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  • Profiling molecular changes induced by hydrogen treatment of lung allografts prior to procurement. 22902635

    We previously demonstrated that donor treatment with inhaled hydrogen protects lung grafts from cold ischemia/reperfusion (I/R) injury during lung transplantation. To elucidate the mechanisms underlying hydrogen's protective effects, we conducted a gene array analysis to identify changes in gene expression associated with hydrogen treatment.Donor rats were exposed to mechanical ventilation with 98% oxygen and 2% nitrogen or 2% hydrogen for 3 h before harvest; lung grafts were stored for 4h in cold Perfadex. Affymetrix gene array analysis of mRNA transcripts was performed on the lung tissue prior to implantation.Pretreatment of donor lungs with hydrogen altered the expression of 229 genes represented on the array (182 upregulated; 47 downregulated). Hydrogen treatment induced several lung surfactant-related genes, ATP synthase genes and stress-response genes. The intracellular surfactant pool, tissue adenosine triphosphate (ATP) levels and heat shock protein 70 (HSP70) expression increased in the hydrogen-treated grafts. Hydrogen treatment also induced the transcription factors C/EBPα and C/EBPβ, which are known regulators of surfactant-related genes.Donor ventilation with hydrogen significantly increases expression of surfactant-related molecules, ATP synthases and stress-response molecules in lung grafts. The induction of these molecules may underlie hydrogen's protective effects against I/R injury during transplantation.
    Document Type:
    Reference
    Product Catalog Number:
    07-623
    Product Catalog Name:
    Anti-Clara Cell Secretory Protein Antibody
  • Preferential localization of human origins of DNA replication at the 5'-ends of expressed genes and at evolutionarily conserved DNA sequences. 21602917

    Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7-1.5 kb obtained from the breast cancer cell line MCF-7 hybridized to a custom tiling array containing 50-60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (greater than 70%) in all cell lines tested; (c) origins are enriched at the 5'ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.
    Document Type:
    Reference
    Product Catalog Number:
    05-623
    Product Catalog Name:
    Anti-RNA polymerase II Antibody, clone CTD4H8
  • cDNA cloning and genomic structure of three genes localized to human chromosome band 5q31 encoding potential nuclear proteins. 11087669

    Loss of a whole chromosome 5, or a del(5q), is a recurring abnormality in malignant myeloid diseases. By cytogenetic and molecular analyses, we delineated previously a 1- to 1.5-Mb region that is deleted in all patients with a del(5q). In our efforts to identify a myeloid tumor suppressor gene within the commonly deleted segment (CDS), we have cloned and characterized the genes encoding three putative nuclear proteins, each of which contains a bipartite nuclear localization signal (NLS). In addition, C5ORF5 contains a putative rhoGAP domain at the N-terminus, C5ORF6 has a proline-rich sequence near the N-terminus, and C5ORF7 has a zinc-finger domain that partially overlaps the NLS. All three genes are ubiquitously expressed and encode novel proteins. The C5ORF5 cDNA is 5.47 kb encoding a protein of 915 amino acids (aa) with a predicted molecular mass of approximately 105 kDa. C5ORF5 has 23 exons spanning over 27 kb. The C5ORF6 transcript is 4.1 kb encoding a protein of 392 aa with a predicted molecular mass of approximately 43 kDa. C5ORF6 has 5 exons and spans approximately 11 kb. The C5ORF7 cDNA is 6.3 kb and encodes a protein of 1417 aa with a predicted molecular mass of approximately 155 kDa. C5ORF7 has 24 exons spanning approximately 64 kb. All three genes were localized to the distal half of the CDS between D5S1983 and D5S500. We evaluated each as a candidate tumor suppressor gene by the analysis of myeloid leukemia cells from patients with -5/del(5q), but no inactivating mutations were identified.
    Document Type:
    Reference
    Product Catalog Number:
    07-1535
  • Anti-Eck/EphA2, clone D7 - 23701

    Document Type:
    Certificate of Analysis
    Lot Number:
    23701
    Product Catalog Number:
    05-480
    Product Catalog Name:
    Anti-Eck/EphA2 Antibody, clone D7
  • Dopaminergic properties and function after grafting of attached neural precursor cultures. 16256357

