Amelioration of amyloid load by anti-Abeta single-chain antibody in Alzheimer mouse model

Parenteral immunization of transgenic mouse models of Alzheimer disease (AD) with synthetic amyloid beta-peptide (Abeta) prevented or reduced Abeta deposits and attenuated their memory and learning deficits. A clinical trial of immunization with synthetic Abeta, however, was halted due to brain inflammation, presumably induced by a toxic Abeta, T-cell- and/or Fc-mediated immune response. Another issue relating to such immunizations is that some AD patients may not be able to raise an adequate immune response to Abeta vaccination due to immunological tolerance or age-associated decline. Because peripheral administration of antibodies against Abeta also induced clearance of amyloid plaques in the model mice, injection of humanized Abeta antibodies has been proposed as a possible therapy for AD. By screening a human single-chain antibody (scFv) library for Abeta immunoreactivity, we have isolated a scFv that specifically reacts with oligomeric Abeta as well as amyloid plaques in the brain. The scFv inhibited Abeta amyloid fibril formation and Abeta-mediated cytotoxicity in vitro. We have tested the efficacy of the human scFv in a mouse model of AD (Tg2576 mice). Relative to control mice, injections of the scFv into the brain of Tg2576 mice reduced Abeta deposits. Because scFvs lack the Fc portion of the immunoglobulin molecule, human scFvs against Abeta may be useful to treat AD patients without eliciting brain inflammation.     

Discovery of 4,5-diphenyl-1,2,4-triazole derivatives as a novel class of selective antagonists for the human V(1A) receptor

In the search for a novel class of selective antagonists for the human V(1A) receptor, high-throughput screening (HTS) of the Yamanouchi chemical library using CHO cells expressing the cloned human V(1A) (hV(1A)) receptor led to the discovery of 5-(4-biphenyl)-4-(2-methoxyphenyl)-3-methyl-1,2,4-triazole (3) which possessed the novel 4,5-diphenyl-1,2,4-triazole structure. Subsequent structure-activity relationships studies on a series of the 4,5-diphenyl-1,2,4-triazole derivatives related to 3 revealed that the 4,5-diphenyl-1,2,4-triazole structure played an essential role in exerting high affinity for the hV(1A) receptor and that introduction of a basic amine moiety to the methoxy part of the 4-phenyl ring was effective in the improvement of both affinity for the hV(1A) receptor and selectivity versus the hV(2) receptor. Compound 3 and the 2-(morphorino)ethoxy derivative (11b) were shown to be antagonists for the hV(1A) receptor, from their effects on AVP-induced [Ca(2+)](i) response in CHO cells expressing the hV(1A) receptor.     

Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome.     

Complement activation plays a key role in antibody-induced infusion toxicity in monkeys and rats

Infusion reactions are a major side effect of the administration of therapeutic Abs and are the result of a complex immune reaction. In this study, we report that substitutions of Fc amino acids in the anti-HLA-DR Ab HD8 reduce its ability to induce infusion reactions in rats and monkeys. We first showed that i.v. administration of IgG1- and IgG2-subclass HD8 Abs induces severe infusion reactions in monkeys. These Abs express strong complement-dependent cytotoxicity (CDC), and in vivo depletion of complement in rats by pretreatment with cobra venom factor abrogated the lethal infusion reactions generated by HD8-IgG1. Thus, the infusion reactions appear to be largely driven by the complement system. To reduce the CDC function of HD8-IgG1, its Fc region was modified by two amino acid substitutions at Pro(331)Ser and Lys(322)Ala. The modified Ab was incapable of expressing CDC in vitro and did not induce severe infusion reactions in rats and monkeys, even at extremely high doses. The modified Ab retained its Ab-dependent cellular cytotoxicity function as well as its antitumor activity in a tumor-bearing mouse model. In summary, complement appears to drive infusion reactions, and modifications that eliminate the CDC activity of an Ab also reduce its ability to induce infusion reactions.     

