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  • Spatial dynamics of DNA damage response protein foci along the ion trajectory of high-LET particles. 21797665

    High-linear energy transfer (LET) ion irradiation of cell nuclei induces complex and severe DNA lesions, and foci of repair proteins are formed densely along the ion trajectory. To efficiently discriminate the densely distributed/overlapping foci along the ion trajectory, a focus recognition algorithm called FociPicker3D based on a local fraction thresholding technique was developed. We analyzed high-resolution 3D immunofluorescence microscopic focus images and obtained the kinetics and spatial development of ?-H2AX, 53BP1 and phospho-NBS1 foci in BJ1-hTERT cells irradiated with 55 MeV carbon ions and compared the results with the dynamics of double-strand break (DSB) distributions simulated using the PARTRAC model. Clusters consisting of several foci were observed along the ion trajectory after irradiation. The spatial dynamics of the protein foci supports that the foci clusters are not formed by neighboring foci but instead originate from the DSB cluster damage induced by high-LET radiations.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-726
    Nombre del producto:
    Anti-53BP1 Antibody, clone BP13

  • Heme oxygenase-1/carbon monoxide induces vascular endothelial growth factor expression via p38 kinase-dependent activation of Sp1. 21115498

    Heme oxygenase-1 (HO-1) is a stress-inducible enzyme catalyzing the oxidative degradation of heme to free iron, CO, and biliverdin. Previous studies demonstrated that HO-1 overexpression promoted VEGF expression and angiogenesis in the ischemic heart. However, the underlying mechanism remained elusive. Here we show that adenovirus-mediated HO-1 transduction of rat primary cardiomyocytes and H9C2 myocytes resulted in significant induction of VEGF expression, and a similar effect was seen in cells directly exposed to CO gas or a CO-releasing compound, tricarbonyldichlororuthenium (II) dimer. HO-1/CO-induced VEGF expression was significantly suppressed by pharmacological inhibition of p38 kinase, but not of AKT, activation. VEGF promoter-luciferase reporter assays, electrophoretic mobility shift assays, supershift assay, and chromatin immunoprecipitation showed that CO-induced VEGF promoter activation requires the binding of the Sp1 transcriptional factor to a cis-regulatory sequence located at the VEGF promoter. Western blot analysis and immunostaining experiments demonstrated that HO-1/CO induced p38-dependent phosphorylation of Sp1 at Thr-453 and Thr-739 both in vitro and in vivo. Overexpression of Sp1 protein with an alanine mutation at Thr-453 or Thr-739 suppressed CO-induced Sp1 binding to the VEGF promoter and its transcriptional activation. Collectively, these data suggest that p38-dependent phosphorylation of Sp1 at Thr-453/Thr-739 is crucial for HO-1/CO-induced VEGF expression in myocytes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Molecular determinants of the stereoselectivity of agonist activity of estrogen receptors (ER) alpha and beta. 12547836

    The two known estrogen receptors, ERalpha and ERbeta, are hormone-inducible transcription factors that have distinct roles in regulating cell proliferation and differentiation. Previously, our laboratory demonstrated that ERalpha exhibits stereoselective ligand binding and transactivation for several structural derivatives and metabolites of the synthetic estrogen diethylstilbestrol. We have previously described the properties of indenestrol A (IA) enantiomers on ERalpha. In the study presented here, the estrogenic properties of the S and R enantiomers of IA, IA-S and IA-R, respectively, were expanded to examine the activity in different cell and promoter contexts using ERalpha and ERbeta. Using human cell lines stably expressing either ERalpha or ERbeta, we found that IA-S was a more potent activator of transcription than IA-R through ERalpha in human endometrial Ishikawa and breast MDA-MB 231 (MDA) cells. Interestingly, IA-R was more potent on ERbeta when compared with ERalpha in MDA, but not in Ishikawa cells, and IA-R exhibited equally low binding affinities to ERalpha and ERbeta in vitro in contrast to its cell line-dependent preferential activation of ERbeta. Alignment of the protein structures of the ligand-binding domains of ERalpha and ERbeta revealed one mismatched residue, Leu-384 in ERalpha and Met-283 in ERbeta, which may be responsible for making contact with the methyl substituent at the chiral carbon of IA-S and IA-R. Mutagenesis and exchange of this one residue showed that the binding of IA-R and IA-S was not affected by this mutation in ERalpha and ERbeta. However, in transactivation studies, IA-R showed higher potency in activating L384M-mutated ERalpha and wild-type ERbeta compared with wild-type ERalpha and M283L-mutated ERbeta in all cell and promoter contexts examined. Furthermore, IA-R-bound ERalpha L384M and wild-type ERbeta displayed enhanced interactions with the nuclear receptor interaction domains of the coactivators SRC-1 and GRIP1. These data demonstrate that a single residue in the ligand-binding domain determines the stereoselectivity of ERalpha and ERbeta for indenestrol ligands and that IA-R shows cell type selectivity through ERbeta.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-629

