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  • Application of multiplex PCR for characterization of human embryonic stem cells (hESCs) and its differentiated progenies. 20530724

    Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Ghrelin vaccination decreases plasma MCP-1 level in LDLR(-/-)-mice. 19751783

    Ghrelin is a novel peptide hormone having growth hormone releasing activity and many endocrine and metabolic functions. In rats and pigs, ghrelin immunizations have recently been shown to induce an antibody response against ghrelin simultaneously with a decrease in body weight gain. Our aim was to test the role of ghrelin immunization on atherosclerosis and weight gain in mice. LDLR(-/-)-mice (n=36) were immunized with ghrelin-PADRE, PADRE alone and PBS and then placed on a high fat diet for 22 weeks. Weight gain and food intake were followed throughout the study. Acylated and total ghrelin, cytokines and MCP-1 were analyzed from plasma using commercial kits. Stomach ghrelin was assessed using qRT-PCR and immunohistochemistry. Atherosclerosis was determined from aorta and cross-sections at the end of study. Mice immunized with ghrelin-PADRE developed high plasma IgG titers to ghrelin simultaneously with a significant increase in plasma acylated and total ghrelin levels. Plasma MCP-1 levels decreased in mice immunized with ghrelin-PADRE compared to mice immunized with PADRE and PBS. There were no differences in atherosclerosis determined from aorta and cross-sections as well as in body weights and food intake in LDLR(-/-)-mice between the different immunization groups. Our data indicates that ghrelin-PADRE vaccination induces a strong exclusive IgG response to ghrelin and increases plasma acylated and total ghrelin levels in mice. Ghrelin vaccination decreases plasma MCP-1 levels even though no effects on developing signs of atherosclerosis or weight gain in mice were observed.
    Document Type:
    Reference
    Product Catalog Number:
    EZRMI-13K
    Product Catalog Name:
    Rat/Mouse Insulin ELISA

  • Activation of platelet-activating factor receptor and pleiotropic effects on tyrosine phospho-EGFR/Src/FAK/paxillin in ovarian cancer. 18632638

    Among the proinflammatory mediators, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a major primary and secondary messenger involved in intracellular and extracellular communication. Evidence suggests that PAF plays a significant role in oncogenic transformation, tumor growth, angiogenesis, and metastasis. However, PAF, with its receptor (PAFR) and their downstream signaling targets, has not been thoroughly studied in cancer. Here, we characterized the PAFR expression pattern in 4 normal human ovarian surface epithelial (HOSE) cell lines, 13 ovarian cancer cell lines, paraffin blocks (n = 84), and tissue microarrays (n = 230) from patients with ovarian cancer. Overexpression of PAFR was found in most nonmucinous types of ovarian cancer but not in HOSE and mucinous cancer cells. Correspondingly, PAF significantly induced cell proliferation and invasion only in PAFR-positive cells (i.e., OVCA429 and OVCA432), but not in PAFR-negative ovarian cells (HOSE and mucinous RMUG-L). The dependency of cell proliferation and invasion on PAFR was further confirmed using PAFR-specific small interfering RNA gene silencing probes, antibodies against PAFR and PAFR antagonist, ginkgolide B. Using quantitative multiplex phospho-antibody array technology, we found that tyrosine phosphorylation of EGFR/Src/FAK/paxillin was coordinately activated by PAF treatment, which was correlated with the activation of phosphatidylinositol 3-kinase and cyclin D1 as markers for cell proliferation, as well as matrix metalloproteinase 2 and 9 for invasion. Specific tyrosine Src inhibitor (PP2) reversibly blocked PAF-activated cancer cell proliferation and invasion. We suggest that PAFR is an essential upstream target of Src and other signal pathways to control the PAF-mediated cancer progression.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Effects of ileal interposition on glucose metabolism in obese rats with diabetes. 22316438

    Ileal interposition (IT), in which the distal ileum is transposed isoperistaltically into the proximal jejunum, is considered as a procedure for metabolic or antidiabetes surgery. Our aim was to study the effects of IT on glycemic control, fat metabolism, and hormonal changes in obese rats with spontaneous diabetes.
    Document Type:
    Reference
    Product Catalog Number:
    EZRMGIP-55K
    Product Catalog Name:
    Rat/Mouse GIP (total) ELISA

  • Mesenchymal stem cells secrete multiple cytokines that promote angiogenesis and have contrasting effects on chemotaxis and apoptosis. 22558198

    We have previously shown that mesenchymal stem cells (MSC) improve function upon integration in ischemic myocardium. We examined whether specific cytokines and growth factors produced by MSCs are able to affect angiogenesis, cellular migration and apoptosis. Conditioned media (CM) was prepared by culturing MSC for 48 hours. CM displayed significantly elevated levels of VEGF, Monocyte Chemoattractant Protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monokine induced by IFN-γ (MIG) compared to control media. MSC contained RNA for these factors as detected by RT-PCR. CM was able to induce angiogenesis in canine vascular endothelial cells. MCP-1 and MIP-1α increased cell migration of MSC while VEGF reduced it. H9c2 cells treated with CM under hypoxic conditions for 24 hours displayed a 16% reduction in caspase-3 activity compared to controls. PI 3-kinase γ inhibitor had no effect on controls but reversed the effect of CM on caspase-3 activity. MCP-1 alone mimicked the protective effect of CM while the PI 3-Kγ inhibitor did not reverse the effect of MCP-1. CM reduced phospho-BAD (Ser112) and phospho-Akt (Ser473) while increasing phospho-Akt (Thr308). MCP-1 reduced the level of phospho-Akt (Ser473) while having no effect on the other two; the PI 3-Kγ inhibitor did not alter the MCP-1 effect. ERK 1/2 phosphorylation was reduced in CM treated H9c2 cells, and inhibition of ERK 1/2 reduced the phosphorylation of Akt (Ser473), Akt (Thr308) and Bad (Ser112). In conclusion, MSC synthesize and secrete multiple paracrine factors that are able to affect MSC migration, promote angiogenesis and reduce apoptosis. While both MCP-1 and PI3-kinase are involved in the protective effect, they are independent of each other. It is likely that multiple pro-survival factors in addition to MCP-1 are secreted by MSC which act on divergent intracellular signaling pathways.
    Document Type:
    Reference
    Product Catalog Number:
    ECM630
    Product Catalog Name:
    Fibrin In Vitro Angiogenesis Assay

