Isolation of connective-tissue-specific genes involved in <Emphasis Type="Italic">Xenopus</Emphasis> intestinal remodeling: thyroid hormone up-regulates Tolloid/BMP-1 expression

To clarify connective-tissue-specific genes involved in adult epithelial development during amphibian intestinal remodeling, we have isolated 16 cDNA clones derived from the anterior part of Xenopus laevis intestine cultured in vitro by using subtractive suppression hybridization. Among four genes identified, the expression of Xtld, a Xenopus homolog of Drosophila Tolloid closely related to bone morphogenic protein-1 (BMP-1), was most remarkably up-regulated during metamorphosis. To further explore the roles of Xtld in intestinal remodeling, we examined its developmental expression in the X. laevis intestine by in situ hybridization and northern blot analysis. Xtld mRNA first became detectable in the connective tissue just before the appearance of adult epithelial primordia. Subsequently, the level of Xtld mRNA reached a high in the connective tissue, concomitantly with adult epithelial development along the anteroposterior axis of the intestine. Thereafter, towards the completion of metamorphosis, the expression of Xtld mRNA was down-regulated. Thus, the expression profile of Xtld mRNA spatiotemporally correlates well with adult epithelial development in vivo. Furthermore, the present culture study has shown that thyroid hormone (TH) up-regulates the expression of Xtld mRNA organ-autonomously in the anterior part of the intestine, but not in its posterior part, and that TH up-regulation of Xtld expression is not mediated by the epithelium. These results suggest that TH directly up-regulates Xtld expression in the connective tissue along the anteroposterior axis, which in turn plays important roles in adult epithelial development during amphibian intestinal remodeling.     

Dynamic modeling of genetic networks using genetic algorithm and S-system

MOTIVATION: The modeling of system dynamics of genetic networks, metabolic networks or signal transduction cascades from time-course data is formulated as a reverse-problem. Previous studies focused on the estimation of only network structures, and they were ineffective in inferring a network structure with feedback loops. We previously proposed a method to predict not only the network structure but also its dynamics using a Genetic Algorithm (GA) and an S-system formalism. However, it could predict only a small number of parameters and could rarely obtain essential structures. In this work, we propose a unified extension of the basic method. Notable improvements are as follows: (1) an additional term in its evaluation function that aims at eliminating futile parameters; (2) a crossover method called Simplex Crossover (SPX) to improve its optimization ability; and (3) a gradual optimization strategy to increase the number of predictable parameters. RESULTS: The proposed method is implemented as a C program called PEACE1 (Predictor by Evolutionary Algorithms and Canonical Equations 1). Its performance was compared with the basic method. The comparison showed that: (1) the convergence rate increased about 5-fold; (2) the optimization speed was raised about 1.5-fold; and (3) the number of predictable parameters was increased about 5-fold. Moreover, we successfully inferred the dynamics of a small genetic network constructed with 60 parameters for 5 network variables and feedback loops using only time-course data of gene expression.     

Natural killer cytolytic activity is associated with the expression of killer cell immunoglobulin-like receptors on peripheral lymphocytes in human

Although it has been shown that killer cell immunoglobulin-like receptors (KIRs) on peripheral lymphocytes are upregulated by interleukin-2 (IL-2), which activates natural killer (NK) activity, it has not been demonstrated whether the expression of KIRs is related to NK activity. Therefore, we investigated the association between the KIR expression on lymphocytes and NK activity. CD158a/b expression on lymphocytes obtained from 37 subjects was analyzed using flow cytometry. Simultaneously, NK activity was measured each sample using a 51Cr-release assay. Additionally, lymphocytes were cultured in RPMI 1640 medium with or without IL-2 for 48 h, and then their CD158a/b expression and NK activity was analyzed. CD158a/b expression was significantly correlated with NK activity. Especially, the percentage of CD16+CD158a+ and CD8+CD158a/b+ cells in lymphocytes showed a highly significant correlation with NK activity. However, analysis of CD8+ and CD16+ cells revealed that there was only a significant correlation between the percentage of CD8+CD158a+ cells among only CD8+ cells and NK activity. The upregulation of CD16+CD158a+/b+ cells in response to IL-2 tended to be related to the increase of NK activity, but the relationship was not significant. In conclusion, the level of KIR expression was correlated with NK activity, and IL-2 treatment resulted in an increase of NK activity as well as KIR expression, suggesting that upregulation of KIRs enhances the ability to sort target cells, such as virus-infected cells from uninfected cells, according to major histocompatibility complex class I expression.     

