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  • Viral vector tropism for supporting cells in the developing murine cochlea. 21530627

    Gene-based therapeutics are being developed as novel treatments for genetic hearing loss. One roadblock to effective gene therapy is the identification of vectors which will safely deliver therapeutics to targeted cells. The cellular heterogeneity that exists within the cochlea makes viral tropism a vital consideration for effective inner ear gene therapy. There are compelling reasons to identify a viral vector with tropism for organ of Corti supporting cells. Supporting cells are the primary expression site of connexin 26 gap junction proteins that are mutated in the most common form of congenital genetic deafness (DFNB1). Supporting cells are also primary targets for inducing hair cell regeneration. Since many genetic forms of deafness are congenital it is necessary to administer gene transfer-based therapeutics prior to the onset of significant hearing loss. We have used transuterine microinjection of the fetal murine otocyst to investigate viral tropism in the developing inner ear. For the first time we have characterized viral tropism for supporting cells following in utero delivery to their progenitors. We report the inner ear tropism and potential ototoxicity of three previously untested vectors: early-generation adenovirus (Ad5.CMV.GFP), advanced-generation adenovirus (Adf.11D) and bovine adeno-associated virus (BAAV.CMV.GFP). Adenovirus showed robust tropism for organ of Corti supporting cells throughout the cochlea but induced increased ABR thresholds indicating ototoxicity. BAAV also showed tropism for organ of Corti supporting cells, with preferential transduction toward the cochlear apex. Additionally, BAAV readily transduced spiral ganglion neurons. Importantly, the BAAV-injected ears exhibited normal hearing at 5 weeks of age when compared to non-injected ears. Our results support the use of BAAV for safe and efficient targeting of supporting cell progenitors in the developing murine inner ear.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB3080
    Nombre del producto:
    Anti-Green Fluorescent Protein Antibody

  • Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells. 15087463

    Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Exceptional total and functional yields of the human adenosine (A2a) receptor expressed in the yeast Saccharomyces cerevisiae. 16289981

    The yeast Saccharomyces cerevisiae was used to express a medically relevant G-protein coupled receptor (GPCR), the human adenosine (A2a) receptor, with a C-terminal green fluorescent protein (GFP) fusion tag. In prior studies, we established an expression system for A2a-GFP. Here, we quantified the total A2a-GFP expression levels by correlating GFP levels as detected by fluorescence and densitometry to A2a-GFP molecules overexpressed in the system. We also quantified A2a-GFP functional levels by classical radioligand binding assays. Approximately, 120,000 functional A2a-GFP molecules per cell were present on the plasma membrane as determined by radioligand binding. Using whole cell GFP fluorescence, 340,000 A2a-GFP molecules per cell were detected; approximately 70% of those molecules were plasma membrane localized, as determined by using confocal microscopy analysis. These results show that a significant portion of the total expressed protein is functional. In addition, the quick and inexpensive whole cell fluorescence appears to provide a good approximation of functional receptor numbers for this case. Importantly, the amount of functionally expressed A2a-GFP per culture ( approximately 4 mg/L) is among the highest reported for any GPCR in any expression system.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB16901
    Nombre del producto:
    Anti-Green Fluorescent Protein Antibody

  • The Role of octopamine and tyramine in Drosophila larval locomotion. 22627970

    The characteristic crawling behavior of Drosophila larvae consists of a series of rhythmic waves of peristalsis and episodes of head swinging and turning. The two biogenic amines octopamine and tyramine have recently been shown to modulate various parameters of locomotion, such as muscle contraction, the time spent in pausing or forward locomotion, and the initiation and maintenance of rhythmic motor patterns. By using mutants having altered octopamine and tyramine levels and by genetic interference with both systems we confirm that signaling of these two amines is necessary for larval locomotion. We show that a small set of about 40 octopaminergic/tyraminergic neurons within the ventral nerve cord is sufficient to trigger proper larval locomotion. Using single-cell clones, we describe the morphology of these neurons individually. Given various potential roles of octopamine and tyramine in the larval brain, such as locomotion, learning and memory, stress-induced behaviors or the regulation of the energy state, functions that are often not easy to discriminate, we dissect here for the first time a subset of this complex circuit that modulates specifically larval locomotion. Thus, these data will help to understand-for a given neuronal modulator-how specific behavioral functions are executed within distinct subcircuits of a complex neuronal network. J. Comp. Neurol. 520:3764-3785, 2012. © 2012 Wiley Periodicals, Inc.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB124

  • Anti-GFP - 3043683

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    3043683
    Referencia del producto:
    AB10145
    Nombre del producto:
    Anti-GFP Antibody

  • Anti-GFP, clone 3F8.2 -2669937

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    2669937
    Referencia del producto:
    MAB1083
    Nombre del producto:
    Anti-GFP Antibody, clone 3F8.2

  • Anti-GFP Polyclonal Antibody

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    2895626
    Referencia del producto:
    AB10145
    Nombre del producto:
    Anti-GFP Antibody

  • Anti-GFP, clone 3F8.2 -2658916

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    2658916
    Referencia del producto:
    MAB1083
    Nombre del producto:
    Anti-GFP Antibody, clone 3F8.2

  • Anti-GFP, clone 2B6 - 4163739

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    4163739
    Referencia del producto:
    MABC1689-100UG
    Nombre del producto:
    Anti-GFP Antibody, clone 2B6

  • Anti-GFP -2798616

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    2798616
    Referencia del producto:
    AB10145
    Nombre del producto:
    Anti-GFP Antibody