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  • Titration of GLI3 repressor activity by sonic hedgehog signaling is critical for maintaining multiple adult neural stem cell and astrocyte functions. 24174682

    Sonic hedgehog (SHH), a key regulator of embryonic neurogenesis, signals directly to neural stem cells (NSCs) in the subventricular zone (SVZ) and to astrocytes in the adult mouse forebrain. The specific mechanism by which the GLI2 and GLI3 transcriptional activators (GLI2(A) and GLI3(A)) and repressors (GLI2(R) and GLI3(R)) carry out SHH signaling has not been addressed. We found that the majority of slow-cycling NSCs express Gli2 and Gli3, whereas Gli1 is restricted ventrally and all three genes are downregulated when NSCs transition into proliferating progenitors. Surprisingly, whereas conditional ablation of Smo in postnatal glial fibrillary acidic protein-expressing cells results in cell-autonomous loss of NSCs and a progressive reduction in SVZ proliferation, without an increase in glial cell production, removal of Gli2 or Gli3 does not alter adult SVZ neurogenesis. Significantly, removing Gli3 in Smo conditional mutants largely rescues neurogenesis and, conversely, expression of a constitutive GLI3(R) in the absence of normal Gli2 and Gli3 abrogates neurogenesis. Thus unattenuated GLI3(R) is a primary inhibitor of adult SVZ NSC function. Ablation of Gli2 and Gli3 revealed a minor role for GLI2(R) and little requirement for GLI(A) function in stimulating SVZ neurogenesis. Moreover, we found that similar rules of GLI activity apply to SHH signaling in regulating SVZ-derived olfactory bulb interneurons and maintaining cortical astrocyte function. Namely, fewer superficial olfactory bulb interneurons are generated in the absence of Gli2 and Gli3, whereas astrocyte partial gliosis results from an increase in GLI3(R). Thus precise titration of GLI(R) levels by SHH is critical to multiple functions of adult NSCs and astrocytes.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition. 25713373

    During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.
    Document Type:
    Reference
    Product Catalog Number:
    04-928
    Product Catalog Name:
    Anti-Histone H3 Antibody, CT, pan, clone A3S, rabbit monoclonal

  • Influenza virus titration, antigenic characterization, and serological methods for antibody detection. 22528152

    This chapter describes some commonly used methods of influenza virus titration, antigenic characterization, and serological methods by antibody detection. These methods are essential not only for virus characterization but also for identifying new antigenic variants, vaccine strain selection, and sero-epidemiologic studies of influenza virus transmission and prevalence. Virus titration methods such as the hemagglutination assay, 50% egg or tissue culture infectious dose, and plaque assay are employed to determine the amount of virus particles in a sample. The hemagglutination inhibition assay is a reliable, relatively simple and inexpensive technique to antigenically characterize isolates of influenza viruses. Serological methods such as virus neutralization and hemagglutination inhibition are the fundamental tools used in sero-epidemiologic studies of influenza virus transmission and prevalence and in the evaluation of vaccine immunogenicity. While serological methods rarely yield an early diagnosis of acute influenza virus infection, well-timed, paired acute, and convalescent serum samples may establish the diagnosis of a recent influenza infection even when attempts to detect the virus are negative.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8257
    Product Catalog Name:
    Anti-Influenza A Antibody, nucleoprotein, clone A1

  • Thermodynamic characterization of the interaction between CAR-RXR and SRC-1 peptide by isothermal titration calorimetry. 17223708

    The constitutive androstane receptor (CAR) enhances transcription of specific target genes that regulate several metabolic pathways. CAR functions as an obligate heterodimer (CAR-RXR) with the retinoid X receptor (RXR). Also part of the active receptor complex is the steroid receptor coactivator-1 (SRC-1) which interacts with the receptor complex via specific receptor interaction domains (RIDs). A peptide derived from SRC-1 RID2 is used to study the thermodynamic properties of the interaction with the CAR-RXR ligand binding domain (LBD) complex. In the absence of ligands for both CAR and RXR, binding of coactivator peptide to the CAR-RXR heterodimer is characterized by a favorable enthalpy change and an unfavorable entropy change. The addition of the CAR agonist, TCPOBOP, increases the affinity for coactivator by decreasing the unfavorable entropy and increasing the favorable intrinsic enthalpy of the interaction. The RXR ligand, 9-cis-RA, generates a second SRC-1 site and increases the affinity by improving the entropic component of binding. There is an additional increase in affinity for one of the two sites in the presence of both ligands. The change in heat capacity (deltaCp) is also investigated. A 2-fold difference in deltaCp is observed between liganded and unliganded CAR-RXR. The observed thermodynamic parameters for binding of SRC-1 peptide to liganded and apo CAR-RXR as well as the difference in the deltaCp data provide evidence that the apo CAR-RXR heterodimer is conformationally mobile. The more favorable enthalpic contribution for TCPOBOP-bound CAR-RXR indicates that preformation of the binding site improves the complementarity of the coactivator-receptor interaction.
    Document Type:
    Reference
    Product Catalog Number:
    AB10303