A novel neurotrophic drug for cognitive enhancement and Alzheimer's disease

Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer''s disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer''s disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.     

Characterization of O-GlcNAcylation in starfish (Asterina pectinifera) development from fertilization to bipinnaria larva

Though O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins has been found in many multicellular organisms, its presence or absence in Echinodermata is unknown. Here we report the occurrence of O-GlcNAcylation in starfish (Asterina pectinifera) oocytes and the apparent O-GlcNAcylation pattern in starfish early development. O-GlcNAcylation might participate in the regulation of starfish development at the mid-blastula stage and thereafter.     

Response of Japanese beech (Fagus japonica Maxim.) sprouts to canopy gaps

The response of Japanese beech (Fagus japonica Maxim.) sprouts to canopy gaps in natural beech forest in central Japan was studied using two contrasted gaps in which tree-ring chronologies of regenerating stems were analyzed. The gaps were created by uprooting of a single Quercus mongolica var. grosseserrata stem (diameter: 50 cm; gap size: 40 m²; 23 years old) and by concurrent uprootings of four F. japonica stools (gap size: 180 m²; 30 years old). Japanese beech sprouts emerged before and after the gap formation and dominated stem populations in both gaps. In gaps, growth of F. japonica sprouts was equal or lower than growth of stems of seed origin, but most sprouts (F. japonica, Acer mono var. marmoratum) appeared a few years before emergence of seedlings. The small gap created by single stem fall was dominated by some beech sprouts from stools adjacent to the gap. The multiple gap was not closed by beech sprouts from stools surrounding the gap, but some dominant beech stems were resprouts from the uprooted beech stools. The existence of a sprout bank under the canopy may play an important role in the closing process of gaps in natural Japanese beech forest.     

Nitrite is an alternative source of NO in vivo

In this study, we investigated whether orally administered nitrite is changed to NO and whether nitrite attenuates hypertension in a dose-dependent manner. We utilized a stable isotope of [15N]nitrite (15NO2-) as a source of nitrite to distinguish between endogenous nitrite and that exogenously administered and measured hemoglobin (Hb)-NO as an index of circulating NO in whole blood using electron paramagnetic resonance (EPR) spectroscopy. When 1 mg/kg Na15NO2 was orally administered to rats, an apparent EPR signal derived from Hb15NO (A(Z) = 23.4 gauss) appeared in the blood. The peak blood HbNO concentration occurred at the first measurement after intake (5 min) for treatment with 1 and 3 mg/kg (HbNO: 4.93 +/- 0.52 and 10.58 +/- 0.40 microM, respectively) and at 15 min with 10 mg/kg (HbNO: 38.27 +/- 9.23 microM). In addition, coadministration of nitrite (100 mg/l drinking water) with N(omega)-nitro-L-arginine methyl ester (L-NAME; 1 g/l) for 3 wk significantly attenuated the L-NAME-induced hypertension (149 +/- 10 mmHg) compared with L-NAME alone (170 +/- 13 mmHg). Furthermore, this phenomenon was associated with an increase in circulating HbNO. Our findings clearly indicate that orally ingested nitrite can be an alternative to L-arginine as a source of NO in vivo and may explain, at least in part, the mechanism of the nitrite/nitrate-rich Dietary Approaches to Stop Hypertension diet-induced hypotensive effects.     

Sensitivity to RNA interference-mediated gene silencing in intact and ligated larvae of the armyworm, Mythimna separata (Lepidoptera: Noctuidae)

