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  • Lysis of leukemic cells by human macrophages: inhibition by 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor. 8830789

    Proteases are known to be involved in regulation of macrophage activation and killing. We examined the effect of a serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), on lysis of leukemic cells by human macrophages. Monocytes, isolated by Histopaque gradients and centrifugal elutriation, were cultured for 5 days in RPMI-1640 medium with 5% AB serum, and then activated with interferon-gamma (IFN-gamma; 100 U/mL) and lipopolysaccharide (LPS) (5 ng/mL), with or without AEBSF, for 2 days. On day 7, macrophages were washed, fresh medium without AEBSF added, and target cells added for 2 days. Lytic activity against two leukemic cell lines (K562 and HL-60) was assessed by an 111indium-releasing assay. Macrophages treated with IFN-gamma + LPS lysed K562 and HL-60 cells. AEBSF (50-150 microM) blocked the killing of these leukemic cells in a concentration-dependent manner. Other protease inhibitors were not effective. AEBSF was nontoxic at the concentrations used, and did not inhibit tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion from the macrophages. The lytic activity against leukemic cells was inhibited by anti-TNF-alpha antibody, but not by anti-IL-1 beta, nor by superoxide dismutase or catalase. However, the leukemic cells were resistant to being killed by recombinant TNF-alpha alone in the absence of macrophages, indicating that TNF-alpha was required for killing, but that other factors that were inhibited by AEBSF were also required. Serum-free culture supernatant of activated macrophages had significant cytotoxic activity against leukemic cells. This cytotoxic activity was not altered by addition of AEBSF to the culture supernatant, suggesting that AEBSF affected macrophage activation, rather than inhibiting cytotoxic proteases secreted by the macrophages, or affecting the target cells themselves. Thus, a protease, which is susceptible to AEBSF, might be involved in the activation of macrophages, and might regulate the secretion of antitumor effector molecules other than TNF-alpha.
    Document Type:
    Reference
    Product Catalog Number:
    20-176
    Product Catalog Name:
    100X GTPγS, 10mM

  • 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

  • Long-term exposure of INS-1 rat insulinoma cells to linoleic acid and glucose in vitro affects cell viability and function through mitochondrial-mediated pathways. 21161439

    Obesity with excessive levels of circulating free fatty acids (FFAs) is tightly linked to the incidence of type 2 diabetes. Insulin resistance of peripheral tissues and pancreatic β-cell dysfunction are two major pathological changes in diabetes and both are facilitated by excessive levels of FFAs and/or glucose. To gain insight into the mitochondrial-mediated mechanisms by which long-term exposure of INS-1 cells to excess FFAs causes β-cell dysfunction, the effects of the unsaturated FFA linoleic acid (C 18:2, n-6) on rat insulinoma INS-1 β cells was investigated. INS-1 cells were incubated with 0, 50, 250 or 500 μM linoleic acid/0.5% (w/v) BSA for 48 h under culture conditions of normal (11.1 mM) or high (25 mM) glucose in serum-free RPMI-1640 medium. Cell viability, apoptosis, glucose-stimulated insulin secretion, Bcl-2, and Bax gene expression levels, mitochondrial membrane potential and cytochrome c release were examined. Linoleic acid 500 μM significantly suppressed cell viability and induced apoptosis when administered in 11.1 and 25 mM glucose culture medium. Compared with control, linoleic acid 500 μM significantly increased Bax expression in 25 mM glucose culture medium but not in 11.1 mM glucose culture medium. Linoleic acid also dose-dependently reduced mitochondrial membrane potential (ΔΨm) and significantly promoted cytochrome c release from mitochondria in both 11.1 mM glucose and 25 mM glucose culture medium, further reducing glucose-stimulated insulin secretion, which is dependent on normal mitochondrial function. With the increase in glucose levels in culture medium, INS-1 β-cell insulin secretion function was deteriorated further. The results of this study indicate that chronic exposure to linoleic acid-induced β-cell dysfunction and apoptosis, which involved a mitochondrial-mediated signal pathway, and increased glucose levels enhanced linoleic acid-induced β-cell dysfunction.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1800

  • 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

  • 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

  • A protein profile study to discriminate CIN lesions from normal cervical epithelium. 21573931

    Cervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN.Cervical punch biopsies from 55 women were incubated for 24 h at 4°C in RPMI1640 medium for protein analysis prior to usual FFPE processing and p16 and Ki67-supported histologic consensus diagnosis was assessed. The biopsy supernatants were subjected to surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) for identifying differentially expressed proteins. Binary logistic regression and classification and regression trees (CART) were used to develop a classification model.The age of the patients ranged from 26 to 40 years (median 29.7). The consensus diagnoses were normal cervical tissue (n = 10) and CIN2-3 (n = 45). The mean protein concentration was 1.00 and 1.09 mg/ml in the normal and CIN2-3 group, respectively. The peak detection and clustering process resulted in 40 protein peaks. Many of these peaks differed between the two groups, but only three had independent discriminating power. The overall classification results were 88%.Water-soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and CIN2-3.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Discrimination of grade 2 and 3 cervical intraepithelial neoplasia by means of analysis of water soluble proteins recovered from cervical biopsies. 21711556

    BACKGROUND:
    Cervical intraepithelial neoplasia (CIN) grades 2 and 3 are usually grouped and treated in the same way as "high grade", in spite of their different risk to cancer progression and spontaneous regression rates. CIN2-3 is usually diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. This procedure virtually eliminates the availability of water-soluble proteins which could have diagnostic and prognostic value.

    AIM:
    To investigate whether a water-soluble protein-saving biopsy processing method followed by a proteomic analysis of supernatant samples using LC-MS/MS (LTQ Orbitrap) can be used to distinguish between CIN2 and CIN3.

    METHODS:
    Fresh cervical punch biopsies from 20 women were incubated in RPMI1640 medium for 24 hours at 4°C for protein extraction and subsequently subjected to standard FFPE processing. P16 and Ki67-supported histologic consensus review CIN grade (CIN2, n = 10, CIN3, n = 10) was assessed by independent gynecological pathologists. The biopsy supernatants were depleted of 7 high abundance proteins prior to uni-dimensional LC-MS/MS analysis for protein identifications.

    RESULTS:
    The age of the patients ranged from 25-40 years (median 29.7), and mean protein concentration was 0.81 mg/ml (range 0.55 - 1.14). After application of multistep identification criteria, 114 proteins were identified, including proteins like vimentin, actin, transthyretin, apolipoprotein A-1, Heat Shock protein beta 1, vitamin D binding protein and different cytokeratins. The identified proteins are annotated to metabolic processes (36%), signal transduction (27%), cell cycle processes (15%) and trafficking/transport (9%). Using binary logistic regression, Cytokeratin 2 was found to have the strongest independent discriminatory power resulting in 90% overall correct classification.

    CONCLUSIONS:
    114 proteins were identified in supernatants from fresh cervical biopsies and many differed between CIN2 and 3. Cytokeratin 2 is the strongest discriminator with 90% overall correct classifications.
    Document Type:
    Reference
    Product Catalog Number:
    07-330
    Product Catalog Name:
    Anti-Partitioning-defective 3 Antibody
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