Structural Basis of α-Catenin Recognition by EspB from Enterohaemorrhagic E. coli Based on Hybrid Strategy Using Low-Resolution Structural and Protein Dissection

Enterohaemorrhagic E. coli (EHEC) induces actin reorganization of host cells by injecting various effectors into host cytosol through type III secretion systems. EspB is the natively partially folded EHEC effector which binds to host α-catenin to promote the actin bundling. However, its structural basis is poorly understood. Here, we characterize the overall structural properties of EspB based on low-resolution structural data in conjunction with protein dissection strategy. EspB showed a unique thermal response involving cold denaturation in the presence of denaturant according to far-UV circular dichroism (CD). Small angle X-ray scattering revealed the formation of a highly extended structure of EspB comparable to the ideal random coil. Various disorder predictions as well as CD spectra of EspB fragments identified the presence of α-helical structures around G41 to Q70. The fragment corresponding to this region indicated the thermal response similar to EspB. Moreover, this fragment showed a high affinity to C-terminal vinculin homology domain of α-catenin. The results clarified the importance of preformed α-helix of EspB for recognition of α-catenin.     

Stomatal closure induced by high vapor pressure deficit limited midday photosynthesis at the canopy top of <Emphasis Type="Italic">Fagus crenata</Emphasis> Blume on Naeba mountain in Japan

Diurnal changes in gas exchange, chlorophyll fluorescence and leaf water potential (_leaf) were measured to determine the environmental and physiological factors that limit carbon gain in the horizontal leaves of Fagus crenata Blume at the canopy top. Although midday depression of the net CO₂ assimilation rate (A_n) and stomatal conductance (gH₂O) were clearly evident on a fine day, the potential quantum yield of PS II (F_v/F_m) was fairly constant around 0.83 throughout the day. This result indicates that the leaves at the canopy top do not suffer from chronic photoinhibition, and the excess energy is dissipated safely. Large reversible increases in non-photochemical quenching (NPQ) were evident on fine days. Therefore, the non-radiative energy dissipation of excess light energy contributed to avoid chronic photoinhibition. The electron transfer rate (ETR) reached maximum during the midday depression, and thus there was no positive relation between ETR and A_n under high light conditions, indicating a high rate of photorespiration and the absence of non-stomatal effect during midday. The protective mechanisms such as non-radiative energy dissipation and photorespiration play an important role in preventing photoinhibitory damage, and stomatal limitation is the main factor of midday depression of A_n. To separate the effect of air to leaf vapor pressure deficit (ALVPD) and leaf temperature (T_leaf) on gas exchange, the dependencies of A_n and gH₂O on ALVPD and T_leaf were measured using detached branches under controlled conditions. A_n and gH₂O were insensitive to an increase in T_leaf. With the increase in ALVPD, A_n and gH₂O exhibited more than a 50% decrease even though water supply was optimum, suggesting the dominant role of high ALVPD in the midday depression of gH₂O. We conclude that midday depression of A_n results from the midday stomatal closure caused by high ALVPD.     

Developmental abnormalities in mouse embryos tetrasomic for chromosome 11: apparent similarity to embryos functionally disomic for the x chromosome

Adopting a mating system involving two different Robertsonian translocations with monobrachial homology, we studied the early development of mouse embryos trisomic or tetrasomic for chromosome 11. A developmental delay of 12-24 hours was evident in trisomic embryos at embryonic day (E)7.5, whereas tetrasomic embryos apparently had stopped growth by E6.5 without formation of extraembryonic structures. This extremely severe developmental abnormality found in tetrasomic embryos is similar to that reported in embryos having two active X chromosomes in extraembryonic cell lineages. Autosomal tetrasomy, but not autosomal trisomy, can lead to such early developmental errors. Thus, a reasonable inference would be that the X chromosome is twice as active as the autosome. Probably, the X chromosome became upregulated in response to the evolutionary necessity of minimizing haplo-insufficiency brought about by miniaturization of the Y chromosome.     

Olfactory responses to natal stream water in sockeye salmon by BOLD fMRI

Many studies have shown that juvenile salmon imprint olfactory memory of natal stream odors during downstream migration, and adults recall this stream-specific odor information to discriminate their natal stream during upstream migration for spawning. The odor information processing of the natal stream in the salmon brain, however, has not been clarified. We applied blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging to investigate the odor information processing of the natal stream in the olfactory bulb and telencephalon of lacustrine sockeye salmon (Oncorhynchus nerka). The strong responses to the natal stream water were mainly observed in the lateral area of dorsal telencephalon (Dl), which are homologous to the medial pallium (hippocampus) in terrestrial vertebrates. Although the concentration of L-serine (1 mM) in the control water was 20,000-times higher than that of total amino acid in the natal stream water (47.5 nM), the BOLD signals resulting from the natal stream water were stronger than those by L-serine in the Dl. We concluded that sockeye salmon could process the odor information of the natal stream by integrating information in the Dl area of the telencephalon.     

