Enhancement of nucleoside phosphorylation activity in an acid phosphatase

Escherichia blattae non-specific acid phosphatase (EB-NSAP) possesses a pyrophosphate-nucleoside phosphotransferase activity, which is C-5'-position selective. Current mutational and structural data were used to generate a mutant EB-NSAP for a potential industrial application as an effective and economical protein catalyst in synthesizing nucleotides from nucleosides. First, Gly74 and Ile153 were replaced by Asp and Thr, respectively, since the corresponding replacements in the homologous enzyme from Morganella morganii reduced the K(m) value for inosine and thus increased the productivity of 5'-IMP. We determined the crystal structure of G74D/I153T, which has a reduced K(m) value for inosine, as expected. The tertiary structure of G74D/I153T was virtually identical to that of the wild-type. In addition, neither of the introduced side chains of Asp74 and Thr153 is directly involved in the interaction with inosine in a hypothetical binding mode of inosine to EB-NSAP, although both residues are situated near a potential inosine-binding site. These findings suggested that a slight structural change caused by an amino acid replacement around the potential inosine-binding site could significantly reduce the K(m) value. Prompted by this hypothesis, we designed several mutations and introduced them to G74D/I153T, to decrease the K(m) value further. This strategy produced a S72F/G74D/I153T mutant with a 5.4-fold lower K(m) value and a 2.7-fold higher V(max) value as compared to the wild-type EB-NSAP.     

Size distribution,growth and inter-year variation in sex expression of Bischofia javanica,an invasive tree

Flowering activity and sex expression of Bischofia javanica Blume were investigated for 3 years. B. javanica is an invasive dioecious tree of subtropical forests on the Bonin Islands in the western Pacific of Japan. The sex ratio showed a significant male bias (1.25-2.33). Smaller trees were significantly male biased, whereas larger trees showed no significant difference in sex expression, suggesting that males tend to be more precocious in sexual reproduction. We found evidence for sex changes in B. javanica; these have not been reported previously. Most of the 1,653 census trees remained non-flowering (58.1 %); 3.7 % of them showed sex changes, and the percentage of trees repeatedly flowering as males and females was 10.5 and 3.4 %, respectively. Sex changes were observed in both directions but a larger percentage of male trees became female. Flowering frequency and sex expression were significantly related to tree size (i.e. diameter at breast height). Over the 3 years, trees that were consistent females were the largest; inconsistent trees (switching sex between years) were intermediate in size, whereas consistent males were the smallest. There were no significant differences in relative growth rate (RGR) among trees of different sex or flowering frequencies. These results suggest that the maintenance of female reproduction is not related to changes in RGR of diameter but to flowering frequency or the reversal to the male form, dependent upon the internal resource status of individual trees.     

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.     

DAP12 ITAM motif regulates differentiation and apoptosis in M1 leukemia cells

DAP12 is an immunoreceptor tyrosine-based activation motif (ITAM)-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. DAP12 is expressed not only in NK cells, but also in myeloid cells. Previously, we reported that DAP12 was likely to be involved in monocyte differentiation to macrophage. In this study, we established the mutant DAP12-M1 transfectants (Y76F-M1) that have mutation at their ITAM motifs. We observed that Y76F-M1 cells could not differentiate to macrophages by stimulation via DAP12, whereas wild type DAP12 transfectants (FDAP-M1) could. Furthermore, we demonstrated that the apoptosis signal mediated by LPS was inhibited in Y76F-M1 cells, but was augmented in FDAP-M1 cells. In contrast to the LPS-mediated apoptosis, the combination of LPS and DAP12 stimulation showed good cell viability in FDAP-M1 cells. Collectively our studies demonstrated that DAP12 has a critical role for macrophage differentiation and LPS induced apoptosis in M1 leukemia cells.     

