Design, synthesis, and biological evaluation of tricyclic heterocycle-tetraamine conjugates as potent NMDA channel blockers

We have developed a new class of N-methyl-d-aspartate (NMDA) channel blockers having a conjugate structure that consists of a nitrogenous heterocyclic head and a tetraamine tail. Among them, dihydrodibenzazepine-homospermine conjugate (8) exhibited potent antagonistic activity at NR1/NR2A or NR1/NR2B NMDA subtype receptors compared with the lead compound, AQ343 (1), or memantine, as well as weak cytotoxicity. Its superior biological profiles compared with known compounds point to its potential use as therapeutic agents for neurological disorders.     

A new method of defense response analysis using a transient expression system in rice protoplasts

We established a new plant defense response assay using a transient expression system in rice protoplasts. The assay system sensitively detected defense induction by flagellin, which had previously been assigned to a specific elicitor. Our assay system provides a rapid and efficient way to dissect rice defense mechanisms.     

Association of cerebrospinal fluid anti-ribosomal P protein antibodies with diffuse psychiatric/neuropsychological syndromes in systemic lupus erythematosus

We explored the relationship of antibodies to the whole ribosomal P proteins (P0, P1, and P2) in cerebrospinal fluid (CSF) with diffuse psychiatric/neuropsychological syndromes in systemic lupus erythematosus (SLE). CSF samples were obtained from 71 SLE patients (52 patients with diffuse psychiatric/neuropsychological syndromes [diffuse NP-SLE] and 19 patients with neurological syndromes or peripheral neuropathy [focal NP-SLE]) as well as from 24 patients with non-inflammatory neurological disease. Immunoglobulin G (IgG) antibodies to the C-terminal 22-amino acid ribosomal P synthetic peptide (anti-P_C22) and those to purified bovine ribosomal P proteins (P0, P1, and P2) (anti-whole P) were determined by enzyme-linked immunosorbent assay; affinity-purified IgG anti-P_C22 were used as the standard. The concentrations of antibodies to epitopes other than the C-terminal 22 amino acids of ribosomal P proteins were calculated by subtracting anti-P_C22 from anti-whole P (anti-P_EX.C22). CSF anti-whole P levels were significantly elevated in diffuse NP-SLE compared with focal NP-SLE or control patients. By contrast, there were no significant differences in CSF anti-P_C22 levels among the three groups. Of note, CSF anti-P_EX.C22 levels were significantly elevated in diffuse NP-SLE compared with the other two groups. CSF anti-P_EX.C22 levels were not significantly correlated with CSF anti-P_C22 levels, but with CSF antibodies against the recombinant ribosomal P0 protein lacking the C-terminal 22 amino acids (C22-depleted rP0). Moreover, levels of CSF anti-P_EX.C22 or CSF anti-C22-depleted rP0, but not CSF anti-P_C22, were significantly correlated with CSF anti-neuronal cell antibodies (anti-N). These results indicate that CSF IgG antibodies to the epitopes other than the C-terminal 22 amino acids of ribosomal P proteins, which might contain one of the major targets of CSF anti-N, are associated with the development of diffuse NP-SLE.     

GFP transgenic mice reveal active canonical Wnt signal in neonatal brain and in adult liver and spleen

In the past decades, the function of the Wnt canonical pathway during embryogenesis has been intensively investigated; however, little survey of neonatal and adult tissues has been made, and the role of this pathway remains largely unknown. To investigate its role in mature tissues, we generated two new reporter transgenic mouse lines, ins-TOPEGFP and ins-TOPGAL, that drive EGFP and beta-galactosidase expression under TCF/beta-catenin, respectively. To obtain the accurate expression pattern, we flanked these transgenes with the HS4 insulator to reduce chromosomal positional effects. Analysis of embryos showed that the reporter genes were activated in regions where canonical Wnt activity has been implicated. Furthermore, their expression patterns were consistent in both lines, indicating the accuracy of the reporter signal. In the neonatal brain, the reporter signal was detected in the mesencephalon and hippocampus. In the adult mice, the reporter signal was found in the mature pericenteral hepatocytes in the normal liver. Furthermore, during inflammation the number of T cells expressing the reporter gene increased in the adult spleen. Thus, in this research, we identified two organs, i.e., the liver and spleen, as novel organs in which the Wnt canonical signal is in motion in the adult. These transgenic lines will provide us broader opportunities to investigate the function of the Wnt canonical pathway in vivo.     

Characterization of the Rv3378c gene product, a new diterpene synthase for producing tuberculosinol and (13R, S)-isotuberculosinol (nosyberkol), from the Mycobacterium tuberculosis H37Rv genome

The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of 1H- and 13C-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.     

Synthesis and antiproliferative evaluation of 3,5-disubstituted 1,2,4-triazoles containing flurophenyl and trifluoromethanephenyl moieties

An efficient 1,3-dipolar cycloaddition method was performed for the synthesis of a series of monofluoro- and trifluoromethane-3,5-disubstituted 1,2,4-triazoles. This efficient cycloaddition method was to react hydrazonoyl hydrochlorides with a series of aldehydes in the presence of NEt(3) as catalytic basic agent to provide the corresponding product in 28-94%. Their growth inhibitory results against cancer cells indicated that some of the fluorine- and trifluoromethane-containing compounds could effectively inhibit the growth of NCI-H226 and T-cell leukemia (Jurkat) cells. Among the compounds, trifluoromethane-containing 1,2,4-triazoles possessed the five-membered ring groups on the C-5 position of the triazolic ring, including cyclopentyl, 3-furyl, 3-thienyl, and 2-pyrrolyl, possessed the significant inhibitory activity for NCI-H226 cancer cells.     

Monoallelic gene expression and its mechanisms

Although the majority of genes are expressed equally from both alleles, some genes are differentially expressed. Monoallelic gene expression, the differential gene expression of the alleles such as genomic imprinting, is reported in several organisms and plays significant roles in proper development and diversity in gene expression and phenotypic variation. Recent studies in flowering plants have greatly increased our understanding of the underlying mechanisms of monoallelic gene expression. They indicate that machineries of gene silencing such as DNA methylation, histone modifications, and noncoding RNAs function in monoallelic gene expression. A combination of genetics and high-throughput technologies expands the scope of study on monoallelic gene expression in flowering plants.     

A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.     

Enamel matrix derivative is a potent inhibitor of breast cancer cell attachment to bone

This study examined whether enamel matrix derivative (EMD) inhibits the adhesion of cancer cells to bone. A typical breast cancer cell line, MCF-7, was used. Conditioned human osteosarcoma cell (Saos-2) medium was used as extracellular bone matrix (ECBM) to measure cell attachment. MCF-7 cells were incubated on ECBM-coated culture plates with or without soluble EMD, Arg-Gly-Asp (RGD) sequence blocking peptides, recombinant bone sialoprotein (rBSP), or specific integrin antibodies, and the attached cells were quantified using toluidine blue staining. EMD markedly reduced the attachment of MCF-7 cells to ECBM in a dose-dependent manner. An RGD peptide (GRGDSP) and recombinant BSP inhibited cell attachment to the same degree as EMD. Similarly, anti-alphavbeta3 integrin antibody strongly reduced cell attachment, whereas anti-alphavbeta5 and anti-beta1 integrin antibodies had less marked effects on cell attachment. These results show that EMD inhibits MCF-7 cell attachment to a bone matrix and that it might be useful as an anti-adhesive agent for breast cancer cells to bone in vivo.