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  • The proximal regulatory element of the interferon-gamma promoter mediates selective expression in T cells. 8943243

    Interferon-gamma (IFN-gamma) is produced by natural killer cells and certain subsets of T cells, but the basis for its selective expression is unknown. Within the region between -108 and -40 base pairs of the IFN-gamma promoter are two conserved and essential regulatory elements, which confer activation-specific expression in T cells. This report describes studies indicating that the most proximal of these two regulatory elements is an important determinant of its restricted expression. The proximal element is a composite site that binds members of the CREB/ATF, AP-1, and octamer families of transcription factors. Jun is essential for activation-induced transcription and binds preferably as a heterodimer with ATF-2. In contrast, CREB appears to dampen transcription from this element. The CpG dinucleotide in this element is selectively methylated in Th2 T cells and other cells that do not express IFN-gamma, and methylation markedly reduces transcription factor binding. As a target for DNA methylation and for binding of transcription factors that mediate or impede transcription, this element appears to play a central role in controlling IFN-gamma expression.
    Document Type:
    Reference
    Product Catalog Number:
    ECM600
    Product Catalog Name:
    uPA Activity Assay Kit

  • Strategies for repair of white matter: influence of osmolarity and microglia on proliferation and apoptosis of oligodendrocyte precursor cells in different basal culture ... 24421756

    The aim of the present study has been to obtain high yields of oligodendrocyte precursor cells (OPCs) in culture. This is a first step in facilitation of myelin repair. We show that, in addition to factors, known to promote proliferation, such as basic fibroblast growth factor (FGF-2) and platelet derived growth factor (PDGF) the choice of the basal medium exerts a significant influence on the yield of OPCs in cultures from newborn rats. During a culture period of up to 9 days we observed larger numbers of surviving cells in Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute Medium (RPMI) compared with Neurobasal Medium (NB). A larger number of A2B5-positive OPCs was found after 6 days in RPMI based media compared with NB. The percentage of bromodeoxyuridine (BrdU)-positive cells was largest in cultures maintained in DMEM and RPMI. The percentage of caspase-3 positive cells was largest in NB, suggesting that this medium inhibits OPC proliferation and favors apoptosis. A difference between NB and DMEM as well as RPMI is the reduced Na(+)-content. The addition of equiosmolar supplements of mannitol or NaCl to NB medium rescued the BrdU-incorporation rate. This suggested that the osmolarity influences the proliferation of OPCs. Plating density as well as residual microglia influence OPC survival, BrdU incorporation, and caspase-3 expression. We found, that high density cultures secrete factors that inhibit BrdU incorporation whereas the presence of additional microglia induces an increase in caspase-3 positive cells, indicative of enhanced apoptosis. An enhanced number of microglia could thus also explain the stronger inhibition of OPC differentiation observed in high density cultures in response to treatment with the cytokines TNF-α and IFN-γ. We conclude that a maximal yield of OPCs is obtained in a medium of an osmolarity higher than 280 mOsm plated at a relatively low density in the presence of as little microglia as technically achievable.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Expansion of natural killer cells with lytic activity against autologous blasts from adult and pediatric acute lymphoid leukemia patients in complete hematologic remissio ... 15951291

    BACKGROUND AND OBJECTIVES: Natural killer (NK) cells constitute an important area of research for hematologic malignancies. The anti-leukemic activity of NK cells against acute myeloid leukemia (AML) blasts has been described, but very few data are available for acute lymphoid leukemia (ALL). The present study was designed to investigate whether: (i) NK effectors could be expanded from adult and pediatric ALL patients in complete remission; (ii) the signal transduction machinery of these cells was preserved; (iii) NK cells showed cytotoxic activity against autologous blasts; (iv) interleukin (IL)-2, IL-12 and IL-15 were able to increase lytic activity in our in vitro model; (v) any differences in cytotoxic activity could be found between expanded effectors from adult and pediatric patients. DESIGN AND METHODS: We co-cultured patients' peripheral blood mononuclear cells (PBMC) with the feeder cell line RPMI 8866 and analyzed the NK cells' expansion capacity by cell count and cytofluorimetric analyses. Signal transduction of expanded effector cells was evaluated by Western blot. 51Cr release assays, before and after stimulation with activating cytokines, were performed to analyze the cytotoxic potential of effector cells against tumor cell lines and autologous blast cells. Data were analyzed with t-tests for paired data. RESULTS: We obtained an average 40-fold increase in NK cells. Signal transduction through the CD16 receptor was preserved. Patients' expanded cells showed cytotoxic activity against target cell lines comparable to that of normal donors. More significantly, these cells also exerted a lytic effect against autologous blasts. In addition, incubating these effectors for 24 hours with IL-2 + IL-15 significantly increased this cytotoxic function. No differences in expansion and cytotoxic activity were found between pediatric and adult patients. INTERPRETATION AND CONCLUSIONS: These findings document for the first time the possibility of expanding ex vivo cytotoxic effectors with autologous killing capacity from ALL patients in remission, and suggest a new potential immunotherapeutic strategy for the management of early disease recurrence or of residual disease.
    Document Type:
    Reference
    Product Catalog Number:
    05-321
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone 4G10®

  • Ad-fRNK and Ad-p53 cooperate to augment drug-induced death of a transformed cell line. 16886630

    BACKGROUND: Transformed cells have abnormalities in the survival pathways contributing to drug resistance. These abnormalities include p53 mutations and increased focal adhesion kinase (FAK) expression. Because FAK regulates cell survival via p53-dependent and -independent pathways, it was hypothesized that combined therapy using wild-type p53 and an inhibitor of FAK (FRNK) would sensitize cells to anticancer drugs. MATERIALS AND METHODS: RPMI 2650 cells were infected with recombinant adenoviruses causing overexpression of p53 (Ad-p53) and FRNK (Ad-FRNK). Cell viability and apoptosis were measured using in vitro assays, whereas protein expression was determined by Western blotting. RESULTS: Co-infection of cells with Ad-p53 and Ad-FRNK induced more cellular apoptosis than transfection with either agent alone. Likewise, the co-transfection of cells with Ad-FRNK and Ad-p53 improved the cytotoxic response to four commonly used anticancer drugs relative to cells transfected with Ad-FRNK alone, Ad-p53 alone, or the equivalent amount of control adenovirus. This effect was associated with loss of endogenous FAK protein.
    Document Type:
    Reference
    Product Catalog Number:
    06-543
    Product Catalog Name:
    Anti-FAK Antibody

  • The roles of nuclear factor of activated T cells and ying-yang 1 in activation-induced expression of the interferon-gamma promoter in T cells. 9857002

    Nuclear factor of activated T cells (NFAT) plays an important role in expression of many cytokine genes including interleukin-2 and interleukin-4. However, its role in interferon-gamma (IFN-gamma) expression is not well understood. In the current studies, two strong NFAT-binding sites in the IFN-gamma promoter were identified by DNase I footprint analysis at positions -280 to -270 and -163 to -155. NFATp bound independently to both sites and was required for the formation of a composite element with AP-1 spanning position -163 to -147. In Jurkat T cells and primary lymphocytes, activation-induced expression of IFN-gamma reporter constructs containing point mutations in either NFAT site or the AP-1 component of the composite site was decreased by approximately 40-65%. Despite elimination of both strong NFAT-binding sites, the IFN-gamma promoter remained completely sensitive to inhibition by cyclosporin. This suggests that other elements in the IFN-gamma promoter, such as the IFN-gamma proximal element, are sufficient for cyclosporin sensitivity of this gene. Ying-Yang 1 (YY1), a potential inhibitor of IFN-gamma expression, binds to sites located between the two NFAT sites. Mutation of the YY1 sites alone had little effect on IFN-gamma promoter activity. However, mutation of both the NFAT and YY1-binding sites abolished activation-induced expression in primary murine splenocytes but not in Jurkat T cells. This suggests that under some conditions, YY1 may play a positive role in activation-induced transcription of IFN-gamma.
    Document Type:
    Reference
    Product Catalog Number:
    ECM600
    Product Catalog Name:
    uPA Activity Assay Kit

  • Pure antiestrogen-induced G1-arrest in myeloma cells results from the reduced kinase activity of cyclin D3/CDK6 complexes whereas apoptosis is mediated by endoplasmic ret ... 18183592

    Multiple myeloma (MM) is a malignancy characterized by the accumulation of tumoral plasma cells in bone marrow. This disease remains incurable and the development of new therapeutic strategies is urgently required. We have studied the effects of 2 selective estrogen receptor disrupters (SERDs), RU 58668 (RU) and ICI 182,780 (ICI) or pure antiestrogens (AEs) on MM cell lines. Both compounds have antimyeloma activity through either cell cycle arrest or induction of apoptosis. To analyze the molecular mechanisms of SERD action, we choose 2 differently responding cell lines as models. In LP-1 cells, RU blocked cell cycle at the G1 phase. RU treatment induced a rapid decrease of c-Myc, an upregulation of p27(Kip1), and the subsequent decreased activity of cyclin-dependent kinase, CDK6 and associated cyclin D3, impairing the inactivation of the retinoblastoma protein (pRb). In RPMI 8226 cells, RU induced apoptosis by recruiting endoplasmic reticulum- as well as mitochondria-associated caspases. Moreover, RU interfered with the NF-kappaB survival pathway, often deregulated in MM malignancy. Antimyeloma activities were observed in dexamethasone (Dex)- and RU-resistant cells when RU was combined with bortezomib; Dex and bortezomib being frequently used in MM therapy. RU induced the death of CD138+ cells purified from MM patients but not CD19+ normal cells obtained from tonsils. Therefore, RU mediates the inhibition of survival, the activation of apoptosis and finally potentiates anticancer drug. Those combinatory effects provide a basis for the potential use of pure AEs in MM treatment.
    Document Type:
    Reference
    Product Catalog Number:
    06-755
    Product Catalog Name:
    Anti-Histone H3 Antibody

  • Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient. 15328152

    We found that supernatants of leukapheresis products (SLPs) of patients mobilized with granulocyte-colony-stimulating factor (G-CSF) or the various components of SLPs (fibrinogen, fibronectin, soluble vascular cell adhesion molecule-1 [VCAM-1], intercellular adhesion molecule-1 [ICAM-1], and urokinase plasminogen activator receptor [uPAR]) increase the chemotactic responses of hematopoietic stem/progenitor cells (HSPCs) to stromal-derived factor-1 (SDF-1). However, alone they do not chemoattract HSPCs, but they do increase or prime the cells' chemotactic responses to a low or threshold dose of SDF-1. We observed that SLPs increased calcium flux, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, secretion of matrix metalloproteinases, and adhesion to endothelium in CD34+ cells. Furthermore, SLPs increased SDF-dependent actin polymerization and significantly enhanced the homing of human cord blood (CB)- and bone marrow (BM)-derived CD34+ cells in a NOD/SCID mouse transplantation model. Moreover, the sensitization or priming of cell chemotaxis to an SDF-1 gradient was dependent on cholesterol content in the cell membrane and on the incorporation of the SDF-1 binding receptor CXCR4 and the small GTPase Rac-1 into membrane lipid rafts. This colocalization of CXCR4 and Rac-1 in lipid rafts facilitated guanosine triphosphate (GTP) binding/activation of Rac-1. Hence, we postulate that CXCR4 could be primed by various factors related to leukapheresis and mobilization that increase its association with membrane lipid rafts, allowing the HSPCs to better sense the SDF-1 gradient. This may partially explain why HSPCs from mobilized peripheral blood leukapheresis products engraft more quickly in patients than do those from BM or CB. Based on our findings, we suggest that the homing of HSPCs is optimal when CXCR4 is incorporated in membrane lipid rafts and that ex vivo priming of HSPCs with some of the SLP-related molecules before transplantation could increase their engraftment.
    Document Type:
    Reference
    Product Catalog Number:
    17-283
    Product Catalog Name:
    Rac1 Activation Assay Kit

  • Cell susceptibility to baculovirus transduction and echovirus infection is modified by protein kinase C phosphorylation and vimentin organization. 23824807

    Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Toxicogenomics in the assessment of immunotoxicity. 17161310

    Microarray analysis is used for simultaneous measurement of expression of thousands of genes in a given sample and as such extends and deepens our understanding of biological processes. Application of the technique in toxicology is referred to as toxicogenomics. The examples of assessment of immunotoxicity by gene expression profiling presented and discussed here, show that microarray analysis is able to detect known and novel effects of a wide range of immunomodulating agents. Besides the elucidation of mechanisms of action, toxicogenomics is also applied to predict consequences of exposing biological systems to toxic agents. Successful attempts to classify compounds using signature gene expression profiles have been reported. These did, however, not specifically focus on immunotoxicity. Databases containing expression profiles can facilitate the applications of toxicogenomics. Platforms and methodologies for gene expression profiling may vary, however, hampering data compiling across different laboratories. Therefore, attention is paid to standardization of the generation, reporting, and management of microarray data. Obtained gene expression profiles should be anchored to pathological and functional endpoints for correct interpretation of results. These issues are also important when using toxicogenomics in risk assessment. The application of toxicogenomics in evaluation of immunotoxicity is thus not yet without challenges. It already contributes to the understanding of immunotoxic processes and the development of in vitro screening assays, though, and is therefore expected to be of value for mechanistic insight into immunotoxicity and hazard identification of existing and novel compounds.
    Document Type:
    Reference
    Product Catalog Number:
    S7110
    Product Catalog Name:
    ApopTag® Fluorescein In Situ Apoptosis Detection Kit

  • Significant impact of survivin on myeloma cell growth. 17315024

    Survivin is a fascinating member of the inhibitor of apoptosis protein (IAP) family with its dual roles in mitosis and apoptosis, and emerges as an attractive target for cancer therapy. Multiple myeloma (MM) is a plasma cell malignancy, characterized by deregulated proliferation, cell-death processes and fatal outcome. We thus investigated survivin expression in myeloma cells and its role in MM biology to evaluate its potential interest as a target in MM treatment. Our results describe the cancer-specific overexpression of survivin in myeloma cells and show a significant correlation between survivin expression at protein level and clinical course of MM. Moreover, survivin knockdown by RNA interference led to growth rate inhibition of myeloma cells related to apoptosis induction and deep cell-cycle disruption. Finally, survivin knockdown sensitized myeloma cells to conventional anti-myeloma agents. Altogether, these data argue for the interest to evaluate survivin antagonists in MM treatment.
    Document Type:
    Reference
    Product Catalog Number:
    LP1
    Product Catalog Name:
    VLDL, human