The miR-199a/Brm/EGR1 axis is a determinant of anchorage-independent growth in epithelial tumor cell lines. Kobayashi, K; Sakurai, K; Hiramatsu, H; Inada, K; Shiogama, K; Nakamura, S; Suemasa, F; Kobayashi, K; Imoto, S; Haraguchi, T; Ito, H; Ishizaka, A; Tsutsumi, Y; Iba, H Scientific reports
5
8428
2015
Show Abstract
In epithelial cells, miRNA-199a-5p/-3p and Brm, a catalytic subunit of the SWI/SNF complex were previously shown to form a double-negative feedback loop through EGR1, by which human cancer cell lines tend to fall into either of the steady states, types 1 [miR-199a(-)/Brm(+)/EGR1(-)] and 2 [miR-199a(+)/Brm (-)/EGR1(+)]. We show here, that type 2 cells, unlike type 1, failed to form colonies in soft agar, and that CD44, MET, CAV1 and CAV2 (miR-199a targets), all of which function as plasma membrane sensors and can co-localize in caveolae, are expressed specifically in type 1 cells. Single knockdown of any of them suppressed anchorage-independent growth of type 1 cells, indicating that the miR-199a/Brm/EGR1 axis is a determinant of anchorage-independent growth. Importantly, two coherent feedforward loops are integrated into this axis, supporting the robustness of type 1-specific gene expression and exemplifying how the miRNA-target gene relationship can be stably sustained in a variety of epithelial tumors. | Western Blotting | 25673149
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Bile acids induce hepatic differentiation of mesenchymal stem cells. Sawitza, I; Kordes, C; Götze, S; Herebian, D; Häussinger, D Scientific reports
5
13320
2015
Show Abstract
Mesenchymal stem cells (MSC) have the potential to differentiate into multiple cell lineages and their therapeutic potential has become obvious. In the liver, MSC are represented by stellate cells which have the potential to differentiate into hepatocytes after stimulation with growth factors. Since bile acids can promote liver regeneration, their influence on liver-resident and bone marrow-derived MSC was investigated. Physiological concentrations of bile acids such as tauroursodeoxycholic acid were able to initiate hepatic differentiation of MSC via the farnesoid X receptor and transmembrane G-protein-coupled bile acid receptor 5 as investigated with knockout mice. Notch, hedgehog, transforming growth factor-β/bone morphogenic protein family and non-canonical Wnt signalling were also essential for bile acid-mediated differentiation, whereas β-catenin-dependent Wnt signalling was able to attenuate this process. Our findings reveal bile acid-mediated signalling as an alternative way to induce hepatic differentiaion of stem cells and highlight bile acids as important signalling molecules during liver regeneration. | | 26304833
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Dextran as a generally applicable multivalent scaffold for improving immunoglobulin-binding affinities of peptide and peptidomimetic ligands. Morimoto, J; Sarkar, M; Kenrick, S; Kodadek, T Bioconjugate chemistry
25
1479-91
2014
Show Abstract
Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors. | | 25073654
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GSE is a maternal factor involved in active DNA demethylation in zygotes. Hatanaka, Y; Shimizu, N; Nishikawa, S; Tokoro, M; Shin, SW; Nishihara, T; Amano, T; Anzai, M; Kato, H; Mitani, T; Hosoi, Y; Kishigami, S; Matsumoto, K PloS one
8
e60205
2013
Show Abstract
After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote. | Western Blotting | 23560077
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Decorin induces rapid secretion of thrombospondin-1 in basal breast carcinoma cells via inhibition of Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase 1. Neill, T; Jones, HR; Crane-Smith, Z; Owens, RT; Schaefer, L; Iozzo, RV The FEBS journal
280
2353-68
2013
Show Abstract
Pathological neovascularization relies on an imbalance between potent proangiogenic agents and equally effective antiangiogenic cues. Collectively, these factors contribute to an angiogenic niche within the tumor microenvironment. Oncogenic events and hypoxia contribute to augmented levels of angiokines, and thereby activate the so-called angiogenic switch to promote aggressive tumorigenic and metastatic growth. Soluble decorin functions as a paracrine pan-inhibitor of receptor tyrosine kinases, such as Met and epidermal growth factor receptor, and thus is capable of suppressing angiogenesis under normoxia. This leads to noncanonical repression of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor A (VEGFA), and concurrent induction of thrombospondin-1. The substantial induction of endogenous tumor cell-derived thrombospondin-1, a potent antiangiogenic effector, led us to the discovery of an unexpected secretory phenotype occurring very rapidly (within 5 min) after decorin treatment of the triple-negative basal breast carcinoma cell line