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  • Proteomic approach to the identification of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme. 18725266

    The expression of the transcription factor DeltaLf is deregulated in cancer cells. Its overexpression provokes cell cycle arrest along with antiproliferative effects and we recently showed that the Skp1 gene promoter was a target of DeltaLf. Skp1 belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination and the proteosomal degradation of numerous cellular regulators. The transcriptional activity of DeltaLf is highly controlled and negatively regulated by O-GlcNAc, a dynamic post-translational modification known to regulate the functions of many intracellular proteins. We, therefore, constructed a DeltaLf-M4 mutant corresponding to a constitutively active DeltaLf isoform in which all the glycosylation sites were mutated. In order to discover novel targets of DeltaLf transcriptional activity and to investigate the impact of the O-GlcNAc regulation on this activity in situ we compared the proteome profiles of DeltaLf- and DeltaLf-M4-expressing HEK293 cells versus null plasmid transfected cells. A total of 14 differentially expressed proteins were visualized by 2D electrophoresis and silver staining and eight proteins were identified by mass spectrometry analyses (MALDI-TOF; LC-MS/MS), all of which were upregulated. The identified proteins are involved in several processes such as mRNA maturation and stability, cell viability, proteasomal degradation, protein and mRNA quality control. Among these proteins, only DcpS and TCPB were also upregulated at the mRNA level. Analysis of their respective promoters led to the detection of a cis-regulating element in the DcpS promoter. The S1(DcpS) is 80% identical to the S1 sequence previously described by He and Furmanski [Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA, Nature 373 (1995) 721-724]. Reporter gene analyses and ChIP assays demonstrated that DeltaLf interacts specifically with the DcpS promoter in vivo. These data established that DcpS, a key enzyme in mRNA decay, is a new target of DeltaLf transcriptional activity.
    Document Type:
    Reference
    Product Catalog Number:
    17-371
    Product Catalog Name:
    EZ-ChIP™

  • Acylation-stimulating protein/C5L2-neutralizing antibodies alter triglyceride metabolism in vitro and in vivo. 17711993

    Acylation-stimulating protein (ASP), a lipogenic hormone, stimulates triglyceride (TG) synthesis and glucose transport upon activation of C5L2, a G protein-coupled receptor. ASP-deficient mice have reduced adipose tissue mass due to increased energy expenditure despite increased food intake. The objective of this study was to evaluate the blocking of ASP-C5L2 interaction via neutralizing antibodies (anti-ASP and anti-C5L2-L1 against C5L2 extracellular loop 1). In vitro, anti-ASP and anti-C5L2-L1 blocked ASP binding to C5L2 and efficiently inhibited ASP stimulation of TG synthesis and glucose transport. In vivo, neither anti-ASP nor anti-C5L2-L1 altered body weight, adipose tissue mass, food intake, or hormone levels (insulin, leptin, and adiponectin), but they did induce a significant delay in TG clearance [P < 0.0001, 2-way repeated-measures (RM) ANOVA] and NEFA clearance (P < 0.0001, 2-way RM ANOVA) after a fat load. After treatment with either anti-ASP or anti-C5L2-L1 antibody there was no change in adipose tissue AMPK activity, but neutralizing antibodies decreased perirenal TG mass (-38.4% anti-ASP, -18.8% anti-C5L2, P < 0.01-0.001) and perirenal LPL activity (-75.6% anti-ASP, -72.5% anti-C5L2, P < 0.05). In liver, anti-C5L2-L1 decreased TG mass (-42.8%, P < 0.05), whereas anti-ASP increased AMPK activity (+34.6%, P < 0.001). In the muscle, anti-C5L2-L1 significantly increased TG mass (+128.0%, P < 0.05), LPL activity (+226.1%, P < 0.001), and AMPK activity (+71.1%, P < 0.01). In addition, anti-ASP increased LPL activity (+164.4, P < 0.05) and AMPK activity (+53.9%, P < 0.05) in muscle. ASP/C5L2-neutralizing antibodies effectively block ASP-C5L2 interaction, altering lipid distribution and energy utilization.
    Document Type:
    Reference
    Product Catalog Number:
    ABS1020
    Product Catalog Name:
    Anti-Acylation Stimulating Protein Antibody

