Nerve and behavioral responses of mice to various umami substances

Food contains various taste substances. Among them, umami substances play an important role with regard to the perception of the taste of food, but, few studies have examined the taste characteristics of representative umami substances other than monosodium L-glutamate (MSG). By conducting mouse behavioral studies (the 48-h 2-bottle preference test and the conditioned taste aversion test) and assessing gustatory nerve responses, we investigated the taste characteristics of unique umami substances, including sodium succinate, L-theanine, betaine, and the enantiomer of MSG, D-MSG. Furthermore, we examined the synergy of umami with inosine 5'-monophoshate (IMP). In the case of the mice, sodium succinate had an umami taste and showed strong synergy with IMP. L-theanine showed synergy with IMP but did not have an umami taste without IMP. In contrast, betaine did not have an umami taste or synergy with IMP. D-MSG might have weak synergy with IMP.     

S(G), S(I), and EGP of exercise-trained middle-aged men estimated by a two-compartment labeled minimal model

To examine the effects of physical training on glucose effectiveness (S(G)), insulin sensitivity (S(I)), and endogenous glucose production (EGP) in middle-aged men, stable-labeled frequently sampled intravenous glucose tolerance tests (FSIGTT) were performed on 11 exercise-trained middle-aged men and 12 age-matched sedentary men. The time course of EGP during the FSIGTT was estimated by nonparametric stochastic deconvolution. Glucose uptake-specific indexes of glucose effectiveness (S(2*)(G) x 10(2): 0.81 +/- 0.08 vs. 0.60 +/- 0.05 dl. min(-1). kg(-1), P < 0.05) and insulin sensitivity [S(2*)(I) x 10(4): 24.59 +/- 2.98 vs. 11.89 +/- 2.36 dl. min(-1). (microU/ml)(-1). kg(-1), P < 0.01], which were analyzed using the two-compartment minimal model, were significantly greater in the trained group than in the sedentary group. Plasma clearance rate (PCR) of glucose was consistently greater in the trained men than in sedentary men throughout FSIGTT. Compared with sedentary controls, EGP of trained middle-aged men was higher before glucose load. The EGP of the two groups was similarly suppressed by approximately 70% within 10 min, followed by an additional suppression after insulin infusion. EGP returned to basal level at approximately 60 min in the trained men and at 100 min in the controls, followed by its overshoot, which was significantly greater in the trained men than in the controls. In addition, basal EGP was positively correlated with S(2*)(G) . The higher basal EGP and greater EGP overshoot in trained middle-aged men appear to compensate for the increased insulin-independent (S(2*)(G)) and -dependent (S(2*)(I)) glucose uptake to maintain glucose homeostasis.     

Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8

The structurally related orphan G-protein-coupled receptors GPR7 and GPR8 are expressed in the central nervous system, and their ligands have not been identified. Here, we report the identification of the endogenous ligand for both of these receptors. We purified the peptide ligand from porcine hypothalamus using stable Chinese hamster ovary cell lines expressing human GPR8 and cloned the cDNA encoding its precursor protein. The cDNA encodes two forms of the peptide ligand with lengths of 23 and 30 amino acid residues as mature peptides. We designated the two ligands neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). The amino acid sequence of NPW23 is completely identical to that of the N-terminal 23 residues of NPW30. Synthetic NPW23 and NPW30 activated and bound to both GPR7 and GPR8 at similar effective doses. Intracerebroventricular administration of NPW23 in rats increased food intake and stimulated prolactin release. These findings indicate that neuropeptide W is the endogenous ligand for both GPR7 and GPR8 and acts as a mediator of the central control of feeding and the neuroendocrine system.     

Production of Anti-carbohydrate Antibodies by Phage Display Technologies: POTENTIAL IMPAIRMENT OF CELL GROWTH AS A RESULT OF ENDOGENOUS EXPRESSION*

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG₁ Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253–262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263–270). Similarly, anti-Le^x phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Le^x)-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.     

Relationships between the photochemical reflectance index (PRI) and chlorophyll fluorescence parameters and plant pigment indices at different leaf growth stages

This study aimed to evaluate the photochemical reflectance index (PRI) for assessing plant photosynthetic performance throughout the plant life cycle. The relationships between PRI, chlorophyll fluorescence parameters, and leaf pigment indices in Solanum melongena L. (aubergine; eggplant) were studied using photosynthetic induction curves both in short-term (diurnal) and long-term (seasonal) periods under different light intensities. We found good correlations between PRI/non-photochemical quenching (NPQ) and PRI/electron transport rate (ETR) in the short term at the same site of a single leaf but these relationships did not hold throughout the life of the plant. In general, changes in PRI owing to NPQ or ETR variations in the short term were <20?% of those that occurred with leaf aging. Results also showed that PRI was highly correlated to plant pigments, especially chlorophyll indices measured by spectral reflectance. Moreover, relationships of steady-state PRI/ETR and steady-state PRI/photochemical yield of photosystem II (Φ(PSII)) measured at uniform light intensity at different life stages proved that overall photosynthesis capacity and steady-state PRI were better correlated through chlorophyll content than NPQ and xanthophylls. The calibrated PRI index accommodated these pigments effects and gave better correlation with NPQ and ETR than PRI. Further studies of PRI indices based on pigments other than xanthophylls, and studies on PRI mechanisms in different species are recommended.     

TNFR-associated factor family protein expression in normal tissues and lymphoid malignancies

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.     

Recruitment to adult habitats in terrestrial hermit crabs on the coast of Ishigakijima Island,Ryukyu Archipelago,Japan

Terrestrial hermit crabs in the family Coenobitidae (genera Coenobita and Birgus) are distributed in tropical and subtropical regions. They occupy various habitats ranging from shore to inland forests, and the two shore‐dwelling species, Coenobita rugosus and C. violascens, possess different distributional characteristics on Ishigakijima Island, Ryukyu Archipelago, Japan. Coenobita rugosus is distributed throughout the coast of the island and is abundant in beach areas, whereas C. violascens has mainly been found in river mouth areas. However, very little is known about the habitats used by the early life stages of coenobitid crabs because identifying the species of recently landed early juveniles is difficult. We tested whether the species compositions of early juveniles of coenobitids differed between beach and river mouth sites on Ishigakijima Island. We collected and identified the early stage coenobitids using PCR–RFLP techniques. A total of 576 early juveniles of five Coenobita species were collected, of which 0.7% were C. brevimanus, 7.3% were C. cavipes, 0.2% were C. purpureus, 70.1% were C. rugosus, and 21.7% were C. violascens. The early juveniles of Birgus latro were not found. The early juveniles of C. rugosus occurred at both beach and river mouth sites, and they were abundant at beach sites. The early juveniles of C. violascens were only found at river mouth sites. These findings indicate that C. rugosus and C. violascens complete their life cycles on land near the localities where they land. The early juveniles of the inland‐dwelling species, C. cavipes, were also mainly collected from river mouth sites, which suggested that juveniles of C. cavipes selected landing sites near river mouth areas and then migrated into the inland forests, passing through riverside areas. Our results highlighted the importance of river mouth areas for recruitment to adult habitats by some coenobitid species.     

Responsiveness of rabbits to superovulation treatment by a single injection of follicle-stimulating hormone with aluminum hydroxide gel

Aluminum hydroxide gel (Al-gel), which is used as an adjuvant, can absorb macromolecules. We investigated the applicability of Al-gel to the sustained release of follicle-stimulating hormone (FSH) as a simplified method of superovulation (SOV) in rabbits. The responsiveness of rabbits to SOV by a single injection of FSH dissolved in Al-gel suspension (3.2 mg Al/ml) and in 10% (w/v) polyvinylpyrrolidone (PVP), and by multiple injections of FSH in saline was examined. The numbers of total and fertilized eggs recovered from rabbits treated with FSH in Al-gel (40.5 and 26.3, respectively) were similar to multiple injections (47.4 and 28.6, respectively) and were significantly greater (P < 0.05) than single injection of FSH with PVP (17.3 and 11.5, respectively). We also compared the plasma FSH levels of rabbits which were induced SOV by multiple or a single injection of Al-gel. Al-gel provided sustained release of FSH to the blood stream at a high enough dose for SOV. Moreover, the developmental competence of the pups of DNA-injected embryos from rabbits treated with a single injection of FSH mixed with Al-gel (18%) was similar to that of DNA-injected embryos, recovered from rabbits treated with FSH dissolved in saline (21%). Two transgenic pups were obtained from embryos recovered from rabbits by a single injection of FSH with Al-gel. These results indicate that a single injection of FSH with Al-gel is an effective method for SOV of rabbit and that this technique is applicable to research requiring large numbers of rabbit embryos such as the production of transgenic rabbits.     

Mechanosensitive ion channels in chara: influence of water channel inhibitors, HgCl2 and ZnCl2, on generation of receptor potential

Characean internodal cells generate receptor potential (ΔE _m) in response to mechanical stimuli. Upon a long-lasting stimulus, the cells generated ΔE _m at the moment of both compression and decompression, and the amplitude of ΔE _m at the moment of decompression, (ΔE _m)_E, was larger than that at compression. The long-lasting stimulus caused a membrane deformation (ΔD _m) having two components, a rapid one, (ΔD _m)_rapid, at the moment of compression and a slower one, (ΔD _m)_slow, during the long-lasting compression. We assumed that (ΔD _m)_slow might have some causal relation with the larger ΔE _m at (ΔE _m)_E. We treated internodal cells with either HgCl₂ or ZnCl₂, water channel inhibitors, to decrease (ΔD _m)_slow. Both inhibitors attenuated (ΔD _m)_slow during compression. Cells treated with HgCl₂ generated smaller (ΔE _m)_E compared to nontreated cells. On the other hand, cells treated with ZnCl₂ never attenuated (ΔE _m)_E but, rather, amplified it. Thus, the amplitude of (ΔD _m)_slow did not always show tight correlation with the amplitude of (ΔE _m)_E. Furthermore, when a constant deformation was applied to an internodal cell in a medium with higher or lower osmotic value, a cell having higher turgor always showed a larger (ΔE _m)_E. Thus, we concluded that changes in tension at the membrane may be the most important factor to induce activation of mechanosensitive Ca²⁺ channel.