Calcium-regulated exocytosis of dense-core vesicles requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide binding site opener at the plasma membrane

Vacuole fusion requires a coordinated cascade of priming, docking, and fusion. SNARE proteins have been implicated in the fusion itself, although their precise role in the cascade remains unclear. We now report that the vacuolar SNAP-23 homologue Vam7p is a mobile element of the SNARE complex, which moves from an initial association with the cis-SNARE complex via a soluble intermediate to the docking site. Soluble Vam7p is specifically recruited to vacuoles and can rescue a fusion reaction poisoned with antibodies to Vam7p. Both the recombinant Vam7p PX domain and a FYVE domain construct of human Hrs block the recruitment of Vam7p and vacuole fusion, demonstrating that phosphatidylinositol 3-phosphate is a primary receptor of Vam7p on vacuoles. We propose that the Vam7p cycle is linked to the availability of a lipid domain on yeast vacuoles, which is essential for coordinating the fusion reaction prior to and beyond docking.     

Natural<Superscript>15</Superscript> N Abundance of Plants and Soil N in a Temperate Coniferous Forest

Measurement of nitrogen isotopic composition (¹⁵N) of plants and soil nitrogen might allow the characteristics of N transformation in an ecosystem to be detected. We tested the measurement of ¹⁵N for its ability to provide a picture of N dynamics at the ecosystem level by doing a simple comparison of ¹⁵N between soil N pools and plants, and by using an existing model. ¹⁵N of plants and soil N was measured together with foliar nitrate reductase activity (NRA) and the foliar NO₃^– pool at two sites with different nitrification rates in a temperature forest in Japan. ¹⁵N of plants was similar to that of soil NO₃^– in the high-nitrification site. Because of high foliar NRA and the large foliar NO₃^– pool at this site, we concluded that plant ¹⁵N indicated a great reliance of plants on soil NO₃^– there. However, many ¹⁵N of soil N overlapped each other at the other site, and ¹⁵N could not provide definitive evidence of the N source. The existing model was verified by measured ¹⁵N of soil inorganic N and it explained the variations of plant ¹⁵N between the two sites in the context of relative importance of nitrification, but more information about isotopic fractionations during plant N uptake is required for quantitative discussions about the plant N source. The model applied here can provide a basis to compare ¹⁵N signatures from different ecosystems and to understand N dynamics.     

Effect of evening exposure to dim or bright light on the digestion of carbohydrate in the supper meal

In a previous study we found that daytime exposure to bright as compared to dim light exerted a beneficial effect on the digestion of the evening meal. This finding prompted us to examine whether the digestion of the evening meal is also affected by evening light intensity. Subjects lived in light of 200 lux during the daytime (08:00-17:00 h) and took their evening meal at 17:00 h under 20 lux (evening dim-light condition: 17:00-02:00 h) or 2000 lux (evening bright-light condition: 17:00-02:00 h) until retiring at 02:00 h. Assessment of carbohydrate digestion of the evening meal was accomplished by a breath hydrogen test that is indicative of the malabsorption of dietary carbohydrate. Hydrogen excretion in the breath in the evening under the dim-light condition was significantly less than under the bright-light condition (p < 0.05). This finding is the opposite to that obtained in previous experiments in which subjects were exposed to the different intensities of light during the daytime, and indicates that the exposure to dim light in the evening exerts a better effect on carbohydrate digestion in the evening meal than does the exposure to bright light.     

Endocranial cast and morphology of the olfactory bulb of<Emphasis Type="Italic"> Amphipithecus mogaungensis</Emphasis> (latest middle Eocene of Myanmar)

A detailed endocranial cast of the olfactory bulb of Amphipithecus mogaungensis, a latest middle Eocene primate from the Pondaung Formation (Myanmar), was studied in comparison with some Paleogene primates, the olfactory bulb of which has been reported. The olfactory bulb of Amphipithecus is located just anterior to the postorbital constriction, that is, within the interorbital septum. It is relatively large and pedunculate, not overlapped by the frontal lobe, and consists of two parallell aligned bodies. The relative volume of the olfactory bulb shows the same pattern as in adapiforms, but the location and bilobed form are more similar to those of omomyoids than of adapiforms. Electronic Publication     

Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.