Studies on the development of water maze-learning ability in rats (3). Effect of the learning schedule on learning acquisition

The effect of different learning schedules massed or distributed practice conditions (3 trials a day for 3 days), on water-filled multiple T-maze learning ability of 8-week-old SPF Wistar-Imamichi rats was investigated. Although the mean number of errors decreased day by day in both groups, the number of errors in a given day and the total number of errors in 3 days did not differ significantly between the two groups. A tendency toward a decrease in the number of errors was observed as the trials proceeded in the group with distributed practice but not in the group with massed practice. The result suggests that a certain time period for rest after each trial is necessary to acquire the memory.     

Modulation of in vivo IgG crystallization in the secretory pathway by heavy chain isotype class switching and N-linked glycosylation

Crystalline bodies (CBs) can develop in the endoplasmic reticulum (ER) of antibody-producing cells. Although this phenotype is often reported in association with plasma cell dyscrasias and other hematological disorders, the details of CB biogenesis and CB's roles in pathophysiology remain poorly understood. Using an imaging-based screening method, we identified a secretion-competent human IgG2/λ clone that develops spindle-shaped intracellular crystals in transiently-transfected HEK293 cells upon Brefeldin A treatment. When stably overexpressed from CHO cells, the IgG2/λ clone spontaneously produced spindle-shaped CBs in the ER. Some CBs were released to the extracellular space while remaining enclosed by the membranes of secretory pathway origin. Structural modeling on the variable-region did not uncover prominent surface characteristics such as charge clusters. In contrast, alterations to the constant domain-encoded properties revealed their modulatory roles in CB-inducing propensities and CB morphology. For example, deletion of the entire Fc domain changed the morphology of CBs into thin filaments. Elimination of an N-linked glycan by a N297A mutation promoted Russell body biogenesis accompanied by marked reduction in IgG secretion. Isotype class switching from the original IgG2 to IgG1 and IgG4 changed the crystal morphology from spindle-shaped to long needle and acicular shaped, respectively. The IgG3 version, in contrast, suppressed the CB formation. Either the HC or LC alone or the Fc-domain alone did not trigger CB biogenesis. An IgG's in vivo crystal morphology and crystallization propensity can thus be modulated by the properties genetically and biochemically encoded in the HC constant region.     

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.     

Differential spontaneous killing of human and murine tumour cell lines by leucocyte subpopulations from carp peripheral blood leucocytes

Cell populations from carp (Cyprinus carpio L.) peripheral blood leucocytes (PBLs) were examined for nonspecific cytotoxicities. By using monoclonal antibodies (MAbs) against carp thrombocytes (TCL-HB8) and both neutrophils and monocytes (TCL-BE8), PBLs with a density of 1.08 g ml-1 were separated into three fractions: thrombocytes, a mixture of neutrophils and monocytes, and other cells (mainly lymphocytes), and the separated cells were tested for cytotoxic activities against mammalian tumour cell lines (K562, HeLa, P815 and Yac-1 cell). Consequently, the mixture of neutrophils and monocytes exhibited cytolysis against these target cells, whereas the lymphocyte-rich and thrombocyte fractions did not show any cytolysis. To isolate only neutrophils, which do not contain monocytes, the MAb (TCL-BE8) positive cells from PBLs with a density of 1.08-1.09 g ml-1 were separated. Pure isolated neutrophils showed cytotoxic activities against K562 cells, but not P815 cells. Furthermore, analysis of the cytolytic mechanisms indicated that killing of these cells depended on H2O2 or HOCl. These results suggest that both neutrophils and monocytes are effectors for nonspecific cytotoxicity in carp PBLs, and neutrophils may be distinct from monocytes in their reactivity in cytolysis, including target cell selectivity and/or target cell sensitivity, and the cytolytic pathway. In carp, cytotoxicity of target cells can be mediated by several populations of their leucocytes which have cytotoxic capacities with various recognition and cytolytic mechanisms.     

Decreased Amyloid-β Pathologies by Intracerebral Loading of Glycosphingolipid-enriched Exosomes in Alzheimer Model Mice

Elevated levels of amyloid-β peptide (Aβ) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular Aβ can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in Aβ metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped Aβ and with the associated Aβ were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid-β precursor protein transgenic mice resulted in marked reductions in Aβ levels, amyloid depositions, and Aβ-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for Aβ binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for Aβ by carrying it on the exosome surface GSLs and suggest a role of exosomes in Aβ clearance in the central nervous system. Improving Aβ clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease.     

Establishment and characterization of Roberts syndrome and SC phocomelia model medaka (Oryzias latipes)

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.     

Smad7 Inhibits Transforming Growth Factor-β Family Type I Receptors through Two Distinct Modes of Interaction

The inhibitory Smads (I-Smads), i.e. Smad6 and Smad7, are negative regulators of transforming growth factor-β (TGF-β) family signaling. I-Smads inhibit TGF-β family signaling principally through physical interaction with type I receptors (activin receptor-like kinases), so as to compete with receptor-regulated Smads (R-Smads) for activation. However, how I-Smads interact with type I receptors is not well understood. In the present study, we found that Smad7 has two modes of interaction with type I receptors. One is through a three-finger-like structure in the MH2 domain, consisting of residues 331–361, 379–387, and the L3 loop. The other is through a basic groove in the MH2 domain (Mochizuki, T., Miyazaki, H., Hara, T., Furuya, T., Imamura, T., Watabe, T., and Miyazono, K. (2004) J. Biol. Chem. 279, 31568–31574). We also found that Smad6 principally utilizes a basic groove in the MH2 domain for interaction with type I receptors. Smad7 thus has an additional mode of interaction with TGF-β family type I receptors not possessed by Smad6, which may play roles in mediating the inhibitory effects unique to Smad7.     

A Similar Expression Pattern of Adhesion Molecules between Intermediate TCR Cells in the Liver and Intraepithelial Lymphocytes in the Intestine

Two major populations of extrathymically differentiated T cells exist in the liver and intestine. Such T cells in the liver have TCR of intermediate intensity (i.e., intermediate TCR cells) and constitutively express IL-2 receptor β-chain (IL-2Rβ), whereas those in the intestine, especially intraepithelial lymphocytes, have TCR of bright intensity, consisting of a mixture of IL-2Rβ⁺ and IL-2Rβ^–. All mature thymocytes and thymus-derived T cells seen in the peripheral immune organs are TCR-bright⁺IL-2Rβ^– under resting conditions. When the expression pattern of adhesion molecules, including CD44, L-selectin, LFA-1 and ICAM-1, was compared among these T-cell populations, they displayed quite unique patterns of expression. All extrathymic T cells in the liver, intestine, and even other organs were CD44⁺L-selectin^– LFA-1⁺⁺ICAM-1⁺, whereas thymocytes and thymus-derived T cells were CD44^– L-selectin⁺LFA-1⁺ICAM-1^–. This inverted expression of adhesion molecules between extrathymic T cells and thymus-derived T cells might be associated with their unique tissue-localization.     

Biological activities of chemically synthesized analogues of the nonreducing sugar moiety of lipid A

Adriamycin-Fe3+ complex catalyzes the formation of hydroxyl radical from hydrogen peroxide but the DNA-adriamycin-iron ternary complex is much more effective. 11-Deoxyadriamycin, which shows no spectral evidence of complex formation with iron, was ineffective. The generation of hydroxyl radical by adriamycin-Fe3+ complex in the presence of DNA correlates with its ability to cleave DNA. Hydroxyl radicals are thus implicated as the reactive oxygen species involved in the DNA damage caused by the adriamycin-Fe3+ complex.