Jul 2011, Volume 2 Issue 7
    

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  • RECOLLECTION
    Baoyuan Zhang, Pangao Xue, Enduo Wang
  • MINI-REVIEW
    Jurg Ott, Jing Wang

    For many psychiatric and other traits, diagnoses are based on a number of different criteria or phenotypes. Rather than carrying out genetic analyses on the final diagnosis, it has been suggested that relevant phenotypes should be analyzed directly. We provide an overview of statistical methods for the joint analysis of multiple phenotypes in case-control association studies.

  • REVIEW
    Adam S. Lazorchak, Bing Su

    Mammalian target of rapamycin complex 2 (mTORC2) is a key downstream mediator of phosphoinositol-3-kinase (PI3K) dependent growth factor signaling. In lymphocytes, mTORC2 has emerged as an important regulator of cell development, homeostasis and immune responses. However, our current understanding of mTORC2 functions and the molecular mechanisms regulating mTORC2 signaling in B and T cells are still largely incomplete. Recent studies have begun to shed light on this important pathway. We have previously reported that mTORC2 mediates growth factor dependent phosphorylation of Akt and facilitates Akt dependent phosphorylation and inactivation of transcription factors FoxO1 and FoxO3a. We have recently explored the functions of mTORC2 in B cells and show that mTORC2 plays a key role in regulating survival and immunoglobulin (Ig) gene recombination of bone marrow B cells through an Akt2-FoxO1 dependent mechanism. Ig recombination is suppressed in proliferating B cells to ensure that DNA double strand breaks are not generated in actively dividing cells. Our results raise the possibility that genetic or pharmacologic inhibition of mTORC2 may promote B cell tumor development as a result of inefficient suppression of Ig recombination in dividing B cells. We also propose a novel strategy to treat cancers based on our recent discovery that mTORC2 regulates Akt protein stability.

  • RESEARCH ARTICLE
    Xiao-Xi Guo, Hanna Kim, Yang Li, Hyungshin Yim, Seung Ki Lee, Ying-Hua Jin

    Sequential activation of cyclin-dependent kinases (Cdks) controls mammalian cell cycle. Here we demonstrate that the upregulation of cyclin-dependent kinase 2 (Cdk2) activity coincides with the loss of mitochondrial membrane potential (MMP) in paclitaxel-induced apoptosis. Ectopic expression of the dominant negative Cdk2 (Cdk2-dn) and a specific Cdk2 inhibitor, p21WAF1/CIP1, effectively suppresses the loss of MMP, the release of cytochrome c, and subsequent activation of caspase-3 in paclitaxel-treated cells. Whereas forced activation of Cdk2 by overexpression of cyclin A dramatically promotes these events. We further show that Cdk2 activation status does not interfere with a procedure that lies downstream of cytochrome c release induced by Bax protein. These findings suggest that Cdk2 kinase can regulate apoptosis at earlier stages than mitochondrial permeability transition and cytochrome c release.

  • RESEARCH ARTICLE
    Michael F. Wangler, Lawrence T. Reiter, Georgianna Zimm, Jennifer Trimble-Morgan, Jane Wu, Ethan Bier

    Alzheimer’s disease (AD) pathogenesis is characterized by senile plaques in the brain and evidence of oxidative damage. Oxidative stress may precede plaque formation in AD; however, the link between oxidative damage and plaque formation remains unknown. Presenilins are transmembrane proteins in which mutations lead to accelerated plaque formation and early-onset familial Alzheimer’s disease. Presenilins physically interact with two antioxidant enzymes thiol-specific antioxidant (TSA) and proliferation-associated gene (PAG) of the peroxiredoxin family. The functional consequences of these interactions are unclear. In the current study we expressed a presenilin transgene in Drosophila wing and sensory organ precursors of the fly. This caused phenotypes typical of Notch signaling loss-of-function mutations. We found that while expression of TSA or PAG alone produced no phenotype, co-expression of TSA and PAG with presenilin led to an enhanced Notch loss-of-function phenotype. This phenotype was more severe and more penetrant than that caused by the expression of Psn alone. In order to determine whether these phenotypes were indeed affecting Notch signaling, this experiment was performed in a genetic background carrying an activated Notch (Abruptex) allele. The phenotypes were almost completely rescued by this activated Notch allele. These results link peroxiredoxins with the in vivo function of Presenilin, which ultimately connects two key pathogenetic mechanisms in AD, namely, antioxidant activity and plaque formation, and raises the possibility of a role for peroxiredoxin family members in Alzheimer’s pathogenesis.

