analyzing the stomach contents of specimens collected in Ariake Bay, Japan. Of 334 stomach specimens, 324 contained food and 10 (3.0%) were empty. The mean percentage weight of stomach contents per unit of body weight was 0.59%. Thirty-seven taxonomic levels of prey were identified. The most common prey was shrimp, followed by fish and mysids. There were no differences in the composition of the diet between sexes, but an ontogenetic dietary shift was observed. Shrimps were the most common prey in all size classes. In addition, smaller individuals frequently ate mysids, and larger individuals often consumed fish. Dietary breadth values increased with size. Trophic level analysis revealed that trophic level increased with size; however, this species is consistently a secondary consumer. Dental sexual dimorphism was also observed. Specifically, mature males had much longer and sharper cusps than females and immature males. Since males and females had similar diets, dental sexual dimorphism may be related to their reproductive behaviour.