Stabilization of protein by replacement of a fluctuating loop: structural analysis of a chimera of bovine alpha-lactalbumin and equine lysozyme

Equine lysozyme is a calcium-binding lysozyme and an evolutional intermediate between non-calcium binding c-type lysozyme and alpha-lactalbumin. We constructed a chimeric protein by substituting the fluctuating loop of bovine alpha-lactalbumin with the D-helix of equine lysozyme. The substitution affects the protection factors not only in the fluctuating loop but also in the antiparallel beta-sheet, the A- and B-helices, and the loop between the B-helix and the beta-sheet. Amide protons in these regions of the chimera are more protected from exchange than are those of bovine alpha-lactalbumin. We used model-free analysis based on 15N nuclear magnetic resonance relaxation measurements to investigate the dynamics of the main chain of the chimera and showed that the fluctuating loop of the chimera is as rigid as three major helices. When we analyzed the chemical shift deviations and backbone HN-H(alpha) scalar coupling constants, we found that the chimera showed an alpha-helical tendency in residues around the fluctuating loop. Our results suggest that the replacement of a highly fluctuating loop in a protein with a rigid structural element in a homologous one may be useful to stabilize the protein structure.     

Real-time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor

Surface plasmon resonance (SPR)-based sensors have been used to detect the binding between interactive molecules. We applied the SPR technology to the analysis of interactions between living cells and molecules reactive to the cells, using mast cells and mast cell-reactive antigens. The exposure of dinitrophenol-human serum albumin (DNP-HSA), an antigen that stimulates mast cells, to IgE-sensitized mast cells induced a robust and long-lasting SPR signal in a dose-dependent manner. The maximal increase in SPR signal induced by 100 ng/ml DNP-HSA was 0.200 +/- 0.120 angle (mean +/- SD, n = 37), about 1000 times larger than the theoretically expected increase for the simple binding of DNP-HSA to Fc(epsilon)RI, the high-affinity IgE receptor. A small, but similarly prolonged signal was observed when the cells were stimulated by an agonist of the adenosine A3 receptor. The signal induced by DNP-HSA was abolished by genistein, and partially inhibited by phorbol 12-myristate 13-acetate and wortmannin. Interestingly, the signal induced by DNP-HSA was only weakly inhibited by DNP-lysine, suggesting that DNP-lysine manifests its action not by inhibiting, but by modulating the crosslinking of Fc(epsilon)RI. We concluded that SPR sensors can detect biologically significant signals in a real-time manner from the interactions between cells and molecules reactive to the cells.     

The effect of interleukin-15 on the expression of killer-cell immunoglobulin-like receptors on peripheral natural killer cells in human

Interleukin (IL)-15 has emerged as a key regulator of both natural killer (NK) cell differentiation and activation. The aim of the present study was to investigate the expansion of the population of cells expressing killer-cell immunoglobulin-like receptors (CD158a and CD158b) in human peripheral lymphocytes by treatment with IL-15. One million peripheral lymphocytes were cultured in RPMI1640 medium alone or in medium containing IL-2 at 100 U/ml or IL-15 at 0.1, 1.0, or 10.0 ng/ml for 48 h. After each incubation, we assessed the natural killing activity and the population of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. IL-15 increased the NK activity and expanded the populations of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. These actions were dose dependent, and the effects of IL-15 at 1.0 ng/ml were close to those of IL-2 at 100 U/ml. These findings suggest that IL-15 induces the effector functions of resting NK cells throughout the body, and thereby plays a critical role in the activation of tissue-associated immune responses.     

