Double PHD Fingers Protein DPF2 Recognizes Acetylated Histones and Suppresses the Function of Estrogen-related Receptor α through Histone Deacetylase 1

Estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily and regulates many physiological functions, including mitochondrial biogenesis and lipid metabolism. ERRα enhances the transactivation function without endogenous ligand by associating with coactivators such as peroxisome proliferator-activated receptor γ coactivator 1 α and β (PGC-1α and -β) and members of the steroid receptor coactivator family. However, the molecular mechanism by which the transactivation function of ERRα is converted from a repressive state to an active state is poorly understood. Here we used biochemical purification techniques to identify ERRα-associated proteins in HeLa cells stably expressing ERRα. Interestingly, we found that double PHD fingers protein DPF2/BAF45d suppressed PGC-1α-dependent transactivation of ERRα by recognizing acetylated histone H3 and associating with HDAC1. DPF2 directly bound to ERRα and suppressed the transactivation function of nuclear receptors such as androgen receptor. DPF2 was recruited to ERR target gene promoters in myoblast cells, and knockdown of DPF2 derepressed the level of mRNA expressed by target genes of ERRα. These results show that DPF2 acts as a nuclear receptor-selective co-repressor for ERRα by associating with both acetylated histone H3 and HDAC1.     

Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein

To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.     

Impacts of icodextrin on integrin-mediated wound healing of peritoneal mesothelial cells

AimsExposure to glucose and its metabolites in peritoneal dialysis fluid (PDF) results in structural alterations of the peritoneal membrane. Icodextrin-containing PDF eliminates glucose and reduces deterioration of peritoneal membrane function, but direct effects of icodextrin molecules on peritoneal mesothelial cells have yet to be elucidated. We compared the impacts of icodextrin itself with those of glucose under PDF-free conditions on wound healing processes of injured mesothelial cell monolayers, focusing on integrin-mediated cell adhesion mechanisms.Main methodsRegeneration processes of the peritoneal mesothelial cell monolayer were investigated employing an in vitro wound healing assay of cultured rat peritoneal mesothelial cells treated with icodextrin powder- or glucose-dissolved culture medium without PDF, as well as icodextrin- or glucose-containing PDF. The effects of icodextrin on integrin-mediated cell adhesions were examined by immunocytochemistry and Western blotting against focal adhesion kinase (FAK).Key findingsCell migration over fibronectin was inhibited in conventional glucose-containing PDF, while icodextrin-containing PDF exerted no significant inhibitory effects. Culture medium containing 1.5% glucose without PDF also inhibited wound healing of mesothelial cells, while 7.5% icodextrin-dissolved culture medium without PDF had no inhibitory effects. Glucose suppressed cell motility by inhibiting tyrosine phosphorylation of FAK, formation of focal adhesions, and cell spreading, while icodextrin had no effects on any of these mesothelial cell functions.SignificanceOur results demonstrate icodextrin to have no adverse effects on wound healing processes of peritoneal mesothelial cells. Preservation of integrin-mediated cell adhesion might be one of the molecular mechanisms accounting for the superior biocompatibility of icodextrin-containing PDF.     

Expression and localization of aquaporins in the kidney of the musk shrew (Suncus murinus).

Expression and localization of members of the aquaporin (AQP) family (AQP1, 2, 3, 4, and 5) in the kidney of the musk shrew (Suncus murinus) was examined by immunohistochemistry. AQP1 was expressed in the proximal tubules and in the thin limb of the loops of Henle. AQP1 was the only water channel expressed in the proximal nephron examined, indicating that AQP1 may be an independent water transporter in the proximal nephron. AQP2 and AQP5 were localized to the apical cytoplasm of the cortical to medullary collecting duct (CD) cells and AQP3 and AQP4 were localized to the basal aspect of the cortical to medullary CD cells. AQP3 expression was weaker in the cortical cells compared with the medullary cells, whereas AQP4 was strongly positive throughout the CD. These indicate that the CD is the main water reabsorption segment of the nephron and is regulated by AQPs. Indeed, apical water transport of CD cells of the musk shrew may be controlled by both AQP2 and AQP5. The characteristic expression pattern of the AQPs in this animal provides a novel animal model for elucidating the regulation of water reabsorption by AQPs in the mammalian kidney.     

Visible-light-induced patterning of Au- and Ag-TiO2 nanocomposite film surfaces on the basis of plasmon photoelectrochemistry.