Determination of timiperone in rat plasma by high-performance liquid chromatography with electrochemical detection

We report a sensitive new method for the determination of timiperone in rat plasma by using high-performance liquid chromatography with electrochemical detection. The method involves extraction of plasma samples with heptane-isoamyl alcohol at pH>8, followed by back-extraction into dilute acetic acid. Separation was accomplished by reversed-phase high-performance liquid chromatography on an ODS column with the mobile phase consisting of 0.1 M phosphate buffer (pH 3.5)-acetonitrile-methanol (65:20:15, v/v). Recovery was greater than 80%. Calibration curve was linear over the concentration range 0.5–50.0 ng/ml. The limit of quantitation of timiperone was 0.5 ng/ml plasma.     

Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana

The location of tryptophan residues in the actin macromolecule was studied on the basis of the known 3D structure. For every tryptophan residue the polarity and packing density of their microenvironments were evaluated. To estimate the accessibility of the tryptophan residues to the solvent molecules it was proposed to analyze the radial dependence of the packing density of atoms in the macromolecule about the geometric center of the indole rings of the tryptophan residues. The proposed analysis revealed that the microenvironment of tryptophan residues Trp-340 and Trp-356 has a very high density. So these residues can be regarded as internal and inaccessible to solvent molecules. Their microenvironment is mainly formed by non-polar groups of protein. Though the packing density of the Trp-86 microenvironment is lower, this tryptophan residue is apparently also inaccessible to solvent molecules, as it is located in the inner region of macromolecule. Tryptophan residue Trp-79 is external and accessible to the solvent. All residues that can affect tryptophan fluorescence were revealed. It was found that in the close vicinity of tryptophan residues Trp-79 and Trp-86 there are a number of sulfur atoms of cysteine and methionine residues that are known to be effective quenchers of tryptophan fluorescence. The most essential is the location of SG atom of Cys-10 near the NE1 atom of the indole ring of tryptophan residue Trp-86. On the basis of microenvironment analysis of these tryptophan residues and the evaluation of energy transfer between them it was concluded that the contribution of tryptophan residues Trp-79 and Trp-86 must be low. Intrinsic fluorescence of actin must be mainly determined by two other tryptophan residues--Trp-340 and Trp-356. It is possible that the unstrained conformation of tryptophan residue Trp-340 and the existence of aromatic rings of tyrosine and phenylalanine and proline residues in the microenvironments of tryptophan residues Trp-340 and Trp-356 are also essential to their blue fluorescence spectrum.     

Initiation of epigenetic reprogramming of the X chromosome in somatic nuclei transplanted to a mouse oocyte

The active and inactive X chromosomes have distinct epigenetic marks in somatic nuclei, which undergo reprogramming after transplantation into oocytes. We show that, despite the disappearance of Xist RNA coating in 30 min, the epigenetic memory of the inactive X persists with the precocious appearance of histone H3 trimethylation of lysine 27 (H3-3meK27), without the expected colocalization with Eed/Ezh2. Subsequently, Xist re-appears on the original inactive X, and the silent Xist on the active X undergoes re-activation, resulting in unusual biallelic Xist RNA domains. Despite this abnormal Xist expression pattern, colocalization of H3-3meK27 and Eed is thereafter confined to a single Xist domain, which is presumably on the original inactive X. These epigenetic events differ markedly from the kinetics of preferential paternal X inactivation in normal embryos. All the epigenetic marks on the X are apparently erased in the epiblast, suggesting that the oocyte and epiblast may have distinct properties for stepwise programming of the genome.     

The Influence of Previous Irradiance on Photosynthetic Induction in Three Species Grown in the Gap and Understory of a Fagus Crenata Forest

Photosynthetic induction responses to a sudden increase in photosynthetic photon flux density (PPFD) from lower background PPFD (0, 25, 50, and 100 mol m^–2 s^–1) to 1 000 mol m^–2 s^–1 were measured in leaves of Fagus crenata, Acer rufinerve Siebold & Zucc., and Viburnum furcatum growing in a gap and understory of a F. crenata forest in the Naeba mountains. In the gap, A. rufinerve exhibited more than 1.2-fold higher maximum net photosynthetic rate (P _Nmax) than F. crenata and V. furcatum. Meanwhile, in the understory F. crenata exhibited the highest P _Nmax among the three species. The photosynthetic induction period required to reach P _Nmax was 3–41 min. The photosynthetic responses to increase in PPFD depended on the background PPFD before increase in PPFD. The induction period required to reach P _Nmax was 2.5–6.5-fold longer when PPFD increased from darkness than when PPFD increased from 100 mol m^–2 s^–1. The induction period was correlated with initial P _N and stomatal conductance (g _s) relative to maximum values before increase in PPFD. The relationship was similar between the gap and the understory. As the background PPFD increased, the initial P _N and g _s increased, indicating that the degrees of biochemical and stomata limitations to dynamic photosynthetic performance decreased. Therefore, photosynthetic induction responses to increase in PPFD became faster with the increasing background PPFD. The differences in time required to reach induction between species, as well as between gap and understory, were mainly due to the varying of relative initial induction states in P _N and g _s at the same background PPFD.