RNA interference (RNAi) is a common tool for analysis of gene function in both model and non-model insects, but it is becoming evident that RNAi efficiency varies considerably from species to species. We examined RNAi efficiency in larvae of the armyworm Mythimna separata (Walker) using multiple genes and tissues. First, we showed that five different target genes exhibited distinct tissue distribution patterns by quantitative determination of mRNA in total hemocytes, foregut, midgut, hindgut, Malpighian tubules and fat body: neuroglian mRNA was most abundant in fat body; inhibitor of apoptosis proteins mRNA was found to be ubiquitous; aquaporin 4 mRNA was most enriched in hindgut; cueball and prophenoloxidase 2 were mainly expressed in hemocytes. Second, we assessed sensitivity to gene silencing by double-strand RNA injection of these five genes in the six different tissues. We found that these genes generally showed refractoriness to double-strand RNA-mediated gene knockdown irrespective of the tissue tested. Finally, we demonstrated that appreciable gene knockdown was achieved at least in the adhering hemocyte fraction when larval isolated abdomen was prepared by ligation and subjected to dsRNA injection. Our study thus added detailed information on the refractoriness of larval tissues of a lepidopteran insect to gene silencing through RNAi and provided a new potential approach to improve RNAi efficiency.     

ATP-Dependent but Proton Gradient-Independent Polyphosphate-Synthesizing Activity in Extraradical Hyphae of an Arbuscular Mycorrhizal Fungus

Arbuscular mycorrhizal (AM) fungi benefit their host plants by supplying phosphate obtained from the soil. Polyphosphate is thought to act as the key intermediate in this process, but little is currently understood about how polyphosphate is synthesized or translocated within arbuscular mycorrhizas. Glomus sp. strain HR1 was grown with marigold in a mesh bag compartment system, and extraradical hyphae were harvested and fractionated by density gradient centrifugation. Using this approach, three distinct layers were obtained: layers 1 and 2 were composed of amorphous and membranous materials, together with mitochondria, lipid bodies, and electron-opaque bodies, and layer 3 was composed mainly of partially broken hyphae and fragmented cell walls. The polyphosphate kinase/luciferase system, a highly sensitive polyphosphate detection method, enabled the detection of polyphosphate-synthesizing activity in layer 2 in the presence of ATP. This activity was inhibited by vanadate but not by bafilomycin A₁ or a protonophore, suggesting that ATP may not energize the reaction through H⁺-ATPase but may act as a direct substrate in the reaction. This report represents the first demonstration that AM fungi possess polyphosphate-synthesizing activity that is localized in the organelle fraction and not in the cytosol or at the plasma membrane.Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that form symbiotic associations with most land plants (29). These fungi promote the growth of host plants via enhanced uptake of phosphate (P_i) and thus play important roles in the terrestrial phosphorus cycle. In the symbiotic phase, AM fungi take up P_i from soil through an extensive network of extraradical hyphae and rapidly accumulate inorganic polyphosphate (polyP). This accumulation was as rapid as that for a polyP-hyperaccumulating bacterium found in activated sludge (6). PolyP is a linear polymer of three to hundreds of molecules of P_i linked by high-energy phosphoanhydride bonds and has been found across all classes