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  • Mapping the Ligand Binding Sites of Kainate Receptors: Molecular Determinants of Subunit-Selective Binding of the Antagonist [3H]UBP310. 20837679

    Kainate receptors (KARs) modulate synaptic transmission and plasticity, and their dysfunction has been linked to several disease states such as epilepsy and chronic pain. KARs are tetramers formed from five different subunits. GluK1-3 are low affinity kainate binding subunits, whereas GluK4/5 bind kainate with high affinity. A number of these subunits can be present in any given cell type, and different combinations of subunits confer different properties to KARs. Here we report the characterization of a new GluK1 subunit-selective radiolabeled antagonist (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione ([(3)H]UBP310) using human recombinant KARs. [(3)H]UBP310 binds to GluK1 with low nanomolar affinity (K(D) = 21 ± 7 nM) but shows no specific binding to GluK2. However, [(3)H]UBP310 also binds to GluK3 (K(D) = 0.65 ± 0.19 μM) but with ∼30-fold lower affinity than that observed for GluK1. Competition [(3)H]UBP310 binding experiments on GluK1 revealed the same rank order of affinity of known GluK1-selective ligands as reported previously in functional assays. Nonconserved residues in GluK1-3 adjudged in modeling studies to be important in determining the GluK1 selectivity of UBP310 were point-mutated to switch residues between subunits. None of the mutations altered the expression or trafficking of KAR subunits. Whereas GluK1-T503A mutation diminished [(3)H]UBP310 binding, GluK2-A487T mutation rescued it. Likewise, whereas GluK1-N705S/S706N mutation decreased, GluK3-N691S mutation increased [(3)H]UBP310 binding activity. These data show that Ala487 in GluK2 and Asn691 in GluK3 are important determinants in reducing the affinity of UBP310 for these subunits. Insights from these modeling and point mutation studies will aid the development of new subunit-selective KAR antagonists.
    Tipo de documento:
    Referencia
    Referencia del producto:
    04-921
    Nombre del producto:
    Anti-GluR6/7 Antibody, clone NL9, rabbit monoclonal

  • Development of micropost force sensor array with culture experiments for determination of cell traction forces. 17342763

    Cell traction forces (CTFs) are critical for cell motility and cell shape maintenance. As such, they play a fundamental role in many biological processes such as angiogenesis, embryogenesis, inflammation, and wound healing. To determine CTFs at the sub-cellular level with high sensitivity, we have developed high density micropost force sensor array (MFSA), which consists of an array of vertically standing poly(dimethylsiloxane) (PDMS) microposts, 2 microm in diameter and 6 microm in height, with a center-to-center distance of 4 microm. In combination with new image analysis algorithms, the MFSA can achieve a spatial resolution of 40 nm and a force sensitivity of 0.5 nN. Culture experiments with various types of cells showed that this MFSA technology can effectively determine CTFs of cells with different sizes and traction force magnitudes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    FAK100
    Nombre del producto:
    Actin Cytoskeleton / Focal Adhesion Staining Kit

  • Input-specific intrasynaptic arrangements of ionotropic glutamate receptors and their impact on postsynaptic responses. 19828804

    To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB363
    Nombre del producto:
    Anti-NMDAR1 Antibody, clone 54.1

  • Filamin is essential for shedding of the transmembrane serine protease, epithin. 16170303

    Epithin is a type II transmembrane serine protease that exists in a soluble and membrane-bound form. Shedding is thought to be important in regulating its action, but little is known regarding the intracellular events that trigger such shedding. Here, we show that phorbol myristate acetate (PMA) causes the release of epithin. It also causes accumulation of the protein at the site of cell-cell contacts, and this accumulation is dependent on the formation of cortical actin. In addition, we have identified the actin-binding protein, filamin, as the linker between epithin and the actin cytoskeleton. The interaction of epithin and filamin was enhanced by PMA, and epithin was not released from filamin-deficient M2 cells. We also show that the release of epithin does not require its own activity and is blocked by a metalloprotease inhibitor, GM6001. These results show that filamin has an essential role in shedding by linking epithin to the as yet unidentified metalloprotease-shedding enzyme(s).
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1678
    Nombre del producto:
    Anti-Filamin A Antibody, clone PM6/317

  • Forkhead transcription factor FoxO1 transduces insulin-like growth factor's signal to p27Kip1 in primary skeletal muscle satellite cells. 12891709