  • Proteomics strategy based on liquid-phase IEF and 2-D DIGE: application to bone marrow mesenchymal progenitor cells. 17516591

    Global comparative proteomics is a promising new approach with broad application in basic and clinical biological science. Recent advances include the development of 2-D DIGE, a proteomic equivalent to mRNA differential display, in which differentially labeled samples are multiplexed and analyzed by high-resolution 2-DE. This study presents a new 2-D DIGE protocol, in which complex protein samples from normal and leukemic human bone marrow mesenchymal progenitor cells were used as model samples for a novel combination of liquid-phase IEF with 2-D DIGE. Using liquid-phase IEF, the normal and leukemic cells were pre-fractionated into five subproteomes after multiplexing but prior to DIGE. Under these conditions, 2-D DIGE resolved >5000 protein-containing spots within the pH range 4.6-7.0. Differential labeling combined with subsequent MALDI-MS/MS identified proteins that were differentially expressed in leukemic cells. This analysis mapped protein identities to 128 mesenchymal progenitor cell proteins with at least one unique peptide match at >95% confidence. Of these proteins, 72 (56%) were expressed more than 1.25-fold higher or lower in leukemic cells compared with normal cells (p<0.05). These data were used to infer gene ontology biological processes that may be altered in leukemic bone marrow mesenchymal progenitor cells.
    Document Type:
    Reference
    Product Catalog Number:
    LP1
    Product Catalog Name:
    VLDL, human

  • Vorinostat increases carboplatin and paclitaxel activity in non-small cell lung cancer cells. 19621389

    We observed a 53% response rate in non-small cell lung cancer (NSCLC) patients treated with vorinostat plus paclitaxel/carboplatin in a Phase I trial. Studies were undertaken to investigate the mechanism (s) underlying this activity. Growth inhibition was assessed in NSCLC cells by MTT assay after 72 hr of continuous drug exposure. Vorinostat (1 muM) inhibited growth by: 17% +/- 7% in A549, 28% +/- 6% in 128-88T, 39% +/- 8% in Calu1 and 41% +/- 7% in 201T cells. Vorinostat addition to carboplatin or paclitaxel led to significantly greater growth inhibition than chemotherapy alone in all 4 cell lines. Vorinostat (1 muM) synergistically increased the growth inhibitory effects of carboplatin/paclitaxel in 128-88T cells. When colony formation was measured after drug withdrawal, vorinostat significantly increased the effects of carboplatin but not paclitaxel. The % colony formation was control 100%; 1 muM vorinostat, 83% +/- 10%; 5 muM carboplatin, 41% +/- 11%; carboplatin/vorinostat, 8% +/- 4%; 2 nM paclitaxel, 53% +/- 11%; paclitaxel/vorinostat, 46% +/- 21%. In A549 and 128-88T, vorinostat potentiated carboplatin induction of gamma-H2AX (a DNA damage marker) and increased alpha-tubulin acetylation (a marker for stabilized mictrotubules). In A549, combination of vorinostat with paclitaxel resulted in a synergistic increase in alpha-tubulin acetylation, which reversed upon drug washout. We conclude that vorinostat interacts favorably with carboplatin and paclitaxel in NSCLC cells, which may explain the provocative response observed in our clinical trial. This likely involves a vorinostat-mediated irreversible increase in DNA damage in the case of carboplatin and a reversible increase in microtubule stability in the case of paclitaxel.
    Document Type:
    Reference
    Product Catalog Number:
    07-164
    Product Catalog Name:
    Anti-phospho-H2A.X (Ser139) Antibody

  • Collagenase-3 (MMP-13) is expressed during human fetal ossification and re-expressed in postnatal bone remodeling and in rheumatoid arthritis. 9166290

