Cloning and characterization of ribonuclease T2 gene (RNHe30) from the basidiomycete,Hericium erinaceum

A gene encoding a ribonuclease T2 (RNase T2) family enzyme, RNHe30, was cloned from Hericium erinaceum by PCR. The deduced amino acid sequence from the complimentary DNA (cDNA) (1074 bp) encodes a 302-aa protein (RNase He30) that has the consensus amino acid sequences of RNase T2 family enzymes including the putative signal peptide. The presence of five introns in the genomic DNA was confirmed by comparison of the cDNA and genomic DNA sequences. The promoter region contains a putative CAAT box and a consensus TATA box. Genes coding homologous enzymes were also identified in various other basidiomycetes. A phylogenetic tree of RNase T2s from these fungi was constructed from a multiple alignment of the deduced amino acid sequences. The tree showed that the enzymes were divided into two main groups.     

Thermophilic and halophilic β-agarase from a halophilic archaeon Halococcus sp. 197A

An agar-degrading archaeon Halococcus sp. 197A was isolated from a solar salt sample. The agarase was purified by hydrophobic column chromatography using a column of TOYOPEARL Phenyl-650 M. The molecular mass of the purified enzyme, designated as Aga-HC, was ~55 kDa on both SDS-PAGE and gel-filtration chromatography. Aga-HC released degradation products in the order of neoagarohexose, neoagarotetraose and small quantity of neoagarobiose, indicating that Aga-HC was a β-type agarase. Aga-HC showed a salt requirement for both stability and activity, being active from 0.3 M NaCl, with maximal activity at 3.5 M NaCl. KCl supported similar activities as NaCl up to 3.5 M, and LiCl up to 2.5 M. These monovalent salts could not be substituted by 3.5 M divalent cations, CaCl₂ or MgCl₂. The optimal pH was 6.0. Aga-HC was thermophilic, with optimum temperature of 70 °C. Aga-HC retained approximately 90 % of the initial activity after incubation for 1 hour at 65–80 °C, and retained 50 % activity after 1 hour at 95 °C. In the presence of additional 10 mM CaCl₂, approximately 17 % remaining activity was detected after 30 min at 100 °C. This is the first report on agarase purified from Archaea.     

Complex latitudinal variation in the morphology of the kleptoparasitic spider Argyrodes kumadai associated with host use and climatic conditions

We examined complex geographical patterns in the morphology of a kleptoparasitic spider, Argyrodes kumadai, across its distributional range in Japan. To disentangle biotic and abiotic factors underlying morphological variation, latitudinal trends were investigated in two traits, body size and relative leg length, across separate transition zones for host use and voltinism. Statistical analyses revealed complex sawtooth clines. Adult body size dramatically changed at the transition zones for host use and voltinism, and exhibited a latitudinal decline following the converse to Bergmann’s cline under the same host use and voltinism in both sexes. A similar pattern was observed for relative leg length in females but not in males. A genetic basis for a part of observed differences in morphology was supported by a common-garden experiment. Our data suggest that local adaptation to factors other than season length such as resource availability (here associated with host use) obscures underlying responses to latitude.     

Ovarian Cancer Stem Cells Are Enriched in Side Population and Aldehyde Dehydrogenase Bright Overlapping Population

Cancer stem-like cells (CSCs)/cancer-initiaiting cells (CICs) are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP) analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDH^Br) cells from ovarian cancer cells. Both SP cells and ALDH^Br cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2), than those of main population (MP) cells and ALDH^Low cells, respectively. We analyzed an SP and ALDH^Br overlapping population (SP/ALDH^Br), and the SP/ALDH^Br population exhibited higher tumor-initiating ability than that of SP cells or ALDH^Br cells, enabling initiation of tumor with as few as 10² cells. Furthermore, SP/ADLH^Br population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDH^Low, MP/ALDH^Br and MP/ALDH^Low cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDH^Br population was detected in several gynecological cancer cells with ratios of 0.1% for HEC—1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDH^Br overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs.     

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans

Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.     

Dynamical Properties of Condensed Charged Polymer Melts

Abstract

Dynamical properties of condensed charged polymer melts are studied with a two-dimensional model and molecular dynamics simulation. Screened Coulombic interactions are assumed for the interactions between the monomer charges as well as the counterions which were introduced to neutralize the total monomer charges of polymer chains. Through molecular dynamics calculations, we have obtained the radial distribution function and velocity auto-correlation function, and their density dependences. As a structural characteristics in condensed charged polymer melts, we find that the monomers tend to form triangular structures locally. The radial distribution function for the center monomers implies that the polymer chains on our two-dimensional model are not entangled. The diffusion properties for both the counter-ions and innermost monomers are studied in detail. In this paper, we have also computed neutral polymer melts to study the effects of the presence of the long-range Coulombic interactions on the dynamical properties of polymer melts. We find that the Coulombic interactions significantly reduce the self diffusion. Snapshots analyses obtained from the molecular dynamics simulation suggest that the reptation model is not a proper model for two-dimensional polymer-chain melts, in which there are little entanglement effects in agreement with the result of the analyses for the radial distribution function.     

Molecular Dynamics Calculations of Wild Type vs. Mutant Protein C: Relationship Between Binding Affinity to Endothelial Cell Protein C Receptor and Hereditary Disease

Abstract

Molecular dynamics simulations of the protein C γ-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 → Lys) in the protein C Gla domain. Our results suggest that the Gla 25 → Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca²⁺ ion.     

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti‐ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C‐terminal src kinase)‐binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent‐resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti‐ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over‐expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314‐phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314‐phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP‐43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.