Improved culture-based isolation of differentiating endothelial progenitor cells from mouse bone marrow mononuclear cells

Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in endothelial differentiation medium separately. Immunological and molecular analyses exhibited more endothelial-like and less monocyte/macrophage-like characteristics in FL cells compared with AT cells. FL cells formed thick/stable tube and hypoxia or shear stress overload further enhanced these endothelial-like features with increased angiogenic cytokine/growth factor mRNA expressions. Finally, FL cells exhibited therapeutic potential in a mouse myocardial infarction model showing the specific local recruitment to ischemic border zone and tissue preservation. These findings suggest that slow adherent (FL) but not fast attached (AT) BMMNCs in culture are EPC-rich population in mouse.     

Gene transfer of PEDF attenuates ischemic brain damage in the rat middle cerebral artery occlusion model

Pigment epithelium-derived factor (PEDF) is a 50-kDa glycoprotein that protects various types of cultured neurons against neurotoxic stimuli, but its precise role in the CNS is not fully understood. In this study, we used rats whose brains were transfected to over-express human PEDF in order to elucidate the neuroprotective effect of PEDF following transient middle cerebral artery occlusion (MCAO). A replication-defective adenoviral vector containing the human PEDF gene (Ad.PEDF) or E. coli β-galactosidase (Ad.LacZ) was directly injected into the right striatum at 7 days prior to 70 min of MCAO in rats. Infarct volume and degree of edema of the Ad.PEDF-treated group were significantly reduced compared to the Ad.LacZ-treated group 24 h after MCAO. Degeneration of neurons, astrocytes, and oligodendrocytes caused by MCAO were attenuated by over-expression of PEDF. The up-regulation of pro-inflammatory genes (TNFα, IL-1β, IL-6, COX-2, and iNOS) and water channel aquaporin 4 after MCAO was significantly reduced in Ad.PEDF-injected striatum. In conclusion, the results from this study provide the first in vivo evidence that PEDF is effective in protecting CNS neurons from ischemic insult, suggesting that PEDF may have a role as an endogenous neuroprotectant in the CNS.     

Effect of exo-β-D-(1→3)-galactanase digestion on complement activating activity of neutral arabinogalactan unit in a pectic arabinogalactan from roots of Angelica acutiloba Kitagawa

A complement activating pectic arabinogalactan (AGIIb-1) has been isolated from roots of Angelica acutiloba Kitagawa, and a neutral arabinogalactan (N-I) unit, which was liberated from a side chain in AGIIb-1 by mild acid hydrolysis, has found to show the most potent complement activating activity among the arabinogalactan side chains of AGIIb-1. In order to clarify the essential carbohydrate side chains in N-I for the activity, neutral arabinogalactan (AF-N-I) unit, which was obtained from exo--L-arabinofuranosidase-digested AGIIb-1, was digested with exo-β-d-(1 → 3)-galactanase from Irpex lacteus. The galactanase digestion of the AF-N-I unit completely stopped its reactivity with β-d-glucosyl-Yariv antigen, and significantly reduced its complement activating activity. The digestion products gave high molecular weight fragments (GN-1A and -1B) and intermediate size fragments in addition to β-d-(1 → 6)-galactosyl mono to pentasaccharides as the side chains of the AF-N-I unit. Among the fragments, GN-1A, GN-1B and intermediate size fragments showed relatively potent complement activating activity, suggesting that these side chains in the N-I unit might be responsible for expression of the activity of the N-I unit.     

Genetic factors and epigenetic factors for autism: Endoplasmic reticulum stress and impaired synaptic function

The molecular pathogenesis of ASD (autism spectrum disorder), one of the heritable neurodevelopmental disorders, is not well understood, although over 15 autistic‐susceptible gene loci have been extensively studied. A major issue is whether the proteins that these candidate genes encode are involved in general function and signal transduction. Several mutations in genes encoding synaptic adhesion molecules such as neuroligin, neurexin, CNTNAP (contactin‐associated protein) and CADM1 (cell‐adhesion molecule 1) found in ASD suggest that impaired synaptic function is the underlying pathogenesis. However, knockout mouse models of these mutations do not show all of the autism‐related symptoms, suggesting that gain‐of‐function in addition to loss‐of‐function arising from these mutations may be associated with ASD pathogenesis. Another finding is that family members with a given mutation frequently do not manifest autistic symptoms, which possibly may be because of gender effects, dominance theory and environmental factors, including hormones and stress. Thus epigenetic factors complicate our understanding of the relationship between these mutated genes and ASD pathogenesis. We focus in the present review on findings that ER (endoplasmic reticulum) stress arising from these mutations causes a trafficking disorder of synaptic receptors, such as GABA (γ‐aminobutyric acid) B‐receptors, and leads to their impaired synaptic function and signal transduction. In the present review we propose a hypothesis that ASD pathogenesis is linked not only to loss‐of‐function but also to gain‐of‐function, with an ER stress response to unfolded proteins under the influence of epigenetic factors.     