    To explore possible physiologic functions for the metalloproteinase collagenase-3, we have examined its temporal and spatial expression during human fetal development. Except for mesenchymal cells in the umbilical cord at 4 weeks of gestation, signal for collagenase-3 mRNA was confined to mineralizing skeletal tissue and detected in hypertrophic chondrocytes and osteoblastic cells involved in ossification beginning at 10 weeks and continuing through gestation. These cells were also immunoreactive with collagenase-3 antiserum, indicating their ability to produce collagenase-3 protein. In osteoblastic cells, the expression of membrane-type 1 metalloproteinase and 72-kd gelatinase mRNA, which have the capacity to activate collagenase-3 in vitro, colocalized with that of collagenase-3. In postnatal tissues, collagenase-3 was re-expressed in processes involving skeletal remodeling, such as bone cysts and ectopic bone and cartilage formation. Multinucleated osteoclasts were consistently negative for collagenase-3. Furthermore, in patients with seropositive rheumatoid arthritis, expression of collagenase-3 was prominent in articular cartilage, and collagenase-3 protein was detected by immunoblotting in synovial fluids. Consistent with its substrate specificities, a plausible function for collagenase-3 in these processes is to preferentially degrade type II collagen, thus serving a role during primary ossification, in skeletal remodeling, and in destructive joint disease.
    Document Type:
    Reference
    Product Catalog Number:
    CC1047
    Product Catalog Name:
    MMP-13, human, proform, his-tagged, E. coli recombinant

  • Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking. 17728239

    In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8702
    Product Catalog Name:
    Anti-Dengue Virus Type II Antibody, clone 3H5-1

  • The RNA polymerase II kinase Ctk1 regulates positioning of a 5' histone methylation boundary along genes. 17088384

    In yeast and other eukaryotes, the histone methyltransferase Set1 mediates methylation of lysine 4 on histone H3 (H3K4me). This modification marks the 5' end of transcribed genes in a 5'-to-3' tri- to di- to monomethyl gradient and promotes association of chromatin-remodeling and histone-modifying enzymes. Here we show that Ctk1, the serine 2 C-terminal domain (CTD) kinase for RNA polymerase II (RNAP II), regulates H3K4 methylation. We found that CTK1 deletion nearly abolished H3K4 monomethylation yet caused a significant increase in H3K4 di- and trimethylation. Both in individual genes and genome-wide, loss of CTK1 disrupted the H3K4 methylation patterns normally observed. H3K4me2 and H3K4me3 spread 3' into the bodies of genes, while H3K4 monomethylation was diminished. These effects were dependent on the catalytic activity of Ctk1 but are independent of Set2-mediated H3K36 methylation. Furthermore, these effects are not due to spurious transcription initiation in the bodies of genes, to changes in RNAP II occupancy, to changes in serine 5 CTD phosphorylation patterns, or to "transcriptional stress." These data show that Ctk1 acts to restrict the spread of H3K4 methylation through a mechanism that is independent of a general transcription defect. The evidence presented suggests that Ctk1 controls the maintenance of suppressive chromatin in the coding regions of genes by both promoting H3K36 methylation, which leads to histone deacetylation, and preventing the 3' spread of H3K4 trimethylation, a mark associated with transcriptional initiation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Depletion of new neurons by image guided irradiation. 21541259

