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  • Visualizing the Replication Cycle of Bunyamwera Orthobunyavirus Expressing Fluorescent Protein-tagged Gc Glycoprotein. 20573824

    The virion glycoproteins Gn and Gc of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family and also the Orthobunyavirus genus, are encoded by the M RNA genome segment, and are involved in both viral attachment and entry. After their synthesis Gn and Gc form a heterodimer in the ER and transport to the Golgi for virus assembly. The N-terminal half of the Gc ectodomain was previously shown to be dispensable for virus replication in cell culture (Shi, X., J. Goli, G. Clark, K. Brauburger, and R. M. Elliott. 2009. J. Gen. Virol. 90:2483-2492). In this study, the coding sequences for fluorescent proteins, either enhanced green fluorescent protein (eGFP) or mCherry fluorescent protein, were fused to the N-terminus of truncated Gc, and two recombinant BUNV (rBUNGc-eGFP and rBUNGc-mCherry) were rescued by reverse genetics. The recombinant viruses showed bright autofluorescence under UV light and were competent for replication in various mammalian cell lines. rBUNGc-mCherry was completely stable over 10 passages whereas internal, in-frame deletions occurred in the chimeric Gc-eGFP protein of rBUNGc-eGFP resulting in loss of fluorescence between passages 5 and 7. Autofluorescence of the recombinant viruses allowed visualization of different stages of the infection cycle, including virus attachment to the cell surface, budding of virus particles in Golgi membranes, and virus-induced morphological changes to the Golgi at later stages of infection. The fluorescent protein tagged viruses will be valuable reagents for live cell imaging studies to investigate virus entry, budding and morphogenesis in real-time.
    Document Type:
    Reference
    Product Catalog Number:
    6500

  • Glucocorticoid ultradian rhythmicity directs cyclical gene pulsing of the clock gene period 1 in rat hippocampus. 20649850

    In vivo glucocorticoid (GC) secretion exhibits a distinctive ultradian rhythmicity. The lipophilic hormone can rapidly diffuse into cells, although only the pulse peak is of sufficient amplitude to activate the low affinity glucocorticoid receptor (GR). Discrete pulses readily access brain regions such as the hippocampus where GR expression is enriched and known to regulate neuronal function, including memory and learning processes. In the present study, we have tested the hypothesis that GR brain targets are responsive to ultradian GC rhythmicity. We have used adrenalectomised rats replaced with pulses of corticosterone to determine the transcriptional effects of ultradian pulses in the hippocampus. Confocal microscopy confirmed that each GC pulse results in transient GR nuclear localisation in hippocampal CA1 neurones. Concomitant GR activation and DNA binding was demonstrated by synthetic glucocorticoid response element oligonucleotide binding, and verified for the Clock gene Period 1 promoter region by chromatin immunoprecipitation assays. Strikingly each GC pulse induced a 'burst' of transcription of Period 1 measured by heterogeneous nuclear RNA quantitative polymerase chain reaction. The net effect of pulsatile GC exposure on accumulation of the mature transcript was also assessed, revealing a plateau of mRNA levels throughout the time course of pulsatile exposure, indicating the pulse timing works optimally for steady state Per1 expression. The plateau dropped to baseline within 120 min of the final pulse, indicating a relatively short half-life for hippocampal Per1. The significance of this strict temporal control is that any perturbation to the pulse frequency or duration would have rapid quantitative effects on the levels of Per1. This in turn could affect hippocampal function, especially circadian related memory and learning processes.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™ - (EZ-ChIP™)

  • c-Myb interacts with the glucocorticoid receptor and regulates its level in pre-B-acute lymphoblastic leukemia cells. 22516378

    Glucocorticoid (GC) hormones are used in the treatment of hematopoietic malignancies. When the GC binds to the glucocorticoid receptor (GR) protein, c-Myb and GR are recruited at the Glucocorticoid Response Unit in the DNA. Here we demonstrate that c-Myb interacts with the GR and that decreasing c-Myb amounts reduces the levels of GR transcripts and protein in 697 pre-B-acute lymphoblastic leukemia (ALL) cells. Furthermore, the auto-upregulation of GR promoter 1C and promoter 1D is blunted at reduced c-Myb levels. Taken together, these data show that c-Myb is a direct, key regulator of the GR. Unexpectedly, the reduction in c-Myb levels increased the sensitivity of the cells to steroid-mediated apoptosis. This was because the reduction in c-Myb itself decreases cell viability, and the residual GR remained above the threshold needed to trigger apoptosis. These studies show the mutual importance of c-Myb and the GR in controlling survival of pre-B ALL cells.
    Document Type:
    Reference
    Product Catalog Number:
    05-175
    Product Catalog Name:
    Anti-Myb Antibody, clone 1-1 - (Anti-Myb Antibody, clone 1-1)