Measles virus infects both polarized epithelial and immune cells by using distinctive receptor-binding sites on its hemagglutinin

Measles is one of the most contagious human infectious diseases and remains a major cause of childhood morbidity and mortality worldwide. The signaling lymphocyte activation molecule (SLAM), also called CD150, is a cellular receptor for measles virus (MV), presumably accounting for its tropism for immune cells and its immunosuppressive properties. On the other hand, pathological studies have shown that MV also infects epithelial cells at a later stage of infection, although its mechanism has so far been unknown. In this study, we show that wild-type MV can infect and produce syncytia in human polarized epithelial cell lines independently of SLAM and CD46 (a receptor for the vaccine strains of MV). Progeny viral particles are released exclusively from the apical surface of these polarized epithelial cell lines. We have also identified amino acid residues on the MV attachment protein that are likely to interact with a putative receptor on epithelial cells. All of these residues have aromatic side chains and may form a receptor-binding pocket located in a different position from the putative SLAM- and CD46-binding sites on the MV attachment protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells by using distinctive receptor-binding sites on the attachment protein corresponding to each of their respective receptors. The ability of MV to infect polarized epithelial cells and its exclusive release from the apical surface may facilitate its efficient transmission via aerosol droplets, resulting in its highly contagious nature.     

Increased aerobic metabolism is essential for the beneficial effects of caloric restriction on yeast life span

Calorie restriction is a dietary regimen capable of extending life span in a variety of multicellular organisms. A yeast model of calorie restriction has been developed in which limiting the concentration of glucose in the growth media of Saccharomyces cerevisiae leads to enhanced replicative and chronological longevity. Since S. cerevisiae are Crabtree-positive cells that present repression of aerobic catabolism when grown in high glucose concentrations, we investigated if this phenomenon participates in life span regulation in yeast. S. cerevisiae only exhibited an increase in chronological life span when incubated in limited concentrations of glucose. Limitation of galactose, raffinose or glycerol plus ethanol as substrates did not enhance life span. Furthermore, in Kluyveromyces lactis, a Crabtree-negative yeast, glucose limitation did not promote an enhancement of respiratory capacity nor a decrease in reactive oxygen species formation, as is characteristic of conditions of caloric restriction in S. cerevisiae. In addition, K. lactis did not present an increase in longevity when incubated in lower glucose concentrations. Altogether, our results indicate that release from repression of aerobic catabolism is essential for the beneficial effects of glucose limitation in the yeast calorie restriction model. Potential parallels between these changes in yeast and hormonal regulation of respiratory rates in animals are discussed. G. A. Oliveira and E. B. Tahara contributed equally to this work.     

Production of enokipodins A, B, C, and D: a new group of antimicrobial metabolites from mycelial culture of Flammulina velutipes

Antimicrobial compounds enokipodins A, B, C, and D were originally isolated from the culture filtrates of Flammulina velutipes mycelial culture. Analysis of antibacterial activity by the paper disk method and quantification of enokipodins A–D by high performance liquid chromatography (HPLC) showed that F. velutipes mycelia produced enokipodins A–D in their late growing phase. Great genetic variability in production of these compounds was observed among ten strains of F. velutipes in analyses of antimicrobial activity by the hole-plate diffusion method and quantification by HPLC. Enokipodins A–D demonstrated antimicrobial activity mainly against the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. Evaluation of minimum inhibitory doses (MIDs) showed that MIDs of enokipodins A and C for B. subtilis were as low as that of the penicillin G antibiotic.     

Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis

The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.     

Isolation of stable (alphabeta)4-Tetraprotomer from Na+/K+-ATPase solubilized in the presence of short-chain fatty acids

Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (alphabeta) 2-diprotomer (D) and alphabeta-protomer (P) of solubilized dog kidney Na (+)/K (+)-ATPase [ Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173 ]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 (5) for H and quantitative Na (+)/K (+)-dependent interconversion among H, D, and P showed that H was an (alphabeta) 4-tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K (+)Prop at pH 5.6 by gel chromatography performed at 0 degrees C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K (+)-congeners constituting K (+)Prop for the maximal amount of T were NH 4 (+) > Rb (+) congruent with K (+) > Tl (+), while Na (+) had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 degrees C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.