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  • DOG1 for the diagnosis of gastrointestinal stromal tumor (GIST): Comparison between 2 different antibodies. 20571340

    Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. Discovered on GIST-1 (DOG1) is a recently described protein expressed in GISTs irrespective of mutation status. The aim of this study was to investigate the immunohistochemical expression of DOG1 using 2 different monoclonal antibodies (DOG1.1 and the commercially available K9 antibody) in 668 GIST cases and to compare the results with the expression of KIT. DOG1 and KIT expression also were studied in most human normal tissues and several nonmesenchymal and mesenchymal tumors other than GIST. KIT was expressed in 643 (96.3%) GISTs. DOG1.1 and K9 were positive in 538 (80.5%) and 642 (96.1%) GIST cases, respectively. In 25 (3.7%) KIT-negative GIST cases, DOG1 was expressed in 5 (20.0%) and 19 (76.0%) using DOG1.1 and K9 antibodies, respectively. Only 0.9% of GISTs were negative for KIT, DOG1.1, and K9. Most normal human tissues did not reveal KIT and DOG1 expression. DOG1.1 was positive in only 2 of 57 synovial sarcomas and 1 of 61 soft tissue leiomyosarcomas. K9 was positive in 5 of 57 synovial sarcomas, 1 of 14 angiosarcomas, 1 of 61 soft tissue leiomyosarcomas, 3 of 4 adenoid cystic carcinomas of the head and neck, and in myoepithelial cells of 9 of 11 fibroadenomas of the breast. In conclusion, the commercially available K9 is of great utility for the diagnosis of most KIT-negative GISTs, and the combination of both KIT and K9 antibody in a panel of immunohistochemistry can define the diagnosis of GIST in more than 99% of cases.
    Document Type:
    Reference
    Product Catalog Number:
    MABC36

  • A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer. 21262398

    Cancer secretomes are a promising source for biomarker discovery. The analysis of cancer secretomes still faces some difficulties mainly related to the intracellular contamination, which hinders the qualification and follow-up validations. This study aimed to establish a high-quality secretome of A549 cells by using the cellular proteome as a reference and to test the merits of this refined secretome for biomarker discovery for non-small cell lung cancer (NSCLC). Using one-dimensional gel electrophoresis followed by liquid-chromatography tandem mass spectrometry, we comprehensively investigated the secretome and the concurrent cellular proteome of A549 cells. A high-quality secretome consisting of 382 proteins was refined from 889 initial secretory proteins. More than 85.3% of proteins were annotated as secreted and 76.8% as extracellular or membrane-bound. The discriminative power of the lung-cancer associated secretome was confirmed by gene expression and serum proteomic data. The elevated level of C4b-binding Protein (C4BP) in NSCLC blood was verified by enzyme-linked immunosorbent assays (ELISA, p = 6.07e-6). Moreover, the serum C4BP level in 89 patients showed a strong association with the clinical staging of NSCLC. Our reference-experiment-driven strategy is simple and widely applicable, and may facilitate the identification of novel promising biomarkers of lung cancer.
    Document Type:
    Reference
    Product Catalog Number:
    09-272
    Product Catalog Name:
    Anti-phospho-p38α (Thr180/Tyr182) Antibody

  • ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta. 18680760

    We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of (14)C-PhIP (2 microM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72+/-0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of (14)C-PhIP from maternal to fetal circulation (FM ratio 0.90+/-0.08 at 6 h, p<0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75+/-0.10, p=0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of (14)C-PhIP (R=-0.81, p<0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: -0.11, p=0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of (14)C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarcinoma cells.
    Document Type:
    Reference
    Product Catalog Number:
    MAB4148
    Product Catalog Name:
    Anti-MRP2 Antibody, clone M2 I-4

  • Effect of myocardial volume overload and heart failure on lactate transport into isolated cardiac myocytes. 12571142

    The purpose of this study was to determine lactate transport kinetics in single isolated rat ventricular cardiac myocytes after 1) 8 wk of myocardial volume overload (MVO) and 2) congestive heart failure (CHF). Twenty male Sprague-Dawley rats were assigned to one of four groups: myocardial hypertrophy (MH), MH sham (MHS), CHF, or CHF sham (CHFS). A chronic MVO was induced in the MH and CHF groups by an infrarenal arteriovenous fistula. Postdeath heart and lung weights were significantly greater (P 0.05) for the MH and CHF groups compared with controls. Isolated cardiac myocytes were loaded with BCECF to determine intracellular pH (pH(i)) changes after the addition of lactate to the extracellular superfusate. Alterations in pH(i) with the addition of varied lactate concentrations were attenuated 72-89% by 5.0 mM alpha-cyano-4-hydroxycinnamate. Significant differences (P 0.05) were found in estimated maximal lactate transport rates between the experimental and sham groups (MH = 19.4 +/- 1.1 nmol x microl(-1) x min(-1) vs. MHS = 15.1 +/- 1.1 nmol x microl(-1) x min(-1); CHF = 20.2 +/- 2.0 nmol x microl(-1) x min(-1) vs. CHFS = 14.0 +/- 0.9 nmol x microl(-1) x min(-1)). Western blot analysis confirmed a 270% increase in monocarboxylate symport protein 1 (MCT1) protein content in CHF compared with CHFS rats. The results of this study suggest that MH and CHF induced by MVO engender a greater maximal lactate transport capacity across the cardiac myocyte sarcolemma along with an increase in MCT1 protein content. These alterations would likely benefit the cell by attenuating intracellular acidification during a period of increased myocardial load.
    Document Type:
    Reference
    Product Catalog Number:
    AB1286

  • Nanoparticle based delivery of hypoxia-regulated VEGF transgene system combined with myoblast engraftment for myocardial repair. 21216458

