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.     

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.     

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.     

Parasporin 1Ac2, a Novel Cytotoxic Crystal Protein Isolated from Bacillus thuringiensis B0462 Strain

Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S¹¹³–R²⁵⁰) and 60 kDa (I²⁵¹–S⁷⁷⁷) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.     

Landslide-facilitated species diversity in a beech-dominant forest

To evaluate the extent to which landslides affect community dynamics and consequent species diversity in a beech-dominated forest, differences in the composition and size structure of tree species were compared between landslide and adjacent stable (control) stands. Demography and changes in size were compared between the two stands over a 5-year period about 60 years after a landslide. In the control stand, replacement occurred even amongst late-successional species, with beech (Fagus crenata)—the most dominant species—increasing in relative abundance. In the landslide stand, very few large individuals of late-successional species occurred, whereas large individuals of early-successional species occurred only in the landslide stand. The traits indicate that the landslide strongly facilitated species diversity, not only by reducing the dominance of late-successional species, but also by promoting recruitment of early-successional species. However, new recruitment of early-successional species was inhibited in the landslide stand, although we observed succeeding regeneration and subsequent population growth of late-successional species there. As a result, the relative dominance of late-successional species increased with succession after the landslide, thus decreasing future species diversity. In beech-dominant forest landscapes in Japan that include communities with different developmental stages, the mosaic of serial stages may facilitate species diversity after a landslide.     

Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects

Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G₁/S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds O-GlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy.     

A leucine zipper-based peptide hybrid delivers functional Nanog protein inside the cell nucleus

We synthesized a pair of compounds containing leucine zipper peptides to deliver protein cargo into cells. One is a cell-penetrating peptide (CPP) with Lz(E), a leucine zipper peptide containing negatively charged amino acids, and the other is a Nanog protein with Lz(K), a leucine zipper peptide containing positively charged amino acids. When cells were treated with these equimolar mixtures, Nanog-Lz(K) hybridized with Lz(E)-CPP was successfully delivered into the cells. Furthermore, Nanog-Lz(K) exerted its proper function after nuclear transport.     

Synanceia quinque,a new species of stonefish (Synanceiidae) from Borneo and Flores

A new species of stonefish, Synanceia quinque (Synanceiidae) is described on the basis of two specimens (61.5–84.4 mm standard length) collected off Sabah (Borneo), Malaysia and Flores, Indonesia. The new species is characterized by 12 pectoral-fin rays, and is most similar to Synanceia alula Eschmeyer and Rama-Rao 1973 (11 pectoral-fin rays in the latter vs. 14 or more in other congeners). Other characters distinguishing S. quinque from S. alula include numbers of pelvic-fin rays [I, 5 in the former vs. I, 3 or 4 (usually I, 4) in the latter], gill rakers (0 + 4 or 5 vs. 0 or 1 + 7), and five preopercular spines/skin flaps (upper three spines relatively well developed, lower two rudimentary) (vs. four preopercle spines, fifth spine or skin flap absent). An updated key to species of Synanceia is provided.

     

Translocation and activation of sphingosine kinase 1 by ceramide-1-phosphate

Sphingosine kinases (SphKs) and ceramide kinase (CerK) phosphorylate sphingosine to sphingosine-1-phosphate (S1P) and ceramide to ceramide-1-phosphate (C1P), respectively. S1P and C1P are bioactive lipids that regulate cell fate/function and human health/diseases. The translocation and activity of SphK1 are regulated by its phosphorylation of Ser ²²⁵ and by anionic lipids such as phosphatidic acid and phosphatidylserine. However, the roles of another anionic lipid C1P on SphK1 functions have not yet been elucidated, thus, we here investigated the regulation of SphK1 by CerK/C1P. C1P concentration dependently bound with and activated recombinant human SphK1. The inhibition of CerK reduced the phorbol 12-myristate 13-acetate-induced translocation of SphK1 to the plasma membrane (PM) and activation of the enzyme in membrane fractions of cells. A treatment with C1P translocated wild-type SphK1, but not the SphK1-S225A mutant, to the PM without affecting phosphorylation signaling. A cationic RxRH sequence is proposed to be a C1P-binding motif in α-type cytosolic phospholipase A ₂ and tumor necrosis factor α-converting enzyme. The mutation of four cationic amino acids to Ala in the 56-RRNHAR-61 domain in SphK1 reduced the phorbol 12-myristate 13-acetate- and C1P-induced translocation of SphK1 to the PM, however, the capacity of C1P to bind with and activate SphK1 was not affected by this mutation. In conclusion, C1P modulates SphK1 functions by interacting with multiple sites in SphK1.     

A 28 kDa protein of the Bacillus thuringiensis serovar shandongiensis isolate 89-T-34-22 induces a human leukemic cell-specific cytotoxicity

A 28 kDa protein that exhibits cytocidal activity specific for human leukemic T (MOLT-4) cells was purified from proteinase K-digested parasporal inclusion of a Bacillus thuringiensis serovar shandongiensis isolate. The N-terminal sequence of the protein was identical with that of the 32 kDa protein, regarded as a protoxin, of the inclusion proteins. The median effective concentration of this protein was 0.23 microg/ml against MOLT-4 cells and its specific activity was 7.9 times greater than that of the whole inclusion proteins. The 28 kDa protein induced necrosis-like cytotoxicity against MOLT-4 cells and the cytopathic effect with the passage of time was characterized by cell swelling, nuclear membrane isolation and chromatin condensation.