MDA-MB-231. Surprisingly, the effect was not mediated by Met receptor antagonism, as initially hypothesized, but required epidermal growth factor receptor signaling to achieve swift and robust thrombospondin-1 release. Furthermore, this effect was ultimately dependent on the prompt degradation of Ras homolog gene family member A, via the 26S proteasome, leading to direct inactivation of Rho-associated coiled-coil containing protein kinase 1. The latter led to derepression of thrombospondin-1 secretion. Collectively, these data provide a novel mechanistic role for Rho-associated coiled-coil containing protein kinase 1, in addition to providing the first conclusive evidence of decorin exclusively targeting a receptor tyrosine kinase to achieve a specific effect. The overall effects of soluble decorin on the tumor microenvironment would cause an immediately-early as well as a sustained antiangiogenic response in vivo. | | 23350987
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Comparison of surface plasmon resonance, resonant waveguide grating biosensing and enzyme linked immunosorbent assay (ELISA) in the evaluation of a dengue virus immunoassay. Hu, D; Fry, SR; Huang, JX; Ding, X; Qiu, L; Pan, Y; Chen, Y; Jin, J; McElnea, C; Buechler, J; Che, X; Cooper, MA Biosensors
3
297-311
2013
Show Abstract
Two label-free biosensor platforms, Resonance Waveguide Grating (RWG) and Surface Plasmon Resonance (SPR), were used to rank a large panel of anti-dengue virus NS1 antibodies. Dengue non-structural 1 (NS1) protein is an established serological marker for the early detection of dengue infection. A variety of commercial dengue NS1 antigen capture immunoassays are available in both ELISA and lateral flow format. However, there is a significant scope to improve both the sensitivity and the specificity of those tests. The interactions of antibody (Ab)-antigen (Ag) were profiled, with weak interactions (KD = 1-0.1 μM) able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR. There were significant differences in the absolute affinities determined by the two technologies, and there was a poor correlation between antibodies best ranked by RWG and the lower limit of detection (LLOD) found by ELISA. Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats. | | 25586260
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Stability of reference proteins in human placenta: general protein stains are the benchmark. D Lanoix,J St-Pierre,A-A Lacasse,M Viau,J Lafond,C Vaillancourt,A A Lacasse Placenta
33
2012
Show Abstract
The stability of reference proteins in semi-quantitative Western blot experiments in normal and diseased placenta has never been studied. This study aims to determine the stability of five reference proteins and two general protein stains in placentas from preeclampsia, gestational diabetes mellitus and matched control pregnancies. The stability of the reference proteins was analysed using indicators of inter-group (P value) and intra-group (coefficient of variation) stability. The effect of different normalization strategies was determined by normalizing serotonin transporter (SERT) expression against the different reference protein markers. Results show significant expression variability of β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolyl isomerase A (PPIA) and α-tubulin, and that amido black staining is the most stable reference protein marker. Furthermore, results show that SERT expression significantly differs according to the reference protein markers used for its normalization. The present study demonstrated the importance of using stable reference protein markers and normalization strategy in order to get correct results in semi-quantitative Western blot experiments in placental tissues. | | 22244735
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Placental melatonin production and melatonin receptor expression are altered in preeclampsia: new insights into the role of this hormone in pregnancy. Dave Lanoix,Pascale Guérin,Cathy Vaillancourt Journal of pineal research
53
2012
Show Abstract
The melatonin system in preeclamptic pregnancies has been largely overlooked, especially in the placenta. We have previously documented melatonin production and expression of its receptors in normal human placentas. In addition, we and others have shown a beneficial role of melatonin in placental and fetal functions. In line with this, decreased maternal blood levels of melatonin are found in preeclamptic compared with normotensive pregnancies. However, melatonin production and expression of its receptors in preeclamptic compared with normotensive pregnancy placentas has never been examined. This study compares (i) melatonin-synthesizing enzyme expression and activity, (ii) melatonin and serotonin, melatonin's immediate precursor, levels and (iii) expression of MT1 and MT2 melatonin receptors in placentas from preeclamptic and normotensive