Identification of effector cells for TNFalpha-mediated cytotoxicity against WEHI164S cells

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4,300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFalpha-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR(int) cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFalpha-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFalpha-mediated cytotoxicity. These results suggest that TNFalpha-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFalpha if there was appropriate stimulation.     

Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions

To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.     

The role of propagule pressure in the invasion success of bluegill sunfish, Lepomis macrochirus, in Japan

The bluegill sunfish, Lepomis macrochirus, is a widespread exotic species in Japan that is considered to have originated from 15 fish introduced from Guttenberg, Iowa, in 1960. Here, the genetic and phenotypic traits of Japanese populations were examined, together with 11 native populations of the USA using 10 microsatellite markers and six meristic traits. Phylogenetic analysis reconfirmed a single origin of Japanese populations, among which populations established in the 1960s were genetically close to Guttenberg population, keeping high genetic diversity comparable to the ancestral population. In contrast, genetic diversity of later-established populations significantly declined with genetic divergence from the ancestral population. Among the 1960s established populations, that from Lake Biwa showed a significant isolation-by-distance pattern with surrounding populations in which genetic bottlenecks increased with geographical distance from Lake Biwa. Although phenotypic divergence among populations was recognized in both neutral and adaptive traits, P(ST)-F(ST) comparisons showed that it is independent of neutral genetic divergence. Divergent selection was suggested in some populations from reservoirs with unstable habitats, while stabilizing selection was dominant. Accordingly, many Japanese populations of L. macrochirus appear to have derived from Lake Biwa population, expanding their distribution with population bottlenecks. Despite low propagule pressure, the invasion success of L. macrochirus is probably because of its drastic population growth in Lake Biwa shortly after its introduction, together with artificial transplantations. It not only enabled the avoidance of a loss in genetic diversity but also formed a major gene pool that supported local adaptation with high phenotypic plasticity.     

Expression, purification and characterization of a photoprotein, clytin, from Clytia gregarium

A novel histidine-tagged secretion vector in Escherichia coli was constructed and large amounts of highly pure clytin, a calcium-binding photoprotein, was prepared. The histidine-tagged apoclytin expressed into the periplasmic space in E. coli was purified by nickel chelate affinity chromatography. Recombinant clytin was regenerated from apoclytin by incubation with coelenterazine in the presence of dithiothreitol and then purified by anion-exchange chromatography and hydrophobic chromatography. The yield of recombinant clytin was 20mg from 2L of cultured cells with purity greater than 95%. Luminescence properties of recombinant clytin were identical to that of native clytin (phialidin). The Ca(2+) sensitivity of recombinant clytin is lower than that of aequorin and clytin is suited for measuring higher concentration of Ca(2+). Semi-synthetic clytins were also prepared with coelenterazine analogues, and the initial intensity, luminescence capacity and half decay time were characterized.     

Effect of alendronate on osteoclast differentiation and bone volume in transplanted bone.

Alendronate, one of the bisphosphonates, is known to have an inhibitory effect on bone resorption. The purpose of this study was to investigate the effects of alendronate on ectopic bone graft resorption and to determine the optimal dose in the mouse. The grafted bone in the control group disappeared due to resorption by osteoclasts within 5 weeks. In the experimental groups, the area of bone tissue decreased by only 20-40% at 5 weeks post-operatively. At 8 and 9 weeks after surgery, the decreased area of bone structure was significantly less in all the 10(-4) M injected alendronate-immersed groups than in the 10(-4) M non-injected alendronate-immersed. At 9 weeks after surgery, the number of osteoclasts were significantly less in the 10(-4) M injected alendronate-treated groups than in the 10(-4) M non-injected alendronate-treated groups. These results suggest that alendronate inhibits resorption of ectopic bone graft at concentrations of 10(-4) and 10(-6) M.     