Distinct roles of overlapping and non-overlapping regions of hub protein interfaces in recognition of multiple partners

Cellular functions of an organism are maintained by protein-protein interactions. Those proteins that bind multiple partners asynchronously (date hub proteins) are important to make the interaction network coordinated. It is known that many date hub proteins bind different partners at overlapping (OV) interfaces. To understand how OV interfaces of date hub proteins can recognize multiple partners, we analyzed the difference between OV and non-overlapping (Non-OV) regions of interfaces involved in the binding of different partners. By using the structures of 16 date hub proteins with various interaction partners (ranging from 5 to 33), we compared buried surface area, compositions of amino acid residues and secondary structures, and side-chain orientations. It was found that buried interface residues are important for recognizing multiple partners, while exposed interface residues are important for determining specificity to a particular ligand. In addition, our analyses reveal that residue compositions in OV and Non-OV regions are different and that residues in OV region show diverse side-chain torsion angles to accommodate binding to multiple targets.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis.

The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA-RNA hybrid as a substrate revealed that the enzymatic properties of SIB1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was approximately 25 degrees C for SIB1 RNase HI and approximately 60 degrees C for E.coli RNase HI. The optimum temperature for the SIB1 RNase HI activity was also shifted downward by 20 degrees C compared with that of E.coli RNase HI. Nevertheless, SIB1 RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10 degrees C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the alphaI-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.     

Immunobiotic Bifidobacteria Strains Modulate Rotavirus Immune Response in Porcine Intestinal Epitheliocytes via Pattern Recognition Receptor Signaling

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-β and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-β production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities.     

Cell-specific delivery of polymeric nanoparticles to carbohydrate-tagging cells

Carbohydrates on cell surfaces contribute a variety of communications between the cell and its environment, and they have been assumed to act as markers for cellular recognition. In this research, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer nanoparticles, which can react with specific carbohydrates of target cells, were newly prepared to serve as novel drug carriers. A water-soluble MPC polymer bearing hydrazide groups (PMBH) was synthesized by conventional radical polymerization. The MPC polymer showed amphiphilic nature and worked as an emulsifier to form nanoparticles. The nanoparticles covered with PMBH were prepared by the solvent evaporation method and exhibited monodispersity. They were approximately 200 nm in diameter and -2.0 mV in surface potential. According to a surface analysis of the nanoparticles, phosphorylcholine and hydrazide groups were observed, and the surface was fully covered with PMBH. Unnatural carbohydrates having ketone groups on human cervical carcinoma cell (HeLa) surfaces were expressed by treatment with levulinoyl mannosamine (ManLev). When the PMBH nanoparticles were in contact with the ManLev-treated HeLa cells, they accumulated in the cells. In contrast, the nanoparticles were not observed in native HeLa cells (without unnatural carbohydrates). These results indicate that the hydrazide groups of the nanoparticles selectively reacted to the ketone groups of the carbohydrates on the cell surface. The PMBH nanoparticles immobilized with anticancer drugs such as doxorubicin or paclitaxel were in contact with either ManLev-treated or untreated HeLa cells. The viability of the ManLev-treated HeLa cells was effectively reduced, but that of the untreated cells was preserved. This indicated that the anticancer drugs were selectively delivered to the ManLev-treated cells. Nonspecific cellular uptake of the nanoparticles was effectively reduced by MPC polymer coating. Furthermore, the immobilization processes of the drugs differed because of the solubility of the drugs. In conclusion, cellular-specific drug delivery by means of the novel nanoparticles was demonstrated with the selective reaction between unnatural carbohydrates on the cell surface and the hydrazide groups bearing the phosphorylcholine polymer nanoparticles.     

Mutational analysis of block and facilitation of HERG current by a class III anti-arrhythmic agent, nifekalant

Chemicals and toxins are useful tools to elucidate the structure-function relationship of various proteins including ion channels. The HERG channel is blocked by many compounds and this may cause life-threatening cardiac arrhythmia. Besides block, some chemicals such as the class III anti-arrhythmic agent nifekalant stimulate HERG at low potentials by shifting its activation curve towards hyperpolarizing voltages. This is called "facilitation". Here, we report mutations and simulations analyzing the association between nifekalant and channel pore residues for block and facilitation. Alanine-scanning mutagenesis was performed in the pore region of HERG. The mutations at the base of the pore helix (T623A), the selectivity filter (V625A) and the S6 helix (G648A, Y652A and F656A) abolished and S624A attenuated both block and facilitation induced by the drug. On the other hand, the mutation of other residues caused either an increase or a decrease in nifekalant-induced facilitation without affecting block. An open-state homology model of the HERG pore suggested that T623, S624, Y652 and F656 faced the central cavity, and were positioned within geometrical range for the drug to be able to interact with all of them at the same time. Of these, S649 was the only polar residue located within possible interaction distance from the drug held in its blocking position. Further mutations and flexible-docking simulations suggest that the size, but not the polarity, of the side chain at S649 is critical for drug induced facilitation.