    The insulin-like growth factor I (IGF-I) stimulates muscle satellite cell proliferation. Chakravarthy et al., (2000, J Biol Chem 275:35942-35952.) previously found that IGF-I-stimulated proliferation of primary satellite cells was associated with the activation of phosphatidylinositol 3'-kinase (PI3K)/Akt and the downregulation of a cell-cycle inhibitor p27Kip1. To understand mechanisms by which IGF-I signals the downregulation of p27Kip1 in rat skeletal satellite cells, the role of Forkhead transcription factor FoxO1 in transcriptional activity of p27Kip1 was examined. When primary rat satellite cells were transfected with a p27Kip1 promoter-reporter gene construct, IGF-I (100 ng/ml) inhibited specific p27Kip1 promoter activity. Addition of LY294002, an inhibitor of PI3K, reversed the IGF-I-mediated downregulation of p27Kip1 promoter activity. Co-transfection of wild type (WT) FoxO1 into satellite cells increased p27Kip1 promoter activity in the absence of IGF-I supplementation. Addition of IGF-I reversed the induction of p27Kip1 promoter activity by WT FoxO1. When a mutated FoxO1 (without Thr24, Ser256, and Ser316 Akt phosphorylation sites) was used, IGF-I was no longer able to reverse the FoxO1 induced stimulation of p27Kip1 promoter activity that had been seen when WT FoxO1 was present. When the satellite cells were treated with IGF-I, phosphorylation of Akt-Ser473 and FoxO1-Ser256 was increased. In addition, when the cells were pre-incubated with LY294002 before IGF-I stimulation, the phosphorylation of Akt-Ser473 and FoxO1-Ser256 was inhibited, implying that phosphorylation of Akt and FoxO1 was downstream of IGF-I-induced PI3K signaling. However, IGF-I did not induce phosphorylation of FoxO1 on residues Thr24 and Ser316. These results suggested that IGF-I induced the phosphorylation of Ser256 and inactivated FoxO1 thereby downregulating the activation of the p27Kip1 promoter. Thus, inactivation of FoxO1 by IGF-I plays a critical role in rat skeletal satellite cell proliferation through regulation of p27Kip1 expression.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Age-related modulation of γ-secretase activity in non-human primate brains. 22817324

    Age-dependent accumulation of the amyloid-β peptide (Aβ) in the brain is a pre-condition for development of Alzheimer's disease. A relative increase in the generation of longer Aβ species such as Aβ42 and Aβ43 is critical for Aβ deposition, but the underlying mechanism remains unresolved. Here, we performed a cell-free assay using microsome fractions of temporal cortex tissues from 42 cynomolgus monkeys and found that Aβ40-generating γ-secretase activity (γ40) decreased with age, whereas Aβ42-generating γ-secretase activity (γ42) was unaltered. In ELISAs, more than 80% of monkeys over 20-years old showed evidence of Aβ accumulation in the temporal cortex. The ratio of γ42 to γ40 increased with age and correlated with the level of accumulated Aβ. These results suggest that γ-secretase activity undergoes age-related, non-genetic modulation and that this modulation may cause Aβ accumulation in aging brains. Similar modulation may predispose aged human brains to Alzheimer's disease.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5232

  • Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1. 20635087

    Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-482
    Nombre del producto:
    Anti-Calmodulin Binding Protein Epitope Tag Antibody

  • A central role for transcription factor C/EBP-beta in regulating CD1d gene expression in human keratinocytes. 19592659

    CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-beta binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-beta caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-beta in regulating CD1d transcription.
    Tipo de documento:
    Referencia
    Referencia del producto:
    17-295
    Nombre del producto:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Identification of pigment epithelium-derived factor as a direct target of the p53 family member genes. 15856012

    p63 and p73 show a high degree of structural homology to p53 and are members of a family of transcriptional factors that can activate transcription of p53-responsive genes. p53 is mutated in more than 50% of human cancers, whereas p63 and p73 are rarely mutated. Studies of knockout mice also revealed an unexpected functional diversity among the p53 family. To determine how p63 and p73 are involved in tumorigenesis and normal development, we used cDNA microarray to examine 9216 genes in human colorectal cancer cells. We discovered that the expression of pigment epithelium-derived factor (PEDF) was specifically induced by either p63 or p73, but not by p53. We also report here that the PEDF gene contains a response element specific for p63 and p73 in its promoter region and is a direct target of p63 and p73. Collectively, p63 and p73 may be involved in cell fate by inducing PEDF expression.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1059
    Nombre del producto:
    Anti-Pigment Epithelium Derived Factor Antibody, clone 10F12.2

  • Selective neuronal vulnerability and the distribution of N-methyl-D-aspartate (NMDA) receptors. 2554170

    N-Methyl-D-Aspartate (NMDA) receptors are believed to play a critical role in excitotoxic cell death in the CNS. The distribution of NMDA-preferring binding sites is compared here with the patterns of selective neuronal death observed in Alzheimer's disease and following transient ischemia. The distribution of NMDA receptors, by itself, is unable to account for the characteristic patterns of selective neuronal vulnerability observed in conjunction with these types of neuropathology.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1557P
    Nombre del producto:
    Anti-NMDAR2B Antibody