of organisms (19). Although polyP is considered to play a central role in long-distance translocation of P_i in AM fungal associations (4, 10, 30, 31), the translocation mechanism, metabolism, and dynamics in the fungi have not been elucidated due to the difficulty in obtaining sufficient fungal material for analysis.Many enzymes/genes involved in polyP synthesis/metabolism have been identified and characterized in prokaryotes (19). For instance, exopolyphosphatase hydrolyzes the terminal high-energy bonds of polyP, and polyphosphate glucokinase (PPGK) transfers the terminal P_i residue to glucose. Polyphosphate kinase 1 (PPK1) is responsible both for polyP synthesis, using ATP as a phosphoryl donor, and for the reverse ATP-generating reaction. This enzyme is bound to the plasma membrane (18) and has been found in a wide range of bacteria (17). Unlike the case for prokaryotes, knowledge of polyP synthesis/metabolism in eukaryotes remains limited. The first eukaryotic PPK genes, DdPPK1 (32) and DdPPK2 (14), were identified from the social slime mold Dictyostelium discoideum. The products of these genes, as known for bacterial PPK1s, are responsible both for polyP synthesis and for the ATP-generating reaction and have been suggested to be associated with vacuoles or small vesicles (14, 32). Although several homologues of bacterial PPK1 genes have now been found in the genomes of eukaryotic microorganisms (17), yeast Candida humicola is the only organism apart from D. discoideum for which PPK-like activity has been confirmed (22). The model organism Saccharomyces cerevisiae is known to accumulate polyP, to up to 10% of its dry weight (19). A unique polyP synthetic pathway different from those of PPK1 has been proposed for S. cerevisiae based on the observation that vacuolar-type H⁺-ATPase (V-ATPase)-defective mutants could not accumulate polyP (23). In this hypothetical pathway, P_i would be polymerized by an analogous system (enzyme) of mitochondrial F₁-ATPase on the vacuolar membrane, using the proton motive force created by V-ATPase (23). On the other hand, Hothorn et al. (16) demonstrated very recently that vacuolar transporter chaperone 4 (VTC4), a small transmembrane protein associated with the membrane, polymerizes P_i by using the γ-P_i residue of ATP as a phosphoryl donor in S. cerevisiae.More than 2 decades ago, Capaccio and Callow (3) reported the presence of polyP-hydrolyzing, -metabolizing (PPGK), and -synthesizing (PPK-like) activities in the soluble (cytosolic) fractions of the hyphae of the AM fungus Glomus mosseae. Recently, polyP-hydrolyzing activity was found in both the cytosolic and insoluble (membrane) fractions and then characterized (8). PPGK activity has also been confirmed in the cytosolic fraction, although the activity was quite low and hexokinase (ATP-hexose phosphotransferase) activity appeared to dominate in the glucose phosphorylation process (9). PPK-like activity, however, could not be detected in the same fraction (10), and this seems likely because all other prokaryotic (reviewed in reference 17) and eukaryotic (14, 16, 22, 32) polyP-synthesizing enzymes, so far, are associated with membranes. These observations suggest that AM fungi possess a polyP-synthesizing enzyme that is probably associated with membranes and that ATP may be essential in the synthesis as a phosphoryl donor or via H⁺-ATPase, as suggested by Ogawa et al. (23). In this study, a cell fractionation technique was applied to demonstrate polyP-synthesizing activity in an AM fungus, and then the role of ATP in the synthesis was investigated.     