  • Flaxseed ameliorates interstitial nephritis in rat polycystic kidney disease. 9987066

    BACKGROUND: Flaxseed has demonstrated useful antiinflammatory properties in a number of animal models and human diseases. We undertook a study to determine if flaxseed would also modify clinical course and renal pathology in the Han:SPRD-cy rat. METHODS: Male Han:SPRD-cy rats were pair fed a 10% flaxseed of control rat chow diet for eight weeks from weaning. Tissue was harvested for analysis of cystic change, apoptosis, cell proliferation, and fibrosis. Tissue was also harvested for lipid analysis using gas chromatography. RESULTS: Animals thrived on both diets. Flaxseed-fed animals had lower serum creatinine (69 vs. 81 mumol/liter, P = 0.02), less cystic change (1.78 vs. 2.03 ml/kg, P = 0.02), less renal fibrosis (0.60 vs. 0.93 ml/kg, P = 0.0009), and less macrophage infiltration (13.8 vs. 16.7 cells/high-power video field) of the renal interstitium than controls. The groups did not differ in renal tubular epithelial cell apoptosis and proliferation. Lipid analysis revealed significant renal enrichment of 18 and 20 carbon omega 3 polyunsaturated fatty acids (total omega 6:omega 3 ratio 3.6 vs. 9.1, P 0.0001). CONCLUSIONS: Flaxseed ameliorates Han:SPRD-cy rat polycystic kidney disease through moderation of the associated chronic interstitial nephritis. The diet alters renal content of polyunsaturated fatty acids in a manner that may promote the formation of less inflammatory classes of renal prostanoids.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1435
    Nombre del producto:
    Anti-Macrophages/Monocytes Antibody, clone ED-1

  • Zerumbone induces heme oxygenase-1 expression in mouse skin and cultured murine epidermal cells through activation of Nrf2. 21367956

    Zerumbone, a sesquiterpene derived from tropical ginger, contains an electrophilic α,β-unsaturated carbonyl moiety and was found to suppress chemically induced papilloma formation in mouse skin. Here, we report that topical application of zerumbone onto dorsal skin of hairless mice induces activation of NF-E2-related factor 2 (Nrf2) and expression of heme oxygenase-1 (HO-1). We compared the levels of HO-1 protein in the skin of zerumbone-treated Nrf2 wild-type and Nrf2 knockout mice, and nrf2-deficient mice expressed HO-1 protein to a much lesser extent than the wild-type animals following topical application of zerumbone. Treatment of mouse epidermal JB6 cells with zerumbone caused a marked increase of Nrf2 nuclear translocation followed by the promoter activity of HO-1, and also enhanced direct binding of Nrf2 to the antioxidant response element. Moreover, knockdown of Nrf2 in JB6 cells diminished the zerumbone-induced upregulation of HO-1. Notably, α-humulene and 8-hydroxy-α-humulene, the structural analogues of zerumbone that lack the α,β-unsaturated carbonyl group, failed to activate Nrf2 and were unable to increase HO-1 expression. Unlike zerumbone, these nonelectrophilic analogues could not suppress the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced JB6 cell transformation and the intracellular accumulation of reactive oxygen species (ROS). Interestingly, when JB6 cells were treated with carbon monoxide-releasing molecule that mimics the HO-1 activity, the TPA-induced ROS production was markedly reduced. Taken together, these findings suggest that upregulation of HO-1 expression by zerumbone is mediated through activation of Nrf2 signaling, which provides a mechanistic basis for the chemopreventive effects of this sesquiterpene on mouse skin carcinogenesis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-371
    Nombre del producto:
    EZ-ChIP™