Identification of two catalytic domains in a luciferase secreted by the copepod Gaussia princeps

Gaussia luciferase secreted by the copepod Gaussia princeps catalyzes the oxidation of coelenterazine to produce blue light. The primary structure of Gaussia luciferase deduced from the cDNA sequence shows two repeat sequences of 71 amino acid residues, suggesting the luciferase consists of two structural domains. Two domains in Gaussia luciferase were expressed independently in Escherichia coli cells, purified and characterized. We found that both domains have luminescence activity with coelenterazine, and the catalytic properties including luminescence spectrum, optimal pH, substrate specificity and luminescence stimulation by halogen ions (Cl⁻, Br⁻ and I⁻) are identical to intact Gaussia luciferase. Thus, Gaussia luciferase has two catalytic domains for the luminescence reaction.     

Heterogeneity of mouse primordial germ cells reflecting the distinct status of their differentiation, proliferation and apoptosis can be classified by the expression of cell surface proteins integrin α6 and c-Kit

Primordial germ cells (PGCs) in mouse embryos likely include heterogeneous cells having distinct cellular properties. In the present study, we found that heterogeneity of PGCs can be defined by the expression of integrin α6 and c-Kit. The changes in integrin α6 and c-Kit expression in PGCs were obvious as embryonic development progressed, and the PGCs became a mixture of populations consisting of cells with distinct levels of cell surface protein expression. The changes and heterogeneity of cell surface protein expression mainly reflected asynchronous differentiation of PGCs. Apoptosis of PGCs was biased in populations of c-Kit or integrin α6 negative PGCs at particular developmental stages, suggesting possible linkage between PGC apoptosis and the levels of expression of these cell surface proteins. Histochemical analysis confirmed the heterogeneous expression of c-Kit and integrin α6 in PGCs in embryonic gonads, and revealed that PGCs showing different levels of integrin α6 or c-Kit expression and the apoptotic PGCs were scattered and did not show specific localization within gonads. The present study enables us to analyze and isolate populations of living PGCs showing a distinct status of differentiation, or different properties of proliferation or of cell death in individual embryos, and provides a new strategy to examine the mechanisms of PGC development.     

Recombinant Gaussia luciferase with a reactive cysteine residue for chemical conjugation: Expression, purification and its application for bioluminescent immunoassays

The mutated recombinant Gaussia luciferase (hgGLase) having the hinge sequence with a reactive cysteine residue at the carboxyl terminal region was purified from Escherichia coli cells by nickel-chelate affinity chromatography and hydrophobic chromatography. The biotinylated hgGLase (Biotin-hgGLase) was prepared by chemical conjugation with a maleimide activated biotin and apply to bioluminescent immunoassay. In the streptavidin and biotin complex system using Biotin-hgGLase, the measurable range of α-fetoprotein as a model analyte was 0.02–100 ng/ml with the coefficient of variation between 2.5% and 5.2%. The sensitivity of Biotin-hgGLase was similar to that by using the detection system of aequorin, alkaline phosophatase and horseradish peroxidase as a label enzyme.     

Interleukin-1 Receptor-associated Kinase-4 (IRAK4) Promotes Inflammatory Osteolysis by Activating Osteoclasts and Inhibiting Formation of Foreign Body Giant Cells

Formation of foreign body giant cells (FBGCs) occurs following implantation of medical devices such as artificial joints and is implicated in implant failure associated with inflammation or microbial infection. Two major macrophage subpopulations, M1 and M2, play different roles in inflammation and wound healing, respectively. Therefore, M1/M2 polarization is crucial for the development of various inflammation-related diseases. Here, we show that FBGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules in vitro. We also found that FBGC formation was significantly inhibited by inflammatory cytokines or infection mimetics in vitro. Interleukin-1 receptor-associated kinase-4 (IRAK4) deficiency did not alter osteoclast formation in vitro, and IRAK4-deficient mice showed normal bone mineral density in vivo. However, IRAK4-deficient mice were protected from excessive osteoclastogenesis induced by IL-1β in vitro or by LPS, an infection mimetic of Gram-negative bacteria, in vivo. Furthermore, IRAK4 deficiency restored FBGC formation and expression of M2 macrophage markers inhibited by inflammatory cytokines in vitro or by LPS in vivo. Our results demonstrate that osteoclasts and FBGCs are reciprocally regulated and identify IRAK4 as a potential therapeutic target to inhibit stimulated osteoclastogenesis and rescue inhibited FBGC formation under inflammatory and infectious conditions without altering physiological bone resorption.