Regulation of galectin-9 expression and release in Jurkat T cell line cells

Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.     

Visualization of rod photoreceptor development using GFP-transgenic zebrafish

Zebrafish retina contains five morphologically distinct classes of photoreceptors, each expressing a distinct type of opsin gene. Molecular mechanisms underlying specification of opsin expression and differentiation among the cell types are largely unknown. This is partly because mutants affected with expression of a particular class of opsin gene are difficult to find. In this study we established the transgenic lines of zebrafish carrying green fluorescent protein (GFP) gene under the 1.1-kb and 3.7-kb upstream regions of the rod-opsin gene. In transgenic fish, GFP expression initiated and proceeded in the same spatiotemporal pattern with rod-opsin gene. The retinal section from adult transgenic fish showed GFP expression throughout the rod cell layer. These results indicate that the proximal 1.1-kb region is sufficient to drive gene expression in all rod photoreceptor cells. These transgenic fish should facilitate screening of mutants affected specifically with rod-opsin expression or rod cell development by visualization of rod cells by GFP.     

Acute graft-versus-host disease does not require alloantigen expression on host epithelium

Alloantigen expression on host antigen-presenting cells (APCs) is essential to initiate graft-versus-host disease (GvHD); therefore, alloantigen expression on host target epithelium is also thought to be essential for tissue damage. We tested this hypothesis in mouse models of GvHD using bone-marrow chimeras in which either major histocompatibility complex class I or class II alloantigen was expressed only on APCs. We found that acute GvHD does not require alloantigen expression on host target epithelium and that neutralization of tumor necrosis factor-alpha and interleukin-1 prevents acute GvHD. These results pertain particularly to CD4-mediated GvHD but also apply, at least in part, to CD8-mediated GvHD. These results challenge current paradigms about the antigen specificity of GvHD effector mechanisms and confirm the central roles of both host APCs and inflammatory cytokines in acute GvHD.     

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Element analysis of the Polysphondylium pallidum gp64 promoter

gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5' deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to -170) and a vegetatively activated, downstream region (VAD, -110 to -63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one fourth of that of the cells fed on bacteria. By the gel retardation, we detected a protein bound to the A1 sequence.     

Synthesis of DNA conjugates by solid phase fragment condensation

Development of a novel method for the synthesis of DNA conjugates is described. Oligonucleotides were successfully conjugated with a variety of functional molecules on a solid phase (Solid Phase Fragment Condensation) using an amino, a hydroxyl, a thiol, and a carboxyl group. DNA-peptide conjugate was obtained as a pure from by a single RPHPLC purification approximately in 20% yield. Moreover, it was demonstrated that the present method was effective for the preparation of conjugate molecules, DNA-sugar, DNA-polyamine, DNA-lipid and so on. The study to create new intelligent DNAs by accumulation various biofunctions on the molecule by SPFC is now in progress in our laboratory.     

Mitochondrials complex I activity is reduced in latent adriamycin-induced cardiomyopathy of rat

We previously reported on the use of enzymatic analysis to impair fatty acid metabolism followed by reduced myocardial energy content, leading to severe heart failure in adriamycin (ADR)-treated rats. The aim of this study is to investigate whether impaired myocardial energy metabolism can also be detected by other methods; i.e. measuring mitochondrial complex I activity and myocardial ¹²⁵I-15-(p-iodophenyl)-3-(R,S)- methylpentadecanoic acid (BMIPP) accumulation in ADR-treated rats. Eight-week-old male Sprague-Dawley rats received 6 intraperitoneal injections of ADR (total 15 mg/kg: group ADR) or saline (control group) over 2 weeks. Left ventricular (LV) ejection fraction was assessed using echocardiography at 3- and 6-weeks after ADR injection (3 weeks and 6 weeks, respectively). Myocardial fatty acid utilization was assessed at 3 weeks and 6 weeks. The myocardial counts of BMIPP were measured after intravenous BMIPP (370 kBq) injection, and ¹²⁵I counts were measured to calculate the uptake ratio. The enzymatic activity of complex I was assessed by monitoring the oxidation of nicotinamide-adenine-dinucleotide-disodium-salt (NADH). In rats treated with ADR, significant decrease in LV ejection fraction was observed only at 6 weeks compared to control (72.5 vs. 84.5%, p < 0.01rpar;. LV ejection fraction at 3 weeks was identical between group ADR and control (81.8 vs. 84.4%). However, at 3 weeks, complex I activity was already reduced significantly in group ADR as compared to control group (p = 0.03), but the reduction in BMIPP accumulation was not (p = 0.15). Our data indicated that reduced complex I activity in a phenomenon occurred in early phase of ADR-induced cardiomyopathy, and it might play an important role in the progression of ADR-induced heart failure.