Ubiquitin-like protein MNSFβ regulates TLR-2-mediated signal transduction

Post-translational modification by monoclonal nonspecific suppressor factor β (MNSFβ) has been involved in the regulation of a variety of cellular processes. Previous studies have demonstrated that MNSFβ covalently binds to the intracellular pro-apoptotic protein Bcl-G and regulates TLR-4-mediated signal transduction. Recently, we found that MNSFβ also covalently conjugates to endophilin II, a member of the endophilin A family, and inhibits the signal pathway upstream of IKK activation, but not downstream of TLR-2 signaling. In this study, we further examined the mechanism of action of MNSFβ in TLR-2-mediated signal transduction in macrophage-like cell line Raw264.7 cells. Although MNSFβ siRNA enhanced Pam(3)CDK(4) (TLR-2-specific ligand)-stimulated TNFα production, Bcl-G siRNA did not affect. MNSFβ cDNA inhibited the Pam(3)CDK(4)-stimulated TNFα production. High-molecular weight (130 kDa) MNSFβ-adduct was induced in Pam(3)CDK(4)-stimulated Raw264.7 cells. This MNSFβ-adduct was not induced by LPS, indicative of the specificity of TLR-2-mediated signal transduction. Similar observations were seen in BALB/c peritoneal macrophages. Interestingly, 40-kDa MNSFβ-adduct was tyrosine phosphorylated by Pam(3)CDK(4) stimulation. Collectively, novel MNSFβ-adducts may regulate TLR-2 signaling pathway in macrophages.     

Channel-dependent permeation of water and glycerol in mouse morulae

The cryosensitivity of mammalian embryos depends on the stage of development. Because permeability to water and cryoprotectants plays an important role in cryopreservation, it is plausible that the permeability is involved in the difference in the tolerance to cryopreservation among embryos at different developmental stages. In this study, we examined the permeability to water and glycerol of mouse oocytes and embryos, and tried to deduce the pathway for the movement of water and glycerol. The water permeability (L(P), microm min(-1) atm(-1)) of oocytes and four-cell embryos at 25 degrees C was low (0.63-0.70) and its Arrhenius activation energy (E(a), kcal/mol) was high (11.6-12.3), which implies that the water permeates through the plasma membrane by simple diffusion. On the other hand, the L(p) of morulae and blastocysts was quite high (3.6-4.5) and its E(a) was quite low (5.1-6.3), which implies that the water moves through water channels. Aquaporin inhibitors, phloretin and p-(chloromercuri) benzene-sulfonate, reduced the L(p) of morulae significantly but not that of oocytes. By immunocytochemical analysis, aquaporin 3, which transports not only water but also glycerol, was detected in the morulae but not in the oocytes. Accordingly, the glycerol permeability (P(GLY), x 10(-3) cm/min) of oocytes was also low (0.01) and its E(a) was remarkably high (41.6), whereas P(GLY) of morulae was quite high (4.63) and its E(a) was low (10.0). Aquaporin inhibitors reduced the P(GLY) of morulae significantly. In conclusion, water and glycerol appear to move across the plasma membrane mainly by simple diffusion in oocytes but by facilitated diffusion through water channel(s) including aquaporin 3 in morulae.     

Regulatory roles for MD-2 and TLR4 in ligand-induced receptor clustering

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe(119) to Lys(132) (Arg(132) in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly(59) was found to be a novel critical amino acid for LPS binding outside the region 119-132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe(126) or Gly(129) impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.     

Hyperactivation of midbrain dopaminergic system in schizophrenia could be attributed to the down-regulation of dysbindin

Extraordinal activation of nigrostriatal and mesolimbic dopaminergic systems (midbrain dopaminergic system) is thought to be one of the most important etiologies for schizophrenia, though the reason why unusual hyperactivation of the dopaminergic system occurs in the schizophrenic brain is quite obscure. Dysbindin, one of the most susceptible genes for schizophrenia, has been reported to be reduced in the schizophrenic brain. In situ hybridization analysis showed the mRNA expression of dysbindin in the mouse substantia nigra. Furthermore, suppression of dysbindin expression in PC12 cells resulted in an increase of the expression of SNAP25, which plays an important role in neurotransmitter release, and increased the release of dopamine. On the other hand, up-regulation of dysbindin expression in PC12 cells showed a tendency to decrease the expression of SNAP25. These data suggest that dysbindin might regulate the dopamine release of the dopaminergic system via modulation of the expression of SNAP25.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