    Ionizing radiation continues to be a relevant tool in both imaging and the treatment of cancer. Experimental uses of focal irradiation have recently been expanded to studies of new neurons in the adult brain. Such studies have shown cognitive deficits following radiation treatment and raised caution as to possible unintentional effects that may occur in humans. Conflicting outcomes of the effects of irradiation on adult neurogenesis suggest that the effects are either transient or permanent. In this study, we used an irradiation apparatus employed in the treatment of human tumors to assess radiation effects on rat neurogenesis. For subjects we used adult male rats (Sprague-Dawley) under anesthesia. The irradiation beam was directed at the hippocampus, a center for learning and memory, and the site of neurogenic activity in adult brain. The irradiation was applied at a dose-rate 0.6 Gy/min for total single-fraction, doses ranging from 0.5 to 10.0 Gy. The animals were returned to home cages and recovered with no sign of any side effects. The neurogenesis was measured either 1 week or 6 weeks after the irradiation. At 1 week, the number of neuronal progenitors was reduced in a dose-dependent manner with the 50% reduction at 0.78 Gy. The dose-response curve was well fitted by a double exponential suggesting two processes. Examination of the tissue with quantitative immunohistochemistry revealed a dominant low-dose effect on neuronal progenitors resulting in 80% suppression of neurogenesis. This effect was partially reversible, possibly due to compensatory proliferation of the remaining precursors. At higher doses (greater than 5 Gy) there was additional, nearly complete block of neurogenesis without compensatory proliferation. We conclude that notwithstanding the usefulness of irradiation for experimental purposes, the exposure of human subjects to doses often used in radiotherapy treatment could be damaging and cause cognitive impairments.
    Document Type:
    Reference
    Product Catalog Number:
    AB1778

  • Temporal disparity in the induction of matrix metalloproteinases and tissue inhibitors of metalloproteinases after thoracic aortic aneurysm formation. 17000289

    BACKGROUND: An important component of matrix remodeling during thoracic aortic aneurysm progression is the balance between matrix metalloproteinases and their endogenous inhibitors (tissue inhibitors of metalloproteinases). However, whether and to what degree matrix metalloproteinase/tissue inhibitor of metalloproteinases profiles change over time with an evolving thoracic aortic aneurysm remains unclear. METHODS: Descending thoracic aortic aneurysms were induced in mice (FVB strain, 15 minutes of 0.5 mol/L CaCl2 exposure) and followed for 24 hours, 72 hours, 1 week, 2 weeks, 4 weeks, or 8 weeks (each group, n = 13). Thoracic aortic aneurysm size was determined by means of video micrometry, and immunoblotting was used to measure aortic matrix metalloproteinase 2, 8, 9, and 12 and tissue inhibitor of metalloproteinases 1 and 4 levels (expressed as a percentage of control values, n = 13). RESULTS: Increased aortic diameter was detected by 72 hours and reached a maximal size at 4 weeks (135% +/- 4% increase from baseline, P .05), which is consistent with thoracic aortic aneurysm progression. Active matrix metalloproteinase 8 (collagenase) levels increased at 72 hours (178% +/- 49%, P .05 from control), and active matrix metalloproteinase 12 (elastase) levels increased by 24 hours (138% +/- 11%, P .05), whereas active matrix metalloproteinase 2 levels increased at 72 hours and 1 week after thoracic aortic aneurysm induction (72 hours: 158% +/- 12%, 1 week: 162% +/- 19%; P .05). At 1 week after thoracic aortic aneurysm induction, active matrix metalloproteinase 9 and 12 levels decrease (matrix metalloproteinase 9: 55% +/- 5%; matrix metalloproteinase 12: 63% +/- 5%; P .05); however, matrix metalloproteinase 9 and 12 levels were increased from these values at 4 and 8 weeks (P .05). Tissue inhibitor of metalloproteinases 1 levels were decreased at 1 week (52% +/- 15%, P .05) and later returned to control values, whereas tissue inhibitor of metalloproteinases 4 levels increased at the late thoracic aortic aneurysm time points (4 weeks: 278% +/- 46%; 8 weeks: 213% +/- 40%; P .05). CONCLUSIONS: These findings show 2 phases of matrix metalloproteinase abundance during murine thoracic aortic aneurysm formation. The late tissue inhibitor of metalloproteinases 4 increase might explain prevention of further aortic dilation past 4 weeks. Unique matrix metalloproteinase/tissue inhibitor of metalloproteinases temporal relationships occurred during the natural history of thoracic aortic aneurysm progression that might hold both diagnostic and therapeutic relevance.
    Document Type:
    Reference
    Product Catalog Number:
    AB770
    Product Catalog Name:
    Anti-TIMP-1 Antibody