Identification of three clones which commonly interact with the kinase domains of highly homologous two receptor-like kinases, RLK902 and RKL1

We have previously reported the characterization of highly homologous two leucine-rich repeat (LRR)-receptor-like kinase (RLK) genes, RLK902 and RKL1, which showed 75% identity at the amino acid sequence level. To investigate the RLK902 and RKL1 mediated signal transduction pathways, we performed yeast two-hybrid screening using the kinase domains of RLK902 and RKL1 as baits. Three clones, Y-1, 2 and 3, were found to interact commonly with the kinase domain of RLK902 and RKL1 and not to interact with the kinase domain of BRI1, a member of LRR-RLKs. This result suggests that RLK902 and RKL1 may have common biochemical functions, especially in their downstream signal transduction. Furthermore, the detail analysis of their responsiveness to various conditions suggests their involvement in such stress conditions as mechanical wounding, treatment with salicylic acid, and pathogen infection.     

Maturation of fermented rice-koji miso can be monitored by an increase in fatty acid ethyl ester

A mixture of steamed soybean and boiled rice with seeded Aspergillus oryzae was naturally fermented without addition of yeasts or Lactobacilli, and kept matured for 12 months at room temperature. Chemical analysis of this rice-koji miso sample for lipid changes during maturation showed that triacylglycerol was gradually decomposed into free fatty acid, with distinct formation of fatty acid ethyl ester which, six months after the start of fermentation, came to account for 35.0% of total lipid. The ester was constituted primarily with linoleic acid (ca. 50%) and oleic acid (ca. 20%), no appreciable change in this proportion being observed during maturation. Also, the proportion was unique in that this did not reflect the fatty acid composition in a mixture of the two materials. It is possible to monitor the maturation of the rice-koji miso by following up the increase with time in fatty acid ethyl ester.     

Modeling the vertical foliage distribution of an individual <Emphasis Type="Italic">Castanopsis cuspidata</Emphasis> (Thunb.) Schottky,a dominant broad-leaved tree in Japanese warm-temperate forest

The vertical foliage distribution of Castanopsis cuspidata (Thunb.) Schottky was examined in trees of various sizes to clarify its variation in relation to tree size and the light environment in a stand. As indices of these parameters, we analyzed crown social position (CSP: percent of stand height) and specific leaf area (SLA). The vertical foliage distribution of trees was expressed by a Weibull function. The variation in the vertical foliage distribution of C. cuspidata could be categorized into three types using crown social position and light environment. In the first type, leaves were concentrated to the top 20% of the tree; such trees are canopy trees that can receive full sunlight. The second type had a large relative crown depth and an asymmetric distribution with the maximum foliage located near the top of the tree; such trees are suppressed trees whose crowns do not receive sufficient light. The third type had a large relative crown depth and a symmetric distribution; such trees occur in high light environments, although their crowns are in the understory layer. The differences in the vertical foliage distribution are related to the strategies used to capture light. Multiple regression analysis showed that CSP and SLA at the top layer of the tree explained successive changes in the vertical foliage distribution. These results will contribute to scaling-up the vertical foliage distribution to the community level in pure stands of C. cuspidata using an individual-based model.     

Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Here, we report that inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerate cell differentiation and cause a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. Our data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells.     

Resonance Raman study on synergistic activation of soluble guanylate cyclase by imidazole, YC-1 and GTP

Soluble guanylate cyclase (sGC), a physiological nitric oxide (NO) receptor, is a heme-containing protein and catalyzes the conversion of GTP to cyclic GMP. We found that 200 mM imidazole moderately activated sGC in the coexistence with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), although imidazole or YC-1 alone had little effect for activation. GTP facilitated this process. Resonance Raman spectra of imidazole complex of native sGC and CO-bound sGC (CO-sGC) have demonstrated that a simple heme adduct with imidazole at the sixth coordination position is not present for both sGC and CO-sGC below 200 mM of the imidazole concentration and that the Fe-CO stretching band (nuFe-CO)) appears at 492 cm(-1) in the presence of imidazole compared with 473 cm(-1) in its absence. Both frequencies fall on the line of His-coordinated heme proteins in the nuFe-CO vs nuC-O plot. However, it is stressed that the CO-heme of sGC becomes apparently photo-inert in a spinning cell in the presence of imidazole, suggesting the formation of five-coordinate CO-heme or of six-coordinate heme with a very weak trans ligand. These observations suggest that imidazole alters not only the polarity of heme pocket but also the coordination structure at the fifth coordination side presumably by perturbing the heme-protein interactions at propionic side chains. Despite the fact that the isolated sGC stays in the reduced state and is not oxidized by O(2), sGC under the high concentration of imidazole (1.2 M) yielded nu4 at 1373 cm(-1) even after its removal by gel-filtration, but addition of dithionite gave the strong nu4 band at 1360 cm(-1). This indicated that imidazole caused autoxidation of sGC.     

Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays an important role in the formation of the viral nucleocapsid and a regulatory protein involved in hepatocarcinogenesis. In this study, we have identified proteasome activator PA28gamma (11S regulator gamma) as an HCV core binding protein by using yeast two-hybrid system. This interaction was demonstrated not only in cell culture but also in the livers of HCV core transgenic mice. These findings are extended to human HCV infection by the observation of this interaction in liver specimens from a patient with chronic HCV infection. Neither the interaction of HCV core protein with other PA28 subtypes nor that of PA28gamma with other Flavivirus core proteins was detected. Deletion of the PA28gamma-binding region from the HCV core protein or knockout of the PA28gamma gene led to the export of the HCV core protein from the nucleus to the cytoplasm. Overexpression of PA28gamma enhanced the proteolysis of the HCV core protein. Thus, the nuclear retention and stability of the HCV core protein is regulated via a PA28gamma-dependent pathway through which HCV pathogenesis may be exerted.