Spatial Analysis of Slowly Oscillating Electric Activity in the Gut of Mice Using Low Impedance Arrayed Microelectrodes

Smooth and elaborate gut motility is based on cellular cooperation, including smooth muscle, enteric neurons and special interstitial cells acting as pacemaker cells. Therefore, spatial characterization of electric activity in tissues containing these electric excitable cells is required for a precise understanding of gut motility. Furthermore, tools to evaluate spatial electric activity in a small area would be useful for the investigation of model animals. We thus employed a microelectrode array (MEA) system to simultaneously measure a set of 8×8 field potentials in a square area of ∼1 mm². The size of each recording electrode was 50×50 µm², however the surface area was increased by fixing platinum black particles. The impedance of microelectrode was sufficiently low to apply a high-pass filter of 0.1 Hz. Mapping of spectral power, and auto-correlation and cross-correlation parameters characterized the spatial properties of spontaneous electric activity in the ileum of wild-type (WT) and W/W^v mice, the latter serving as a model of impaired network of pacemaking interstitial cells. Namely, electric activities measured varied in both size and cooperativity in W/W^v mice, despite the small area. In the ileum of WT mice, procedures suppressing the excitability of smooth muscle and neurons altered the propagation of spontaneous electric activity, but had little change in the period of oscillations. In conclusion, MEA with low impedance electrodes enables to measure slowly oscillating electric activity, and is useful to evaluate both histological and functional changes in the spatio-temporal property of gut electric activity.     

Stabilization of G-quadruplex structure on vascular endothelial growth factor gene promoter depends on CpG methylation site and cation type

Background

DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure.

Analysis of the acrolein-modified sites of apolipoprotein B-100 in LDL

We have reported that acrolein-conjugated low-density lipoprotein (Acro-LDL) uptake by scavenger receptor class A type 1 (SR-A1) can mediate macrophage foam cell formation. The purpose of this study was to determine which amino acid residues of apoB protein in LDL are conjugated with acrolein. Acro-apoB was prepared by incubation of LDL with acrolein (10 to 60 μM) at 37 °C for 7 days. Identification of acrolein-conjugated amino acid residues in apoB was performed using LC-MS/MS. The levels of acrolein-conjugated amino acid residues of apoB as well as crosslinking apoB increased in proportion to acrolein concentration. The level of LDL uptake by macrophages was parallel with the acrolein-conjugated monomer apoB. Acrolein-conjugated amino acid residues in apoB were C212, K327, K742, K949, K1087, H1923, K2634, K3237 and K3846. The NH₂-teriminal four amino acid residues (C212, K327, K742 and K949) were located at the scavenger receptor SR-A1 recognition site, suggesting that these four acrolein-conjugated amino acids are involved in the rapid uptake of Acro-LDL by macrophages. It is proposed that the rapid uptake of LDL by macrophages is dependent on acrolein conjugation of four amino acids residues at the scavenger receptor recognition site of apoB in LDL.     

Synthesis and structural and pharmacological properties of cyclopropane-based conformationally restricted analogs of 4-methylhistamine as histamine H3/H4 receptor ligands

On the basis of the previous results on a histamine H₄ receptor agonist 4-methylhistamine and a cyclopropane-based conformationally restricted analog CEIC (3) with potent H₃/H₄ receptor antagonistic effect, 4-methylhistamine analogs 4 and 5 of CEIC were designed and synthesized. Compound 4 showed strong affinity (K_i = 38.7 nM) for the H₃ receptor, which was more potent than a well-known H₃ antagonist thioperamide. Stable tautomer and conformation of 3 and 4, which can affect the pharmacological activity, were analyzed by ab initio calculations.     

Regulation of intracellular Mg2+ by superoxide in amnion cells.