    Generation of dopaminergic (DA) neurons from multipotent embryonic progenitors represents a promising therapeutical strategy for Parkinson's disease (PD). Aim of the present study was the establishment of enhanced cell culture conditions, which optimize the use of midbrain progenitor cells in animal models of PD. In addition, the progenitor cells were characterized during expansion and differentiation according to morphological and electrophysiological criteria and compared to primary tissue. Here, we report that CNS precursors can be expanded in vitro up to 40-fold and afterwards be efficiently differentiated into DA neurons. After 4-5 days under differentiation conditions, more than 70% of the neurons were TH+, equivalent to 30% of the total cell population. Calcium imaging revealed the presence of calcium-permeable AMPA receptors in the differentiated precursors which are capable to contribute to many developmental processes. The overall survival rate, degree of reinnervation and the behavioral performance after transplantation of 4 days in-vitro-differentiated cells were similar to results after direct grafting of E14 ventral mesencephalic cells, whereas after shorter or longer differentiation periods, respectively, less effects were achieved. Compared to the amount of in-vitro-generated DA neurons, the survival rate was only 0.8%, indicating that these cells are very vulnerable. Our results suggest that expanded and differentiated DA precursors from attached cultures can survive microtransplantation and integrate within the striatum in terms of behavioral recovery. However, there is only a short time window during in vitro differentiation, in which enough cells are already differentiated towards a DA phenotype and simultaneously not too mature for implantation. However, additional factors and/or genetical manipulation of these expanded progenitors will be required to increase their in vivo survival in order to improve both the ethical and the technical outlook for the use of fetal tissue in clinical transplantation.
    Document Type:
    Reference
    Product Catalog Number:
    AB5954
  • MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages. 16237101

    Alveolar macrophages (AMs) avidly bind and ingest inhaled environmental particles and bacteria. To identify the particle binding receptor(s) on human AMs, we used functional screening of anti-human AM hybridomas and isolated a mAb, PLK-1, which inhibits AM binding of unopsonized particles (e.g., TiO2, latex beads; 63 +/- 5 and 67 +/- 4% inhibition, respectively, measured by flow cytometry; n = 11) and unopsonized bacteria ( approximately 84 and 41% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PLK-1, respectively). The PLK-1 Ag was identified as the human class A scavenger receptor (SR) MARCO (macrophage receptor with collagenous structure) by observing specific immunolabeling of COS cells transfected with human MARCO (but not SR-AI/II) cDNA and by immunoprecipitation by PLK-1 of a protein of appropriate molecular mass (approximately 70 kDa) from both normal human bronchoalveolar lavage cells (>90% AMs) and human MARCO-transfected COS cells. PLK-1 also specifically inhibited particle binding by COS cells, only after transfection with human MARCO cDNA. Immunostaining showed specific labeling of AMs within human lung tissue, bronchoalveolar lavage samples, as well as macrophages in other sites (e.g., lymph node and liver). Using COS transfectants with different truncated forms of MARCO, allowed epitope mapping for the PLK-1 Ab to MARCO domain V between amino acid residues 420 and 431. A panel of Abs to various SRs identified expression on AMs, but failed to inhibit TiO2 or S. aureus binding. The data support a dominant role for MARCO in the human AM defense against inhaled particles and pathogens.
    Document Type:
    Reference
    Product Catalog Number:
    AB5486
  • Insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery by A-type lamin-associated pY19-Caveolin-2 in the inner nuclear membrane. 25753664

    Insulin controls transcription to sustain its physiologic effects for the organism to adapt to environmental changes added to genetic predisposition. Nevertheless, insulin-induced transcriptional regulation by epigenetic factors and in defined nuclear territory remains elusive. Here we show that inner nuclear membrane (INM)-integrated caveolin-2 (Cav-2) regulates insulin-response epigenetic activation of Egr-1 and JunB genes at the nuclear periphery. INM-targeted pY19-Cav-2 in response to insulin associates specifically with the A-type lamin, disengages the repressed Egr-1 and JunB promoters from lamin A/C through disassembly of H3K9me3, and facilitates assembly of H3K9ac, H3K18ac and H3K27ac by recruitment of GCN5 and p300 and the subsequent enrichment of RNA polymerase II (Pol II) on the promoters at the nuclear periphery. Our findings show that Cav-2 is an epigenetic regulator of histone H3 modifications, and provide novel mechanisms of insulin-response epigenetic activation at the nuclear periphery.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple