Interaction between trehalose and alkaline-earth metal ions

We investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H-1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4' of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.     

Tryptamine-based human beta3-adrenergic receptor agonists. Part 3: improved oral bioavailability via modification of the sulfonamide moiety

The continued SAR investigation of tryptamine-based human beta(3)-adrenergic receptor (AR) agonists is reported. Prior efforts resulted in the identification of 2 as a potent beta(3)-AR agonist. Further modification of the left side arylsulfonamide portion in 2 provided compounds with good cell permeability, which have potent agonistic activity for beta(3)-AR. Cinnamylamine analog 16i exhibited an excellent agonistic profile in vitro and good oral bioavailability in rats.     

Similarity and difference in the mechanisms of neonatally induced tolerance and cyclophosphamide-induced tolerance in mice

The mechanisms of cyclophosphamide (CP)-induced tolerance were investigated by comparing with those of neonatally induced tolerance. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were given i.v. either AKR/J Sea (AKR; H-2k, Mls-1a) or (AKR x C3H)F1 (AKC3F1; H-2k, Mls-1a/b) spleen cells and treated i.p. with CP 2 days later, a long-lasting skin allograft tolerance to AKR was induced in each case without any signs of graft-vs-host disease (GVHD). However, typical signs of GVHD were observed in the C3H mice neonatally tolerized with AKR spleen cells, but not in those tolerized with AKC3F1 spleen cells. The expression of TCR V beta 6, which is strongly correlated with the reactivity to Mls-1a Ag (of donor AKR origin), in the periphery was quite different between the two types of tolerant C3H mice. Namely, in the lymph nodes of the C3H mice tolerized with AKR spleen cells and CP, only CD4(+)-V beta 6+, but not CD8(+)-V beta 6+, T cells selectively disappeared, whereas both of them were abrogated in the lymph nodes of the C3H mice neonatally tolerized of AKR. By contrast, in the thymus of the two types of tolerant C3H mice, both CD4+CD8- and CD4-CD8+ single-positive thymocytes expressing TCR V beta 6 were clonally deleted, suggesting that the thymic involvement was the same in each type of tolerance. These results suggest that the preferential disappearance of the CD4(+)-V beta 6+ T cells (of host origin) and the effector T cells of GVHD (of donor origin) occurred only in the periphery of the C3H mice tolerized with AKR spleen cells plus CP and was attributable to the destruction of Ag-stimulated T cells by the CP treatment. In contrast, the intrathymic clonal deletion of immature V beta 6+ T cells was a common mechanism for both of the tolerance induction systems.     

Structure of the F‐spondin domain of mindin,an integrin ligand and pattern recognition molecule

Mindin (spondin‐2) is an extracellular matrix protein of unknown structure that is required for efficient T‐cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N‐terminal F‐spondin (FS) domain and C‐terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8‐Å resolution. The structure revealed an eight‐stranded antiparallel β‐sandwich motif resembling that of membrane‐targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C‐mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.     

Comparison of stream benthic invertebrate assemblages among forest types in the temperate region of Japan

We compared benthic invertebrate assemblages among headwater streams in several forest types in Japan. Forests were divided into three clusters based on vegetation composition: old-growth broad-leaved forest, planted coniferous forest, and mixed forest. The numbers of individuals and families and the diversity (Shannon-Wiener) of benthic invertebrate assemblages did not differ significantly among the three forest clusters. However, principal components analysis of family abundance showed differences in the benthic invertebrate assemblages among the three forest clusters. No environmental factors were correlated with these differences. Benthic invertebrate assemblages differed depending on forest composition. The abundances of Taeniopterygidae and Athericidae in old-growth broad-leaved forest were significantly greater than in planted coniferous forest. The abundances of Heptageniidae, Baetiidae, Stenopsychidae, Uenoidae, Chironomidae, and Potamidae in planted coniferous forest were significantly greater than in old-growth broad-leaved forest. If the remaining old-growth broad-leaved forest were to be converted to coniferous plantation, species that inhabit old-growth forest may become extinct.     

Parasporin-2Ab,a Newly Isolated Cytotoxic Crystal Protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC₅₀ values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.     

Sexual variations in food quality and gastrointestinal features of sika deer (Cervus nippon) in Japan during winter: implications for feeding strategy

We assessed sexual variation in food quality and gut macrostructure in adult male and pregnant female sika deer, Cervus nippon (Temminck, 1838), in Japan during winter. These variations might have important implications relative to sexual differences in habitat use, forage acquisition, and digestive strategy. According to the sexual dimorphism-body size hypothesis the larger males would feed on poorer forage and have heavier stomach contents and heavier intestine contents and longer intestines than smaller females. However, the food quality in rumen contents of males was higher than, or at least similar with, that of pregnant females. In correspondence to food quality, the relative weights of stomach contents and intestines with contents, the relative lengths of intestines to the lengths of body and total intestines in pregnant females were similar to adult males. The relative weights of omasum and abomasum tissues in pregnant females were greater than in males. Our findings suggest sexual differences in feeding strategy in sika deer in Japan during winter. To meet greater nutritional demands of high metabolic rate and gestation, pregnant females seemed to maintain a greater volume of digesta in guts and had more stomach tissues than expected by the sexual dimorphism-body size hypothesis to compensate for poorer forage quality.     

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCα^S657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCα^S657 and FoxO1.     

Increase in IP3 and intracellular Ca2+ induced by phosphate depletion in LLC-PK 1 cells

The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.