    A regulated promoter system to control gene expression is desirable for safe and efficacious over-expression of therapeutic transgene. Combined with skeletal myoblast (SkMs), we report the efficacy of hypoxia-regulated VEGF gene delivery for myocardial repair during acute myocardial infarction (AMI). A hypoxia-regulated VEGF plasmid (pHRE-VEGF) was developed. After optimization, ∼30% SkMs were transfected using polyethyleneimine (PEI) nanoparticles. The peak VEGF expression was higher in pHRE-VEGF transfected SkMs ((VEGF)SkMs) under hypoxia (151.34 ± 8.59 ng/ml) than that with normoxia (16.92 ± 2.74 ng/ml). The efficacy of hypoxia-regulated gene expression system was assessed in a rabbit model of AMI. The animals were grouped to receive basal M199 without cells (group-1) or containing non-transfected SkMs (group-2) or (VEGF)SkMs (group-3). In group-4, (VEGF)SkMs were injected into normal heart to serve as normoxia control. Improved SkM survival was observed in group-3 and -4 (p < 0.05 vs group-2) at day-3 and 7 after transplantation. Blood vessel density was 20.1 ± 1.3 in group-3 which was significantly higher than any other groups (p < 0.05) at 2 weeks after treatment. Improved blood flow (ml/min/g) in the left ventricle (LV) anterior wall was observed in group-3 (1.28 ± 0.09, p < 0.05) as compared with group-1 (0.76 ± 0.05) and group-2 (0.96 ± 0.06), and similar to group-4 (1.26 ± 0.05). LV ejection fraction was best preserved in group-3 (58.4 ± 1.75%) which was insignificantly different from group-4 (61.1 ± 1.8%), and group-2 (52.8 ± 1.4%), but significantly improved compared with group-1 (44.7 ± 2.2%, p < 0.05). The study demonstrates that nanoparticle based delivery of hypoxia-regulated VEGF transgene combined with SkMs during AMI effectively preserves LV regional blood flow and contractile function of the heart.Copyright © 2010 Elsevier Ltd. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1628
    Product Catalog Name:
    Anti-Myosin Antibody, slow muscle, clone NOQ7.5.4D

  • Ageing is associated with deterioration of calcium homeostasis in isolated human right atrial myocytes. 25712961

    Ageing-related cardiac disorders such as heart failure and atrial fibrillation often present with intracellular calcium homeostasis dysfunction. However, knowledge of the intrinsic effects of ageing on cellular calcium handling in the human heart is sparse. Therefore, this study aimed to analyse how ageing affects key mechanisms that regulate intracellular calcium in human atrial myocytes.Whole membrane currents and intracellular calcium transients were measured in isolated human right atrial myocytes from 80 patients with normal left atrial dimensions and no history of atrial fibrillation. Patients were categorized as young (less than 55 years, n = 21), middle aged (55-74 years, n = 42), and old (≥75 years, n = 17). Protein levels were determined by western blot. Ageing was associated with the following electrophysiological changes: (i) a 3.2-fold decrease in the calcium transient (P less than 0.01); (ii) reduction of the L-type calcium current (ICa) amplitude (2.4 ± 0.3 pA/pF vs. 1.4 ± 0.2 pA/pF, P less than 0.01); (iii) lower levels of L-type calcium channel alpha-subunit (P less than 0.05); (iv) lower rates of both fast (14.5 ± 0.9 ms vs. 20.9 ± 1.9, P less than 0.01) and slow (73 ± 3 vs. 120 ± 12 ms, P less than 0.001) ICa inactivation; and (v) a decrease in the sarcoplasmic reticulum calcium content (10.1 ± 0.8 vs. 6.4 ± 0.6 amol/pF, P less than 0.005) associated with a significant decrease in both SERCA2 (P less than 0.05) and calsequestrin-2 (P less than 0.05) protein levels. In contrast, ageing did not affect spontaneous sarcoplasmic reticulum calcium release.Ageing is associated with depression of SR calcium content, L-type calcium current, and calcium transient amplitude that may favour a progressive decline in right atrial contractile function with age.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Vector Choice Determines Immunogenicity and Potency of Genetic Vaccines Against Angola Marburgvirus in Nonhuman Primates. 20660192

    The immunogenicity and durability of genetic vaccines is influenced by the composition of gene inserts and choice of delivery vector. DNA vectors are a promising vaccine approach showing efficacy when combined in prime-boost regimens with recombinant protein or viral vectors, but they have shown limited comparative efficacy as a stand-alone platform in primates, due possibly to suboptimal gene expression or cell targeting. Here, regimens using DNA plasmids modified for optimal antigen expression and rAd vectors, all encoding the glycoprotein (GP) gene from Marburg virus (MARV) Angola were compared for their ability to provide immune protection against lethal MARV Angola infection. Heterologous DNA-GP/rAd5-GP prime-boost and single modality rAd5-GP, as well as the DNA-GP only vaccine prevented death in all vaccinated subjects after challenge with a lethal dose of MARV Angola. The DNA/DNA vaccine induced humoral responses comparable to a single inoculation with rAd5-GP, as well as CD4(+) and CD8(+) cellular immune responses, with skewing toward CD4(+) T-cell activity against MARV GP. Vaccine regimens containing rAd-GP, alone or as a boost, exhibited cellular responses with CD8(+) T cell dominance. Across vaccine groups, CD8(+) T-cell subset dominance comprising cells exhibiting a TNF-alpha and IFN-gamma double-positive functional phenotype was associated with an absence or low frequency of clinical symptoms, suggesting that both the magnitude and functional phenotype of CD8(+) T-cells may determine vaccine efficacy against infection by MARV Angola.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit