Molecular evidence for the expression of nicotinic acetylcholine receptor alpha-chain in mouse thymus.

The presence and structure of nicotinic acetylcholine receptor (nAChR) in the thymus has been a subject of interest for many years because of its possible role in the pathogenesis of the autoimmune disease myasthenia gravis. Using the polymerase chain reaction with primers specific for the alpha-chain of nAChR (nAChR-alpha), an 880-bp homologous band was found after amplification of cDNA prepared from mouse thymus, thymic medullary and cortical epithelial cell lines, but not from thymocytes or kidney. Sequencing of the polymerase chain reaction product from the thymus and thymic medullary and cortical epithelial lines showed identity with skeletal muscle nAChR-alpha over the region examined. This region includes the domains of the molecule on which B cell and T cell autoantigenic targets have been described. No evidence was found in mouse tissue for the exon 3A, which has been described in human muscle and the human rhabdomyosarcoma cell line TE671. Our results provide evidence at the RNA level for the expression of the nAChR-alpha on stromal cells but not on thymocytes in normal murine thymus and are consistent with a role for intrathymic autoantigen expression in the pathogenesis of myasthenia gravis.     

Fc gamma receptor-mediated interplatelet activation by a monoclonal antibody against beta 2 microglobulin.

Three different mAb directed against beta 2 microglobulin (two IgG1 and one IgG2a) were tested for their ability to activate human platelets. Although all three antibodies bound to platelets, only one of them, B2.62.2, of the IgG1 subclass, induced platelet activation. This activation is similar to the activation by SYB-1, a CD9 antibody of the same subclass previously described as activating platelets through platelet Fc gamma R. These similarities include serotonin secretion, a lag time preceding aggregation and the induction of a strong intracellular calcium mobilization from storage pools. As with CD9 antibodies, the F(ab')2 fragments of B2.62.2 did not induce activation but blocked the activation by the native antibody, by preventing the binding to beta 2 microglobulin. Also, this activation was inhibited by pretreating the platelet with IV-3, a mAb that blocks the Fc binding site of the FcR. Inasmuch as the same antibody does not prevent the binding of B2.62.2 on platelets, we conclude that the activation by B2.62.2 is mediated by the FcR. Nevertheless, there were differences with the activation by SYB-1. B2.62.2 activation was more dependent on thromboxane A2 formation and no cytoplasmic alkalinization was detected. Finally, contrary to SYB-1, B2.62.2 activation proved to be sensitive to platelet count, suggesting that it involves the formation of immune complexes consisting of antibodies and platelets, that activate nearby platelets.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

Human-Induced Pluripotent Stem Cells: In Quest of Clinical Applications

In the field of regenerative medicine, the development of induced pluripotent stem (iPS) cells may represent a potential strategy to overcome the limitations of human embryonic stem cells (ESCs). iPS cells have the potential to mimic human disease, since they carry the genome of the donor. Hypothetically, with iPS cell technology it is possible to screen patients for a genetic cause of disease (genetic mutation), develop cell lines, reprogram them back to iPS cells, finally differentiate them into one or more cell types that develop the disease. Although the creation of multiple lineages with iPS cells can seem limitless, a number of challenges need to be addressed in order to effectively use these cell lines for disease modeling. These include the low efficiency of iPS cell generation without genetic alterations, the possibility of tumor formation in vivo, the random integration of retroviral-based delivery vectors into the genome, and unregulated growth of the remaining cells that are partially reprogrammed and refractory to differentiation. The establishment of protein or RNA-based reprogramming strategies will help generate human iPS cells without permanent genetic alterations. Finally, direct reprogramming strategies can provide rapid production of models of human ??diseases in a dish??, without first passing the cells through a pluripotent state, so avoiding the challenges of time-consumming and labor-intensive iPS cell line generation. This review will overview methods to develop iPS cells, current strategies for direct reprogramming, and main applications of iPS cells as human disease model, focusing on human cardiovascular diseases, with the aim to be a potential information resource for biomedical scientists and clinicians who exploit or intend to exploit iPS cell technology in a range of applications.     

The systemic administration of neural stem cells expressing an inducible and soluble form of growth arrest specific 1 inhibits mammary gland tumor growth and the formation of metastases

Background aimsMetastasis to different organs is the major cause of death in breast cancer patients. The poor clinical prognosis and lack of successful treatments for metastatic breast cancer patients demand the development of new tumor-selective therapies. Thus, it is necessary to develop treatments capable of releasing therapeutic agents to both primary tumors and metastases that avoid toxic side effects in normal tissue, and neural stem cells are an attractive vehicle for tracking tumor cells and delivering anti-cancer agents. The authorspreviously demonstrated that a soluble form of growth arrest specific 1 (GAS1) inhibits the growth of triple-negative breast tumors and glioblastoma.MethodsIn this study, the authors engineered ReNcell CX (EMD Millipore, Temecula, CA, USA) neural progenitor cells to express truncated GAS1 (tGAS1) under a tetracycline/on inducible system using lentiviral vectors.ResultsHere the authors show that treatment with ReNcell-tGAS1 in combination with tetracycline decreased primary tumor growth and inhibited the formation of metastases in tumor-bearing mice by diminishing the phosphorylation of AKT and ERK1/2 in orthotopic mammary gland tumors. Moreover, the authors observed that ReNcell-tGAS1 prolonged the survival of 4T1 tumor-bearing mice.ConclusionsThese data suggest that the delivery of tGAS1 by ReNcell cells could be an effective adjuvant for the treatment of triple-negative breast cancer.     

Purification and crystallization of a complex between human interferon γ receptor (extracellular domain) and human interferon γ

X-ray diffraction quality crystals have been obtained from a complex between interferon γ and the extracellular domain of its high-affinity cell surface receptor. The crystals were obtained from interferon γ/interferon γ receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon γ. These studies used interferon γ receptor engineered with an eight amino acid N-terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6₅22 with unit cell dimensions a = 145.9 Å and c = 180.3 Å. These crystals diffract to at least 2.9 Å resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon γ and the receptor were present. Analysis of the x-ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent. © 1996 Wiley-Liss, Inc.     

Recognition of Carbohydrate Epitopes Specific for Electron-Dense Granule Antigens from Entamoeba histolytica by Monoclonal Antibodies in the Cecal Content of Infected Hamsters

Saccharomyces cerevisiae CGR1 encodes a 120-amino acid protein with a predominant nucleolar localization. In this study we report the identification and cloning of the ortholog, cgrA, from Aspergillus nidulans. The cgrA gene is comprised of three exons on A. nidulans Chromosome 7. The cDNA contains a single open reading frame (ORF) that would encode a protein of 114 amino acids with 44% sequence identity to yeast Cgr1p. A plasmid expressing cgrA complemented the impaired growth phenotype of a yeast strain that can be inducibly depleted of CGR1, and a green fluorescent protein (GFP)-tagged CgrA protein had the same nucleolar localization as the corresponding yeast protein. These results identify cgrA as the A. nidulans ortholog of yeast CGR1 and suggest evolutionary conservation of nucleolar localization mechanisms used by these proteins. Received: 14 September 2000 / Accepted: 13 November 2000     

Fatty acid composition of the postmortem prefrontal cortex of adolescent male and female suicide victims

Prior epidemiological, prospective intervention, and peripheral and central fatty acid composition studies suggest that omega-3 fatty acid deficiency may be associated with the pathoaetiology of depression and suicide. In the present study, we determined the fatty acid composition of the postmortem prefrontal cortex (PFC) of adolescent male and female suicide victims and age-matched controls. Fatty acid composition (wt% total fatty acids) and concentrations (μmol/g) were determined in the postmortem PFC (Brodmann area 10) of male and female adolescent (aged 13–20 years) suicide victims (n=20) and age-matched controls (n=20) by gas chromatography. None of the major polyunsaturated fatty acids including the principle brain omega-3 fatty acid, docosahexaenoic acid (DHA), monounsaturated fatty acids, or saturated fatty acids differed significantly between adolescent suicide victims and controls before or after segregation by gender. The arachidonic acid (AA, 20:4n-6): DHA ratio and adrenic acid (22:4n-6) composition were negatively correlated with age at death in controls but not in suicides, and males exhibited a greater AA:DHA ratio irrespective of cause-of-death. These results demonstrate that adolescent male and female suicide victims do not exhibit DHA deficits in the postmortem PFC relative to age-matched controls, and suggest that suicide victims do not exhibit the normal age-related decrease in adrenic acid composition and the AA:DHA ratio.     

Stable-isotope probing and metagenomics reveal predation by protozoa drives E. coli removal in slow sand filters

Stable-isotope probing and metagenomics were applied to study samples taken from laboratory-scale slow sand filters 0.5, 1, 2, 3 and 4 h after challenging with ¹³C-labelled Escherichia coli to determine the mechanisms and organisms responsible for coliform removal. Before spiking, the filters had been continuously operated for 7 weeks using water from the River Kelvin, Glasgow as their influent source. Direct counts and quantitative PCR assays revealed a clear predator–prey response between protozoa and E. coli. The importance of top-down trophic-interactions was confirmed by metagenomic analysis, identifying several protozoan and viral species connected to E. coli attrition, with protozoan grazing responsible for the majority of the removal. In addition to top-down mechanisms, indirect mechanisms, such as algal reactive oxygen species-induced lysis, and mutualistic interactions between algae and fungi, were also associated with coliform removal. The findings significantly further our understanding of the processes and trophic interactions underpinning E. coli removal. This study provides an example for similar studies, and the opportunity to better understand, manage and enhance E. coli removal by allowing the creation of more complex trophic interaction models.