Evaluations of substrate specificity and inhibition at PR/p3 cleavage site of HTLV-1 protease

Core sequences necessary for substrate recognition and its inhibition at the PR/p3 site of HTLV-1 protease were clarified for the first time. From the cleavage rates of peptides containing a part of the PR/p3 site, a heptapeptide was found to be the minimal sequence required for substrate recognition. The use of synthetic inhibitors containing hydroxyethylamine dipeptide isostere indicated that a tetrapeptide sequence was necessary to achieve potent inhibition.     

Cell-surface modification of non-GMO without chemical treatment by novel GMO-coupled and -separated cocultivation method

We developed a novel method to coat living non-genetically modified (GM) cells with functional recombinant proteins. First, we prepared GM yeast to secrete constructed proteins that have two domains: a functional domain and a binding domain that recognizes other cells. Second, we cocultivated GM and non-GM yeasts that share and coutilize the medium containing recombinant proteins produced by GM yeasts using a filter-membrane-separated cultivation reactor. We confirmed that GM yeast secreted enhanced green fluorescent protein (EGFP) fusion proteins to culture medium. After cocultivation, EGFP fusion proteins produced by GM yeast were targeted to non-GM yeast (Saccharomyces cerevisiae BY4741ΔCYC8 strain) cell surface. Yeast cell-surface engineering is a useful method that enables the coating of GM yeast cell surface with recombinant proteins to produce highly stable and accumulated protein particles. The results of this study suggest that development of cell-surface engineering from GM organisms (GMOs) to living non-GMOs by our novel cocultivation method is possible.     

Structural characteristics and <Emphasis Type="Italic">in vitro</Emphasis> macrophage activation of acetyl fucoidan from <Emphasis Type="Italic">Cladosiphon okamuranus</Emphasis>

We investigated a structural characteristics of acetyl fucoidan (CAF) isolated from commercially cultured Cladosiphon okamuranus. The CAF-induced macrophage activation and its signaling pathways in murine macrophage cell line, RAW 264.7 were also investigated. From the results of methylation analysis, CAF consisted of α-1→3 linked l-fucosyl residues and substituted sulfate and acetyl groups at C-4 on the main chain. CAF induced production of nitric oxide (NO), tumor necrosis factor-α and interleukin-6 in RAW 264.7 cells. Sulfate and acetyl groups of CAF involved in CAF-induced NO production. Neutralizing anti-Toll-like receptor 4 (TLR4), anti-CD14 and anti-scavenger receptor class A (SRA) but not anti-complement receptor type 3 monoclonal antibodies decreased CAF-induced NO production. The results of immunoblot analysis indicated that CAF activated mitogen-activated protein kinases (MAPKs) such as p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). SB203580 (p38 MAPK inhibitor) and SP600125 (SAPK/JNK inhibitor), but not U0126 (MAPK/ERK kinase 1/2 inhibitor) decreased CAF-induced NO production. The results suggested that CAF induced macrophage activation through membrane receptors TLR4, CD14 and SRA, and MAPK signaling pathways.     

Activation of NFAT signal by p53-K120R mutant

The tumor suppressor p53 is activated by phosphorylation and/or acetylation. We constructed 14 non-phosphorylated, 11 phospho-mimetic, and 1 non-acetylated point p53 mutations and compared their transactivation ability in U-87 human glioblastoma cells by the luciferase reporter assay. Despite mutations at the phosphorylation sites, only the p53-K120R and p53-S9E mutants had marginally reduced activities. On the other hand, the Nuclear factor of activated T-cells (NFAT)-luciferase reporter was more potently activated by p53-K120R than by wild-type p53 and other mutants in glioblastoma, hepatoma and esophageal carcinoma cells. This suggests that acetylation at Lys-120 of p53 negatively regulates a signaling pathway leading to NFAT activation.