pregnancies. Protein and mRNA expression of aralkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT), the melatonin-synthesizing enzymes, as well as MT1 and MT2 receptors were determined by RT-qPCR and Western blot, respectively. The activities of melatonin-synthesizing enzymes were assessed by radiometric assays while melatonin levels were determined by LC-MS/MS. There is a significant inhibition of AANAT, melatonin's rate-limiting enzyme, expression and activity in preeclamptic placentas, correlating with decreased melatonin levels. Likewise, MT1 and MT2 expression is significantly reduced in preeclamptic compared with normotensive pregnancy placentas. We propose that reduced maternal plasma melatonin levels may be an early diagnostic tool to identify pregnancies complicated by preeclampsia. This study indicates a clinical utility of melatonin as a potential treatment for preeclampsia in women where reduced maternal plasma levels have been identified. | | 22686298
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Expression of gap junction protein connexin36 in multiple subtypes of GABAergic neurons in adult rat somatosensory cortex. Ma, Y; Hioki, H; Konno, M; Pan, S; Nakamura, H; Nakamura, KC; Furuta, T; Li, JL; Kaneko, T Cerebral cortex (New York, N.Y. : 1991)
21
2639-49
2011
Show Abstract
To characterize connexin36 (Cx36)-expressing neurons of the adult rat somatosensory cortex, we examined fluorescence signals for Cx36 messenger RNA (mRNA) in 3 nonoverlapping subpopulations of γ-aminobutyric acid (GABA)ergic interneurons, which showed immunoreactivity for 1) parvalbumin (PV); 2) somatostatin (SOM); and 3) either calretinin (CR), vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), or choline acetyltransferase (ChAT). About 80% of PV-, 52% of SOM-, 37% of CR/VIP/CCK/ChAT-immunoreactive cells displayed Cx36 signals across all cortical layers, and inversely 64%, 25%, and 9% of Cx36-expressing neurons were positive for PV, SOM, or CR/VIP/CCK/ChAT, respectively. Notably, although almost all Cx36-expressing neurons in layer (L) 4, L5, and L6 were positive for one of these markers, a substantial proportion of those in L1 (91%) and L2/3 (10%) were negative for the markers tested, suggesting that other types of neurons might express Cx36. We further investigated the colocalization of Cx36 mRNA and α-actinin2 immunoreactivity, as a marker for late-spiking GABAergic neurons, by using mirror-image sections. Surprisingly, more than 77% of α-actinin2-positive cells displayed Cx36 signals in L1-L3, and about 49% and 13% of Cx36-expressing neurons were positive for α-actinin2 in L1 and L2/3, respectively. These findings suggest that all the subtypes of GABAergic interneurons might form gap junctions in the neocortex. | | 21467210
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Prion protein oligomers in Creutzfeldt-Jakob disease detected by gel-filtration centrifuge columns. Haruhiko Minaki, Kensuke Sasaki, Hiroyuki Honda, Toru Iwaki Neuropathology : official journal of the Japanese Society of Neuropathology
29
536-42
2009
Show Abstract
Prion diseases are diagnosed by the detection of accumulation of abnormal prion protein (PrP) using immunohistochemistry or the detection of protease-resistant abnormal PrP (PrP(res)). Although the abnormal PrP is neurotoxic by forming aggregates, recent studies suggest that the most infectious units are smaller than the amyloid fibrils. In the present study, we developed a simplified method by applying size-exclusion gel-filtration chromatography to examine PrP oligomers without proteinase K digestion in Creutzfeldt-Jakob disease (CJD) samples, and evaluated the correlation between disease severity and the polymerization degree of PrP. Brain homogenates of human CJD and non-CJD cases were applied to the gel-filtration spin columns, and fractionated PrP molecules in each fraction were detected by western blot. We observed that PrP oligomers could be detected by the simple gel-filtration method and distinctly separated from monomeric cellular PrP (PrP(c)). PrP oligomers were increased according to the disease severity, accompanied by the depletion of PrP(c). The separated PrP oligomers were already protease-resistant in the case with short disease duration. In the cases with quite severe pathology the oligomeric PrP reached a plateau, which may indicate that PrP molecules could mostly develop into amyloid fibrils in the advanced stages. The increase of PrP oligomers correlated with the degree of histopathological changes such as spongiosis and gliosis. The decrease of monomeric PrP(c) was unexpectedly obvious in the diseased cases. Dynamic changes of both oligomerization of the human PrP and depletion of normal PrP(c) require further elucidation to develop a greater understanding of the pathogenesis of human prion diseases. | | 19389076
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