Gene expression of arachidonate cyclooxygenase pathway leading to the delayed synthesis of prostaglandins E2 and F2alpha in response to phorbol 12-myristate 13-acetate and action of these prostanoids during life cycle of adipocytes

Several types of prostaglandin (PG)s are synthesized in adipocytes and involved differently in the control of adipogenesis. To elucidate how the PG synthesis is regulated at different stages in the life cycle of adipocytes, we examined the gene expression of arachidonate cyclooxygenase (COX) pathway leading to the delayed synthesis of PGE2 and PGF2alpha and their roles in adipogenesis after exposure of cultured cells to phorbol 12-myristate 13-acetate (PMA), which is a useful system for monitoring mitogen-induced changes. While the expression of COX-1 remained constitutive, mRNA and protein levels of COX-2 were up-regulated by treatment with PMA. Preadipocytes exhibited higher gene expression of cytosolic phospholipase A2alpha (cPLA2alpha) and PGF synthase. In contrast, three isoforms of PGE synthase are expressed constitutively during all phases. The delayed synthesis of PGE2 and PGF2alpha following the stimulation for 24 with a mixture of PMA and calcium ionophore A23187 was the highest in preadipocytes, reflecting the increased expression levels of cPLA2alpha and COX-2. Cultured cells treated with PMA during the differentiation phase and then exposed to the maturation medium, or cells treated with PMA in the maturation medium after the differentiation phase showed the suppression of adipogenesis in adipocytes. The attenuating effect of PMA was additionally enhanced when the cell were treated along with A32187 during the differentiation phase, suggesting the involvement of endogenous PGs. The cells at the stages of the differentiation and maturation phases were highly sensitive to exogenous PGE2 and PGF2alpha, respectively, resulting in the marked suppression of the stored fats in adipocytes. Taken together, these results provided the evidence for the distinct gene expression of isoformic enzymes in the COX pathway leading to the synthesis of PGE2 and PGF2alpha and the specific action of these prostanoids at different cycle stages of adipocytes.     

Differential gene expression profiling between wild-type and ALAS2-null erythroblasts: identification of novel heme-regulated genes

To identify erythroid-specific heme-regulated genes, we performed differential expression analysis between wild-type and heme-deficient erythroblasts, which had been prepared from wild-type and erythroid-specific delta-aminolevulinate synthase-null mouse ES cells, respectively. Among 8737 clones on cDNA array, 40 cDNA clones, including 34 unknown ESTs, were first selected by their high expression profiles in wild-type erythroblasts, and evaluated further for their erythroid-lineage specificity, expression in hematopoietic tissues in vivo, and heme-dependent expression, which yielded 11, 4, and 4 genes, respectively. Because of the selection strategy employed, the final 4 were considered as the newly identified erythroid-specific heme-regulated genes. These 4 genes were uncoupling protein 2, nucleolar spindle-associated protein, cellular nucleic acid-binding protein, and a novel acetyltransferase-like protein. These findings thus suggest that heme may regulate a wide variety of hitherto unrecognized genes, and further analysis of these genes may clarify their role in erythroid cell differentiation.     

CTCF-dependent chromatin insulator is linked to epigenetic remodeling

Chromatin insulators are boundary elements between distinctly regulated, neighboring chromosomal domains, and they function by blocking the effects of nearby enhancers in a position-dependent manner. Here, we show that the SNF2-like chromodomain helicase protein CHD8 interacts with the insulator binding protein CTCF. Chromatin immunoprecipitation analysis revealed that CHD8 was present at known CTCF target sites, such as the differentially methylated region (DMR) of H19, the locus control region of beta-globin, and the promoter region of BRCA1 and c-myc genes. RNA interference-mediated knockdown of CHD8 significantly abolished the H19 DMR insulator activity that depends highly on CTCF, leading to reactivation of imprinted IGF2 from chromosome of maternal origin. Further, the lack of CHD8 affected CpG methylation and histone acetylation around the CTCF binding sites, adjacent to heterochromatin, of BRCA1 and c-myc genes. These findings provide insight into the role of CTCF-CHD8 complex in insulation and epigenetic regulation at active insulator sites.     

6-Carboxy-5,7-diarylcyclopenteno[1,2-b]pyridine derivatives: a novel class of endothelin receptor antagonists

Compounds (2-5) with a 6-carboxy-5,7-diarylcyclopentenopyridine skeleton were designed, synthesized, and identified as a new class of potent non-peptide endothelin receptor antagonists. The regio-isomer 2 was found to show potent inhibitory activity with an IC(50) value of 2.4 nM against (125)I-labeled ET-1 binding to human ET(A) receptors and a 170-fold selectivity for ET(A) over ET(B) receptors. Furthermore, 2 displayed more potent in vivo activity than did the indan-type compound 1 in a mouse ET-1 induced lethality model, suggesting the potential of 2 as a new lead structure. Derivatization on substituted phenyl groups at the 5- and 7-positions of 2 revealed that a 3,4-methylenedioxyphenyl group at the 5-position and a 4-methoxyphenyl group at the 7-position were optimal for binding affinity. Further derivatization of 2 by incorporating a substituent into the 2-position of the 4-methoxyphenyl group led to the identification of a more potent ET(A) selective antagonist 2p with an IC(50) value of 0.87 nM for ET(A) receptors and a 470-fold selectivity. In addition, 2p showed highly potent in vivo efficacy (AD(50): 0.04 mg/kg) in the lethality model.     

XopR, a type III effector secreted by Xanthomonas oryzae pv. oryzae, suppresses microbe-associated molecular pattern-triggered immunity in Arabidopsis thaliana

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. The XopR protein, secreted into plant cells through the type III secretion apparatus, is widely conserved in xanthomonads and is predicted to play important roles in bacterial pathogenicity. Here, we examined the function of XopR by constructing transgenic Arabidopsis thaliana plants expressing it under control of the dexamethasone (DEX)-inducible promoter. In the transgenic plants treated with DEX, slightly delayed growth and variegation on leaves were observed. Induction of four microbe-associated molecular pattern (MAMP)-specific early-defense genes by a nonpathogenic X. campestris pv. campestris hrcC deletion mutant were strongly suppressed in the XopR-expressing plants. XopR expression also reduced the deposition of callose, an immune response induced by flg22. When transiently expressed in Nicotiana benthamiana, a XopR::Citrine fusion gene product localized to the plasma membrane. The deletion of XopR in X. oryzae pv. oryzae resulted in reduced pathogenicity on host rice plants. Collectively, these results suggest that XopR inhibits basal defense responses in plants rapidly after MAMP recognition.     

Crosstalk between adenosine A1 and β1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.     

Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2-3μm-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.