  • A thermo extraction–UV/Vis spectrophotometric method for total iodine quantification in soils and sediments 17924101

    Iodine in soils and sediments is a difficult element to analyze due to its volatility in acidic conditions. Traditionally it has been quantified using neutron activation analysis techniques, which, unfortunately, requires access to a nuclear reactor. We present here a simple method for solid-phase iodine analysis by thermo extraction at 1000°C and quantification by UV/Vis photometry. Samples are combusted in an oxygen stream and trapped in Milli-Q water. The extracts are then quantified by an As3+–Ce4+ spectrometric method whereby iodide catalyzes the oxidation of As3+ to As5+ and reduction of Ce4+ to Ce3+. Three standard reference materials were analyzed with excellent recoveries (97–113%) and RSDs (<5%). Moreover, the detection limit was less than 50 ng absolute iodine with a confidence limit of 95%. When applied to carbonate-rich samples from sediment traps deployed in Lake Constance we found very low iodine levels (0.8–2 mg kg-1). Despite the low concentrations, the precision of the method was consistently better than 5% RSD. However, the method needed to be slightly modified for organic and iodine-rich sediments (20–30% organic carbon) from a lake in the Black Forest by increasing the oxygen flow rate and decreasing the combustion time. Using the modified method we were able to achieve RSDs lower than 5%.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo

  • Region-specific deficits in dopamine, but not norepinephrine, signaling in a novel A30P α-synuclein BAC transgenic mouse. 24121116

    Parkinson's disease (PD) is a neurodegenerative disorder classically characterized by the death of dopamine (DA) neurons in the substantia nigra pars compacta and by intracellular Lewy bodies composed largely of α-synuclein. Approximately 5-10% of PD patients have a familial form of Parkinsonism, including mutations in α-synuclein. To better understand the cell-type specific role of α-synuclein on DA neurotransmission, and the effects of the disease-associated A30P mutation, we generated and studied a novel transgenic model of PD. We expressed the A30P mutant form of human α-synuclein in a spatially-relevant manner from the 111kb SNCA genomic DNA locus on a bacterial artificial chromosome (BAC) insert on a mouse null (Snca-/-) background. The BAC transgenic mice expressed α-synuclein in tyrosine hydroxylase-positive neurons and expression of either A30P α-synuclein or wildtype α-synuclein restored the sensitivity of DA neurons to MPTP in resistant Snca-/- animals. A30P α-synuclein mice showed no Lewy body-like aggregation, and did not lose catecholamine neurons in substantia nigra or locus coeruleus. However, using cyclic voltammetry at carbon-fiber microelectrodes we identified a deficit in evoked DA release in the caudate putamen, but not in the nucleus accumbens, of SNCA-A30P Snca-/- mice but no changes to release of another catecholamine, norepinephrine (NE), in the NE-rich ventral bed nucleus of stria terminalis. SNCA-A30P Snca-/- mice had no overt behavioral impairments but exhibited a mild increase in wheel-running. In summary, this refined PD mouse model shows that A30P α-synuclein preferentially perturbs the dopaminergic system in the dorsal striatum, reflecting the region-specific change seen in PD.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB152
    Nombre del producto:
    Anti-Tyrosine Hydroxylase Antibody