Changes of intracellular free Mg2+ concentration ([Mg2+]i) in human amnion cells induced by superoxide anion were determined using a highly Mg(2+)-sensitive fluorescent dye Mg(2+)-fura2 or Mg(2+)-indol. Superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, induced decrease of [Mg2+]i. The decrease was significantly inhibited by an anion channel blocker, 4,4'diisothiocyano-2,2' disulfonic acid stilbene (DIDS). Superoxide dismutase (SOD), injected into cells by cell fusion, also inhibited the change of [Mg2+]i, but catalase did not. Superoxide anion induced prompt increase of intracellular pH (pHi) as well as decrease of [Mg2+]i and subsequently activated the increase of intracellular free Ca2+ ([Ca2+]i) and the release of arachidonate. In contrast to superoxide anion, NH4Cl which induces increase of pHi in amnion cells increased [Mg2+]i. The elevation of basal level of [Mg2+]i by Mg(2+)-ionophore inhibited the change of [Ca2+]i and the release of arachidonate induced by superoxide anion. These results suggest that superoxide anion, transported through anion channels into cells, decreases [Mg2+]i directly, not due to a pH-effect and that the decrease of [Mg2+]i may regulate biological functions of the cells via increase of [Ca2+]i.     

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2⁺ cells after intravenous injection and in CD11c⁺ dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8⁺ T-cell response in HLA-A2 transgenic mice and stimulated a CD4⁺ T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A^b. As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.     

Characterization of a mosquitocidal Bacillus thuringiensis serovar sotto strain isolated from Okinawa, Japan

AIMS: To characterize the mosquitocidal activity of parasporal inclusions of the Bacillus thuringiensis serovar sotto strain 96-OK-85-24, for comparison with two well-characterized mosquitocidal strains. METHODS AND RESULTS: The strain 96-OK-85-24 significantly differed from the existing mosquitocidal B. thuringiensis strains in: (1) lacking the larvicidal activity against Culex pipiens molestus and haemolytic activity, and (2) SDS-PAGE profiles, immunological properties and N-terminal amino acid sequences of parasporal inclusion proteins. CONCLUSIONS: It is clear from the results that the strain 96-OK-85-24 synthesizes a novel mosquitocidal Cry protein with a unique toxicity spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the occurrence of a mosquitocidal B. thuringiensis strain with an unusual toxicity spectrum, lacking the activity against the culicine mosquito.     

The critical region for Behçet disease in the human major histocompatibility complex is reduced to a 46-kb segment centromeric of HLA-B, by association analysis using refined microsatellite mapping

The HLA-B51 allele is known to be associated with Behçet disease. Recently, we found a higher risk for Behçet disease in the MICA gene, 46 kb centromeric of HLA-B, by investigation of GCT repetitive polymorphism within exon 5 of MICA. The pathogenic gene causing Behçet disease, however, has remained uncertain. Here, eight polymorphic microsatellite markers, distributed over a 900-kb region surrounding the HLA-B locus, were subjected to association analysis for Behçet disease. Statistical studies of associated alleles detected on each microsatellite locus showed that the pathogenic gene for Behçet disease is most likely found within a 46-kb segment between the MICA and HLA-B genes. The results of this mapping study, and the results of an earlier study of ours, suggest that MICA is a strong candidate gene for the development of Behçet disease.     

Fine-scale spatial structure of genets and sexes in the dioecious plant <Emphasis Type="Italic">Dioscorea japonica</Emphasis>, which disperses by both bulbils and seeds

To understand the evolution of clonal reproduction and the diversity of clonal plants, it is necessary to clarify the characteristics of each clonal habit. There has been little research on whether bulbils alter spatial genetic structure (SGS) because of the lack of connection to maternal ramets. We used simple-sequence-repeat (SSR) markers to determine the fine-scale SGS of the dioecious plant Dioscorea japonica, which disperses both as bulbils and as seeds. We also evaluated the contributions of sexual and clonal reproduction and tested for spatial sex segregation (SSS). We discovered 111 genets from 394 ramets in a 2.8-ha plot. Genotypic richness (R = 0.28) and clonal diversity (Simpson’s D = 0.94, Fager’s E = 0.90) were high. We did not find SSS, suggesting that the population does not suffer from a shortage of mating pairs due to clonal reproduction. The Sp values revealed moderate SGS at the genet level (Sp = 0.013–0.014), and the genets intermingled at a local scale. Significant SGS at the ramet level showed that ramets within the same genet tended to aggregate. We also found a skewed clonal spatial distribution. The spatial extent of genets was positively correlated with the number of ramets within a genet. The contribution of bulbil production to the variance of parent–offspring gene dispersal was about one–fifth the contribution from sexual reproduction. These results suggest that the dispersal via bulbils affects the SGS in D. japonica, although its contribution to gene dispersal is small compared to the contribution of sexual reproduction.