A novel technique for patterning based on visible light at Au-TiO2 and Ag-TiO2 nanocomposite film surfaces has been developed for the first time. TiO2 films loaded with Au and Ag nanoparticles were modified with hydrophobic thiols to obtain hydrophobic surfaces. The surfaces were converted to hydrophilic by visible light irradiation. Hydrophobic/hydrophilic patterning was also possible by visible light irradiation through a photomask. The patterning was due to removal of the thiol based on plasmon photoelectrochemistry. Visible-light-induced plasmon resonance at the Au and Ag nanoparticles gives rise to charge separation and redox reactions. The thiol is removed from the Au-TiO2 film probably by oxidative desorption, and from the Ag-TiO2 film owing chiefly to oxidation of Ag nanoparticles to Ag+.     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis.     

Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells

A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.     

Hepatocyte Growth Factor-induced Asef-IQGAP1 Complex Controls Cytoskeletal Remodeling and Endothelial Barrier

Hepatocyte growth factor (HGF) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via Rac-dependent enhancement of the peripheral actin cytoskeleton. However, the precise mechanisms of HGF effects on the peripheral cytoskeleton are not well understood. This study evaluated a role for Rac/Cdc42-specific guanine nucleotide exchange factor Asef and the multifunctional Rac effector, IQGAP1, in the mechanism of HGF-induced EC barrier enhancement. HGF induced Asef and IQGAP1 co-localization at the cell cortical area and stimulated formation of an Asef-IQGAP1 functional protein complex. siRNA-induced knockdown of Asef or IQGAP1 attenuated HGF-induced EC barrier enhancement. Asef knockdown attenuated HGF-induced Rac activation and Rac association with IQGAP1, and it abolished both IQGAP1 accumulation at the cell cortical layer and IQGAP1 interaction with actin cytoskeletal regulators cortactin and Arp3. Asef activation state was essential for Asef interaction with IQGAP1 and protein complex accumulation at the cell periphery. In addition to the previously reported role of the IQGAP1 RasGAP-related domain in the Rac-dependent IQGAP1 activation and interaction with its targets, we show that the IQGAP1 C-terminal domain is essential for HGF-induced IQGAP1/Asef interaction and Asef-Rac-dependent activation leading to IQGAP1 interaction with Arp3 and cortactin as a positive feedback mechanism of IQGAP1 activation. These results demonstrate a novel feedback mechanism of HGF-induced endothelial barrier enhancement via Asef/IQGAP1 interactions, which regulate the level of HGF-induced Rac activation and promote cortical cytoskeletal remodeling via IQGAP1-Arp3/cortactin interactions.     

Chemical analysis of the female sex pheromone in Palpita nigropunctalis (Lepidoptera: Crambidae)

The lilac pyralid, Palpita nigropunctalis Bremer (Lepidoptera: Crambidae), is a common pest of Oleaceae plants. A crude extract of the female sex pheromone glands was examined by gas chromatography-electroantennogram detection (GC-EAD) and GC coupled to a mass spectrometer (GC/MS). The GC-EAD analysis revealed three EAG-active components (I–III) in a ratio of 1:0.2:0.01 (I: II: III). GC/MS analysis successfully recorded the mass spectra of I and II. For I, ions at m/z 238 (M⁺) and 220 ([M-18]⁺) indicated the structure of a monoenyl aldehyde with a 16-carbon chain. For II, M⁺ was not detected, but ions at m/z 222 ([M-60]⁺) and 61 ([AcOH+1]⁺) suggested that II was a monoenyl acetate with a 16-carbon chain. Further GC/MS analysis of the extract treated with dimethyl disulfide revealed that the double bonds in both I and II are located at the same position of 11th-carbon. In addition, the pheromone extract was examined by GC/Fourier transform-infrared spectrophotometer (GC/FT-IR). An IR spectrum of I showed characteristic absorption at 1716 and 966?cm^?1, indicating a formyl group and E configuration of the double bond, respectively. In the case of II, absorption at 1745 and 968?cm^?1 indicated an ester carbonyl and E configuration, respectively. Taken together and by comparison with authentic standards, I and II were confirmed as (E)-11-hexadecenal and (E)-11-hexadecenyl acetate, respectively; while III was speculated as (E)-11-hexadecen-1-ol. The synthetic I, II and III all coincided well with those of the natural components in chemical data, and elicited strong electroantennographic activity in male P. nigropunctalis.