  • Regulation of natriuretic peptide receptor-A gene expression and stimulation of its guanylate cyclase activity by transcription factor Ets-1. 18651838

    ANP (atrial natriuretic peptide) exerts its biological effects by binding to GC (guanylate cyclase)-A/NPR (natriuretic peptide receptor)-A, which generates the second messenger cGMP. The molecular mechanism mediating Npr1 (coding for GC-A/NPRA) gene regulation and expression is not well understood. The objective of the present study was to elucidate the mechanism by which Ets-1 [Ets (E twenty-six) transformation-specific sequence] contributes to the regulation of Npr1 gene transcription and expression. Chromatin immunoprecipitation and gel-shift assays confirmed the in vivo and in vitro binding of Ets-1 to the Npr1 promoter. Overexpression of Ets-1 enhanced significantly Npr1 mRNA levels, protein expression, GC activity and ANP-stimulated intracellular accumulation of cGMP in transfected cells. Depletion of endogenous Ets-1 by siRNA (small interfering RNA) dramatically decreased promoter activity by 80%. Moreover, methylation of the Npr1 promoter region (-356 to +55) reduced significantly the promoter activity and hypermethylation around the Ets-1 binding sites directly reduced Ets-1 binding to the Npr1 promoter. Collectively, the present study demonstrates that Npr1 gene transcription and GC activity of the receptor are critically controlled by Ets-1 in target cells.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™ - (EZ-ChIP™)

  • Terminal field specificity of forebrain efferent axons to brainstem gustatory nuclei. 19028464

    Rostral forebrain structures like the gustatory cortex (GC), bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), and lateral hypothalamus (LH) send projections to the nucleus of solitary tract (NST) and the parabrachial nucleus (PBN) that modulate taste-elicited responses. However, the proportion of forebrain-induced excitatory and inhibitory effects often differs when taste cell recording changes from the NST to the PBN. The present study investigated whether this descending influence originates from a shared or distinct population of forebrain neurons. Under electrophysiological guidance, the retrograde tracers fast blue (FB) and fluorogold (FG) or green (GFB) and red (RFB) fluorescent latex microbeads were injected iontophoretically or by pressure pulses (10 ms at 20 psi) into the taste-responsive regions of the NST and the ipsilateral PBN in six rats. Seven days later, the animals were euthanized and tissue sections containing the LH, CeA, BNST, and GC were processed for co-localization of FB and FG or GFB and RFB. The results showed that the CeA is the major source of input to the NST (82.3+/-7.6 cells/section) and the PBN (76.7+/-11.5), compared to the BNST (31.8+/-4.5; 37.0+/-4.8), the LH (35.0+/-5.4; 33.6+/-5.7), and the GC (27.5+/-4.0; 29.0+/-4.6). Of the total number of retrogradely labeled cells, the incidence of tracer co-localization was 17+/-3% in the GC, 17+/-2% in the CeA, 15+/-3% in the BNST and 16+/-1% in the LH. Thus, irrespective of forebrain source the majority of descending input to the gustatory NST and PBN originates from distinct neuronal populations. This arrangement provides an anatomical substrate for differential modulation of taste processing in the first and second central relays of the ascending gustatory system.
    Document Type:
    Reference
    Product Catalog Number:
    AB153

  • Genome-wide DNA methylation maps in follicular lymphoma cells determined by methylation-enriched bisulfite sequencing. 20927367

    Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL.We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+) B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+) B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+) B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells.This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Interaction and functional interference of glucocorticoid receptor and SOCS1. 18524780

    Cytokine and glucocorticoid (GC) hormone signaling act in an integrated fashion to control inflammation and immune response. Here we establish a new mode of interaction of these two pathways and propose Suppressor of Cytokine Signaling (SOCS)-1 as an essential player in mediating cross-talk. We observed that glucocorticoid receptor (GR) and SOCS1 form an intracellular complex through an interaction, which required the SH2 domain of SOCS1 and the ligand binding domain of GR. Furthermore, GC stimulation was found to increase the nuclear level of SOCS1. SOCS1 binding to the GR did not require ligand binding of the receptor; however, it was abolished after long term GC stimulation, suggesting a functional role of the interaction for the early phase of GC action. The interaction between GR and SOCS1 appeared to negatively influence the transcription of the two GR-regulated genes, FKBP5 and MKP1, because the GC-dependent expression of these genes was inhibited by the SOCS1 inducer IFNgamma and enhanced in SOCS1-deficient murine embryonic fibroblasts as compared with IFNgamma treated wild-type cells. Our results suggest a prominent role of SOCS1 in the early phase of cross-talk between GR and cytokine signaling.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5 - (Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5)