  • The beta-catenin axis integrates multiple signals downstream from RET/papillary thyroid carcinoma leading to cell proliferation. 19223551

    RET/papillary thyroid carcinoma (RET/PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase domain with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC stimulates the beta-catenin pathway. By stimulating PI3K/AKT and Ras/extracellular signal-regulated kinase (ERK), RET/PTC promotes glycogen synthase kinase 3beta (GSK3beta) phosphorylation, thereby reducing GSK3beta-mediated NH(2)-terminal beta-catenin (Ser33/Ser37/Thr41) phosphorylation. In addition, RET/PTC physically interacts with beta-catenin and increases its phosphotyrosine content. The increased free pool of S/T(nonphospho)/Y(phospho)beta-catenin is stabilized as a result of the reduced binding affinity for the Axin/GSK3beta complex and activates the transcription factor T-cell factor/lymphoid enhancer factor. Moreover, through the ERK pathway, RET/PTC stimulates cyclic AMP-responsive element binding protein (CREB) phosphorylation and promotes the formation of a beta-catenin-CREB-CREB-binding protein/p300 transcriptional complex. Transcriptional complexes containing beta-catenin are recruited to the cyclin D1 promoter and a cyclin D1 gene promoter reporter is active in RET/PTC-expressing cells. Silencing of beta-catenin by small interfering RNA inhibits proliferation of RET/PTC-transformed PC Cl3 thyrocytes, whereas a constitutively active form of beta-catenin stimulates autonomous proliferation of thyroid cells. Thus, multiple signaling events downstream from RET/PTC converge on beta-catenin to stimulate cell proliferation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • The medicinal plant goldenseal is a natural LDL-lowering agent with multiple bioactive components and new action mechanisms. 16885565

    Our previous studies have identified berberine (BBR), an alkaloid isolated from the Chinese herb huanglian, as a unique cholesterol-lowering drug that upregulates hepatic low density lipoprotein receptor (LDLR) expression through a mechanism of mRNA stabilization. Here, we demonstrate that the root extract of goldenseal, a BBR-containing medicinal plant, is highly effective in upregulation of liver LDLR expression in HepG2 cells and in reducing plasma cholesterol and low density lipoprotein cholesterol (LDL-c) in hyperlipidemic hamsters, with greater activities than the pure compound BBR. By conducting bioassay-driven semipurifications, we demonstrate that the higher potency of goldenseal is achieved through concerted actions of multiple bioactive compounds in addition to BBR. We identify canadine (CND) and two other constituents of goldenseal as new upregulators of LDLR expression. We further show that the activity of BBR on LDLR expression is attenuated by multiple drug resistance-1 (MDR1)-mediated efflux from liver cells, whereas CND is resistant to MDR1. This finding defines a molecular mechanism for the higher activity of CND than BBR. We also provide substantial evidence to show that goldenseal contains natural MDR1 antagonist(s) that accentuate the upregulatory effect of BBR on LDLR mRNA expression. These new findings identify goldenseal as a natural LDL-c-lowering agent, and our studies provide a molecular basis for the mechanisms of action.
    Document Type:
    Reference
    Product Catalog Number:
    ECM910
    Product Catalog Name:
    MDR1 Efflux Assay

  • Polycomb target genes are silenced in multiple myeloma. 20634887

    Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb-targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Increases in mitochondrial biogenesis impair carcinogenesis at multiple levels. 21855427

    Although mitochondrial respiration is decreased in most cancer cells, the role of this decrease in carcinogenesis and cancer progression is still unclear. To better understand this phenomenon, instead of further inhibiting mitochondrial function, we induced mitochondrial biogenesis in transformed cells by activating the peroxisome proliferator-activated receptors (PPARs)/peroxisome proliferator-activated receptor gamma co-activator 1? (PGC-1?) pathways. This was achieved by treating the cells with bezafibrate, a PPARs panagonist that also enhances PGC-1? expression. We confirmed that bezafibrate treatment led to increased mitochondrial proteins and enzyme functions. We found that cells with increased mitochondrial biogenesis had decreased growth rates in glucose-containing medium. In addition, they became less invasive, which was directly linked to the reduced lactate levels. Surprisingly, even though bezafibrate-treated cells had higher levels of mitochondrial markers, total respiration was not significantly altered. However, respiratory coupling, and ATP levels were. Our data show that by increasing the efficiency of the mitochondrial oxidative phosphorylation system, cancer progression is hampered by decreases in cell proliferation and invasiveness.
    Document Type:
    Reference
    Product Catalog Number:
    ECM555
    Product Catalog Name:
    QCM ECMatrix Cell Invasion Assay, 96-well (8 µm), fluorimetric