Reduction of hepatitis C virus NS5A phosphorylation through its interaction with amphiphysin II

Hepatitis C virus non-structural protein 5A (NS5A) is a pleiotropic protein with key roles in viral RNA replication, modulation of cellular-signaling pathways and interferon (IFN) responses. To search for possible host factors involved in mediating these functions of NS5A, we adopted an affinity purification approach coupled with mass spectrometry to examine protein-protein interactions, and found that human amphiphysin II (also referred to as Bin1) specifically interacts with NS5A in mammalian cells. Pull-down assays showed that the Src homology 3 (SH3) domain of amphiphysin II is required for NS5A interaction and that c-Src also interacts with NS5A in cells. IFN-alpha treatment reduced the interaction of NS5A with c-Src, but not amphiphysin II, suggesting that the latter is independent of the IFN-signaling pathway. NS5A is a phosphoprotein and its phosphorylation status is considered to have an effect on viral RNA replication. In vitro kinase assays demonstrated that its interaction with amphiphysin II inhibits phosphorylation of NS5A. These results suggest that amphiphysin II participates in the HCV life cycle by modulating the phosphorylation of NS5A.     

Effect of hydrostatic pressure on conformational changes of canine milk lysozyme between the native, molten globule, and unfolded states

The effect of pressure on the unfolding of the native (N) and molten globule (MG) state of canine milk lysozyme (CML) was examined using ultraviolet (UV) spectroscopy at pH 4.5 and 2.0, respectively. It appeared that the thermally induced unfolding was promoted by the increase of pressure from atmospheric to 100 MPa, which indicates that both the N and MG states of CML unfolded with the decrease of the partial molar volume change (DeltaV). The volume changes needed for unfolding were estimated from the free energy change vs. pressure plots, and these volume changes became less negative from 20 to 60 degrees C. The DeltaV values at 25 degrees C were obtained for the N-MG (-46 cm3/mol) and MG-unfolded-state (U) transition (-40 cm3/mol). With regards to the MG-U transition, this value is contrastive to that of bovine alpha-lactalbumin (BLA) (0.9 cm3/mol), which is homologous to CML. Previous studies revealed that the MG state of CML was significantly more stable, and closer to the N state in structure, than that of BLA. In contrast to the swollen hydrophobic core of the MG state of BLA, our results suggest that the MG state of CML possesses a tightly packed hydrophobic core into which water molecules cannot penetrate.     

Thermodynamic analysis of the activation mechanism of the GCSF receptor induced by ligand binding

The granulocyte colony-stimulating factor receptor (GCSFR), containing the Ig-like domain (Ig) and cytokine receptor homologous region (CRH), was prepared as a preformed dimer (Ig-CRH-Fc)(2) after fusion to the mouse Fc region via an eight-residue linker (approximately 55 A). Monomer Ig-CRH was also prepared after the Fc region was removed from (Ig-CRH-Fc)(2). GCSF binding to Ig-CRH and (Ig-CRH-Fc)(2) was investigated using light scattering and isothermal titration calorimetry. The average molecular mass determined by light scattering showed that both Ig-CRH and (Ig-CRH-Fc)(2) formed a 2:2 dimer with GCSF. Moreover, isothermal titration calorimetry showed that the thermodynamic parameters upon binding of GCSF to Ig-CRH and (Ig-CRH-Fc)(2) were comparable, suggesting a similar binding stoichiometry and interface [including similar buried surface area (5700-6000 A(2))] despite the presence of the eight-residue linker. The buried surface area is much larger than that calculated from our previous report of the crystal structure of the GCSF-CRH complex [Aritomi, M., et al. (1999) Nature 401, 713-717], suggesting a substantial contribution of the Ig domain to GCSF binding. The data also indicate that the distance (55 A) between two CRH domains in the 2:2 complex is much shorter than in our previous model (approximately 90 A) predicted from the same crystal structure of the GCSF-CRH complex.