  • RESEARCH ARTICLE
    Sangun Lee, Yanhai Wang, Sung Ouk Kim, Jiahuai Han

    The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW 264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.

  • RESEARCH ARTICLE
    Guoxin Wu, Sethu Sankaranarayanan, Donna L. Montgomery, Adam J. Simon, Zhiqiang An, Mary J. Savage

    We have previously described a novel artificial NFEV β-secretase (BACE1) cleavage site, which when introduced into the amyloid-β precursor protein (APP), significantly enhances APP cleavage by BACE1 in in vitro and cellular assays. In this study, we describe the identification and characterization of a single chain fragment of variable region (scFv), specific to the EV neo-epitope derived from BACE1 cleavage of the NFEV-containing peptide, and its conversion to IgG1. Both the scFv displayed on phage and EV-IgG1 show exquisite specificity for binding to the EV neoepitope without cross-reactivity to other NFEV containing peptides or WT-APP KMDA cleavage products. EV-IgG1 can detect as little as 0.3 nmol/L of the EV peptide. EV-IgG1 antibody was purified, conjugated with alkaline phosphatase and utilized in various biological assays. In the BACE1 enzymatic assay using NFEV substrate, a BACE1 inhibitor MRK-3 inhibited cleavage with an IC50 of 2.4 nmol/L with excellent reproducibility. In an APP_NFEV stable SH-SY5Y cellular assay, the EC50 for inhibition of EV-Aβ peptide secretion with MRK-3 was 236 nmol/L, consistent with values derived using an EV polyclonal antibody. In an APP_NFEV knock-in mouse model, both Aβ_EV40 and Aβ_EV42 peptides in brain homogenate showed excellent gene dosage dependence. In conclusion, the EV neoepitope specific monoclonal antibody is a novel reagent for BACE1 inhibitor discovery for both in vitro, cellular screening assays and in vivo biochemical studies. The methods described herein are generally applicable to novel synthetic substrates and enzyme targets to enable robust screening platforms for enzyme inhibitors.

  • RESEARCH ARTICLE
    Jizhou Lv, Ning Tao, Hao Wu, Xiaoman Liu, Xia Xu, Yingxin Xu, Zhihai Qin

    Mounting evidence has demonstrated that CD4+ T cells play an important role in anti-tumor immune responses. Thus, adoptive transfer of these cells may have great potential for anti-cancer therapy. However, due to the difficulty to generate sufficient tumor-specific CD4+ T cells, the use of CD4+ T cells in tumor therapy is limited. It has been found that IL-15 transfection enhances the proliferation and anti-tumor activity of tumor-specific CD8+ T cells, but the effect of IL-15 transfection on CD4+ T cells remains unknown. Here, the effects of retrovirus-mediated IL-15 expression in Ova-specific CD4+ T cells from Do11.10 mice were evaluated and it was discovered that IL-15 transfected CD4+ T cells expressed both soluble and membrane-bound IL-15. Retrovirus-mediated IL-15 expression led to a selective expansion of antigen-specific CD4+ T cells by inhibiting their apoptosis. Invivo IL-15 transfected CD4+ T cells were more effective in suppressing tumor growth than control retroviral vector transfected ones. To ensure the safety of the method, the employment of thymidine kinase gene made it possible to eliminate these transgenic CD4+ T cells following ganciclovir treatment. Together, we show that IL-15 transfection induced a selective expansion of antigen-specific CD4+ T cells ex vivo and enhanced their tumor-suppression effects in vivo. This has an important significance for improving the efficacy of adoptive T cell therapy.