Inhibitory effect of hot-water extract of quince (<Emphasis Type="Italic">Cydonia oblonga</Emphasis>) on immunoglobulin E-dependent late-phase immune reactions of mast cells

We evaluated the effect of a crude hot-water extract (HW) of quince (Cydonia oblonga Miller) fruit on immunoglobulin E (IgE)-dependent late-phase immune reactions of mast cells using in vitro system. Mast cell-like RBL-2H3 cells were treated with quince HW and late-phase reaction was then induced by stimulation with IgE + Antigen. Quince HW reduced the elevation of interleukin-13 and tumor necrosis factor-α expression level. Furthermore, quince HW suppressed these cytokine expressions of mouse bone marrow-derived mast cells (BMMCs), a normal mast cell model. Leukotriene C₄ and prostaglandin D₂ production in BMMCs after 1 and 6 h of stimulation, respectively, were also reduced by treating the cells with quince HW. We found that the induction of intracellular cyclooxygenase (COX)-2 expression but not COX-1 expression in BMMCs was reduced by quince HW. These results suggest that quince HW has an inhibitory effect on broad range of the late-phase immune reactions of mast cells.     

Effects of voluntary resistance exercise on heme biosynthesis in rats given glucocorticoid-injections

To identify the effects of a tower-climbing exercise on bone marrow heme biosynthesis and hematological status in rats given glucocorticoid-injections as a model of aging, 29 male Sprague-Dawley rats, 10 weeks of age, were assigned to three groups: a saline control (C, n = 9), a glucocorticoid-sedentary (GS, n = 10) group, and a glucocorticoid-exercise (GE, n = 10) group. The GS and GE groups were given 2 mg/kg prednisolone daily, and the C group was given 2 ml of saline daily, subcutaneously. Each group was meal-fed commercial rat chow isoenergetically and given free access to water for 8 weeks. The GE group were allowed to climb a 200-cm tower to drink water from a bottle set at the top of it. Weight gain during the 8-week experimental period was greater in the C group than in the GS and GE groups. The gastrocnemius and tibialis anterior muscles were heavier in the C group than in other groups. The hematological parameters were not influenced by glucocorticoid administration with or without climbing exercise. Bone marrow delta-aminolevulinic acid dehydratase activity was significantly higher in the GE group than in the C group. These results suggest that 8 weeks of climbing exercise increases heme biosynthesis without alteration of hematological status in rats given glucocorticoid-injections. Resistance exercise may be a preventive therapy for iron-deficiency anemia associated with aging.     

Cryopreserved autologous nipple-areola complex transfer to the reconstructed breast

If a patient's nipple-areola complex is available for grafting after mastectomy, it is the best material to use for nipple-areola reconstruction. The authors performed delayed autologous nipple-areola complex transfer to reconstructed breasts in 10 patients (mean age, 47 years; range, 40 to 53 years). The nipple-areola complex was cryopreserved with a programmed freezer after mastectomy. Histological examination of the tissue surrounding the nipple and areola eliminated the possibility of cancer invasion. At the time of transfer, the cryopreserved nipple-areola complex was thawed in 37 degrees C water and grafted on a projection made by a denuded dermal flap on the reconstructed breast. Each patient underwent immediate breast reconstruction using an innervated pedicled transverse rectus abdominis musculocutaneous (TRAM) flap. The patients' postoperative courses were uneventful. The timing of transfer ranged from 3 months to 1 year (mean, 5.8 months) after breast reconstruction. Nipple projection was made by the "four" dermal flap in five cases, a round dermal flap in three cases, a double dermal flap in one case, and a denuded skate flap in one case. The follow-up period ranged from 5 to 36 months (mean, 21.8 months). All grafts were adapted. The final evaluation of nipple-areola complex adaptation was good in four cases, fair in four cases, and poor in two cases. Histological examination of the hematoxylin and eosin stains showed no remarkable destruction of the skin of the nipple and areola, and electron microscopic examination of the areola skin revealed no significant change. However, electron microscopic examination of the nipple skin showed serious damage to skin components, including elongation of the desmosome, widening of the intercellular space at the prickle cell and basal layers, and shrinking of prickle and basal cells. Although further development of the freezing process and cryopreservation technique is needed to prevent depigmentation of the nipple and areola, cryopreserved nipple-areola complex transfer to a reconstructed breast could be an alternative method of nipple-areola reconstruction.     

Importance of topography and soil texture in the spatial distribution of two sympatric dipterocarp trees in a Bornean rainforest

Relationships between spatial distributions and site conditions, namely topography and soil texture, were analyzed for two congeneric emergent trees, Dryobalanops aromatica and Dryobalanops lanceolata (Dipterocarpaceae), in a tropical rainforest in Sarawak, East Malaysia. A 52-ha permanent plot was divided into 1300 quadrats measuring 20m×20m; for each Dryobalanops species, the number and total basal area of trees 1cm in d.b.h. were compared among groups of quadrats with different site conditions. Because spatial distributions of both Dryobalanops and site-condition variables were aggregated, Monte-Carlo permutation tests were applied to analyze the relationships. Both single and multifactor statistical tests showed that the density and basal area distributions of the two species were significantly non-random in relation to soil texture and topographic variables. D.aromatica was significantly more abundant at higher elevations, in sandy soils, and on convex and steep slopes. In contrast, D.lanceolata preferred lower elevations and less sandy soils. In the study plot, there were very few sites (3 of 1150 quadrats tested) where the models of Hayashis method predicted the co-occurrence of the two species. These results suggest that between-species differences in habitat preferences are so large that they alone explain the spatially segregated distributions of these two species within the 52-ha study plot.